Charles W Putnam

Interests

Research

As described earlier, my current research interests include: the genomic basis of early onset colorectal carcinoma, targeted immunotherapy of cancer, chemotherapeutic drug synergism with repurposed agents, and the role of so-called "14-3-3 proteins" in oncogenesis. Since joining the ICT, my work focuses on islet cell transplantation for Type 1 diabetes.

Teaching

Although I no longer have formal teaching or course assignments, I continue to mentor individuals at every academic level from high school students to senior faculty.

Courses

No activities entered.

Scholarly Contributions

Journals/Publications

• Einstein, S. A., Steyn, L. V., Weegman, B. P., Suszynski, T. M., Sambanis, A., O???Brien, T. D., Avgoustiniatos, E. S., Firpo, M. T., Graham, M. L., Janecek, J., Eberly, L. E., Garwood, M., Putnam, C. W., & Papas, K. K. (2023). Hypoxia within subcutaneously implanted macroencapsulation devices limits the viability and functionality of densely loaded islets. Frontiers in Transplantation, 2.
• Papas, K. K., Putnam, C. W., Johnson, R. C., & Sambanis, A. (2023). Encapsulated Cells for the Treatment of Diabetes: Danger of Acute Hypoglycemia Following Injury?. Cell transplantation, 32, 9636897231163233.
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  Transplants comprised of encapsulated islets have shown promise in treating insulin-dependent diabetes. A question raised in the scientific and clinical communities is whether the insulin released from an implanted encapsulation device damaged in an accident could cause a serious hypoglycemic event. In this commentary, we consider the different types of damage that a device can sustain, including the encapsulation membrane and the islets within, and the amount of insulin released in each case. We conclude that the probability that device damage would cause an adverse hypoglycemic event is indeed very low.
• Putnam, C. W. (2023).

  116.2: Dynamic insulin/glucagon measurements from human islet perifusion

  . Transplantation. doi:10.1097/01.tp.0000993832.82292.21
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  Introduction: The islets of Langerhans of the pancreas (multicellular spheroidal organoids constituting 1-3% of the pancreas volume) are the primary endocrine cells regulating glucose homeostasis via two primary hormones, insulin, and glucagon. Insulin is secreted from beta cells and glucagon from alpha cells. The two hormones have opposing effects on glucose homeostasis: insulin promotes glucose uptake into several cell types and thus lowers blood glucose levels; glucagon drives glucose production in the liver, thus raising glucose levels. Secretion of both hormones is a highly dynamic process, involving complex mechanisms including crosstalk within islets, which are not fully understood. Most of these dynamic processes have been studied primarily with rodent islets, whereas human islets studies are limited. Methods: For our study, we used a Biorep perifusion system to collect samples for the concurrent measurements of insulin and glucagon secreted from human islets exposed to two glucose concentrations, as well as KCl or arginine stimulation. Human islets were isolated at the Institute for Cellular Transplantation at the University of Arizona. Perifusions were performed on 25 individual islet isolations in which insulin and glucagon were simultaneously measured using a multiplex assay. Results: Glucose stimulation (16.7mM) caused a biphasic insulin response with the 1st phase peaking ~ 2 minutes and lasting 6-8 minutes, followed by a sustained 2nd phase. As predicted, glucagon levels decreased during glucose stimulation and remained repressed until islets were again exposed to low glucose (2.8mM). KCL (30mM) was used to mimic depolarization, which induced a monophasic peak for both glucagon and insulin secretion. Arginine stimulation (10mM) under low glucose conditions initiated a biphasic glucagon response and a modest monophasic increase in insulin. Conclusions: Our data provides a physiological template to assess islet function for clinical and research use, as well as a method to test novel drugs to potentiate/inhibit secretion of these hormones. We would like to thank the members of the Institute for Cellular Transplantation at the University of Arizona for their technical support and contributions to this research. Partial funding from NIH/NIDDK (1DP3DK106933-01, 1R43DK113537-01) and JDRF (1-PNF-2018-520-SB, 1-PNF-2018-519-SB).
• Putnam, C. W. (2023).

  401.2: Effects of hyperoxia on human and rat islets

  . Transplantation, 107(10S2), 159-159. doi:10.1097/01.tp.0000994636.20851.f5
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  Background: Islet transplantation of encapsulated cells within an immunoprotective device is a promising approach to cure Type 1 diabetes without the need for lifelong immunosuppression. Unfortunately, in order to use a device of a reasonable size, the large number of cells required for diabetes reversal dictates high-cell packing density, which results in oxygen limitations affecting the viability and functionality of the encapsulated cells. Oxygen at a high concentration delivered to encapsulated cells can help reduce the hypoxia induced reduction of encapsulated cell viability and function. However, hyperoxia can also be detrimental to cells, so the toxicity of high oxygen exposure needs to be investigated so as to establish safe levels of oxygen exposure. In this study, we investigated rat and human islets exposed to different concentrations of oxygen and evaluated the viability and function of the cells at various time points. Methods: Human and rat islets were exposed to normoxic and hyperoxic conditions (40, 60, and 95%) for up to 7 days. Islet viability was assessed by oxygen consumption rate (OCR) and membrane integrity staining with fluorescein diacetate and propidium iodide. Islet function was assessed by dynamic glucose stimulated insulin secretion (GSIS) via perifusion (Biorep). Results: In hyperoxic 40% culture, neither OCR nor GSIS were changed in human or rat islets for as long as 7 days of exposure relative to normoxic control. In hypoxic 60% culture, neither OCR nor GSIS were changed in rat islets relative to normoxic control; however, OCR was reduced by ~15% after 1 day of exposure and by ~30% after 7 days of exposure in human islets. GSIS was also significantly reduced as early as 1 day of exposure. Hyperoxic 95% culture significantly reduced both OCR and GSIS in both rat and human islets after 1 day of exposure and with a very small fraction of surviving cells after 7 days of exposure. Conclusions: Supplemental oxygen, particularly in the peri-transplant period, will be essential to support enough insulin secreting cells within a reasonably sized encapsulation device for the treatment of Type 1 diabetes in a research animal model or human patients. Our data indicates that up to 40% oxygen can be supplied to rat and human islets and it is not detrimental to viability or function. However, hyperoxia of >60% can be toxic to human islets with as little as 24 hours of exposure. Future work will be aimed at further defining the “safe” oxygen exposure limits at which islet viability and function are maintained for primary and stem cell derived islets. We would like to thank all of the members of the Papas Laboratory at ICT including Jose Cano and Barry Huey. Work was performed with funding from NIH/NIDDK (1DP3DK106933-01, 1R43DK113537-01), JDRF (3-SRA-2015-40-Q-R, 1-PNF-2018-520-SB, 1-PNF-2018-519-SB), and Procyon Technologies LLC.
• Putnam, C. W. (2023).

  401.3: The development of a sheep model for encapsulated cell therapies

  . Transplantation, 107(10S2), 159-159. doi:10.1097/01.tp.0000994640.18377.bb
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  Introduction: Cell therapy has become increasingly studied for therapeutic use in drug delivery and regenerative medicine. Immunoisolating devices intended for implantation of cells require construction of membranes that can safely contain transplanted cells, provide a barrier to the host immune system, and induce close vessel formation. Sheep make an excellent model for translational research because of their nearly comparable size, physiology, and disease profile with humans, as well as their docility, capacity for repeated blood draws, ability to tolerate numerous implants during a single operation, and affordability. In this study, sheep were implanted with devices containing allogeneic primary fetal sheep or xenogeneic Rat-2 fibroblasts, to determine cell survival in immunoisolating devices and immune response. Methods: Primary fetal sheep fibroblasts were isolated from a healthy fetus and cultured in RPMI. Rat-2 fibroblasts (ATCC) were cultured in DMEM. On the day of transplant, cells were aliquoted and loaded into devices at different densities. Sheep underwent allogeneic or xenogeneic transplantation with cell-loaded devices, as well as empty control and perforated devices. Devices were implanted subcutaneously through small incisions along the flank within individual pockets made by blunt dissection. Devices remained in vivo until explant at days 3, 7, 21 and 38 to evaluate host responses, vascularization, and cell survival. Explanted devices were fixed, embedded, and sectioned for histological evaluation. Results: Primary fetal sheep cells survived at different densities as assessed by histology, confirming alloprotection with good vascularization and absence of a major foreign body response or extensive inflammation around the device host interface. Devices at the highest densities were overloaded and exhibited some host immune responses. Perforated devices exhibited a greater immune response that infiltrated the device. A robust inflammatory response was seen around devices loaded with xenogeneic Rat-2 fibroblasts with no encapsulated cells surviving. Conclusion: Sheep make a valuable large animal model in cell therapy research. The results of this study concluded alloprotection in immunoisolating devices, allorejection in purposely perforated devices, as well as host immune response to xenogeneic transplantation, representing a feasible model for translational development of cell therapies. Future work with this accessible and relevant platform will include not only allogeneic studies but also avoiding xenogeneic rejection by incorporating immunomodulatory technologies using genetically edited cells or factors. We would like to thank all of the members of the Papas Laboratory at ICT and the Limesand Laboratory at the William J. Parker Agricultural Research Center. Partial funding from JDRF (2-SRA-2020-870-S-B and 2-SRA-2018-685-S-B) and Procyon Technologies LLC.
• Putnam, C. W. (2023).

  401.4: Sustained hyperoxic exposure decreases cell viability and reduces cell growth in pancreatic beta cell lines

  . Transplantation. doi:10.1097/01.tp.0000994644.91754.36
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  Background. Immunoprotective encapsulation devices hold promise for enabling cell-based insulin replacement therapies without immunosuppression, for example with human islets or stem cell–derived β cells. However, for the encapsulation device to be of reasonable size, cell densities within the device must be high; this contributes to a hypoxic environment within the device. Supplemental oxygen to an encapsulation device would reduce the highly detrimental effects of low oxygen on the cells residing within it. However, hyperoxia can also be toxic; this study investigates the effects of various oxygen concentrations on pancreatic β-cell lines in order to identify a safe oxygen exposure level. Methods. Pancreatic β-cell lines were exposed to normoxic or hyperoxic culture conditions (40, 60, and 95%) for 7 days, after which viability was assessed by oxygen consumption rate (OCR) and with acridine orange staining. Cell counts were performed after nuclear DNA staining with DAPI. Results. In hyperoxia of only 40% O2, MIN6 cells showed reduced OCR (P
• Putnam, C. W. (2023).

  Accelerated absorption of regular insulin administered via a vascularizing permeable microchamber implanted subcutaneously in diabetic Rattus norvegicus

  . PLoS ONE. doi:10.1371/journal.pone.0278794
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  In Type 1 diabetes patients, even ultra-rapid acting insulins injected subcutaneously reach peak concentrations in 45 minutes or longer. The lag time between dosing and peak concentration, as well as intra- and inter-subject variability, render prandial glucose control and dose consistency difficult. We postulated that insulin absorption from subcutaneously implantable vascularizing microchambers would be significantly faster than conventional subcutaneous injection. Male athymic nude R . norvegicus rendered diabetic with streptozotocin were implanted with vascularizing microchambers (single chamber; 1.5 cm 2 surface area per side; nominal volume, 22.5 μl). Plasma insulin was assayed after a single dose (1.5 U/kg) of diluted insulin human (Humulin®R U-100), injected subcutaneously or via microchamber. Microchambers were also implanted in additional animals and retrieved at intervals for histologic assessment of vascularity. Following conventional subcutaneous injection, the mean peak insulin concentration was 22.7 (SD 14.2) minutes. By contrast, when identical doses of insulin were injected via subcutaneous microchamber 28 days after implantation, the mean peak insulin time was shortened to 7.50 (SD 4.52) minutes. Peak insulin concentrations were similar by either route; however, inter-subject variability was reduced when insulin was administered via microchamber. Histologic examination of tissue surrounding microchambers showed mature vascularization on days 21 and 40 post-implantation. Implantable vascularizing microchambers of similar design may prove clinically useful for insulin dosing, either intermittently by needle, or continuously by pump including in “closed loop” systems, such as the artificial pancreas.
• Steyn, L. V., Drew, D., Vlachos, D., Huey, B., Cocchi, K., Price, N. D., Johnson, R., Putnam, C. W., & Papas, K. K. (2023). Accelerated absorption of regular insulin administered via a vascularizing permeable microchamber implanted subcutaneously in diabetic Rattus norvegicus. PloS one, 18(6), e0278794.
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  In Type 1 diabetes patients, even ultra-rapid acting insulins injected subcutaneously reach peak concentrations in 45 minutes or longer. The lag time between dosing and peak concentration, as well as intra- and inter-subject variability, render prandial glucose control and dose consistency difficult. We postulated that insulin absorption from subcutaneously implantable vascularizing microchambers would be significantly faster than conventional subcutaneous injection. Male athymic nude R. norvegicus rendered diabetic with streptozotocin were implanted with vascularizing microchambers (single chamber; 1.5 cm2 surface area per side; nominal volume, 22.5 μl). Plasma insulin was assayed after a single dose (1.5 U/kg) of diluted insulin human (Humulin®R U-100), injected subcutaneously or via microchamber. Microchambers were also implanted in additional animals and retrieved at intervals for histologic assessment of vascularity. Following conventional subcutaneous injection, the mean peak insulin concentration was 22.7 (SD 14.2) minutes. By contrast, when identical doses of insulin were injected via subcutaneous microchamber 28 days after implantation, the mean peak insulin time was shortened to 7.50 (SD 4.52) minutes. Peak insulin concentrations were similar by either route; however, inter-subject variability was reduced when insulin was administered via microchamber. Histologic examination of tissue surrounding microchambers showed mature vascularization on days 21 and 40 post-implantation. Implantable vascularizing microchambers of similar design may prove clinically useful for insulin dosing, either intermittently by needle, or continuously by pump including in "closed loop" systems, such as the artificial pancreas.
• Han, S., Lim, K. S., Blackburn, B. J., Yun, J., Putnam, C. W., Bull, D. A., & Won, Y. W. (2022). The Potential of Topoisomerase Inhibitor-Based Antibody-Drug Conjugates. Pharmaceutics, 14(8).
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  DNA topoisomerases are essential enzymes that stabilize DNA supercoiling and resolve entanglements. Topoisomerase inhibitors have been widely used as anti-cancer drugs for the past 20 years. Due to their selectivity as topoisomerase I (TOP1) inhibitors that trap TOP1 cleavage complexes, camptothecin and its derivatives are promising anti-cancer drugs. To increase accumulation of TOP1 inhibitors in cancer cells through the targeting of tumors, TOP1 inhibitor antibody-drug conjugates (TOP1-ADC) have been developed and marketed. Some TOP1-ADCs have shown enhanced therapeutic efficacy compared to prototypical anti-cancer ADCs, such as T-DM1. Here, we review various types of camptothecin-based TOP1 inhibitors and recent developments in TOP1-ADCs. We then propose key points for the design and construction of TOP1-ADCs. Finally, we discuss promising combinatorial strategies, including newly developed approaches to maximizing the therapeutic potential of TOP1-ADCs.
• Han, S., Jung, M., Kim, A. S., Lee, D. Y., Cha, B. H., Putnam, C. W., Lim, K. S., Bull, D. A., & Won, Y. W. (2021). Peptide Adjuvant to Invigorate Cytolytic Activity of NK Cells in an Obese Mouse Cancer Model. Pharmaceutics, 13(8).
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  Cancer patients who are overweight compared to those with normal body weight have obesity-associated alterations of natural killer (NK) cells, characterized by poor cytotoxicity, slow proliferation, and inadequate anti-cancer activity. Concomitantly, prohibitin overexpressed by cancer cells elevates glucose metabolism, rendering the tumor microenvironment (TME) more tumor-favorable, and leading to malfunction of immune cells present in the TME. These changes cause vicious cycles of tumor growth. Adoptive immunotherapy has emerged as a promising option for cancer patients; however, obesity-related alterations in the TME allow the tumor to bypass immune surveillance and to down-regulate the activity of adoptively transferred NK cells. We hypothesized that inhibiting the prohibitin signaling pathway in an obese model would reduce glucose metabolism of cancer cells, thereby changing the TME to a pro-immune microenvironment and restoring the cytolytic activity of NK cells. Priming tumor cells with an inhibitory the prohibitin-binding peptide (PBP) enhances cytokine secretion and augments the cytolytic activity of adoptively transferred NK cells. NK cells harvested from the PBP-primed tumors exhibit multiple markers associated with the effector function of active NK cells. Our findings suggest that PBP has the potential as an adjuvant to enhance the cytolytic activity of adoptively transferred NK cells in cancer patients with obesity.
• Khandelwal Gilman, K. A., Han, S., Won, Y. W., & Putnam, C. W. (2021). Complex interactions of lovastatin with 10 chemotherapeutic drugs: a rigorous evaluation of synergism and antagonism. BMC cancer, 21(1), 356.
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  Evidence bearing on the role of statins in the prevention and treatment of cancer is confounded by the diversity of statins, chemotherapeutic agents and cancer types included in the numerous published studies; consequently, the adjunctive value of statins with chemotherapy remains uncertain.
• Centuori, S. M., Gomes, C. J., Kim, S. S., Putnam, C. W., Larsen, B. T., Garland, L. L., Mount, D. W., & Martinez, J. D. (2018). Double-negative (CD27IgD) B cells are expanded in NSCLC and inversely correlate with affinity-matured B cell populations. Journal of translational medicine, 16(1), 30.
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  The presence of B cells in early stage non-small cell lung cancer (NSCLC) is associated with longer survival, however, the role these cells play in the generation and maintenance of anti-tumor immunity is unclear. B cells differentiate into a variety of subsets with differing characteristics and functions. To date, there is limited information on the specific B cell subsets found within NSCLC. To better understand the composition of the B cell populations found in NSCLC we have begun characterizing B cells in lung tumors and have detected a population of B cells that are CD79ACD27IgD. These CD27IgD (double-negative) B cells have previously been characterized as unconventional memory B cells and have been detected in some autoimmune diseases and in the elderly population but have not been detected previously in tumor tissue.
• Putnam, C. W., DiMarco, J., & Cairns, C. B. (2018). Recruitment of Dual-Career Academic Medicine Couples. Academic medicine : journal of the Association of American Medical Colleges, 93(11), 1604-1606.
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  Today it is not uncommon to discover that a candidate for a faculty position has a partner or spouse who is also an academician, adding complexity to the recruitment process. Here, the authors address two practical obstacles to the recruitment of faculty who have an academic partner: dual recruitment and conflict of interest. The authors have found that tandem recruitment works best when suitable positions for both spouses are first identified so that recruitment can proceed synchronously. This approach decreases misperceptions of favoritism toward either's candidacy. Managing conflict of interest, generated by the appointment of one spouse in a supervisory position over the other, requires a proactive, transparent, well-designed plan. After canvassing human resource policies and conducting interviews with national academic leaders, the authors have developed an administrative structure that places "key" decisions (hiring and retention; promotion and tenure; salary, bonuses, and benefits; performance evaluations; and disciplinary matters) regarding the supervised spouse in the jurisdiction of an alternative administrator or committee. The authors also offer suggestions both for mitigating misperceptions of bias in day-to-day decisions and for the support and mentoring of the supervised partner or spouse.
• Gomes, C. J., Centuori, S. M., Harman, M. W., Putnam, C. W., Wolgemuth, C. W., & Martinez, J. D. (2017). The induction of endoreduplication and polyploidy by elevated expression of 14-3-3γ. Genes & cancer, 8(11-12), 771-783.
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  Several studies have demonstrated that specific 14-3-3 isoforms are frequently elevated in cancer and that these proteins play a role in human tumorigenesis. 14-3-3γ, an isoform recently demonstrated to function as an oncoprotein, is overexpressed in a variety of human cancers; however, its role in promoting tumorigenesis remains unclear. We previously reported that overexpression of 14-3-3γ caused the appearance of polyploid cells, a phenotype demonstrated to have profound tumor promoting properties. Here we examined the mechanism driving 14-3-3γ-induced polyploidization and the effect this has on genomic stability. Using FUCCI probes we showed that these polyploid cells appeared when diploid cells failed to enter mitosis and subsequently underwent endoreduplication. We then demonstrated that 14-3-3γ-induced polyploid cells experience significant chromosomal segregation errors during mitosis and observed that some of these cells stably propagate as tetraploids when isolated cells were expanded into stable cultures. These data lead us to conclude that overexpression of the 14-3-3γ promotes endoreduplication. We further investigated the role of 14-3-3γ in human NSCLC samples and found that its expression is significantly elevated in polyploid tumors. Collectively, these results suggests that 14-3-3γ may promote tumorigenesis through the production of a genetically unstable polyploid intermediate.
• Putnam, C. W. (2017).

