Bonnie J LaFleur

Interests

Research

Immunobiology of AgingMetrology - diagnostics and longitudinal trends of measurement Methods for Data Integration Normative Cognitive Aging

Teaching

Statistical MethodsStatistical PredictionBiomarker Directed Clinical Trials Reproducible Research

Courses

Scholarly Contributions

Journals/Publications

• Liang, J., Hussainy, S., Lee, S. M., Wu, G., Bautista, N., Ding, M., Wang, H., LaFleur, B., Perry, G., Wang, X., & , f. A. (2025). Association of Polygenic Risk Score for 5 Diseases With Alzheimer Disease Progression, Biomarkers, and Amyloid Deposition. Neurology, 104(4), e210250.
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  Alzheimer disease (AD) is a heterogeneous neurodegenerative disorder influenced by genetic and environmental factors. Conditions such as type 2 diabetes (T2D), cardiovascular disease, obesity, depression, and obstructive sleep apnea (OSA) increase AD risk and progression. This study aimed to examine the genetic predisposition to these conditions and their effect on AD pathophysiology, risk, and progression.
• Asghari, M., Elali, K., Sullivan, A., LaFleur, B., Madigan, M. L., & Toosizadeh, N. (2024). Assessing the role of ankle and hip joint proprioceptive information in balance recovery using vibratory stimulation. Heliyon, 10(4), e25979.
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  Previous work suggests that proprioceptive information from ankle and hip are crucial in maintaining balance during upright standing; however, the contribution of these proprioceptive information during stepping balance recovery in not clear. The goal of the current study was to assess the role of ankle and hip proprioceptive information on balance recovery performance by manipulating type 1a afferent in muscle spindles using vibratory stimulation.
• Liang, J., LaFleur, B., Hussainy, S., & Perry, G. (2024). Gene Co-Expression Analysis of Multiple Brain Tissues Reveals Correlation of FAM222A Expression with Multiple Alzheimer???s Disease-Related Genes.
• Narendran, N., Volpe, S., Ramadan, I., Herbert, J. R., LaFleur, B., Samargandy, S., Le, C. H., & Chang, E. H. (2024). A prospective longitudinal study assessing the impact of rhinovirus and bacterial infections in acute exacerbations of chronic rhinosinusitis. International forum of allergy & rhinology, 14(12), 1914-1920.
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  Acute exacerbations of chronic rhinosinusitis (AECRS) are thought to arise from common viral infections progressing to secondary bacterial infections. However, the pathophysiology of AECRS remains poorly understood due to a lack of prospective longitudinal studies.
• Porter, W. T., Gade, L., Montfort, P., Mihaljevic, J. R., Bowers, J. R., Willman, A., Klimowski, B. A., LaFleur, B. J., Sunenshine, R. H., Collins, J., Adame, G., Brady, S., Komatsu, K. K., Williams, S., Toda, M., Chiller, T., Litvintseva, A. P., & Engelthaler, D. M. (2024). Understanding the exposure risk of aerosolized Coccidioides in a Valley fever endemic metropolis. Scientific reports, 14(1), 1311.
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  Coccidioides is the fungal causative agent of Valley fever, a primarily pulmonary disease caused by inhalation of fungal arthroconidia, or spores. Although Coccidioides has been an established pathogen for 120 years and is responsible for hundreds of thousands of infections per year, little is known about when and where infectious Coccidioides arthroconidia are present within the ambient air in endemic regions. Long-term air sampling programs provide a means to investigate these characteristics across space and time. Here we present data from > 18 months of collections from 11 air sampling sites across the Phoenix, Arizona, metropolitan area. Overall, prevalence was highly variable across space and time with no obvious spatial or temporal correlations. Several high prevalence periods were identified at select sites, with no obvious spatial or temporal associations. Comparing these data with weather and environmental factor data, wind gusts and temperature were positively associated with Coccidioides detection, while soil moisture was negatively associated with Coccidioides detection. These results provide critical insights into the frequency and distribution of airborne arthroconidia and the associated risk of inhalation and potential disease that is present across space and time in a highly endemic locale.