  Recruitment of Dual-Career Academic Medicine Couples

  . Academic Medicine. doi:10.1097/acm.0000000000002084
• Centuori, S. M., Garland, L. L., Gomes, C. J., Kim, S., Larsen, B. T., Martinez, J. D., Mount, D. B., & Putnam, C. W. (2016).

  Abstract 713: Determining the role of tumor-infiltrating B cells in NSCLC

  . Cancer Research, 76(14_Supplement), 713-713. doi:10.1158/1538-7445.am2016-713
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  Recent studies in human non-small cell lung cancer (NSCLC) have shown that upregulation of B cell-associated genes strongly correlate with early stage patient survival. After analyzing a gene expression database of lung tumors we showed that CD79A, a pan-B cell marker, had the strongest predictive value, suggesting that B cells are playing a crucial role in NSCLC immunity. In order to evaluate this we first examined where this genetic signature was originating from. We observed that patients with high levels of B cell-related genes also showed high numbers of CD79A+ B cells intimately associated with the tumor, but not being expressed by tumor cells themselves. A closer look at tumor-infiltrating B cells (TIL-B cells) by IHC and flow cytometry of fresh patient tumor samples has confirmed their presence and allowed us to begin elucidating their phenotype. Interestingly, we have found that not all early stage patients have detectable levels of TIL-B cells, but in those that do, TIL-B cells represent a marked proportion of the lymphocytic infiltration. Further evaluation of T cell populations in the same samples indicate that T cell numbers remain relatively consistent but that the numbers of B-cells, especially CD79A+ B-cells, does vary from patient to patient. These data indicate that CD79A+ TIL-B cells are contributing to the generation of an efficient immune response in some but not all patients, and are greatly influencing disease survivability. This information may allow us to stratify patients into low and high risk groups based on the presence or absence of TIL-B cells, respectively. Citation Format: Sara M. Centuori, Samuel Kim, Cecil Gomes, Charles Putnam, David Mount, Linda Garland, Brandon Larsen, Jesse D. Martinez. Determining the role of tumor-infiltrating B cells in NSCLC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 713.
• Centuori, S. M., Gomes, C. J., Trujillo, J., Borg, J., Brownlee, J., Putnam, C. W., & Martinez, J. D. (2016). Deoxycholic acid mediates non-canonical EGFR-MAPK activation through the induction of calcium signaling in colon cancer cells. Biochimica et biophysica acta, 1861(7), 663-70.
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  Obesity and a western diet have been linked to high levels of bile acids and the development of colon cancer. Specifically, increased levels of the bile acid deoxycholic acid (DCA), an established tumor promoter, has been shown to correlate with increased development of colorectal adenomas and progression to carcinoma. Herein we investigate the mechanism by which DCA leads to EGFR-MAPK activation, a candidate mechanism by which DCA may promote colorectal tumorigenesis. DCA treated colon cancer cells exhibited strong and prolonged activation of ERK1/2 when compared to EGF treatment alone. We also showed that DCA treatment prevents EGFR degradation as opposed to the canonical EGFR recycling observed with EGF treatment. Moreover, the combination of DCA and EGF treatment displayed synergistic activity, suggesting DCA activates MAPK signaling in a non-canonical manner. Further evaluation showed that DCA treatment increased intracellular calcium levels and CAMKII phosphorylation, and that blocking calcium with BAPTA-AM abrogated MAPK activation induced by DCA, but not by EGF. Finally we showed that DCA-induced CAMKII leads to MAPK activation through the recruitment of c-Src. Taken together, we demonstrated that DCA regulates MAPK activation through calcium signaling, an alternative mechanism not previously recognized in human colon cancer cells. Importantly, this mechanism allows for EGFR to escape degradation and thus achieve a constitutively active state, which may explain its tumor promoting effects.
• Centuori, S. M., Garland, L. L., Gomes, C. J., Kim, S., Martinez, J. D., Mount, D. B., & Putnam, C. W. (2015).

  Abstract 1299: Tumor-infiltrating B cells are predictive of human lung squamous cell survival

  . Immunology, 75(15_Supplement), 1299-1299. doi:10.1158/1538-7445.am2015-1299
• Mount, D. W., Putnam, C. W., Centouri, S. M., Manziello, A. M., Pandey, R., Garland, L. L., & Martinez, J. D. (2014). Using logistic regression to improve the prognostic value of microarray gene expression data sets: application to early-stage squamous cell carcinoma of the lung and triple negative breast carcinoma. BMC medical genomics, 7, 33.
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  Numerous microarray-based prognostic gene expression signatures of primary neoplasms have been published but often with little concurrence between studies, thus limiting their clinical utility. We describe a methodology using logistic regression, which circumvents limitations of conventional Kaplan Meier analysis. We applied this approach to a thrice-analyzed and published squamous cell carcinoma (SQCC) of the lung data set, with the objective of identifying gene expressions predictive of early death versus long survival in early-stage disease. A similar analysis was applied to a data set of triple negative breast carcinoma cases, which present similar clinical challenges.
• Radhakrishnan, V. M., Putnam, C. W., & Martinez, J. D. (2012). Activation of phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling and the consequent induction of transformation by overexpressed 14-3-3γ protein require specific amino acids within 14-3-3γ N-terminal variable region II. The Journal of biological chemistry, 287(52), 43300-11.
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  Members of the 14-3-3 superfamily regulate numerous cellular functions by binding phosphoproteins. The seven human isoforms (and the myriad of other eukaryotic 14-3-3 proteins) are highly conserved in amino acid sequence and secondary structure, yet there is abundant evidence that the various isoforms manifest disparate as well as common functions. Several of the human 14-3-3 isoforms are dysregulated in certain cancers and thus have been implicated in oncogenesis; experimentally, 14-3-3γ behaves as an oncogene, whereas 14-3-3σ acts as a tumor suppressor. In this study, we sought to localize these opposing phenotypes to specific regions of the two isoforms and then to individual amino acids therein. Using a bioinformatics approach, six variable regions (VRI-VRVI) were identified. Using this information, two sets of constructs were created in which N-terminal portions (including either VRI-IV or only VRI and VRII) of 14-3-3γ and 14-3-3σ were swapped; NIH3T3 cells overexpressing the four chimeric proteins were tested for transformation activity (focus formation, growth in soft agar) and activation of PI3K and MAPK signaling. We found that the specific phenotypes of 14-3-3γ are associated with the N-terminal 40 amino acids (VRI and VRII); in like fashion, VRI and VRII of 14-3-3σ dictated its tumor suppressor function. Using individual amino acid substitutions within the 14-3-3γ VRII, we identified two residues required for and two contributing to the γ-specific phenotypes. Our observations suggest that isoform-specific phenotypes are dictated by a relatively few amino acids within variable regions.
• Putnam, C. W., Martinez, J. D., & Radhakrishnan, V. M. (2011).

  Abstract A61: 14-3-3gamma transforming ability is determined by a motif outside the conserved phosphoamio-acid binding domain

  . Cancer Research, 71(18_Supplement), A61-A61. doi:10.1158/1538-7445.fbcr11-a61
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  The 14-3-3 proteins are a set of seven highly conserved scaffolding proteins that have been implicated as having a role in human tumorigenesis. However, despite the striking similarity of amino acid sequences and structures of the eukaryotic 14-3-3 family of proteins, there is increasing evidence that the seven human isoforms manifest disparate functions. For instance 14-3-3gamma functions as an oncogene and 14-3-3sigma acts as a tumor suppressor. To gain insight into how specific functions are manifested we constructed chimeric proteins by swaping various regions between gamma and sigma. Chimeric proteins were then utilized in NIH3T3 cell transformation assays. We found that transforming ability was associated with a small variable 40 amino acid region in the N-terminus of the gamma protein and that the transforming ability of gamma was dominant to the tumor suppressor activity of sigma. Importantly, the transforming motif was located outside the conserved 14-3-3 phosphoamino acid binding motif. Hence, transforming ability of 14-3-3 proteins is determined by a variable region that does not involve the phosphoamino acid binding site. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr A61.
• Radhakrishnan, V. M., Putnam, C. W., Qi, W., & Martinez, J. D. (2011). P53 suppresses expression of the 14-3-3 gamma oncogene. BMC cancer, 11, 378.
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  14-3-3 proteins are a family of highly conserved proteins that are involved in a wide range of cellular processes. Recent evidence indicates that some of these proteins have oncogenic activity and that they may promote tumorigenesis. We previously showed that one of the 14-3-3 family members, 14-3-3 gamma, is over expressed in human lung cancers and that it can induce transformation of rodent cells in vitro.
• Nyberg, K. A., Michelson, R. J., Putnam, C. W., & Weinert, T. A. (2002). Toward maintaining the genome: DNA damage and replication checkpoints. Annual review of genetics, 36, 617-56.
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  DNA checkpoints play a significant role in cancer pathology, perhaps most notably in maintaining genome stability. This review summarizes the genetic and molecular mechanisms of checkpoint activation in response to DNA damage. The major checkpoint proteins common to all eukaryotes are identified and discussed, together with how the checkpoint proteins interact to induce arrest within each cell cycle phase. Also discussed are the molecular signals that activate checkpoint responses, including single-strand DNA, double-strand breaks, and aberrant replication forks. We address the connection between checkpoint proteins and damage repair mechanisms, how cells recover from an arrest response, and additional roles that checkpoint proteins play in DNA metabolism. Finally, the connection between checkpoint gene mutation and genomic instability is considered.
• Gardner, R., Putnam, C. W., & Weinert, T. (1999). RAD53, DUN1 and PDS1 define two parallel G2/M checkpoint pathways in budding yeast. The EMBO journal, 18(11), 3173-85.
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  Eukaryotic checkpoint genes regulate multiple cellular responses to DNA damage. In this report, we examine the roles of budding yeast genes involved in G2/M arrest and tolerance to UV exposure. A current model posits three gene classes: those encoding proteins acting on damaged DNA (e.g. RAD9 and RAD24), those transducing a signal (MEC1, RAD53 and DUN1) or those participating more directly in arrest (PDS1). Here, we define important features of the pathways subserved by those genes. MEC1, which we find is required for both establishment and maintenance of G2/M arrest, mediates this arrest through two parallel pathways. One pathway requires RAD53 and DUN1 (the 'RAD53 pathway'); the other pathway requires PDS1. Each pathway independently contributes approximately 50% to G2/M arrest, effects demonstrable after cdc13-induced damage or a double-stranded break inflicted by the HO endonuclease. Similarly, both pathways contribute independently to tolerance of UV irradiation. How the parallel pathways might interact ultimately to achieve arrest is not yet understood, but we do provide evidence that neither the RAD53 nor the PDS1 pathway appears to maintain arrest by inhibiting adaptation. Instead, we think it likely that both pathways contribute to establishing and maintaining arrest.
• Hunter, G. C., Henderson, A. M., Westerband, A., Kobayashi, H., Suzuki, F., Yan, Z. Q., Sirsjo, A., Putnam, C. W., & Hansson, G. K. (1999). The contribution of inducible nitric oxide and cytomegalovirus to the stability of complex carotid plaque. Journal of vascular surgery, 30(1), 36-49; discussion 50.
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  Although the association between inflammation and atherosclerosis is well established, the biologic events that trigger the local inflammatory response within plaque are not fully understood. Cytotoxic free radicals and infectious agents, both of which are associated with an inflammatory response, have previously been implicated in the initiation and progression of atherosclerosis. In this study, we analyzed carotid plaque for evidence of oxidative vascular injury by determining the presence and distribution of inducible nitric oxide synthase (iNOS) expression and nitrotyrosine formation and for evidence of infection with cytomegalovirus.
• Bull, D. A., Karwande, S. V., Hawkins, J. A., Neumayer, L. A., Taylor, D. O., Jones, K. W., Renlund, D. G., Putnam, C. W., & Putnam, C. W. (1996). Long-term results of cardiac transplantation in patients older than sixty years. UTAH Cardiac Transplant Program. The Journal of thoracic and cardiovascular surgery, 111(2), 423-7; discussion 427-8.
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  Advanced age has traditionally been a contraindication to cardiac transplantation. We have, however, offered cardiac transplantation to patients older than 60 years with end-stage heart failure if they were otherwise acceptable candidates. From 1985 to 1994, 527 patients underwent cardiac transplantation. Among these patients, 101 were older than 60 years at transplantation. The mean follow-up of this group is 6 years. Patients older than 60 years had significantly fewer rejection episodes per patient than those who were younger than 60 years at transplantation (1.9 +/- 1.3 vs 2.6 +/- 1.8, p = 0.009). No difference in the number of infectious complications per patient was detected between the two groups. Both short-term and long-term survival after transplantation were significantly lower for patients who were older than 60 years at transplantation than for younger patients (p < 0.05). The 6-year actuarial survival after transplantation for patients older than 60 years was 54% compared with 72% for patients younger than 60 years at transplantation (p < 0.05). Patients older than 60 years at transplantation were more likely to die of infectious complications or malignant disease after transplantation (p < 0.05). We believe caution is warranted in offering cardiac transplantation to patients older than 60 years. This group of patients should be carefully observed for the development of potentially life-threatening infectious complications or new malignant tumors after transplantation.
• Hunter, G. C., Smyth, S. H., Aguirre, M. L., Baxter, B. T., Bull, D. A., King, D. D., Wang, Y. P., Hall, K. A., & Putnam, C. W. (1996). Incidence and histologic characteristics of blebs in patients with abdominal aortic aneurysms. Journal of vascular surgery, 24(1), 93-101.
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  Aortic blebs-focal outpouchings within aortic aneurysms-may contribute to their eventual rupture. In this study we determine the incidence of aortic blebs and describe their microscopic features.
• Killion, S. L., Hunter, G. C., Eskelson, C. D., Dubick, M. A., Putnam, C. W., Hall, K. A., Luedke, C. A., Misiorowski, R. L., Schilling, J. D., & McIntyre, K. E. (1996). Vitamin E levels in human atherosclerotic plaque: the influence of risk factors. Atherosclerosis, 126(2), 289-97.
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  Recent studies suggest that vitamin E may be an important preventative factor in the development and progression of atherosclerosis. In order to more clearly define the role of vitamin E in atherosclerosis, we measured vitamin E, conjugated diens, and lipid flurochromes, as well as cholesterol, triglycerides and phospholipid in arterial and venous tissue of 83 patients. Serum cholesterol and triglyceride levels were significantly higher (P < 0.05) in patients with aortic occlusive (AIOD) and aneurysmal (AAA) disease than in control organ donors (OD). Tissue cholesterol concentrations were significantly elevated in AAA tissue when compared to OD and tissue from patients with peripheral occlusive disease (POD). Tissue from patients with AIOD contained greater concentrations of phospholipid (PL) than were measured in patients with POD and in OD. Vitamin E concentrations were highest in POD tissue and approximately 3.0, 2.0, and 1.6 fold greater than OD, AIOD and AAA tissue respectively. Diene conjugates and lipid flurochromes, measures of early and intermediate products of lipid peroxidation, were markedly elevated in all diseased arterial tissue compared to controls. There were no significant differences in tissue or serum lipid levels between saphenous vein (SVBG) and diseased vein grafts (DVG). However, conjugated diene concentrations were elevated in DVG compared to SVBG. Vitamin E levels were significantly elevated in diseased arterial and venous tissue (AIOD, AAA, POD, DVG) removed from patients with diabetes (P = 0.013) and hypertension (P = 0.049) compared to those without these risk factors. Diabetes was the only risk factor associated with significantly increased (P = 0.005) levels of vitamin E when only data from atherosclerotic arterial tissue (AAA, POD, AIOD) were analyzed. These preliminary data provide additional evidence of altered vitamin E metabolism and free radical processes in the tissues of patients with various manifestations of atherosclerosis.
• Bull, D. A., Hunter, G. C., Holubec, H., Aguirre, M. L., Rappaport, W. D., & Putnam, C. W. (1995). Cellular origin and rate of endothelial cell coverage of PTFE grafts. The Journal of surgical research, 58(1), 58-68.
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  To determine the origin, cell type present, and rate of endothelial cell coverage of PTFE grafts, 5-cm segments of 4-mm-diameter, 60-microns PTFE grafts were implanted end-to-end bilaterally in the carotid arteries of greyhound dogs. An external jugular vein wrap was applied to the outer surface of one of the PTFE grafts; the contralateral PTFE graft, which was unwrapped, served as its control. Two dogs each were sacrificed at 3, 5, 7, 14, 21, 28, and 35 days postimplantation. Anastomotic endothelial ingrowth was analyzed using scanning electron microscopy. Microvessel ingrowth was documented in longitudinal H&E sections. Cell identity was established by immunohistochemistry with factor VIII antibody, Ulex europaes, leukocyte common antigen, and antibodies to alpha-actin, desmin, vimentin, and basic fibroblast growth factor. All grafts were patent at the time of harvest. Endothelial cell migration from the native artery adjacent to the anastomosis commenced at 7 days, extended to 5 mm beyond the proximal and distal anastomoses by 14 days and to 1.0 cm by 35 days. Endothelialization of the mid-portion of the wrapped grafts occurred via microvessel ingrowth, a process which began at 7 days. Microvessels reached the luminal surface by 28 days and an endothelial cell monolayer was established by 35 days. Wrapping the external surface of the graft with vein increased the rate of graft healing. Basic fibroblast growth factor was detectable by immunohistochemistry at the vein wrap-graft interface in the first 14 days.
• Hall, K. A., Peters, B., Smyth, S. H., Warneke, J. A., Rappaport, W. D., Putnam, C. W., & Hunter, G. C. (1995). Abdominal wall hernias in patients with abdominal aortic aneurysmal versus aortoiliac occlusive disease. American journal of surgery, 170(6), 572-5; discussion 575-6.
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  This study was undertaken to determine the incidence of ventral incisional hernias (VIHs) and inguinal hernias (IHs) in patients with abdominal aortic aneurysmal (AAA) versus those with aortoiliac occlusive disease (AIOD).
• Bull, D. A., Neumayer, L. A., Venerus, B. J., Putnam, C. W., Rosado, L., Lund, P., McIntyre, K. E., Bernhard, V. M., Copeland, J. G., & Sethi, G. K. (1994). The effects of improved hemodynamics on aortic dimensions in patients undergoing heart transplantation. Journal of vascular surgery, 20(4), 539-44; discussion 544-5.
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  Retrospective studies have demonstrated an accelerated growth rate of abdominal aortic aneurysms in heart transplant patients. This prospective study was undertaken to define the relationship between cardiac hemodynamics and posttransplant aortic dilation.
• Daller, J. A., Buckley, A. R., Van Hook, F. W., Buckley, D. J., & Putnam, C. W. (1994). Suramin, a protein kinase C inhibitor, impairs hepatic regeneration. Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research, 5(7), 761-7.
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  Previously we have shown that partial hepatectomy (PH) or exposure of the liver to the mitogen prolactin induces activation of hepatic protein kinase C (PKC). Here, we used suramin, an antitrypanosomal and chemotherapeutic drug which inhibits that enzyme, as a probe of PKC signal transduction in the regenerative response after PH in the rat. Suramin was administered i.p. in nonhepatotoxic doses of 20 to 160 mg/kg 14 days prior to PH. Three measures of hepatic DNA synthesis or cell division, thymidine kinase activity, [3H]thymidine incorporation, and mitotic index were inhibited in a dose-dependent fashion. Baseline PKC activity, in both the cytosolic and particulate fractions, was unchanged by suramin. After PH, PKC activation, signalled by an increase in activity in the particulate fraction, was observed in control rats at 30 and 60 min. However, rats which had previously received suramin demonstrated dose-dependent inhibition of PKC activation. Suramin is known to also disrupt the binding of certain growth factors to their receptors. But if inhibition of PKC activation were conferred by interference with growth factor-receptor binding by suramin, then the generation of diacylglycerol, the second messenger for PKC activation, should likewise be impaired. However, we observed that the diacylglycerol mass generated at 15, 30, and 45 min after PH was not altered by suramin pretreatment. We conclude that the diminution in DNA synthesis after PH by suramin is likely the consequence of direct inhibition of PKC, suggesting that PKC activation is an important, perhaps obligatory, signal transduction event in liver regeneration.
• Sawyer, J. S., Daller, J. A., Brendel, K., Yohem, K., & Putnam, C. W. (1994). The hepatotoxicities of endotoxin and ethanol comparisons in vitro using the precision-cut rat liver slice model. Life sciences, 55(18), 1407-17.
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  Using the precision-cut rat liver slice model, the in vitro toxicities of endotoxin and ethanol, independently and in combination, were evaluated. Hepatotoxicity was assessed by two measures: the leakage of LDH from slice to medium and the ability of slices to reduce a tetrazolium compound, MTT. Ethanol, in concentrations of 1% and greater, exhibited a time and dose dependent hepatotoxicity; MTT reductive capacity was more profoundly affected than LDH leakage. Endotoxin (0.1 to 100 micrograms/ml), however, had only a modest effect on MTT reduction and did not perturb LDH leakage. When combined in vitro, the toxicities of 2% ethanol and various concentrations of endotoxin were additive. Slices prepared from the livers of rats injected ip with endotoxin one day previously sustained considerably more injury (compared to normal rat liver slices) upon subsequent in vitro exposure to either endotoxin or ethanol. Prior in vivo exposure to ethanol, however, did not affect the subsequent in vitro toxicity of endotoxin. Thus, while endotoxin exhibits only subtle toxic effects upon liver slices in vitro, exposure to endotoxin in vivo renders the liver more susceptible to subsequent direct injury by endotoxin or ethanol.
• Wong, R. W., Rappaport, W. D., Witzke, D. B., Putnam, C. W., & Hunter, G. C. (1994). Factors influencing the safety of colostomy closure in the elderly. The Journal of surgical research, 57(2), 289-92.
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  Although colostomy closure is a commonly performed surgical procedure, there remains concern that the attendant risks may be misjudged, especially in the elderly. The purpose of this study was to evaluate the safety of performing colostomy closure and to define the factors that may influence morbidity and mortality in patients over the age of 70. Three hundred seven patients (178 males, 129 females) underwent colostomy closure over a 5-year period. The mean age was 52 years and 84 (27%) of the patients were 70 years or older. The indications for colostomy included diverticulitis, 115 (38%); malignancy, 47 (15%); perforation 35 (11%); trauma, 34 (11%); congenital anomalies, 26 (8%); obstruction, 13 (4%); bleeding, 6 (2%); colovesical fistulae, 6 (2%); polyps, 2 (0.7%); and miscellaneous, 23 (8%). An end colostomy was performed in 193 (63%) patients and a transverse loop colostomy in the remaining 114 (37%). There were no deaths. Complications occurred in 27 (9%) patients: 17 were directly related to colostomy closure (8 wound infections, 3 intraabdominal abscesses, 3 small bowel obstructions, 2 anastomotic strictures, and 1 anastomotic leak) while 10 were nonsurgical. Risk factors statistically associated with increased morbidity included age > 70 years (13% versus 5%), end versus loop colostomy (10% versus 2%), an operative time > 2 hr, and estimated blood loss > or = 500 ml (P < 0.05). ASA classification was only predicative of postoperative complications in patients over 70 years of age. We conclude that although colostomy closure can be performed without mortality, the increased morbidity associated with this procedure in patients 70 years or older necessitates careful preoperative assessment.
• Bull, D. A., Hunter, G. C., Crabtree, T. G., Bernhard, V. M., & Putnam, C. W. (1993). Hepatic ischemia, caused by celiac axis compression, complicating pancreaticoduodenectomy. Annals of surgery, 217(3), 244-7.