• Schepps, S., Xu, J., Yang, H., Mandel, J., Mehta, J., Tolotta, J., Baker, N., Tekmen, V., Nikbakht, N., Fortina, P., Fuentes, I., LaFleur, B., Cho, R. J., & South, A. P. (2024). Skin in the game: a review of single-cell and spatial transcriptomics in dermatological research. Clinical chemistry and laboratory medicine, 62(10), 1880-1891.
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  Single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics (ST) are two emerging research technologies that uniquely characterize gene expression microenvironments on a cellular or subcellular level. The skin, a clinically accessible tissue composed of diverse, essential cell populations, serves as an ideal target for these high-resolution investigative approaches. Using these tools, researchers are assembling a compendium of data and discoveries in healthy skin as well as a range of dermatologic pathophysiologies, including atopic dermatitis, psoriasis, and cutaneous malignancies. The ongoing advancement of single-cell approaches, coupled with anticipated decreases in cost with increased adoption, will reshape dermatologic research, profoundly influencing disease characterization, prognosis, and ultimately clinical practice.
• Jergović, M., Watanabe, M., Bhat, R., Coplen, C. P., Sonar, S. A., Wong, R., Castaneda, Y., Davidson, L., Kala, M., Wilson, R. C., Twigg, H. L., Knox, K., Erickson, H. E., Weinkauf, C. C., Bime, C., Bixby, B. A., Parthasarathy, S., Mosier, J. M., LaFleur, B. J., , Bhattacharya, D., et al. (2023). T-cell cellular stress and reticulocyte signatures, but not loss of naïve T lymphocytes, characterize severe COVID-19 in older adults. GeroScience, 1-16.
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  In children and younger adults up to 39 years of age, SARS-CoV-2 usually elicits mild symptoms that resemble the common cold. Disease severity increases with age starting at 30 and reaches astounding mortality rates that are ~330 fold higher in persons above 85 years of age compared to those 18-39 years old. To understand age-specific immune pathobiology of COVID-19, we have analyzed soluble mediators, cellular phenotypes, and transcriptome from over 80 COVID-19 patients of varying ages and disease severity, carefully controlling for age as a variable. We found that reticulocyte numbers and peripheral blood transcriptional signatures robustly correlated with disease severity. By contrast, decreased numbers and proportion of naïve T-cells, reported previously as a COVID-19 severity risk factor, were found to be general features of aging and not of COVID-19 severity, as they readily occurred in older participants experiencing only mild or no disease at all. Single-cell transcriptional signatures across age and severity groups showed that severe but not moderate/mild COVID-19 causes cell stress response in different T-cell populations, and some of that stress was unique to old severe participants, suggesting that in severe disease of older adults, these defenders of the organism may be disabled from performing immune protection. These findings shed new light on interactions between age and disease severity in COVID-19.
• LaFleur, B., Curiel-Lewandrowski, C., Tapia, E., Parker, J., White, L., Chow, H. S., & South, A. P. (2023). Characterizing dermal transcriptional change in the progression from sun-protected skin to actinic keratosis. The Journal of investigative dermatology.
• Lafleur, B. J., White, L., Dake, M. D., Nikolich, J. Z., Sprissler, R., & Bhattacharya, D. (2023). No Evidence That Analgesic Use after COVID-19 Vaccination Negatively Impacts Antibody Responses. ImmunoHorizons, 7(12), 834-841.
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  Uptake of mRNA vaccines, especially booster immunizations, against COVID-19 has been lower than hoped, perhaps in part due to their reactogenicity. Analgesics might alleviate symptoms associated with vaccination, but they might also impact immune responses. We semiquantitatively measured Ab responses following COVID-19 vaccination in 2354 human participants surveyed about analgesic use after vaccination. Participants who used nonsteroidal anti-inflammatory drugs or acetaminophen after vaccination showed elevated Ab levels against the receptor-binding domain of Spike protein relative to those who did not use analgesics. This pattern was observed for both mRNA-1273 and BNT162b2 and across age groups. Participants who used analgesics more frequently reported fatigue, muscle aches, and headaches than did those who did not use painkillers. Among participants who reported these symptoms, we observed no statistically significant differences in Ab levels irrespective of analgesic use. These data suggest that elevated Ab levels are associated with symptoms and inflammatory processes rather than painkiller use per se. Taken together, we find no evidence that analgesic use reduces Ab responses after COVID-19 vaccination. Recommendation of their use to alleviate symptoms might improve uptake of booster immunizations.