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  In the course of pancreaticoduodenectomy, profound hepatic ischemia developed in two patients (one with ampullary carcinoma, the other with chronic pancreatitis). This article addresses the diagnosis and correction of the celiac axis compression responsible in this complication.
• Bull, D. A., Seftor, E. A., Hendrix, M. J., Larson, D. F., Hunter, G. C., & Putnam, C. W. (1993). Putative vascular endothelial cell chemotactic factors: comparison in a standardized migration assay. The Journal of surgical research, 55(5), 473-9.
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  While a number of chemoattractants of vascular endothelial cells have now been identified in vitro, differences in methodology preclude comparisons of substances evaluated in different assays. Here, we report a standardized chemotactic assay in which the migration of calf pulmonary artery endothelial cells in a 48-well microchemotaxis chamber was determined. Nonstimulated (control) migration was remarkably constant (mean +/- SD, 96 +/- 14) from plate to plate, thus allowing the indexing of relative migration of stimulated cells to that of nonstimulated cells in the control wells of that plate. Based on the relative migrations observed in response to each of the substances evaluated, those proving to be stimulatory of migration were placed in rank order by potency. The growth factors epidermal growth factor, transforming growth factor-alpha, and basic fibroblast growth factor (followed by pentosan polysulfate, plasmin, fibronectin, fibrinogen, granulocyte-macrophage colony stimulating factor heparin, adenosine, and MgSO4) were the most potent. Only the platelet factors platelet-derived growth factor-BB and platelet activating factor proved inhibitory of migration. Combining fibrinogen with other chemoattractants produced either stimulation or inhibition in comparison to the migration observed with fibrinogen alone, suggesting that more than one signal transduction mechanism was, in all likelihood, invoked by the various agents. This assay will allow the rapid screening and rank ordering of additional putative chemoattractants, will facilitate the study of the biochemical mechanisms involved in endothelial cell migration, and will permit the evaluation of pharmacologic agents capable of modulating stimulated or unstimulated migration.
• Buckley, A. R., Montgomery, D. W., Hendrix, M. J., Zukoski, C. F., & Putnam, C. W. (1992). Identification of prolactin receptors in hepatic nuclei. Archives of biochemistry and biophysics, 296(1), 198-206.
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  Prolactin is a trophic hormone which may act directly at the hepatocyte nucleus. In this study, specific prolactin binding sites were sought in purified rat liver nuclei. Saturable and specific, high affinity 125I-prolactin binding sites were demonstrated to be on or within the nucleus. Prolactin binding was competitively inhibited by rat and ovine prolactins but not by rat growth hormone. Using immunogold electron microscopy, we detected prolactin receptors throughout the nucleus, in association with heterochromatin. Furthermore, endogenous immunoreactive prolactin was demonstrated to be within hepatic nuclei. We conclude that rat liver nuclei possess prolactin binding sites which likely participate in hormone-directed growth processes.
• Bull, D. A., Hunter, G. C., Copeland, J. G., Bernhard, V. M., Rosado, L. J., McIntyre, K. E., Sethi, G. K., & Putnam, C. W. (1992). Peripheral vascular disease in heart transplant recipients. Journal of vascular surgery, 16(4), 546-53; discussion 553-4.
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  The improved longevity of heart transplant recipients demands heightened awareness of the long-term complications of the procedure. Between 1979 and 1990, 232 patients received 241 heart transplants at our institution. Accelerated coronary atherosclerosis occurred in 45 (19%) of the 232 patients, typically appearing within 2 years of transplantation, whereas peripheral vascular disease (PVD) appeared in 23 (10%) of the 232 patients, usually within 3 years of transplantation. In the patients with PVD, 13 had occlusive disease, nine had aneurysms, and one patient suffered a vertebral artery dissection. Accelerated coronary atherosclerosis afflicted 12 (52%) of the 23 patients affected by PVD (p < 0.05) and preceded the development of PVD in all 12. Logistic regression analysis revealed risk factors predictive of the development of PVD after transplantation to be a pretransplant history of ischemic cardiomyopathy and posttransplant hypertension and hypertriglyceridemia (p < 0.05), with the presence of more than one risk factor increasing the probability of development of PVD. Those patients thus identified as at risk should be closely monitored for the development of PVD. Aggressive medical management of hypertension and hyperlipidemia in this subpopulation may forestall or prevent the development of peripheral vascular disease after heart transplantation.
• Bull, D. A., Neumayer, L. A., Hunter, G. C., Sethi, G. K., McIntyre, K. E., Bernhard, V. M., & Putnam, C. W. (1992). Improved sterile technique diminishes the incidence of positive line cultures in cardiovascular patients. The Journal of surgical research, 52(2), 106-10.
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  In order to determine the incidence of positive line cultures, especially as affected by differing protocols for line insertion, cultures were obtained from lines in residence for up to 4 days from cardiac patients (who received Cefuroxime) and vascular patients (who received Cefazolin) as prophylaxis perioperatively. Positive cultures were obtained from 95 (19%) of 496 lines in cardiac patients and 83 (31%) of 261 lines in vascular patients. There was a linear relationship between duration of line residence and the incidence of positive line cultures, increasing from 14% on Day 1 to 33% on Day 4. The use of full sterile technique at the time of insertion halved the incidence of subsequent positive line cultures. Four of the 403 (1%) patients each had a single episode of postoperative line sepsis. Another four patients developed wound infections with the same organisms as cultured from their lines. One patient has had a vascular graft infection with the same organism cultured as was isolated from a Swan-Ganz line 1 year previously. These data suggest that monitoring lines should be inserted using full sterile technique and removed as soon as the patient is hemodynamically stable.
• Hall, K. A., Dole, E. J., Hunter, G. C., Zukoski, C. F., & Putnam, C. W. (1992). Hyperpyrexia-related ventricular tachycardia during OKT3 induction therapy. Transplantation, 54(6), 1112-3.
• Holubec, H., Hunter, G. C., Putnam, C. W., Bull, D. A., Rappaport, W. D., & Chvapil, M. (1992). Effect of surgical manipulation of polytetrafluoroethylene grafts on microstructural properties and healing characteristics. American journal of surgery, 164(5), 512-6.
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  The effects on graft healing of alterations in the microstructure of polytetrafluoroethylene (PTFE) grafts induced by surgical instruments have not been fully elucidated. This study evaluates changes in the structural and physical properties of PTFE grafts resulting from the intentional application of commonly used surgical instruments and the influence of these changes on cellular ingrowth. The extent of cellular ingrowth into intact (10, 30, and 60 microns unreinforced and 30 microns reinforced [R]) and structurally compromised PTFE grafts (30 reinforced and 60 microns nonreinforced) implanted subcutaneously in Sprague-Dawley (n = 14) rats was evaluated at 7 and 21 days. The thrombogenicity of 10-, 30-, 60-, and 80-microns intact graft segments was determined gravimetrically after suspension in the internal jugular vein of dogs for 90 minutes. Cellular ingrowth consisting of fibroblasts, macrophages, and microvessels was directly related to porosity and was most extensive in 60-microns uncompromised graft segments, being 7-, 17-, and 20-fold greater than was observed in 60- and 30R-microns compromised grafts and undamaged 10-microns grafts, respectively. There was a direct relationship between porosity and thrombogenicity of intact graft segments suspended in the jugular vein. The amount of thrombus adherent to 80-microns graft segments was eightfold greater compared with 10-microns grafts. Manipulation of PTFE with surgical instruments significantly impairs healing and may be a possible etiologic factor in the poor long-term performance of these grafts.
• Rappaport, W. D., Putnam, C. W., Witzke, D., & Amil, B. (1992). Helping residents' families cope. Academic medicine : journal of the Association of American Medical Colleges, 67(11), 761.
• Valente, J., Rappaport, W., Neumayer, L., Witzke, D., & Putnam, C. W. (1992). Influence of spousal opinions on residency selection. American journal of surgery, 163(6), 596-8.
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  Fourth-year medical students face the difficult task of choosing a residency consistent with their career goals. Our study investigates the input of the spouse on the residency selection. From July 1, 1988, to July 1, 1990, questionnaires were sent to all 69 spouses of fourth-year medical students at the University of Arizona Medical Center. Fifty-six were returned for a response rate of 81%. Of the 16 women and 40 men who responded (mean age: 27 years), 55 (98%) spouse stated that there had been family discussions on the choice of a residency program, and 41 (73%) respondents thought that they had significant input. When asked to rank the items that most influenced their support for a particular training program, career goals of the medical student (68%) and lifestyle (21%) were most important, whereas prestige, earning capacity, and program length were ranked lowest. Specific concerns expressed by spouses on the selection of a surgical residency included time commitment as the most commonly cited (79%), followed by fatigue (48%). A statistically significant correlation existed between those spouses actively discouraging the choice of general surgery and those objecting to the time commitment during residency (p less than 0.05). We conclude that spouses have significant preferences regarding the choice of a training program following medical school. Career goals and lifestyle appear to be the most important factors; however, despite concern about the time commitment, the majority of spouses are supportive of the selection of a surgical residency.
• Buckley, A. R., Crowe, P. D., Bauman, P. A., Neumayer, L. A., Laird, H. E., Russell, D. H., & Putnam, C. W. (1991). Prolactin-provoked alterations of cytosolic, membrane, and nuclear protein kinase C following partial hepatectomy. Digestive diseases and sciences, 36(9), 1313-9.
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  The adenohypophyseal polypeptide hormone prolactin is a potent liver mitogen, stimulating cell cycle progression, an effect that appears coupled to increasing protein kinase C activity in membrane and nuclear fractions. Here, we examine whether hepatocyte proliferation, stimulated by partial hepatectomy, is associated with altered serum prolactin or protein kinase C activation. Within 5-15 min of liver resection, serum prolactin concentrations elevate significantly. Protein kinase C activity in hepatic cytosol decreases significantly, and membrane and nuclear PKC activity increase by 30 min. Hypophysectomy prior to partial hepatectomy abrogates any effect of liver resection on protein kinase C activation in the hepatic remnant. Based upon these data, it is suggested that the rapid increase in serum prolactin seen after partial hepatectomy may be linked to protein kinase C activation, which in turn stimulates the hepatic proliferative response that is essential for hepatic regeneration.
• Daller, J. A., Erstad, B., Rosado, L., Otto, C., & Putnam, C. W. (1991). Aminophylline antagonizes the neuromuscular blockade of pancuronium but not vecuronium. Critical care medicine, 19(7), 983-5.
• Fisher, R., Barr, J., Zukoski, C. F., Putnam, C. W., Sipes, I. G., Gandolfi, A. J., & Brendel, K. (1991). In-vitro hepatotoxicity of three dichlorobenzene isomers in human liver slices. Human & experimental toxicology, 10(5), 357-63.
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  1 The cytotoxicity of dichlorobenzenes in cultured rat liver slices has previously been shown to be strain specific and biotransformation related. 2 In order to extrapolate animal models to humans, the dichlorobenzenes were incubated with human liver slices to try to clarify their hepatotoxic potential in man. 3 The degree of hepatotoxicity observed with the dichlorobenzenes depended on whether Waymouth's or Krebs-Henseleit was used as the incubation medium. 4 All three dichlorobenzenes (1 mM) produced no significant differences from control when incubated in Waymouth's medium. However, in the Krebs-Henseleit buffer there was a substantial increase in cytotoxicity. 5 In both incubation mediums the dichlorobenzene isomers exhibited the following rank order 1,3-DCB greater than 1,2-DCB greater than 1,4-DCB. 6 1,2-dichlorobenzene hepatotoxicity was blocked by metyrapone, 1,3-dichlorobenzene toxicity was blocked by SKF 525-A and neither one of these inhibitors could block the 1,4-dichlorobenzene cytotoxicity. 7 The use of human liver tissues to evaluate potential toxicants merits consideration since the hepatotoxicity of xenobiotics and drugs in man is the ultimate question.
• Fisher, R., Nau, H., Gandolfi, A. J., Putnam, C. W., & Brendel, K. (1991). Valproic acid hepatotoxicity in human liver slices. Drug and chemical toxicology, 14(4), 375-94.
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  Precision cut human liver slices were incubated in organ culture with valproic acid (VPA) to identify patterns of sensitivity to VPA-induced hepatotoxicity. The slices were incubated in Krebs-HEPES buffer supplemented with 25mM glucose and 84 micrograms/ml gentamycin. At 2, 4, 6, 12, 18 and 24 hr slices were taken and analyzed for K+ retention, synthesis of protein and LDH leakage. All three of these viability indicators showed that certain human livers were more susceptible to VPA-induced hepatotoxicity than others. In the limited group of human livers investigated (n = 9) we found one to be particularly sensitive and two relatively insensitive to VPA toxicity. The remaining tissues were of intermediate sensitivity towards VPA. At this time there is no correlation between the human livers that were susceptible to VPA induced hepatotoxicity and age or sex. This study was designed to show that VPA does induce hepatotoxicity in vitro at therapeutically relevant concentrations. Future studies will show whether VPA hepatotoxicity correlates with VPA metabolism, nutritional status or concomitant therapy.
• Fisher, R., Putnam, C. W., Koep, L. J., Sipes, I. G., Gandolfi, A. J., & Brendel, K. (1991). Cryopreservation of pig and human liver slices. Cryobiology, 28(2), 131-42.
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  The ability to cryopreserve human liver slices would greatly enhance the opportunities to test potentially hepatotoxic drugs and environmental contaminants as well as the metabolism of these compounds. This study focused on trying to cryopreserve pig and human liver slices. Since the acquisition of human liver tissue is unpredictable and scarce, an animal model was sought to predict problems associated with cryopreservation of human tissue. The pig liver was chosen because of its anatomical and physiological resemblance to human liver. The human liver tissues that did become available were obtained through the Arizona Organ Bank and the National Disease Research Interchange and from surgical liver resections. An in vitro culture system that employed precision-cut liver slices was used in this study. Different types and concentrations of cryoprotectants, cooling rates, and culture media were all tried in an attempt to cryopreserve pig and human liver slices. The viabilities of fresh and cryopreserved liver slices were evaluated using slice K+ retention and protein synthesis. Pig liver slices following cryopreservation retained between 80 and 85% of intracellular K+ content and protein synthesis as compared to controls using 1.4 M Me2SO, a 12 degrees C/min cooling rate, and a rapid rewarming rate of direct submersion of the slice into 37 degrees C fetal calf serum. Human liver slices following cryopreservation retained between 54 and 89% of intracellular K+ content and protein synthesis as compared to controls using the same protocol as for pigs, except that lower cooling rates were giving better results. The large variation seen in cryopreserved human liver slices was due to the length of warm and cold ischemia to which the tissue was exposed before arriving at the laboratory. This study indicated that pig and human liver slices can be cryopreserved and used for future toxicological and metabolic studies.
• Halldorsson, A., Hunter, G. C., Zukoski, C. F., & Putnam, C. W. (1991). The possible role of cyclosporine in nonocclusive mesenteric ischemia in a renal transplant patient. Transplantation, 51(6), 1298-301.
• Robertson, G. A., Valente, J. F., Hunter, G. C., Bernhard, V. M., & Putnam, C. W. (1991). Iliac-enteric fistula following Dacron patch angioplasty. Annals of vascular surgery, 5(5), 467-9.
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  We report a patient who developed fistulization between the jejunum and a Dacron patch placed at the bifurcation of the right common iliac artery during endarterectomy 14 years previously. The patient was successfully managed by resection of the iliac bifurcation and a segment of the involved bowel. While arterio-enteric fistulae typically involve prosthetic grafts, they may occur in association with artificial materials used to patch endarterectomy sites; hence, knowledge of technical detail is important in the diagnosis of this complication following endarterectomy.
• Wishnies, S. M., Parrish, A. R., Sipes, I. G., Gandolfi, A. J., Putnam, C. W., Krumdieck, C. L., & Brendel, K. (1991). Biotransformation activity in vitrified human liver slices. Cryobiology, 28(3), 216-26.