• Liang, J., LaFleur, B., Hussainy, S., & Perry, G. (2023). Gene Co-Expression Analysis of Multiple Brain Tissues Reveals Correlation of FAM222A Expression with Multiple Alzheimer's Disease-Related Genes. Journal of Alzheimer's disease : JAD.
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  Alzheimer's disease (AD) is the most common form of dementia in the elderly marked by central nervous system (CNS) neuronal loss and amyloid plaques. FAM222A, encoding an amyloid plaque core protein, is an AD brain atrophy susceptibility gene that mediates amyloid-β aggregation. However, the expression interplay between FAM222A and other AD-related pathway genes is unclear.
• Quirk, G. E., Schoenle, M. V., Peyton, K. L., Uhrlaub, J. L., Lau, B., Burgess, J. L., Ellingson, K., Beitel, S., Romine, J., Lutrick, K., Fowlkes, A., Britton, A., Tyner, H. L., Caban-Martinez, A. J., Naleway, A., Gaglani, M., Yoon, S., Edwards, L., Olsho, L., , Dake, M., et al. (2023). Determinants of B cell responses to drifted epitopes in post-vaccination SARS-CoV-2 infections. medRxiv : the preprint server for health sciences.
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  Vaccine-induced immunity may impact subsequent responses to drifted epitopes in SARS-CoV-2 variants, but this has been difficult to quantify due to the challenges in recruiting unvaccinated control groups whose first exposure to SARS-CoV-2 is a primary infection. Through local, statewide, and national SARS-CoV-2 testing programs, we were able to recruit cohorts of individuals who had recovered from either primary or post-vaccination infections by either the Delta or Omicron BA.1 variants. Regardless of variant, we observed greater Spike-specific and neutralizing antibody responses in post-vaccination infections than in those who were infected without prior vaccination. Through analysis of variant-specific memory B cells as markers of responses, we observed that Delta and Omicron BA.1 infections led to a marked shift in immunodominance in which some drifted epitopes elicited minimal responses, even in primary infections. Prior immunity through vaccination had a small negative impact on these responses, but this did not correlate with cross-reactive memory B cells, arguing against competitive inhibition of naïve B cells. We conclude that dampened B cell responses against drifted epitopes are mostly a function of altered immunodominance hierarchies that are apparent even in primary infections, with a more modest contribution from pre-existing immunity, perhaps due to accelerated antigen clearance.
• Srinivasan, J., Vasudev, A., Shasha, C., Selden, H. J., Perez, E., LaFleur, B., Sinari, S. A., Krueger, A., Richie, E. R., & Ehrlich, L. I. (2023). The initial age-associated decline in early T-cell progenitors reflects fewer pre-thymic progenitors and altered signals in the bone marrow and thymus microenvironments. Aging cell, 22(8), e13870.
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  Age-related thymus involution results in decreased T-cell production, contributing to increased susceptibility to pathogens and reduced vaccine responsiveness. Elucidating mechanisms underlying thymus involution will inform strategies to restore thymopoiesis with age. The thymus is colonized by circulating bone marrow (BM)-derived thymus seeding progenitors (TSPs) that differentiate into early T-cell progenitors (ETPs). We find that ETP cellularity declines as early as 3 months (3MO) of age in mice. This initial ETP reduction could reflect changes in thymic stromal niches and/or pre-thymic progenitors. Using a multicongenic progenitor transfer approach, we demonstrate that the number of functional TSP/ETP niches does not diminish with age. Instead, the number of pre-thymic lymphoid progenitors in the BM and blood is substantially reduced by 3MO, although their intrinsic ability to seed and differentiate in the thymus is maintained. Additionally, Notch signaling in BM lymphoid progenitors and in ETPs diminishes by 3MO, suggesting reduced niche quality in the BM and thymus contribute to the early decline in ETPs. Together, these findings indicate that diminished BM lymphopoiesis and thymic stromal support contribute to an initial reduction in ETPs in young adulthood, setting the stage for progressive age-associated thymus involution.