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  In vitro testing of human liver for biotransformation or xenobiotic metabolism studies has been limited by unpredictable acquisition of samples. Consequently, it has become necessary to consider methods to cryopreserve and store these samples whenever they do become available for culture of the revived tissue at a more convenient time. Human liver slices were cryopreserved by vitrification, which allows for the transfer of aqueous media to low temperatures (-196 degrees C) without the formation of ice crystals. Human liver slices were exposed to increasing concentrations of 1,2-propanediol up to a final concentration of 4.76 M in fetal calf serum. Slices were then vitrified by direct immersion into liquid nitrogen and warmed by submersion in 37 degrees C fetal calf serum. Warming was done either immediately or after 4 and 8 weeks of storage under liquid nitrogen. The effects of vitrification, storage time, and warming on biotransformation were determined by assessing the integrated metabolism of 7-ethoxycoumarin (7-EC). Vitrified or fresh human liver slices were exposed to 50 microM 7-EC and its primary metabolite 7-hydroxycoumarin (7-HC) in organ culture for up to 6 hr. Metabolite production of both fresh and vitrified liver slices was compared. Retention of the inherent biotransformation rate was usually high and seemed independent of storage time. Integration of both cytochrome P450-mediated and secondary conjugation processes was retained in cryopreserved tissue. Vitrification offers a way to cryopreserve human liver slices for the study of xenobiotic metabolism in humans.
• Gerrish, K. E., Putnam, C. W., & Laird, H. E. (1990). Prolactin-stimulated mitogenesis in the Nb2 rat lymphoma cell: lack of protoporphyrin IX effects. Life sciences, 47(18), 1647-53.
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  Pharmacological characterization of the Nb2 cell peripheral-type benzodiazepine receptor (PBR) was determined using selected 1,4-benzodiazepines, PK 11195, and protoporphyrin IX (PPIX) to compete for specific [3H] Ro5-4864 binding. These data suggest that PPIX possesses an affinity for the Nb2 cell PBR (Ki = 142 nM). We have previously reported that the peripheral benzodiazepine ligands, Ro5-4864 and PK 11195, modulate prolactin-stimulated mitogenesis in the Nb2 cell(1). In contrast, PPIX, a putative endogenous ligand for the PBR had no effect on prolactin-stimulated mitogenesis in the Nb2 cell over the concentration range from 10(-15) M to 10(-6) M. Taken together these data show that PPIX has an affinity for the Nb2 cell PBR but does not modulate prolactin-stimulated mitogenesis at concentrations which should bind to the Nb2 cell PBR.
• Neumayer, L. A., Bull, D. A., Mohr, J. D., & Putnam, C. W. (1990). The acutely affected abdomen in paraplegic spinal cord injury patients. Annals of surgery, 212(5), 561-6.
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  The records of 145 paraplegic or quadriplegic patients were reviewed to identify those factors useful in the correct diagnosis of the acute abdomen in this population. Twenty-one patients had 22 episodes of acute or subacute abdominal problems. Presenting complaints, physical findings, and laboratory results were useful in various ways. However appropriate radiographic studies led to the correct diagnosis in 77% of patients. Although paraplegic and quadriplegic patients are predisposed to a distinct constellation of medical problems, including urinary tract infection and calculi, they also may present with other abdominal conditions that cause significant morbidity and mortality if not promptly recognized.
• Rappaport, W. D., Hunter, G. C., McIntye, K. E., Ballard, J. L., Malone, J. M., & Putnam, C. W. (1990). Gastric outlet obstruction caused by traumatic pseudoaneurysm of superior mesenteric artery. Surgery, 108(5), 930-2.
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  Traumatic pseudoaneurysms of the superior mesenteric artery (SMA) are extremely rare. We describe two cases of posttraumatic proximal SMA pseudoaneurysms with symptoms of gastric outlet obstruction. Repair was accomplished by aorta-SMA bypass with saphenous vein. Injuries to the proximal SMA are easily missed at laparotomy, especially if intestinal ischemia or hematomas are absent. Recognition and repair are stressed to avoid the complications associated with pseudoaneurysm formation.
• Rappaport, W. D., McIntyre, K. E., Stanton, C., Carmona, R., Witzke, D., Fulginitti, J., Putnam, C. W., & Witte, C. L. (1990). The effect of alcohol in isolated blunt splenic trauma. The Journal of trauma, 30(12), 1518-20.
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  The effect of alcohol on trauma patients is controversial, with numerous authors citing no difference in mortality in acutely intoxicated patients. The purpose of our study was to retrospectively investigate the effect of alcohol in adult patients with isolated blunt splenic injury. From 1980 through 1989, 47 adult patients with splenic trauma as the only major injury were admitted to the Trauma Service. There were 37 males and ten females with a mean age of 29 years (range, 15 to 61). Motor vehicle accidents were responsible for 44 (94%) of the injuries. Group 1 consisted of 24 patients with a mean blood alcohol level of 185 mg/dl (range, 15 to 380). In Group 2 there were 23 patients without detectable blood alcohol. There were no statistically significant differences between the two groups in age, Abbreviated Injury Severity Score, initial hematocrit, and grade of splenic injury. Hypotension was present in 13 patients (55%) in Group 1 versus six patients (26%) in Group 2 (p less than 0.05). Significant abnormalities in clotting studies were present on admission in six patients (25%) in the alcohol-detected group versus one in the other group (p less than 0.05). Blood transfusion requirements in the first 24 hours were significantly greater in Group 1 (mean, 3.9 units) versus Group 2 (mean, 0.5 units) (p less than 0.001). If alcohol was present, there was much less chance for splenic conservation, as 18 patients (75%) underwent splenectomy versus seven patients (30%) in the nonalcohol group (p less than 0.05). There was one death and this occurred in a patient acutely intoxicated who suffered a cerebral infarct.(ABSTRACT TRUNCATED AT 250 WORDS)
• Buckley, A. R., Putnam, C. W., & Russell, D. H. (1989). Phorbol ester-induced attenuation of tissue DNA synthesis: antagonism by prolactin in liver and thymus. Biochemical pharmacology, 38(7), 1192-4.
• Laird, H. E., Gerrish, K. E., Duerson, K. C., Putnam, C. W., & Russell, D. H. (1989). Peripheral benzodiazepine binding sites in Nb 2 node lymphoma cells: effects on prolactin-stimulated proliferation and ornithine decarboxylase activity. European journal of pharmacology, 171(1), 25-35.
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  [3H]Ro 5-4864 binds to Nb 2 node lymphoma cells in a specific saturable and reversible fashion. Scatchard analysis of specific binding data reveals a single, homogeneous class of whole cell binding sites with a Kd of 3.94 +/- 0.22 nM and a Bmax value of 155 +/- 11 fmol (Ro 5-4864 bound)/2 x 10(6) cells. Ro 5-4864, a reported peripheral benzodiazepine receptor agonist both inhibits (10(-6) M) and potentiates (10(-9) M) the mitogenic action of prolactin on the Nb 2 node lymphoma cells. Interestingly, PK 11195, an antagonist, potentiates (10(-9) M) the mitogenic activity of prolactin in these cells. The actions of both Ro 5-4864 and PK 11195 seem to be mediated through a common receptor type since a 10(-6) M concentration of either agent will block the others potentiating action. Furthermore, the simultaneous addition of a 10(-9) M concentration of Ro 5-4864 and PK 11195 does not further increase the effect on prolactin stimulated mitogenesis. Clonazepam, a central benzodiazepine receptor agonist has no effect on prolactin-stimulated mitogenesis in this system. These data suggest that the Nb 2 node lymphoma cells possess a peripheral-type benzodiazepine receptor. In these cells, this receptor seems to serve the function of modulating the ability of the growth factor, prolactin to initiate the mitogenic process. These studies also suggest that Ro 5-4864 is functioning as a partial agonist rather than a 'pure' agonist for the peripheral benzodiazepine receptor in this system.
• Parola, A. L., Putnam, C. W., Russell, D. H., & Laird, H. E. (1989). Solubilization and characterization of the liver peripheral-type benzodiazepine receptor. The Journal of pharmacology and experimental therapeutics, 250(3), 1149-55.
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  The rat liver membrane-bound and digitonin-solubilized peripheral-type benzodiazepine receptors (mPBZR and dsPBZR, respectively) were characterized. Forty percent of the receptors were solubilized from a liver homogenate with 0.25% digitonin. Scatchard analysis of saturation data for the mPBZR and the dsPBZR showed Kd = 1.5 nM and maximum number of binding sites = 3.12 pmol/mg of protein and Kd = 9.2 nM and maximum number of binding sites = 1.10 pmol/mg of protein, respectively. Estimates of Kd calculated from kinetic data agree with estimates from Scatchard analysis. The affinity of the PBZR for [3H]Ro5-4864 was not affected by guanosine 5'0-(3-thiotriphosphate) which suggests the receptor is not coupled to a G-protein. Competition for specific [3H]Ro5-4864 binding by various ligands demonstrated the same rank order potency of binding inhibition for the membrane bound and solubilized receptors (PK-11195 greater than or equal to Ro5-4864 greater than diazepam greater than clonazepam). Thus, the soluble receptor had ligand binding characteristics similar to those of the membrane PBZR. [3H]PK-14105 was used to photoaffinity label the PBZR in a rat liver homogenate. Labeling was specific for the PBZR and the molecular weight of the digitonin-solubilized photoaffinity-labeled receptor was estimated to be 170 kDa by gel filtration chromatography. Estimation of the molecular weight of the [3H]PK-14105 labeled receptor by sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrated a single protein corresponding to 19 kDa.
• Buckley, A. R., Putnam, C. W., & Russell, D. H. (1988). Prolactin as a mammalian mitogen and tumor promoter. Advances in enzyme regulation, 27, 371-91.
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  Cellular proliferation and differentiation of the mammalian mammary gland requires a medley of hormones including the anterior pituitary hormone, PRL. Recent evidence extends the role of PRL as a mammalian mitogen to cells in several physiological systems not directly involved in reproductive functions, such as liver and lymphocytes. PRL administration induces biochemical markers expressed during the G1 phase of cell cycle and activates DNA synthesis in rat liver. Chronic PRL treatment causes hepatomegaly, reflecting its stimulation of the proliferative process. In vitro, a lactogen-dependent cell line, the Nb2 rat node lymphoma cell, serves as a useful paradigm to study PRL action on mitogenesis. These cells, when cultured in the presence of lactogens, proliferate in a dose-dependent manner. The effects of various pharmacological agents on discrete phases of the cell cycle may be readily assessed in these cells since PRL-stimulated entry into cycle is signalled by an elevation of ODC activity at 6 hr and entry into S-phase at 6-12 hr. The parallel effects of phorbol ester tumor promoters and PRL on cell cycle progression in Nb2 lymphoma cells and in hepatic proliferation suggest that PRL may likewise mediate proliferation in aberrant growth conditions such as neoplasia. The data presented support the hypothesis that PRL is capable of promoting hepatocarcinogenesis. Its chronic administration after a hepatic initiating agent stimulated the development of histochemical and biochemical markers characteristic of preneoplasia. Further, the effect of PRL was comparable to that of the hepatocarcinogen when either was administered alone. Thus, hyperprolactinemia may serve to promote the development of hepatic tumors. Phorbol esters are thought to promote tumorigenesis by directly activating PKC. In the Nb2 lymphoma cell model, tumor promoting phorbol esters mimic the effects of PRL. Similarly, PRL-stimulated enzyme induction in liver is mirrored by phorbol ester treatment, and inhibitors of PKC block PRL-stimulated mitogenesis in Nb2 cells. Further, PRL or TPA administration to rats causes translocation of PKC activity from the hepatic cytosol to the membrane fraction, reflecting kinase activation. Therefore, PRL activation of PKC appears to be a physiological phenomenon of general significance, occurring as the result of lactogen receptor stimulation and serving to transmit intracellular signals linked to the regulation of mitogenesis. Further study is required to more fully define the scope of PRL-mediated mitogenic actions as well as its effects on the expression of differentiated products in tissues and cells.
• Buckley, A. R., Russell, D. H., Montgomery, D. W., & Putnam, C. W. (1988). Prolactin as a hepatotrophic hormone. Transplantation proceedings, 20(1 Suppl 1), 706-9.
• Duerson, K. C., Buckley, A. R., Putnam, C. W., Russell, D. H., & Laird, H. E. (1988). Binding characterization of the peripheral type benzodiazepine receptor (P-BZ) in rat liver. Proceedings of the Western Pharmacology Society, 31, 133-5.
• Hunter, G. C., Steinkirchner, T., Burbige, E. J., Guernsey, J. M., & Putnam, C. W. (1988). Venous complications of sclerotherapy for esophageal varices. American journal of surgery, 156(6), 497-501.
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  Although endoscopic sclerotherapy is effective in controlling bleeding from esophageal varices, the effects of sclerosing agents on the extrahepatic portal and splenic veins have not previously been investigated. This study of 21 men with portal hypertension and variceal bleeding compares the morphology of the portal and splenic veins in 11 who had received endoscopic sclerotherapy versus 10 patients who did not. The mean number of injections per patient was 11 +/- 5, the mean volume of 1.5 percent sodium tetradecyl injected was 23 +/- 15 ml, and the interval between the last injection and surgery was 15 +/- 6.5 days. Among the 11 patients who had endoscopic sclerotherapy, portal vein thrombosis occurred in 4 (36 percent). Two of these patients died from acute liver failure; the other two had shunt procedures. Histologic changes included intimal thickening and medial fibrosis in seven patients, thrombus in four patients, and destruction of the venous architecture in two patients. Of the 10 patients with portal hypertension who did not have endoscopic sclerotherapy, all had medial fibrosis of the portal vein, with thrombus and intimal thickening present in only 1. These findings suggest that endoscopic sclerotherapy for esophageal varices should be used cautiously in patients who may later require a shunt. Moreover, further studies are necessary to evaluate the long-term effects of injecting sclerosing agents into the portal circulation before widespread use of prophylactic sclerotherapy can be recommended.
• Parola, A. L., Buckley, A. R., Putnam, C. W., Russell, D. H., & Laird, H. E. (1988). Solubilization of a peripheral type benzodiazepine binding site from rat liver. Proceedings of the Western Pharmacology Society, 31, 49-52.
• Peterson, C. L., Duerson, K. C., Buckley, A. R., Putnam, C. W., Russell, D. H., & Laird, H. E. (1988). Embryological development of peripheral benzodiazepine binding sites in the chick liver. Proceedings of the Western Pharmacology Society, 31, 263-4.
• Buckley, A. R., Putnam, C. W., Evans, R., Laird, H. E., Shah, G. N., Montgomery, D. W., & Russell, D. H. (1987). Hepatic protein kinase C: translocation stimulated by prolactin and partial hepatectomy. Life sciences, 41(26), 2827-34.
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  Prolactin stimulates a hepatotrophic response similar to that caused by phorbol esters or partial hepatectomy in rats. Since phorbol esters, which activate protein kinase C, mimic prolactin action in liver, the relationship between prolactin administration and subsequent hepatic protein kinase C translocation was assessed. Prolactin administration rapidly stimulated a 4-fold elevation of membrane protein kinase C activity. The effect of prolactin on hepatic protein kinase C was specific for lactogenic hormones but could be duplicated by phorbol esters. Further, an increase in serum prolactin was demonstrated subsequent to partial hepatectomy and preceding hepatic protein kinase C translocation. Therefore, translocation of hepatic protein kinase C appears important for hepatic proliferation in response to prolactin administration and to partial hepatectomy.
• Russell, D. H., Buckley, A. R., Montgomery, D. W., Larson, N. A., Gout, P. W., Beer, C. T., Putnam, C. W., Zukoski, C. F., & Kibler, R. (1987). Prolactin-dependent mitogenesis in Nb 2 node lymphoma cells: effects of immunosuppressive cyclopeptides. Journal of immunology (Baltimore, Md. : 1950), 138(1), 276-84.
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  Prolactin (PRL)-stimulated ornithine decarboxylase (ODC) activity and subsequent proliferation are inhibited by the cyclopeptides cyclosporine (CsA) and didemnin B (DB) in Nb 2 node lymphoma cells. Similar concentrations of these agents also inhibit 125I-PRL binding, suggesting that their inhibitory effects on these PRL-dependent physiologic responses are mediated at least in part at the level of PRL receptor interactions. The phorbol ester TPA stimulated ODC activity and [3H]thymidine incorporation to 54% and 31% that of a near-optimal mitogenic concentration of PRL (10 ng/ml), suggesting that mitogenesis in these cells is coupled to some degree to the activation of protein kinase C (PKC). The calcium ionophore A23187 increased ODC activity only slightly and actually decreased [3H]thymidine incorporation to a value below the "cells only" controls. The addition of TPA plus A23187 did not further enhance the effects of TPA to elevate ODC activity and [3H]thymidine incorporation. However, A23187 significantly elevated PRL-stimulated ODC activity with a subsequent inhibition of [3H]thymidine incorporation, suggesting a block of entry into S phase. Both cyclopeptides decreased the elevation of ODC activity in G1 phase of cell cycle in response to PRL, suggestive of a site of action for these agents in early G1, a conclusion compatible with their ability to inhibit PRL binding to these cells. Addition of CsA or DB 2 hr after PRL had no effect on PRL-stimulated ODC activity detectable at 6 hr, but addition of either as late as 6 hr still affected the extent of mitogenesis. This is in line with the requirement for PRL to be present in the culture medium for a minimum of 3 to 6 hr to invoke a maximal effect on mitogenesis. Addition of either cyclopeptide after the cells were in S phase had no effect on the extent of [3H]thymidine incorporation. An inhibitor of the cyclooxygenase pathway (indomethacin) enhanced both PRL-stimulated ODC activity and proliferation, whereas inhibition of the lipoxygenase pathway by NDGA attenuated only proliferation, suggesting that in Nb 2 cells, products of the lipoxygenase pathway may contribute to the mechanism of PRL-stimulated mitogenesis. Because Nb 2 lymphoma cells were derived from estrogenized rats, estrogen was tested as a mitogen. By itself it was not mitogenic, but in conjunction with PRL, estradiol-17 beta elevated the ODC response and inhibited proliferation. Inhibitors of PKC known to have minimal effects on RNA synthesis, quercetin and gossypol, totally inhibited both the elevations of ODC activity and [3H]thymidine incorporation in response to PRL in Nb 2 lymphoma cells.(ABSTRACT TRUNCATED AT 400 WORDS)
• Buckley, A. R., Montgomery, D. W., Kibler, R., Putnam, C. W., Zukoski, C. F., Gout, P. W., Beer, C. T., & Russell, D. H. (1986). Prolactin stimulation of ornithine decarboxylase and mitogenesis in Nb2 node lymphoma cells: the role of protein kinase C and calcium mobilization. Immunopharmacology, 12(1), 37-51.
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  The tumor promotor 12-O-tetradecanoylphorbol-13-acetate (TPA) in combination with calcium ionophores has been shown to bypass the requisite antigen- or lectin-induced signal for lymphocyte mitogenesis. This suggests that protein kinase C activation and calcium mobilization may be early events required for lymphocyte proliferation. Therefore, the relationship(s) of protein kinase C activation and calcium mobilization to ornithine decarboxylase induction and cellular proliferation were examined in a rat node lymphoma cell line (Nb2) which is dependent upon prolactin (PRL) for mitogenesis. TPA enhanced PRL-stimulated Nb2 node lymphoma cell ornithine decarboxylase induction and [3H]thymidine incorporation. Addition of a calcium ionophore (A23187) to cultures containing TPA plus PRL increased ornithine decarboxylase above PRL alone or PRL plus TPA but inhibited proliferation compared to the PRL plus TPA regimen. Exposure of cells to TPA or TPA plus A23187 increased [3H]thymidine incorporation in a similar manner to that demonstrated for low-dose PRL. However, optimal concentrations were only 20-25% as effective as mitogens as was optimal PRL. Protein kinase C and calmodulin antagonists inhibited PRL-stimulated ornithine decarboxylase induction and proliferation. Ca2+ chelation or cation channel antagonism inhibited both PRL-stimulated responses. The cyclic AMP analogue, 8Br-cAMP, inhibited PRL-stimulated ornithine decarboxylase activity as well as cellular proliferation processes assessed by [3H]thymidine incorporation. Finally, tumor-promoting phorbol esters inhibited 125I-rPRL binding. These data strongly suggest that protein kinase C activation and calcium mobilization are requisite events for PRL-stimulated ornithine decarboxylase induction and cellular proliferation in Nb2 node lymphoma cells. An additional component that is linked to alterations in K+ channeling is also implicated. These data support a role for protein kinase C in PRL-coupled mitogenesis. However, other critical Ca2+ and/or ion-induced events are also required.
• Buckley, A. R., Putnam, C. W., Montgomery, D. W., & Russell, D. H. (1986). Prolactin administration stimulates rat hepatic DNA synthesis. Biochemical and biophysical research communications, 138(3), 1138-45.
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  Prolactin is an important growth modulatory hormone in fetal and adult tissues. Its administration stimulates enzymatic markers of the G1 phase of cell cycle in rat liver and other tissues. To determine the effects of prolactin administration on hepatic DNA synthesis (S phase), rats received prolactin at 12 hour intervals for 48 hours and DNA synthesis was assessed by [3H]-thymidine incorporation. Prolactin administration stimulated DNA synthesis 2-4 fold above controls in the livers of adult and weanling animals. Increased incorporation of radiolabel was associated with the nucleus of hepatoparenchymal cells. These data support the hypothesis that prolactin may be a physiological regulator of hepatic DNA synthesis. Further, since stress stimulates prolactin secretion, we suggest that prolactin may participate in the hepatic compensatory hyperplasia elicited by the stress associated with partial hepatectomy.
• Buckley, A. R., Putnam, C. W., & Russell, D. H. (1985). In vivo induction of rat hepatic ornithine decarboxylase and plasminogen activator by 12-O-tetradecanoylphorbol 13-acetate. Biochimica et biophysica acta, 841(1), 127-30.
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  Rapid and substantial elevations in ornithine decarboxylase and plasminogen activator have been linked to tumor promotion in mouse epidermis and in vitro. Systemic administration of 12-O-tetradecanoylphorbol 13-acetate (TPA) rapidly increased both enzymic activities in rat liver. Pretreatment with either cycloheximide or actinomycin D attenuated both enzyme inductions. It is concluded that: (1) systemic TPA rapidly induces plasminogen activator and ornithine decarboxylase activities in rat liver; and (2) both inductions reflect de novo enzyme synthesis.
• Buckley, A. R., Putnam, C. W., & Russell, D. H. (1985). Prolactin is a tumor promoter in rat liver. Life sciences, 37(26), 2569-75.
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  Since prolactin, like the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate, induces ornithine decarboxylase and plasminogen activator activities, biochemical markers of a trophic response, this hormone might likewise promote neoplasia. To test this theory, rats were initiated with a hepatocarcinogen followed by six weeks of ovine prolactin. This regimen caused hepatomegaly and the development of enzyme-altered foci. Promotion with prolactin for 23 weeks further increased the numbers of enzyme-altered foci. We suggest that prolactin is an endogenous tumor promoter for chemically initiated cells.
• Smith, P. F., Gandolfi, A. J., Krumdieck, C. L., Putnam, C. W., Zukoski, C. F., Davis, W. M., & Brendel, K. (1985). Dynamic organ culture of precision liver slices for in vitro toxicology. Life sciences, 36(14), 1367-75.
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  The lack of a reproducible method for the production of thin tissue slices has hindered the use of liver slices as an in vitro tool for hepatotoxicity studies. Fresh human, rat, and rabbit liver was processed using a mechanical slicer. With this instrument, precision (5% of thickness) liver slices in the submillimeter range could be produced at a rapid rate. Slices were prepared from fresh livers in chilled, oxygenated buffer to minimize trauma. Following incubation for up to 20 h in a dynamic organ culture system, histology of incubated slices suggested that 250 m precision-cut slices were optimum in regard to morphology relative to liver slices incubated under conventional organ culture conditions. Addition of bromobenzene to the culture showed time-dependent hepatotoxicity based on two classic parameters of cell degeneration. Histological evidence is presented which suggests the usefulness of this system for hepatotoxicity studies and the production of focal necrosis in vitro.
• Buckley, A. R., Putnam, C. W., & Russell, D. H. (1984). Rapid elevation of plasminogen activator activity in rat tissues by prolactin. Biochemical and biophysical research communications, 122(3), 1005-11.
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  Administration of ovine prolactin resulted in a rapid and significant induction of plasminogen activator activity within two hours in the rat adrenal gland, heart, aorta and liver. Prolactin exposure reduced baseline proteolytic activity in lung and skeletal muscle at 6-8 hours following hormonal challenge. This was coincident with a return toward baseline levels in enzyme activity demonstrated in other tissues. Pretreatment with actinomycin D or cycloheximide ameliorated the enzymic induction in the liver and adrenal and partially inhibited the aortic response. These data suggest that prolactin exposure leads to increased proteolytic activity through de novo biosynthesis in a variety of solid organs. In addition, pro-enzyme activation may also occur in vascular tissue.
• Karrer, F. M., Rhenman, B., Buckley, A. R., Steinbronn, K. K., & Putnam, C. W. (1984). A reproducible large animal model of acute hepatic failure. Current surgery, 41(6), 464-7.
• Putnam, C. W., Buckley, A. R., Warneke, J. A., Karrer, F. M., Rhenman, B., & Steinbronn, K. (1984). The mechanism of hepatoprotection by epsilon aminocaproic acid and putrescine. Surgery, 96(2), 214-22.
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  Hepatic neutral serine proteases (including plasminogen activator) and ornithine decarboxylase (ODC) are induced by the hepatotoxin galactosamine (GALN). We examined the hepatoprotection conferred by epsilon-aminocaproic acid (EACA), a fibrinolytic inhibitor, putrescine (PUTR), the polyamine generated from ornithine by ODC, and alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC. GALN, 450 mg/kg, was administered intraperitoneally to Wistar-Lewis rats (group I). Groups II, III, and IV were also given EACA (80 mg/kg), PUTR (0.3 mmol/kg), or DFMO (0.3 mmol/kg), respectively, 1 hour before and 3, 7, and 12 hours after GALN. Rats were killed 2 hours after an intraperitoneal dose of 3H-thymidine was administered, 30 or 45 hours after GALN. EACA and PUTR were effective protectants against necrosis as judged by enzymes and histologic findings. Neither increased thymidine incorporation above the levels seen with GALN only. DFMO offered no protection even though thymidine incorporation at 45 hours was increased. Both EACA and PUTR, which have similar chemical structures, possessed significant antiprotease activity in vitro, suggesting that they act by inhibiting toxin-induced neutral serine protease activity and not by accelerating regeneration.
• Rhenman, B., Putnam, C. W., Steinbronn, K., Buckley, A. R., & Karrer, F. M. (1984). A reproducible large animal model of acute hepatic failure.. Current Surgery.
• Schnellmann, R. G., Volp, R. F., Putnam, C. W., & Sipes, I. G. (1984). The hydroxylation, dechlorination, and glucuronidation of 4,4'-dichlorobiphenyl (4-DCB) by human hepatic microsomes. Biochemical pharmacology, 33(21), 3503-9.
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  Since chlorine placement and the degree of chlorination of the biphenyl nucleus play an important role in the metabolism and ultimate elimination of polychlorinated biphenyls (PCBs), we have studied the metabolism of 4,4'-dichlorobiphenyl (4-DCB) by human hepatic microsomes. This low molecular weight PCB congener is substituted at the preferred site of metabolism (para-position). 4-DCB was metabolized by human microsomes with a Km of 0.43 microM and a Vmax of 1.2 pmoles/mg microsomal protein/min. Six metabolites were identified: 4,4'-dichloro-3,3'-biphenyldiol, 4'-chloro-3-biphenylol, 4'-chloro-4-biphenylol, 4,4'-dichloro-2-biphenylol, 4,4'-dichloro-3-biphenylol (most abundant), and 3,4'-dichloro-4-biphenylol. [14C]-4-DCB equivalents were found to covalently bind to microsomal protein. Addition of a 1 mM concentration of reduced glutathione decreased the degree of covalent binding. These data suggest that human microsomes metabolize this PCB through an arene oxide and that an "NIH shift" occurs. When UDPGA was added to the incubation, human microsomal glucuronosyltransferase catalyzed the formation of the glucuronide of the major metabolite, 4,4'-dichloro-3-biphenylol. These and previous in vitro results show that the biotransformation of PCBs by humans is governed by the same principles established for the in vivo biotransformation of PCBs by the rat, mouse and monkey. That is, PCBs without two adjacent unsubstituted carbon atoms are poorly metabolized and that an unsubstituted para-position facilitates metabolism.
• Buckley, A. R., & Putnam, C. W. (1983). A new colorimetric assay for plasminogen activator activity. Proceedings of the Western Pharmacology Society, 26, 73-6.
• McIntyre, K. E., & Putnam, C. W. (1983). A new dialysis access device. Surgery, gynecology & obstetrics, 156(6), 804-7.
• Putnam, C. W. (1983). Techniques of ultrasonic dissection in resection of the liver. Surgery, gynecology & obstetrics, 157(5), 474-8.
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  The ultrasonic device allows dissection of hepatic parenchyma without damage to vascular or ductal structures. With it, both standard hepatic resections and more radical ones are made safer. It is an instrument useful for hepatic operations.
• Schnellmann, R. G., Putnam, C. W., & Sipes, I. G. (1983). Metabolism of 2,2',3,3',6,6'-hexachlorobiphenyl and 2,2',4,4',5,5'-hexachlorobiphenyl by human hepatic microsomes. Biochemical pharmacology, 32(21), 3233-9.
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  Since the metabolism of polychlorinated biphenyls (PCBs) is the critical factor that determines whether or not they accumulate in adipose tissue, we have studied the metabolism of two hexachlorobiphenyls (HCBs), 2,2'3,3',6,6'-hexachlorobiphenyl (236-HCB) and 2,2'4,4',5,5'-hexachlorobiphenyl (245-HCB), by human hepatic microsomes. Human microsomes were isolated from patients undergoing liver resection and were found to have cytochrome P-450 levels (0.28 nmoles/mg microsomal protein) and cytochrome P-450-dependent enzymatic activities similar to those reported by other workers. 245-HCB was not metabolized by human microsomes under various conditions, while 236-HCB was metabolized with an apparent Km of 8.8 microM and a Vmax of 5.1 pmoles/mg microsomal protein/min. Two major metabolites were formed and identified by gas chromatography-mass spectrometry as 2,2',3,3',6,6'-hexachloro-4-biphenylol and 2,2',3,3'6,6'-hexachloro-5-biphenylol. [14C]236-HCB equivalents were found to covalently bind to microsomal protein. Addition of 1 or 5 mM reduced glutathione decreased the degree of covalent binding. These data suggest that HCBs are metabolized through an arene oxide. The fact that 245-HCB was not metabolized explains why it is the predominant PCB found in human adipose tissue.
• George, W. L., Rolfe, R. D., Harding, G. K., Klein, R., Putnam, C. W., & Finegold, S. M. (1982). Clostridium difficile and cytotoxin in feces of patients with antimicrobial agent-associated pseudomembranous colitis. Infection, 10(4), 205-8.
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  Thirty patients with antimicrobial agent-associated pseudomembranous colitis (PMC) were studied for the presence of Clostridium difficile and its cytotoxin in feces. Either colonoscopy or barium enema radiography was required in three patients for the diagnosis of PMC because of nondiagnostic findings at sigmoidoscopy. Both the organism and cytotoxin were detected in 27 of the 30 patients; Staphylococcus aureus was excluded as the cause of PMC in two of the remaining patients. Eighteen of 19 patients with C. difficile-induced PMC who were treated with oral vancomycin had a salutary response; seven patients, however, had a relapse of colitis following the discontinuation of vancomycin. In general, relapses of colitis responded to retreatment with vancomycin. The implication of C. difficile as a cause of diarrhea is best achieved by the demonstration of colonic mucosal plaques or of a pseudomembrane. The value of fecal culture for C. difficile and cytotoxin assay is limited by the existence of asymptomatic carriers.
• Putnam, C. W., & Wangensteen, S. L. (1981). Pancreatic pseudocyst after distal splenorenal shunt. Annals of surgery, 194(5), 667.
• Starzl, T. E., Koep, L. J., Weil, R., Lilly, J. R., Putnam, C. W., & Aldrete, J. A. (1980). Right trisegmentectomy for hepatic neoplasms. Surgery, gynecology & obstetrics, 150(2), 208-14.
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  Thirty patients had right trisegmentectomy for 19 primary hepatic malignant tumors, seven localized liver metastases and four benign lesions. A technical refinement that aided resection of bulky posterior and superior tumors was intrahepatic identification and control of the right hepatic vein. The operative mortality was 3.3 per cent. Late hepatic insufficiency was not observed. More than one-half of the patients operated upon a year or more ago for primary hepatic malignant growths had a tumor-free state at the 12 month follow-up period. Beyond this time, there was only one recurrence. The results in children were twice as good as in adults. The results in treating localized liver metastases from distant primary sites were inferior to those in treating primary hepatic tumors. A hypothetical case was made for combining hepatic resection with adjuvant chemotherapy, even though our experience could not be construed as direct support for this practice.
• Corman, J. L., Putnam, C. W., Iwatsuki, S., Redeker, A. G., Porter, K. A., Peters, R. L., Schröter, G., & Starzl, T. E. (1979). Liver allograft. Its use in chronic active hepatitis with macronodular cirrhosis, hepatitis B surface antigen. Archives of surgery (Chicago, Ill. : 1960), 114(1), 75-8.
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  A patient suffering from chronic active hepatitis with macronodular cirrhosis, positive for hepatitis B surface antigen (HBsAg), was treated with an orthotopic liver allograft. The HBs antigenemia, as measured with several precipitation tests and by complement fixation, became negative after transplantation and remained so for about 2 1/2 months. During the interval, very low titers of the antigen were detectable by radioimmunoassay. At about three months after transplantation, she had an attack of acute hepatitis, at which time HBsAg became detectable by all tests. She recovered, but progressive liver disease developed during the remaining 1 1/2 years of her life. She died of disseminated nocardiosis and candidiasis with deteriorating hepatic function. The homograft at autopsy showed no evidence of rejection, but was the site of chronic active liver disease, although of a different pathologic pattern than that affecting her native liver. The differences in histology may reflect the influence of chronic immunosuppression on the features of chronic active hepatitis.
• Kootstra, G., West, J. C., Dryburgh, P., Krom, R. A., Putnam, C. W., & Weil, R. (1978). Pediatric cadaver kidneys for transplantation. Surgery, 83(3), 333-7.
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  A group of 24 kidneys from donors ranging in age from 1 1/2 to 10 years were transplanted singly into adults and were compared to a group of 44 adult cadaveric kidneys transplanted into adults. There were no vascular complications in either group. There were two urological complications in the 24 pediatric donor cases and none with the adult donor cases. During the first month after transplantation, the mean creatinine clearance was lower in the pediatric donor group; later the function of the pediatric donor kidneys was at least as good as the function of the adult donor grafts. In the group of pediatric donor kidneys, the outcome using kidneys from donors younger than 3 years of age was less satisfactory than for donors 3 to 10 years of age. These data suggest that transplantation of a single pediatric kidney into an adult, particularly if the pediatric donor is at least 3 years of age, will provide satisfactory renal function.
• Putnam, C. W. (1978).