• Borden, E. S., Ghafoor, S., Buetow, K. H., LaFleur, B. J., Wilson, M. A., & Hastings, K. T. (2022). NeoScore Integrates Characteristics of the Neoantigen:MHC Class I Interaction and Expression to Accurately Prioritize Immunogenic Neoantigens. Journal of immunology (Baltimore, Md. : 1950).
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  Accurate prioritization of immunogenic neoantigens is key to developing personalized cancer vaccines and distinguishing those patients likely to respond to immune checkpoint inhibition. However, there is no consensus regarding which characteristics best predict neoantigen immunogenicity, and no model to date has both high sensitivity and specificity and a significant association with survival in response to immunotherapy. We address these challenges in the prioritization of immunogenic neoantigens by (1) identifying which neoantigen characteristics best predict immunogenicity; (2) integrating these characteristics into an immunogenicity score, the NeoScore; and (3) demonstrating a significant association of the NeoScore with survival in response to immune checkpoint inhibition. One thousand random and evenly split combinations of immunogenic and nonimmunogenic neoantigens from a validated dataset were analyzed using a regularized regression model for characteristic selection. The selected characteristics, the dissociation constant and binding stability of the neoantigen:MHC class I complex and expression of the mutated gene in the tumor, were integrated into the NeoScore. A web application is provided for calculation of the NeoScore. The NeoScore results in improved, or equivalent, performance in four test datasets as measured by sensitivity, specificity, and area under the receiver operator characteristics curve compared with previous models. Among cutaneous melanoma patients treated with immune checkpoint inhibition, a high maximum NeoScore was associated with improved survival. Overall, the NeoScore has the potential to improve neoantigen prioritization for the development of personalized vaccines and contribute to the determination of which patients are likely to respond to immunotherapy.
• Goff, P. H., Riolobos, L., LaFleur, B. J., Spraker, M. B., Seo, Y. D., Smythe, K. S., Campbell, J. S., Pierce, R. H., Zhang, Y., He, Q., Kim, E. Y., Schaub, S. K., Kane, G. M., Mantilla, J. G., Chen, E. Y., Ricciotti, R., Thompson, M. J., Cranmer, L. D., Wagner, M. J., , Loggers, E. T., et al. (2022). Neoadjuvant therapy induces a potent immune response to sarcoma, dominated by myeloid and B cells. Clinical cancer research : an official journal of the American Association for Cancer Research.
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  To characterize changes in the soft tissue sarcoma tumor immune microenvironment induced by standard neoadjuvant therapy with the goal of informing neoadjuvant immunotherapy trial design.
• Jergović, M., Uhrlaub, J. L., Watanabe, M., Bradshaw, C. M., White, L. M., LaFleur, B. J., Edwards, T., Sprissler, R., Worobey, M., Bhattacharya, D., & Nikolich-Žugich, J. (2022). Competent immune responses to SARS-CoV-2 variants in older adults following two doses of mRNA vaccination. Nature communications, 13(1), 2891.
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  Aging is associated with a reduced magnitude of primary immune responses to vaccination. mRNA-based SARS-CoV-2 vaccines have shown efficacy in older adults but virus variant escape is still unclear. Here we analyze humoral and cellular immunity against an early-pandemic viral isolate and compare that to the P.1 (Gamma) and B.1.617.2 (Delta) variants in two cohorts (55 age) of mRNA vaccine recipients. We further measure neutralizing antibody titers for B.1.617.1 (Kappa) and B.1.595, with the latter SARS-CoV-2 isolate bearing the spike mutation E484Q. Robust humoral immunity is measured following second vaccination, and older vaccinees manifest cellular immunity comparable to the adult group against early-pandemic SARS-CoV-2 and more recent variants. More specifically, the older cohort has lower neutralizing capacity at 7-14 days following the second dose but equilibrates with the younger cohort after 2-3 months. While long-term vaccination responses remain to be determined, our results implicate vaccine-induced protection in older adults against SARS-CoV-2 variants and inform thinking about boost vaccination.
• Mai, A. S., Lee, A. R., Tay, R. Y., Shapiro, L., Thakkar, A., Halmos, B., Grinshpun, A., Herishanu, Y., Benjamini, O., Tadmor, T., Shroff, R. T., LaFleur, B. J., Bhattacharya, D., Peng, S., Tey, J., Lee, S. C., Chai, L. Y., Soon, Y. Y., Sundar, R., & Lee, M. X. (2022). Booster doses of COVID-19 vaccines for patients with haematological and solid cancer: a systematic review and individual patient data meta-analysis. European journal of cancer (Oxford, England : 1990), 172, 65-75.
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  Patients with cancer have an increased risk of severe disease and mortality from COVID-19, as the disease and antineoplastic therapy cause reduced vaccine immunogenicity. Booster doses have been proposed to enhance protection, and efficacy data are emerging from several studies.
• Patel, S. I., Zareba, W., LaFleur, B., Couderc, J. P., Xia, X., Woosley, R., Patel, I. Y., Combs, D., Mashaqi, S., Quan, S. F., & Parthasarathy, S. (2022). Markers of ventricular repolarization and overall mortality in sleep disordered breathing. Sleep medicine, 95, 9-15.
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  Variability and prolongation of ventricular repolarization - measured by changes in QT interval and QT variability are independently associated with ventricular arrhythmias, sudden death, and mortality but such studies did not examine the role of sleep-disordered breathing. We aimed to determine whether sleep-disordered breathing moderated the association between measures of ventricular repolarization and overall mortality.
• Sonar, S. A., Uhrlaub, J. L., Coplen, C. P., Sempowski, G. D., Dudakov, J. A., van den Brink, M. R., LaFleur, B. J., Jergović, M., & Nikolich-Žugich, J. (2022). Early age-related atrophy of cutaneous lymph nodes precipitates an early functional decline in skin immunity in mice with aging. Proceedings of the National Academy of Sciences of the United States of America, 119(17), e2121028119.
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  Secondary lymphoid organs (SLOs) (including the spleen and lymph nodes [LNs]) are critical both for the maintenance of naive T (TN) lymphocytes and for the initiation and coordination of immune responses. How they age, including the exact timing, extent, physiological relevance, and the nature of age-related changes, remains incompletely understood. We used “time stamping” to indelibly mark newly generated naive T cells (also known as recent thymic emigrants) (RTEs) in mice, and followed their presence, phenotype, and retention in SLOs. We found that SLOs involute asynchronously. Skin-draining LNs atrophied by 6 to 9 mo in life, whereas deeper tissue-draining LNs atrophied by 18 to 20 mo, as measured by the loss of both TN numbers and the fibroblastic reticular cell (FRC) network. Time-stamped RTEs at all ages entered SLOs and successfully completed postthymic differentiation, but the capacity of older SLOs to maintain TN numbers was reduced with aging, and that trait did not depend on the age of TNs. However, in SLOs of older mice, these cells exhibited an emigration phenotype (CCR7loS1P1hi), which correlated with an increase of the cells of the same phenotype in the blood. Finally, upon intradermal immunization, RTEs generated in mice barely participated in de novo immune responses and failed to produce well-armed effector cells detectable in blood as early as by 7 to 8 mo of age. These results highlight changes in structure and function of superficial secondary lymphoid organs in laboratory mice that are earlier than expected and are consistent with the long-appreciated reduction of cutaneous immunity with aging.
• Watanabe, M., Jergovic, M., Davidson, L., LaFleur, B. J., Castaneda, Y., Martinez, C., Smithey, M. J., Stowe, R. P., Haddad, E. K., & Nikolich-Žugich, J. (2022). Inflammatory and immune markers in HIV-infected older adults on long-term antiretroviral therapy: Persistent elevation of sCD14 and of proinflammatory effector memory T cells. Aging cell, 21(9), e13681.