  Acute Neurological Complications After Liver Transplantation with Particular Reference to Intraoperative Cerebral Air Embolus

  . Ann. Surg.. doi:10.1097/00000658-197803000-00003
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  Nine of 48 adult patients who underwent orthotopic liver transplantation developed significant clinical neurological abnormalities recognized shortly after operation. Decrease in consciousness occurred with resultant coma, focal and generalized seizures and the occasional appearance of a state of akinetic mutism. Neuropathological abnormalities consisted of multifocal areas of infarction in cerebral cortex and basal ganglia in five patients, central pontine myelinolysis in five (often more extensive than usually reported), Wernicke's encephalopathy in three, glial nodules in two, and fungal abscesses in one. Alzheimer II astrocytosis was found in all brains available for retrospective study. There was direct evidence in two of the patients that air embolization from the homografts had occurred. Correlation of this with the brain infarcts in these and other cases seems reasonable. The ease with which air passed to the systemic circulation is explicable by the right to left venous-arterial shunts that are common in chronic liver disease. With the delineation of this cause for the neurologic complications, measures to prevent it in future cases have been described.
• Starzl, T. E., Putnam, C. W., & Koep, L. J. (1978). Portacaval shunt and hyperlipidemia. Archives of surgery (Chicago, Ill. : 1960), 113(1), 71-4.
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  The use of a completely diverting portacaval shunt for the purpose of lowering the serum concentration of cholesterol and low-density lipoproteins is reviewed and a recommendation provided that such a shunt for hyperlipidemia should be considered for type II individuals.
• Starzl, T. E., Schneck, S. A., Mazzoni, G., Aldrete, J. A., Porter, K. A., Schröter, G. P., Koep, L. J., & Putnam, C. W. (1978). Acute neurological complications after liver transplantation with particular reference to intraoperative cerebral air embolus. Annals of surgery, 187(3), 236-40.
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  Nine of 48 adult patients who underwent orthotopic liver transplantation developed significant clinical neurological abnormalities recognized shortly after operation. Decrease in consciousness occurred with resultant coma, focal and generalized seizures and the occasional appearance of a state of akinetic mutism. Neuropathological abnormalities consisted of multifocal areas of infarction in cerebral cortex and basal ganglia in five patients, central pontine myelinolysis in five (often more extensive than usually reported), Wernicke's encephalopathy in three, glial nodules in two, and fungal abscesses in one. Alzheimer II astrocytosis was found in all brains available for retrospective study. There was direct evidence in two of the patients that air embolization from the homografts had occurred. Correlation of this with the brain infarcts in these and other cases seems reasonable. The ease with which air passed to the systemic circulation is explicable by the right to left venous--arterial shunts that are common in chronic liver disease. With the delination of this cause for the neurologic complications, measures to prevent it in future cases have been described.
• Starzl, T. E., Watanabe, K., Porter, K. A., & Putnam, C. W. (1978). Failure of renal homograft protection with RNA pretreatment. Transplantation, 25(6), 345-6.
• Buerk, C. A., Putnam, C. W., & Starzl, T. E. (1977). Major hepatic resection and portal pressure. Surgery, gynecology & obstetrics, 144(6), 853-4.
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  Although other reports have stressed the inevitability of portal hypertension and splanchnic pooling following major liver resections, clinical observations during 30 major hepatic resections and measurements of portal pressure in three consecutive trisegmentectomies fail to support this contention. If the remaining liver is normal and careful anatomic dissection is used, major resections can be performed without inducing portal hypertension.
• Putnam, C. W., & Starzl, T. E. (1977). Simplified biopsy of the liver in dogs. Surgery, gynecology & obstetrics, 144(5), 759.
• Putnam, C. W., & Starzl, T. E. (1977). Transplantation of the liver. The Surgical clinics of North America, 57(2), 361-73.
• Putnam, C. W., Halgrimson, C. G., Koep, L., & Starzl, T. E. (1977). Progress in liver transplantation. World journal of surgery, 2(1), 165-75.
• Putnam, C. W., Porter, K. A., Peters, R. L., Ashcavai, M., Redeker, A. G., & Starzl, T. E. (1977). Liver replacement for alpha1-antitrypsin deficiency. Surgery, 81(3), 258-61.
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  A 16-year-old girl with advanced cirrhosis and severe alpha 1-antitrypsin deficiency of the homozygous Pi ZZ phenotype was treated by orthotopic liver transplantation. After replacement of the liver with a homograft from a donor with the normal Pi MM phenotype, the alpha 1-antitrypsin concentration in the recipient's serum rose to normal; it had the Pi MM phenotype. Two and a third years later, chronic rejection necessitated retransplantation. Insertion of a homograft from a heterozygous Pi MZ donor was followed by the identification of that phenotype in the recipient's serum. Neither liver graft developed the alpha 1-antitrypsin glycoprotein deposits seen with the deficiency state. These observations confirm that this hepatic-based inborn error of metabolism is metabolically cured by liver replacement.
• Schröter, G. P., Hoelscher, M., Putnam, C. W., Porter, K. A., & Starzl, T. E. (1977). Fungus infections after liver transplantation. Annals of surgery, 186(1), 115-22.
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  The problem of fungus infections after liver transplantation was studied. In 100 consecutive recipients of orthotopic liver homografts there were 10 and 8 examples, respectively, of localized and disseminated infections caused by Candida species. Candidemia was demonstrated in 8 of these 18 patients. One patient who had a localized Candida infection also had disseminated cryptococcosis. An additional 31 patients were infested in that Candida could be cultured from sites where it is not normally found, such as the blood (8 examples), urine (8), ascitic fluid (8), and wounds (22). This exorbitant incidence of monilial infections and infestations was associated with a high frequency of complications involving the homograft as well as the hosts' gastrointestinal tract during the post-transplantation period. The yeasts found in blood, urine, ascitic fluid and elsewhere were thought to have originated from the gut. Ten of the 100 patients had aspergillosis which was localized in 7 instances and disseminated in 3. The lung was the most frequently affected organ. The fungus infections played a contributory role in the downhill course of our patients but in the event of death more fundamental and more frequent causes of failure were technical complications involving the homografts, difficulties in controlling rejection with reasonable immunosuppressive doses and bacterial sepsis. Suggestions have been made for the better control of fungal infections in liver recipients.
• Stables, D. P., Taubman, J., Hilton, J. W., Ronai, P. M., & Putnam, C. W. (1977). The radiology of ex vivo renal perfusion and autotransplantation. AJR. American journal of roentgenology, 128(2), 217-24.
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  The radiologic findings in 16 patients subjected to ex vivo renal perfusion and autotransplantation are reviewed. Preoperative excretion urography and renal arteriography are essential to define ureteral and arterial anatomy; renal scintiangiography and renography are useful for baseline studies. Plain radiographs should be obtained during nephrolithotomy. Intraoperative renal arteriography should be reserved for patients who require more precise definition of vascular anatomy or verification of adequate repair after a difficult dissection. Postoperative scintiangiography is required on the first day to exclude arterial occlusion. Renography and urography to evaluate autotransplant anatomy and function should be deferred for about 3 weeks, unless there is clinical evidence of a complication.
• Starzl, T. E., Porter, K. A., Francavilla, J. A., Benichou, J., & Putnam, C. W. (1977). A hundred years of the hepatotrophic controversy. Ciba Foundation symposium, 111-29.
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  Venous blood returning from the splanchnic viscera has liver-supporting (hepatotrophic) qualities not found to the same degree in other kinds of arterial or venous blood. The effects of portal blood have been noted in animals with two livers (or a differential portal blood supply to different regions of one liver) to include hypertrophy, glycogen storage, hyperplasia, capacity for regeneration, increase of several synthetic functions, and maintenance of normal structure. The main splanchnic venous hepatotrophic factors are endogenous hormones of which the single most important is insulin. Thus, the foregoing portal hepatotrophic effects are largely eliminated with the diabetes produced by alloxan or total pancreatectomy. The injury of portacaval shunt is caused by the diversion of the hormones around the liver. Accordingly, the atrophy, injury to the organelles, and loss of the capacity for cell renewal is minimized if insulin is infused into the portally deprived liver. In these and other experiments, exogenous glucagon alone or the addition of glucagon to insulin has had no effect, but this may be because of the masking presence of gut glucagon and other hormonal or non-hormonal substances in our models. At present, the effects on the liver of exogenous insulin, glucagon, epidermal growth factor, and numerous other hormones are being determined by their intraportal infusion into eviscerated dogs in which other endogenous splanchnic factors have been eliminated.
• Starzl, T. E., Putnam, C. W., & Koep, L. J. (1977). Current status of liver transplantation. Southern medical journal, 70(4), 389-90.
• Starzl, T. E., Putnam, C. W., Hansbrough, J. F., Porter, K. A., & Reid, H. A. (1977). Biliary complications after liver transplantation: with special reference to the biliary cast syndrome and techniques of secondary duct repair. Surgery, 81(2), 212-21.
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  In 93 consecutive cases of orthotopic liver transplantation, there were 24 example of biliary obstruction and eight of bile fistula formation. Six of the obstructed livers developed biliary cast formation so extensive that the smaller intrhepatic ducts became plugged to an extent that they could no longer have been treated by surgical mena. In each of the six cases, the most important causative factor was neglected obstruction of the large bile ducts with the intrahepatic lesions apparently being late and secondary. Stone and/or cast formation also occurred in other obstructed livers in the presence of bile fustulas, but these deposits were limited to the large ducts where they could have been or were removed. Although homograft bile undoubtedly has increased lithogenicity at certain posoperative times, the data from the present study have shown that biliary sludge formation essentially is always associated with defective bile duct reconstruction, and the observations have underscored the urgency with which reoperation must be considered. Techniques of secondary intervention have been described, with emphasis on conversion of cholecystojejunostomy to choledochojejunostomy. This operation has permitted salvage of homografts in eight of nine trials and the survival of seven patients.
• Starzl, T. E., Putnam, C. W., Porter, K. A., & Benichou, J. (1977). Portacaval shunt for glycogen storage disease and hyperlipidaemia. Ciba Foundation symposium, 311-25.
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  Complete portacaval shunt was used to treat 10 patients with glycogen storage disease. A favourable effect was noted on body growth and a number of metabolic abnormalities. More recently, continous night feedings with an intermittently placed gastric tube or through a gastrostomy has been shown to be helpful either before or after portacaval shunts. Such alimentation techniques may eliminate the need for shunts in some patients and be of adjuvant benefit in others. Portacaval shunt was also used for three children who had homozygous Type II hyperlipidaemia. Substantial reductions in serum cholesterol concentration were observed, as well as resorption of xanthomas. Reversal of some cardiovascular lesions has been documented. The benefits of portacaval shunt in these disorders is probably due to the change in the hormone climate of the liver and the whole organism brought about by diversion of the hormone-rich splanchnic venous blood around the liver.
• Starzl, T. E., Weil, R., & Putnam, C. W. (1977). Kidney transplantation. Modern trends in kidney transplantation. Transplantation proceedings, 9(1), 1-8.
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  Trends in renal transplantation stem from recognition of the virtues and drawbacks of this kind of treatment and from a better appreciation of the interrelationship between transplantation and dialysis.
• Yoshioka, H., McCalmon, R. T., Putnam, C. W., & Terman, D. S. (1977). Detection of porcine-specific canine antibodies by a radioimmunological technique. Transplantation, 23(1), 60-4.
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  A sensitive radioimmunological technique is described in which the primary interaction of 125I porcine membrane antigens with naturally occurring canine antibodies is measured by precipittion with 20% polyethylene glycol. Porcine membrane antigens were isolated, radiolabeled, and found to be maximally soluble in 20% polyethylene glycol solution. Binding of canine antisera to porcine antigens could be reduced by absorption with porcine lymphocytes. An eluate was obtained from a porcine kidney that was perfused in vivo for 330 min with oxygenated canine plasma and was identified as canine IgG by immunoelectrophoresis. When added to normal dog serum, the eluate markedly augmented the binding titer. Samples of dog serum taken after extracorporeal perfusion showed a significant decline in binding with no significant changes in lymphocytoxicity titer. These results suggest that this primary binding assay is a more sensitive indicator of the presence of graft-specific humoral antibodies and may be of value in evaluating prospective heterograft donors as well as in following the course of therapy.
• Yoshioka, H., McCalmon, R. T., Putnam, C. W., McIntosh, R. M., & Terman, D. S. (1977). Attenuation of hyperacute xenograft rejection in unmodified host by extracorporeal plasma perfusion. Transplantation, 24(1), 78-81.
• Beart, R. W., Putnam, C. W., & Starzl, T. E. (1976). Use of a U tube in the treatment of biliary disease. Surgery, gynecology & obstetrics, 142(6), 912-4.
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  This surgical technique has permitted re-entry into the intrahepatic and extrahepatic biliary tree for the purpose of dilatation and the manual propulsion of debris through a ductal anastomosis. The method undoubtedly has other applications in the treatment of complicated biliary duct problems.
• Putnam, C. W., Beart, R. W., Bell, R. H., & Starzl, T. E. (1976). Hepatic transplantation, 1975. Postgraduate medical journal, 52(5 Suppl), 104-8.
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  This report reviews experience with 97 patients given liver transplants. We regard out survival statistics as unsatisfactory, but fell they should encourage further work since 22 patients have survived at least one year with a maximum survival of 5 13 YEARS. The Achilles' heel of liver transplantation os bile duct reconstruction. We presently rely upon Roux-en-Y reconstruction, or alternatively, duct-to-duct anastomosis with a T-tube stent. The prime indication for liver replacement is non-neoplastic liver disease, but a favourable malignancy for treatment may prove to be small intrahepatic duct cell carcinomas.
• Putnam, C. W., Bell, R. H., Beart, R. W., Anderson, J. T., & Weil, R. (1976). Past experience and future studies with antilymphocyte globulin in recipients of kidney homografts. Postgraduate medical journal, 52(5 Suppl), 59-63.
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  We remain convinced that antilymphocyte globulin (ALG) is a potent immunosuppressive agent in humans. For that reason, we have used it in essentially every case of renal transplantation since May of 1966; now, however, we are about to embark on a controlled clinical trial of ALG in kidney transplantation and await with tremendous interest the results of other such trials from other centres around the world.
• Putnam, C. W., Porter, K. A., & Starzl, T. E. (1976). Hepatic encephalopathy and light and electron micrographic changes of the baboon liver after portal diversion. Annals of surgery, 184(2), 155-61.
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  Six baboons of varying weights and estimated ages had complete portal diversion. All animals became emaciated and lost hair. Five of the 6 developed hepatic encephalopathy so serious that it either killed them or required their sacrifice after an average of 109 days. One exceptional animal which lived for 208 days without encephalopathy had markedly elevated blood ammonia levels. In one brain that was examined, greatly increased numbers of Alzeheimer's Type II astrocytes were diffusely distributed in the cerebral cortex. Changes in liver function tests were similar to those reported by many authors in dogs. The 6 baboon's livers underwent striking atrophy during the 49 to 208 days of postoperative observation. With some variations in degree, the same light and electron microscopic changes were observed that have now also been seen after completely diverting portacaval shunt in rats, dogs and humans. Thru the hepatic injury of Eck fistula is common to all species so far studied although most of the metabolic consequences of the procedure seem to selectively spare rats and man.
• Putnam, C. W., Porter, K. A., Weil, R., Reid, H. A., & Starzl, T. E. (1976). Liver transplantation of Budd-Chiari syndrome. JAMA, 236(10), 1142-3.
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  Orthotopic liver transplantation was accomplished in a 22-year-old woman dying of the Budd-Chiari syndrome. She is well and has normal liver function 16 months postoperatively. In view of the good early result, it will be appropriate to consider liver replacement for this disease in further well-selected cases.
• Roddy, H., Putnam, C. W., & Fennell, R. H. (1976). Pathology of liver transplantation. Transplantation, 22(6), 625-30.
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  Experience with renal transplantation indicates failure of the graft is usually due to immunological rejection. In a previous study of human liver transplantation, rejection was the major cause of transplant failure in 4 of 17 patients )24%); in this review of 76 liver transplantations, 64 of which survived the first postoperative week, rejection was the primary cause of graft failure in only 4 of these 64 cases (6%). The two most common causes of transplant failure were technical difficulties with the operative procedure and sepsis; these accounted for 47 (62%) graft failures of the total of 76 transplants. Biliary obstruction and sepsis are more common causes of liver failure than rejection, and patients with recurrent jaundice are now studied intensively for evidence of obstruction. Only after obstruction is excluded, is immunosuppression intensified. These results are a basis for optimism concerning the future of liver transplantation in management of potentially fatal liver disease.
• Schröter, G. P., Hoelscher, M., Putnam, C. W., Porter, K. A., Hansbrough, J. F., & Starzl, T. E. (1976). Infections complicating orthotopic liver transplantation: a study emphasizing graft-related septicemia. Archives of surgery (Chicago, Ill. : 1960), 111(12), 1337-47.
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  In 93 recipients of 102 orthotopic liver homografts, the incidence of bacteremia or fungemia exceeded 70%. The graft itself was usually an entry site for systemic infection after both immunologic and nonimmunologic parenchymal injury, especially if there was defective biliary drainage. The role of the homograft itself as the special infectious risk factor has prompted increased use of defunctionalized jejunal Roux limbs to reduce graft contamination. It has also stimulated very aggressive postoperative diagnostic efforts to rule out remedial mechanical complications of the transplant.
• Starzl, T. E., & Putnam, C. W. (1976). Surgical approaches to primary and metastatic liver neoplasms. International journal of radiation oncology, biology, physics, 1(9-10), 959-64.
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  Providing the anatomic conditions are appropriate, removal of as much as 85–90% of the liver is a relatively safe technique for the treatment of benign or malignant primary hepatic tumors and of isolated hepatic metastases. There have been apparent cures of malignant disease by this approach, but the paucity of concentrated experience at any one center and the failure so far to pool data from different centers effectively make a valid prognosis impossible. Total hepatectomy and liver replacement has resulted in the apparent core of some patients with primary hepatic malignancy. However, there has been an extremely high rate of recurrence of tumor. Small cancers which are lethal because of their strategic locations may provide an unusually good indication for liver replacement. Liver transplantation would rarely, if ever, be indicated for metastases to the liver.
• Starzl, T. E., Porter, K. A., & Putnam, C. W. (1976). Insulin, glucagon, and the control of hepatic structure, function, and capacity for regeneration. Metabolism: clinical and experimental, 25(11 Suppl 1), 1429-34.
• Starzl, T. E., Porter, K. A., Putnam, C. W., Schroter, G. P., Halgrimson, C. G., Weil, R., Hoelscher, M., & Reid, H. A. (1976). Orthotopic liver transplantation in ninety-three patients. Surgery, gynecology & obstetrics, 142(4), 487-505.
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  During the 11 1/2 year period ending 13 months ago, 93 consecutive patients were treated with orthotopic liver transplantation. Fifty-six of the recipients were 18 years old or younger, and the other 37 were adults. The most common indications for operation were biliary atresia, primary hepatic malignant tumor, chronic aggressive hepatitis and alcoholic cirrhosis. There has been a gradual improvement in results throughout the period of study, although to a satisfactory level. Twenty-seven of the 93 patients survived for at least one year after liver replacement with a maximum of six years, and 16 are still alive after 13 to 71 months. The 11 late deaths after one to six years were caused by chronic rejection, biliary obstruction, recurrence of hepatoma, systemic infection or hepatitis of the homograft. Rejection of the liver as judged by classical histopathologic criteria played a surprisingly small role in the heavy over-all mortality, accounting for less than 10 per cent of the deaths. Technical or mechanical problems, especially those of biliary duct reconstruction, were a far greater cause of failure, as were systemic infections. Six of the 37 adult recipients had lethal cerebrovascular accidents during, or just after, operation. When abnormalities of liver function developed in the postoperative period, the nearly automatic diagnosis of homograft rejection, in retrospect, proved to have been wrong in most instances. Further development of liver transplantation depends upon two kinds of progress. There must be reduction of operative and early postoperative accidents and complications by more discriminating patient selection, purely technical improvement and better standardization of biliary duct reconstruction. The second area will be in sharpening the criteria for the differnetial diagnosis of postoperative hepatic malfunction, including the liberal use of transhepatic cholangiography and needle biopsy. Only then can better decisions be made about changes in medication or about the need for secondary corrective surgical procedures.
• Starzl, T. E., Watanabe, K., Porter, K. A., & Putnam, C. W. (1976). Effects of insulin, glucagon, and insuling/glucagon infusions on liver morphology and cell division after complete portacaval shunt in dogs. Lancet (London, England), 1(7964), 821-5.
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  Insulin, glucagon, and insulin/glucagon mixtures have been infused for four days into the left portal vein of dogs after portacaval shunt. In the left but not in the right liver lobes, insulin alone reduced atrophy, preserved hepatocyte ultrastructure, and trebled cell renewal. Glucagon alone had no effect. In small doses, glucagon did not potentiate the action of insulin and in large doses it may have reduced the insulin benefit. These studies explain the development of the previously mysterious Eck fistula syndrome, provide clues about in-vivo cell growth control by hormones, and suggests new lines of inquiry about the pathogenesis and/or treatment of several human disease processes.
• Weil, R., Putnam, C. W., Porter, K. A., & Starzl, T. E. (1976). Transplantation in children. The Surgical clinics of North America, 56(2), 467-76.
• Beart, R. W., Putnam, C. W., Porter, K. A., & Starzl, T. E. (1975). Letter: Liver transplantation for Wilson's disease. Lancet (London, England), 2(7926), 176-7.
• Lilly, J. R., Pfister, R. R., Putnam, C. W., Kosloske, A. M., & Starzl, T. E. (1975). Bench surgery and renal autotransplantation in the pediatric patient. Journal of pediatric surgery, 10(5), 623-30.
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  A new operative procedure in renal surgery has evolved from the extensive recent experience in kidney transplantation. Bench surgery and autotransplantation have not been, as yet, fully exploited by surgeons caring for children. This approach to reconstruction of renal substance and renal vessels has as its greatest dividend conservation of kidney tissue. The operation has specific applicability for selected cases of: (1) renovascular hypertension; (2) congenital obstructive uropathy; (3) bilateral Wilms' tumor, and; (4) renal trauma in children.
• Putnam, C. W., Halgrimson, C. G., Groth, C. G., Kashiwagi, N., Porter, K. A., & Starzl, T. E. (1975). Immunosuppression with cyclophosphamide in the dog. Clinical and experimental immunology, 22(2), 323-9.