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  HIV-positive patients whose viral loads are successfully controlled by active antiretroviral therapy (ART) show no clinical signs of AIDS. However, their lifespan is shorter compared with individuals with no HIV infection and they prematurely exhibit a multitude of chronic diseases typically associated with advanced age. It was hypothesized that immune system aging may correlate with, and provide useful biomarkers for, this premature loss of healthspan in HIV-positive subjects. Here, we tested whether the immune correlates of aging, including cell numbers and phenotypes, inflammatory status, and control of human cytomegalovirus (hCMV) in HIV-positive subjects on long-term successful ART (HIV+) may reveal increased "immunological age" compared with HIV-negative, age-matched cohort (HIV-) in participants between 50 and 69 years of age. Specifically, we expected that younger HIV+ subjects may immunologically resemble older individuals without HIV. We found no evidence to support this hypothesis. While T cells from HIV+ participants displayed differential expression in several differentiation and/or inhibitory/exhaustion markers in different T cell subpopulations, aging by a decade did not pronounce these changes. Similarly, while the HIV+ participants exhibited higher T cell responses and elevated inflammatory marker levels in plasma, indicative of chronic inflammation, this trait was not age-sensitive. We did find differences in immune control of hCMV, and, more importantly, a sustained elevation of sCD14 and of proinflammatory CD4 and CD8 T cell responses across age groups, pointing towards uncontrolled inflammation as a factor in reduced healthspan in successfully treated older HIV+ patients.
• Akinbami, L. J., Akinbami, L. J., Petersen, L. R., Petersen, L. R., Sami, S., Sami, S., Vuong, N., Vuong, N., Lukacs, S. L., Lukacs, S. L., Mackey, L., Mackey, L., Atas, J., Atas, J., LaFleur, B. J., & LaFleur, B. J. (2021). COVID-19 symptoms and SARS-CoV-2 antibody positivity in a large survey of first responders and healthcare personnel, May-July 2020. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.
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  A SARS-CoV-2 serosurvey among first responder/healthcare personnel showed that loss of taste/smell was most predictive of seropositivity; percent seropositivity increased with number of COVID-19 symptoms. However, 22.9% with nine symptoms were seronegative, and 8.3% with no symptoms were seropositive. These findings demonstrate limitations of symptom-based surveillance and importance of testing.
• Borden, E. S., Adams, A. C., Buetow, K. H., Wilson, M. A., Bauman, J. E., Curiel-Lewandrowski, C., Chow, H. S., LaFleur, B. J., & Hastings, K. T. (2021). Shared Gene Expression and Immune Pathway Changes Associated with Progression from Nevi to Melanoma. Cancers, 14(1).
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  There is a need to identify molecular biomarkers of melanoma progression to assist the development of chemoprevention strategies to lower melanoma incidence. Using datasets containing gene expression for dysplastic nevi and melanoma or melanoma arising in a nevus, we performed differential gene expression analysis and regularized regression models to identify genes and pathways that were associated with progression from nevi to melanoma. A small number of genes distinguished nevi from melanoma. Differential expression of seven genes was identified between nevi and melanoma in three independent datasets. C1QB, CXCL9, CXCL10, DFNA5 (GSDME), FCGR1B, and PRAME were increased in melanoma, and SCGB1D2 was decreased in melanoma, compared to dysplastic nevi or nevi that progressed to melanoma. Further supporting an association with melanomagenesis, these genes demonstrated a linear change in expression from benign nevi to dysplastic nevi to radial growth phase melanoma to vertical growth phase melanoma. The genes associated with melanoma progression showed significant enrichment of multiple pathways related to the immune system. This study demonstrates (1) a novel application of bioinformatic approaches to aid clinical trials of melanoma chemoprevention and (2) the feasibility of determining a gene signature biomarker of melanomagenesis.
• Hale, L. P., Cheatham, L., Macintyre, A. N., LaFleur, B., Sanders, B., Troy, J., Kurtzberg, J., & Sempowski, G. D. (2021). T cell-depleted cultured pediatric thymus tissue as a model for some aspects of human age-related thymus involution. GeroScience.