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  Cyclophosphamide significantly diminished the canine humoral antibody response to sheep red blood cells and tended to prevent arterial lesions in renal homografts. However, cyclophosphamide failed to prolong renal homograft survival when administered to dogs as the sole immunosuppressive agent, and it did not add to the effectiveness of azathioprine when given as a supplement to the azathioprine and administered simultaneously or sequentially.
• Putnam, C. W., Halgrimson, C. G., Stables, D. P., Pfister, R., Beart, R. W., Kootstra, G., Haberal, M., Atkins, D., & Starzl, T. E. (1975). Ex vivo renal perfusion and autotransplantation in treatment of calculous disease or abdominal aortic aneurysm. Urology, 05(3), 337-42.
• Starzl, T. E., & Putnam, C. W. (1975). Portal diversion. Treatment for glycogen storage disease and hyperlipemia. JAMA, 233(9), 955-7.
• Starzl, T. E., Bell, R. H., Beart, R. W., & Putnam, C. W. (1975). Hepatic trisegmentectomy and other liver resections. Surgery, gynecology & obstetrics, 141(3), 429-37.
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  Trisegmentectomy, extended right hepatic lobectomy, is the removal of the true right lobe of the liver in continuity with most or all of the medial segment of the left lobe. Some important features of the operation have not been well described previously. To perform trisegmentectomy safely, a fusion of liver tissue covering the umbilical fissure at the level of the falciform ligament must first be split open in many patients. The left branches of the portal triad structures are mobilized from the undersurface of the liver nearly to but not into the umbilical fissure. The blood supply and duct drainage of the medial segment originate within the umbilical fissure and feed back toward the right side buried in liver substance. They are found with blunt dissection just to the right of the falciform ligament, encircled and ligated. Failure to appreciate this switch back anatomic arrangement may lead to injury of the blood supply or biliary drainage of the residual lateral segment. Parenthetically, the mirror image operation of lateral segmentectomy could result in devascularization of the medial segment if dissection and ligation were performed within the umbilical fissure instead of well to the left of this landmark. In most trisegmentectomies, the left portion of the caudate lobe is not removed. This small piece of tissue is interposed between the lateral segment and the inferior vena cave into which it drains by small tributaries. If the left portion of the caudate lobe is to be excised, it is necessary to ligate the last two posteriorly running branches before the main left trunks of the portal triad structures reach the umbilical fissure. Once this step is taken and if the caudate removal is completed, the remaining lateral segment usually has only one remaining outflow, that of the left hepatic vein. The other principles of trisegmentectomy are the same as with less radical subtotal hepatic resection. These include vascular suture closure of the main outflow veins, avoidance of parasegmental planes that leave behind a strip of devitalized tissue, preservation of intersegmental or interlobar veins, omission of techniques that sew shut or otherwise cover the raw surface of the remnant and provision of adequate drainage of dead space. After trisegimentectomy and also after true lobectomy, this last objective is usually met by leaving part of the operative incision open. Using these guidelines, there has been no mortality with 27 hepatic resections carried out since 1963, including 14 trisegmentectomies.
• Starzl, T. E., Lee, I. Y., Porter, K. A., & Putnam, C. W. (1975). The influence of portal blood upon lipid metabolism in normal and diabetic dogs and baboons. Surgery, gynecology & obstetrics, 140(3), 381- 96.
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  Complete diversion of portal blood in dogs caused sustained falls in serum cholesterol and phospholipid concentrations an declines in hepatic cholesterol and triglyceride synthesis. The hepatocytes in these canine livers were deglycogenated, and they atrophied to about half of their original size within two months. At the same time, there was evidence of increased mitoses. Ultrastructurally, the dominant change in the hepatocytes was in the rough endoplasmic reticulum which decreased in amount, underwent marked dilatation, and became depleted of ribosomes. There was also marked loss of glycogen granules, variable mitochondrial abnormalities, and widespread accumulation in the hepatocyte cytoplasm of lipid vacuoles. Bypass of intestinal venous return around the liver through a mesenteric caval shunt did not influence the serum lipid concentrations in dogs and baboons, although cholesterol synthesis was depressed in the canine livers and significant morphologic changes, including atrophy, were produced. In both species, the addition of a second stage central portacaval shunt which diverted venous return from the pancreaticogastroduosplenic area caused declines in serum cholesterol and phospholipid concentrations. After the second operation, hepatic cholesterol synthesis in the dogs was further reduced, and triglyceride synthesis was markedly depressed. The eventual ultrastructural changes were similar to those after one stage portal diversion. In other experiments on dogs, discrete regions of the liver were provided with portal perfusion from different splanchnic sources during a two month period. When the right lobes received pancreatiogastroduodenosplenic venous blood and the left lobes received intestinal venous effluent, in vivo cholesterol and triglyceride synthesis were higher in the hormone-enriched right lobes. This advantage was eliminated with pre-existing alloxan-induced diabetes or by the concomitant performance of total pancreatectomy in dogs that were treated during the ensuing two months with subcutaneously administered insulin. The nutrient-enriched left lobes had the higher lipid synthesis. In a final series of experiments, the right lobes of dogs were given the total splanchnic flow, and the left lobes were perfused with systemic venous blood by anastomosing the left portal vein to the suprarenal vena cava. The right lobar advantage in lipid synthesis could not be eliminated in this preparation with alloxan-induced diabetes or total pancreatectomy. These results indicate that a reduction of hepatic lipid synthesis is an important, although not necessarily the sole, factor in the antilipidemic influence of portacaval shunt. The effects upon synthesis and blood lipids apparently are due more to the diversion of endogenous hormones than to the bypass of intestinal nutrients. The substances in portal venous blood that subserve hepatic lipid metabolism are presumably largely the same as the hepatotropic factors which have been described before as profoundly affecting hepatic structure, function, and the capacity for regeneration. These portal blood factors are multiple and interrelated, but the single most important one seems to be insulin.
• Starzl, T. E., Porter, K. A., & Putnam, C. W. (1975). Intraportal insulin protects from the liver injury of portacaval shunt in dogs. Lancet (London, England), 2(7947), 1241-2.
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  4 days after portacaval shunt, the livers of normal dogs had pronounced atrophy and other structural abnormalities. These changes were greatly reduced in the left liver lobes, but not in the right, by a constant infusion to the left portal vein of insulin in non-hypoglycaemic doses. These experimental findings should have implications in clinical medicine.
• Starzl, T. E., Porter, K. A., Kashiwagi, N., Lee, I. Y., Russsell, W. J., & Putnam, C. W. (1975). The effect of diabetes mellitus on portal blood hepatotrophic factors in dogs. Surgery, gynecology & obstetrics, 140(4), 549-62.
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  Ten nondiabetic dogs were submitted to a procedure called splanchnic division which directed the nutrient rich venous return from the intestines into the left lobes of the liver and the hormone rich pancreaticogastroduodenosplenic venous return into the right lobes. Two months later, the right lobes had undergone the expected gross and microscopic hypertrophy. Compared with the abnormal shrunken and glycogen-depleted hepatocytes of the left lobes, the large and otherwise normal hepatocytes of the right lobes had a higher rate of cell division as judged by microscopic examination, measurements of deoxyribonucleic acid synthesis and the results of autoradiography. Both sides had greater cell replication than in the livers of normal unaltered dogs. The dominance of the right lobes following splanchnic division was almost completely eliminated by the prior creation of alloxan-induced diabetes in four dogs and by the performance of total pancreatectomy at the same time as splanchnic division in six dogs. In these ten diabetic dogs, which were treated with subcutaneously administered insulin for the two month period of the post operative study, hepatic lobar and cell size were nearly equal on both sides. By light and electron microscopy, the hepatocytes on both sides had abnormalities, somewhat less pronounced on the right. However, the most active cell division was now transferred to the left lobes. The results with alloxan-induced diabetes were similar to those after total pancreatectomy, except the lipid deposits were less on both liver sides in the alloxan experiments, and the glycogen was selectively reduced in the right lobes. The latter finding presumably was due to the continued action of glucagon in dogs made diabetic with alloxan. Twelve nondiabetic dogs had a procedure called partial portacaval transposition which directed systemic venous blood from the hindquarters, kidneys and adrenal glands into the left lobes of the liver and the total splanchnic venous return into the right lobes. Two months later, the degree of relative hypertrophy and hyperplasia of the glycogen rich right lobes was even greater than after splanchnic division, as was the morphologic damage to the left lobar hepatocytes. The degree of right lobar hypertrophy following partial portacaval transposition was reduced but not eliminated by pre-existing alloxan-induced diabetes in four dogs and by concomitant total pancreatectomy in six more dogs. The dogs were subcutaneously treated with insulin. Structurally, the hepatocytes on the right side after two months were in better condition than were those on the left, although both were abnormal. The dominance of cell division on the right side was reduced, as judged by standard microscopy and by autoradiography, but there was not a shifting of sides. The biochemical analyses reflected the presence or absence of glucagon. These findings are consistent with our earlier multifactorial hypothesis which holds that portal hepatotrophic factors are mainly interreacting hormones generated by splanchnic organs and delivered straight to the liver and that the hormone interrelationships might have augmented significance because of the high concentration of nutritional substrate in the same venous blood. The observations also substantiate by direct testing the suggestion that insulin is the most important hepatotropic factor and that it profoundly affects many aspects of liver cell structure, division and function.
• Starzl, T. E., Putnam, C. W., Groth, C. G., Corman, J. L., & Taubman, J. (1975). Alopecia, ascites, and incomplete regeneration after 85 to 90 per cent liver resection. American journal of surgery, 129(5), 587-90.
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  A nineteen year old female underwent 85 to 90 per cent partial hepatectomy to treat a minimal deviation hepatoma. Observations afterwards suggested that the limit of resection compatible with survival had been reached. She recovered perfect health after many months, although liver regeneration was not complete. Severe but eventually reversible alopecia and ascites developed postoperatively, undoubtedly as a complication of the massive hepatic resection.
• Starzl, T. E., Putnam, C. W., Ishikawa, M., Picache, R., Husberg, B., Halgrimson, C. G., Schroter, G., & Porter, K. A. (1975). Current policies in hepatic transplantation: candidacy of patients with alcoholic liver disease or preformed antidonor antibodies and a reappraisal of biliary duct reconstruction. Annals of the New York Academy of Sciences, 252, 145-58.
• Corby, D. G., Putnam, C. W., & Greene, H. L. (1974). Impaired platelet function in glucose-6-phosphatase deficiency. The Journal of pediatrics, 85(1), 71-6.
• Groth, C. G., Hathaway, W. E., Gustafsson, A., Geis, W. P., Putnam, C. W., Björkén, C., Porter, K. A., & Starzl, T. E. (1974). Correction of coagulation in the hemophilic dog by transplantation of lymphatic tissue. Surgery, 75(5), 725-33.
• Starzl, T. E., Chase, H. P., Putnam, C. W., & Nora, J. J. (1974). Letter: Follow-up of patient with portacaval shunt for the treatment of hyperlipidaemia. Lancet (London, England), 2(7882), 714-5.
• Starzl, T. E., Chase, H. P., Putnam, C. W., Nora, J. J., Fennell, R. H., & Porter, K. A. (1974). Letter: Portacaval shunt in hyperlipidaemia. Lancet (London, England), 2(7891), 1263.
• Starzl, T. E., Ishikawa, M., Putnam, C. W., Porter, K. A., Picache, R., Husberg, B. S., Halgrimson, C. G., & Schroter, G. (1974). Progress in and deterrents to orthotopic liver transplantation, with special reference to survival, resistance to hyperacute rejection, and biliary duct reconstruction. Transplantation proceedings, 6(4 Suppl 1), 129-39.
• Starzl, T. E., Porter, K. A., Halgrimson, C. G., Husberg, B. S., Penn, I., & Putnam, C. W. (1974). A decade followup in early cases of renal homotransplantation. Annals of surgery, 180(4), 606-16.
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  Sixty-four consecutive patients underwent renal homotransplantation 10 1/6 to 11(1/2) years ago, 46 from related and 18 from nonrelated living donors. Thirty-six of these recipients were alive when this series was presented to the American Surgical Assocation in 1965. Now, nine years later, 26 (72%) of the 36 still survive, in 22 instances with function of their original grafts. The 10 who died in the interim tended to have subnormal renal function or graft failure. However, the actual causes of death included 2 or more examples each of myocardial infarction, hepatitis, or other systemic infections. The prognosis for achieving a one decade survival was not obviously related to HL-A tissue match. The best results were with related kidneys, within which subgroup 24 (52%) of the original recipients are still alive. However, there was no particular category of consanguineous donor that had a marked superiority. Only 2 of 18 nonrelated recipients are still alive. All 36 patients who were alive in 1965 had a biopsy of their renal homograft. Kidneys that were destined to function for a decade tended to have relatively minor histopathologic abnormalities. If serious glomerular lesions were found, the outlook for long graft survival was grave. Vascular lesions had a somewhat less serious import. Mononuclear cell infiltration, tubular atrophy, and interstitial fibrosis proved prognostically to be the least significant. Long-term followup of these early cases has shown the durability of chronic renal homografts, particularly if these are from related donors, and has demonstrated the very high degree of rehabilitation that could be achieved even in the early days of renal homotransplantation.
• Starzl, T. E., Porter, K. A., Husbeg, B. S., Ishikawa, M., & Putnam, C. W. (1974). Renal homotransplantation. I. Current problems in surgery, 3-59.
• Corman, J. L., Kashiwagi, N., Porter, K. A., Andres, G., Iwatsuki, S., Putnam, C. W., Popovtzer, M., Penn, I., & Starzl, T. E. (1973). Unsuccessful attempts to control hyperacute rejection of human renal homografts with F(ab') 2 and citrate organ pretreatment. Transplantation, 16(1), 60-3.
• Halgrimson, C. G., Penn, I., Booth, A., Groth, C., Putnam, C. W., Corman, J., & Starzl, T. E. (1973). Eight- to ten-year follow-up in early cases of renal homotransplantation. Transplantation proceedings, 5(1), 787-91.
• Iwatsuki, S., Popovtzer, M. M., Corman, J. L., Ishikawa, M., Putnam, C. W., Katz, F. H., & Starzl, T. E. (1973). Recovery from "hepatorenal syndrome" after orthotopic liver transplantation. The New England journal of medicine, 289(22), 1155-9.
• Starzl, T. E., Francavilla, A., Halgrimson, C. G., Francavilla, F. R., Porter, K. A., Brown, T. H., & Putnam, C. W. (1973). The origin, hormonal nature, and action of hepatotrophic substances in portal venous blood. Surgery, gynecology & obstetrics, 137(2), 179-99.
• Starzl, T. E., Groth, C. G., Putnam, C. W., Corman, J., Halgrimson, C. G., Penn, I., Husberg, B., Gustafsson, A., Cascardo, S., Geis, P., & Iwatsuki, S. (1973). Cyclophosphamide for clinical renal and hepatic transplantation. Transplantation proceedings, 5(1), 511-6.
• Starzl, T. E., Putnam, C. W., Chase, H. P., & Porter, K. A. (1973). Portacaval shunt in hyperlipoproteinaemia. Lancet (London, England), 2(7835), 940-4.
• Starzl, T. E., Putnam, C. W., Porter, K. A., Halgrimson, C. G., Corman, J., Brown, B. I., Gotlin, R. W., Rodgerson, D. O., & Greene, H. L. (1973). Portal diversion for the treatment of glycogen storage disease in humans. Annals of surgery, 178(4), 525-39.
• Amemiya, H., Yokoyama, T., Putnam, C. W., Torisu, M., & Starzl, T. E. (1972). The nature of antiplatelet activity in antilymphoblast ALG--with special reference to cross-reacting antibody, immunochemical characterization, and Coombs' positive thrombocytopenia in ALG-treated renal recipients. Clinical and experimental immunology, 10(3), 417-26.
• Amemiya, H., Yokoyama, T., Torisu, M., Putnam, C. W., Anderson, J. T., & Starzl, T. E. (1972). The reduction of antigenicity of heterologous antilymphocyte serum with acid Taka-protease. Clinical and experimental immunology, 11(1), 67-81.
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  Horse anti-dog and anti-mouse ALS were digested for varying intervals with acid Taka-protease, a proteolytic enzyme. A progressive reduction in antigenicity was demonstrated, as determined by precipitin reactions with standard antisera and by passive cutaneous and systemic anaphylaxis tests. Leukoagglutinins and lymphocytotoxins were conserved in anti-dog ALS but there was a marked diminution in anti-platelet and anti-erthrocyte activity. Both anti-red cell and anti-white cell antibodies of anti-mouse ALS were reduced. Digestion appeared to completely degrade some protein fractions such as albumin and to alter the immunoglobulin molecule, possibly by cleavage of the terminal portion of the Fc fragment. Digestion of ALS with Taka-protease may prove to be a useful procedure particularly since it was demonstrated that immunosuppressive potency was partially retained. For further evaluation a better standardized Taka-protease will be required.
• Groth, C. G., Kashiwagi, N., Moore, G. E., Husberg, B., Bjorken, C., Putnam, C. W., & Starzl, T. E. (1972). Production of a Standardized Anti-Lymphocyte Globulin. Behring Institute Mitteilungen, 51, 86-89.
• Martineau, G., Porter, K. A., Corman, J., Launois, B., Schroter, G. T., Palmer, W., Putnam, C. W., Groth, C. G., Halgrimson, C. G., Penn, I., & Starzl, T. E. (1972). Delayed biliary duct obstruction after orthotopic liver transplantation. Surgery, 72(4), 604-10.
• Putnam, C. W., & Starzl, T. E. (1972). Clinical Experience with ALG.. Behring Institute Mitteilungen.
• Putnam, C. W., Penn, I., Halgrimson, C. G., Corman, J., Kashiwagi, N., Groth, C. G., & Starzl, T. E. (1972). Clinical experience with horse antihuman ALG.. Transplantation Proceedings.
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  There will be little point in dwelling on our clinical experience with ALG, though this has been extensive. The reason is that we have not carried out a contemporaneous control study omitting ALG. We do not have, as a consequence, proof that our results were improved by the clinical introduction of ALG. Nevertheless, there is evidence supporting this point of view which we will briefly describe.
• Starzl, T. E., & Putnam, C. W. (1972). Chronic immunosuppression, Australia antigenemia, and hepatitis. Transplantation proceedings, 4(4), 685-6.
• Starzl, T. E., & Putnam, C. W. (1972). Clinical Experience with ALG. Behring Institute Mitteilungen, 124-125.
• Starzl, T. E., Corman, J., Groth, C. G., Halgrimson, C. G., Penn, I., Putnam, C. W., Schroter, G., & Gustafsson, A. (1972). Personal experience with orthotopic liver transplantation. Transplantation proceedings, 4(4), 759-71.
• Starzl, T. E., Groth, C. G., Kashiwagi, N., Putnam, C. W., Corman, J. L., Halgrimson, C. G., & Penn, I. (1972). Clinical experience with horse antihuman ALG. Transplantation proceedings, 4(4), 491-5.
• Starzl, T. E., Putnam, C. W., Halgrimson, C. G., Groth, C. G., Booth, A. S., & Penn, I. (1972). Renal transplantation under cyclophosphamide. Transplantation proceedings, 4(4), 461-4.
• Torisu, M., Yokoyama, T., Durst, A. L., Schroter, G., Putnam, C. W., Halgrimson, C. G., & Starzl, T. E. (1972). Detection of Australia antigen by biological assay in 'Au negative' kidney homograft recipients with hepatic dysfunction. Clinical and experimental immunology, 10(3), 409-16.
• Halgrimson, C. G., Rapaport, F. T., Terasaki, P. I., Porter, K. A., Andres, G., Penn, I., Putnam, C. W., & Starzl, T. E. (1971). Net histocompatibility ratios (NHR) for clinical transplantation. Transplantation proceedings, 3(1), 140-4.
• Starzl, T. E., Giles, G., Lilly, J. R., Takagi, H., Martineau, G., Schroter, G., Halgrimson, C. G., Penn, I., & Putnam, C. W. (1971). Indications for orthotopic liver transplantation: with particular reference to hepatomas, biliary atresia, cirrhosis, Wilson's disease and serum hepatitis. Transplantation proceedings, 3(1), 308-12.
• Starzl, T. E., Halgrimson, C. G., Penn, I., Martineau, G., Schroter, G., Amemiya, H., Putnam, C. W., & Groth, C. G. (1971). Cyclophosphamide and human organ transplantation. Lancet (London, England), 2(7715), 70-4.
• Starzl, T. E., Penn, I., Putnam, C. W., Groth, C. G., & Halgrimson, C. G. (1971). Iatrogenic alterations of immunologic surveillance in man and their influence on malignancy. Transplantation reviews, 7, 112-45.
• Starzl, T. E., Putnam, C. W., Halgrimson, C. G., Schroter, G. T., Martineau, G., Launois, B., Corman, J. L., Penn, I., Booth, A. S., Groth, C. G., & Porter, K. A. (1971). Cyclophosphamide and whole organ transplantation in human beings. Surgery, gynecology & obstetrics, 133(6), 981-91.
• Torisu, M., Yokoyama, T., Amemiya, H., Kohler, P. F., Schroter, G., Martineau, G., Penn, I., Palmer, W., Halgrimson, C. G., Putnam, C. W., & Starzl, T. E. (1971). Immunosuppression, liver injury, and hepatitis in renal, hepatic, and cardiac homograft recipients: with particular reference to the Australia antigen. Annals of surgery, 174(4), 620-39.
• Ameniya, H., Kashiwagi, N., Putnam, C. W., & Starzl, T. E. (1970). Cross-reactivity studies of horse, goat and rabbit anti-lymphocyte globulin. Clinical and experimental immunology, 6(2), 279-89.
• Starzl, T. E., Brettschneider, L., & Putnam, C. W. (1970). Transplantation of the liver. Progress in liver diseases, 3, 495-542.
• Starzl, T. E., Groth, C. G., Putnam, C. W., Penn, I., Halgrimson, C. G., Flatmark, A., Gecelter, L., Brettschneider, L., & Stonington, O. G. (1970). Urological complications in 216 human recipients of renal transplants. Annals of surgery, 172(1), 1-22.
• Starzl, T. E., Porter, K. A., Andres, G., Groth, C. G., Putnam, C. W., Penn, I., Halgrimson, C. G., Starkie, S. J., & Brettschneider, L. (1970). Thymectomy and renal homotransplantation. Clinical and experimental immunology, 6(6), 803-14.
• Starzl, T. E., Porter, K. A., Andres, G., Halgrimson, C. G., Hurwitz, R., Giles, G., Terasaki, P. I., Penn, I., Schroter, G. T., Lilly, J., Starkie, S. J., & Putnam, C. W. (1970). Long-term survival after renal transplantation in humans: (with special reference to histocompatibility matching, thymectomy, homograft glomerulonephritis, heterologous ALG , AND RECIPIENT MALIGNANCY). Annals of surgery, 172(3), 437-72.
• Starzl, T. E., Putnam, C. W., Brettschneider, L., & Penn, I. (1970). The prospect of organ transplantation in cancer surgery. Proceedings. National Cancer Conference, 6, 45-55.
• Smith, G. V., Kolff, J., Kashiwagi, N., Putnam, C. W., & Starzl, T. E. (1969). Modification of established rejection of canine kidney and liver homografts with antilymphocyte gamma-G globulin. Surgery, 66(3), 546-9.
• Starzi, T. E., Brettschneider, L., Penn, I., Giles, G., Picache, R., & Putnam, C. W. (1969). Clinical liver transplantation. Transplantation reviews, 2, 3-68.
• Starzl, T. E., Brettschneider, L., Penn, I., Bell, P., Groth, C. G., Blanchard, H., Kashiwagi, N., & Putnam, C. W. (1969). Orthotopic liver transplantation in man. Transplantation proceedings, 1(1), 216-22.
• Starzl, T. E., Brettschneider, L., Penn, I., Schmidt, R. W., Bell, P., Kashiwagi, N., Townsend, C. M., & Putnam, C. W. (1969). A trial with heterologous antilymphocyte globulin in man. Transplantation proceedings, 1(1), 448-54.
• Starzl, T. E., Porter, K. A., Brettschneider, L., Penn, I., Bell, P., Putnam, C. W., & McGuire, R. L. (1969). Clinical and pathologic observations after orthotopic transplantation of the human liver. Surgery, gynecology & obstetrics, 128(2), 327-39.
• Fulginiti, V. A., Scribner, R., Groth, C. G., Putnam, C. W., Brettschneider, L., Gilbert, S., Porter, K. A., & Starzl, T. E. (1968). Infections in recipients of liver homografts. The New England journal of medicine, 279(12), 619-26.
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  Seventeen patients received liver homografts between 1963 and May, 1968. The eight treated before July, 1967, died within 34 days; seven had progressive infections with gram-negative bacilli, and cytomegalovirus. The infections were similar to but more fulminating than those after renal homotransplantation. In nine later cases, there was more discriminating donor selection, improved immunosuppression, and better organ preservation. In the first five of these nine patients, all infants, lobar hepatic gangrene apparently secondary to delayed right hepatic arterial thrombosis developed. Two died within a few days, two and three and a half months after transplantation. The three who did not die immediately subsequently had multiple bacteremias, fungemias and cytomegalovirus pulmonary infections. One of these children is alive twelve months after transplantation; the others died after four and a half and six months. In contrast, the last four patients, in whom septic liver infarctions were avoided, have been free of serious infections for two to five and a half months.
• Starzl, T. E., Groth, C. G., Terasaki, P. I., Putnam, C. W., Brettschneider, L., & Marchioro, T. L. (1968). Heterologous antilymphocyte glubulin, histoincompatiblity matching, and human renal homotransplantation. Surgery, gynecology & obstetrics, 126(5), 1023-35.
• Putnam, C. W., Kashiwagi, N., Iwasaki, Y., Terasaki, P. I., Marchioro, T. L., & Starzl, T. E. (1967). Interspecies reactivity and intraspecies specificity of antilymphoid globulin. Surgery, 61(6), 951-4.