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  Human age-related thymus involution is characterized by loss of developing thymocytes and the thymic epithelial network that supports them, with replacement by adipose tissue. The mechanisms that drive these changes are difficult to study in vivo due to constant trafficking to and from the thymus. We hypothesized that the loss of thymocytes that occurs during human thymic organ cultures could model some aspects of thymus involution and begin to identify mechanisms that drive age-related changes in the thymic microenvironment. Potential mechanistically important candidate molecules were initially identified by screening conditioned media from human thymus organ cultures using antibody microarrays. These candidates were further validated using cultured tissue extracts and conditioned media. Results were compared with gene expression studies from a panel of well-characterized (non-cultured) human thymus tissues from human donors aged 5 days to 78 years. L-selectin released into conditioned media was identified as a biomarker for the content of viable thymocytes within the cultured thymus. Levels of the chemokines CCL21 and CXCL12, likely produced by surviving thymic epithelial cells, increased markedly in conditioned media as thymocytes were lost during culture. Native non-cultured thymus from adults older than 18 years also showed a strong trend toward increased CCL21 expression, in conjunction with significant decreases in thymocyte-related mRNAs compared with thymus from subjects younger than 18 years. Together, these findings demonstrate that use of postnatal human thymus organ cultures can model some aspects of human age-related thymic involution.
• Lutrick, K., Ellingson, K. D., Baccam, Z., Rivers, P., Beitel, S., Parker, J., Hollister, J., Sun, X., Gerald, J. K., Komatsu, K., Kim, E., LaFleur, B., Grant, L., Yoo, Y. M., Kumar, A., Mayo Lamberte, J., Cowling, B. J., Cobey, S., Thornburg, N. J., , Meece, J. K., et al. (2021). COVID-19 Infection, Reinfection, and Vaccine Effectiveness in a Prospective Cohort of Arizona Frontline/Essential Workers: The AZ HEROES Research Protocol. JMIR research protocols.
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  The Arizona Healthcare, Emergency Response, and Other Essential workers Study (AZ HEROES) aims to examine the epidemiology of SARS-CoV-2 infection and COVID-19 illness among adults with high occupational exposure risk.
• Schroeder, B. A., LaFranzo, N. A., LaFleur, B. J., Gittelman, R. M., Vignali, M., Zhang, S., Flanagan, K. C., Rytlewski, J., Riolobos, L., Schulte, B. C., Kim, T. S., Chen, E., Smythe, K. S., Wagner, M. J., Mantilla, J. G., Campbell, J. S., Pierce, R. H., Jones, R. L., Cranmer, L. D., & Pollack, S. M. (2021). CD4+ T cell and M2 macrophage infiltration predict dedifferentiated liposarcoma patient outcomes. Journal for immunotherapy of cancer, 9(8).
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  Dedifferentiated liposarcoma (DDLPS) is one of the most common soft tissue sarcoma subtypes and is devastating in the advanced/metastatic stage. Despite the observation of clinical responses to inhibitors, little is known about the immune microenvironment in relation to patient prognosis.
• Shroff, R. T., Chalasani, P., Wei, R., Pennington, D., Quirk, G., Schoenle, M. V., Peyton, K. L., Uhrlaub, J. L., Ripperger, T. J., Jergović, M., Dalgai, S., Wolf, A., Whitmer, R., Hammad, H., Carrier, A., Scott, A. J., Nikolich-Žugich, J., Worobey, M., Sprissler, R., , Dake, M., et al. (2021). Immune responses to two and three doses of the BNT162b2 mRNA vaccine in adults with solid tumors. Nature medicine, 27(11), 2002-2011.
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  Vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have shown high efficacy, but immunocompromised participants were excluded from controlled clinical trials. In this study, we compared immune responses to the BNT162b2 mRNA Coronavirus Disease 2019 vaccine in patients with solid tumors (n = 53) who were on active cytotoxic anti-cancer therapy to a control cohort of participants without cancer (n = 50). Neutralizing antibodies were detected in 67% of patients with cancer after the first immunization, followed by a threefold increase in median titers after the second dose. Similar patterns were observed for spike protein-specific serum antibodies and T cells, but the magnitude of each of these responses was diminished relative to the control cohort. In most patients with cancer, we detected spike receptor-binding domain and other S1-specific memory B cell subsets as potential predictors of anamnestic responses to additional immunizations. We therefore initiated a phase 1 trial for 20 cancer cohort participants of a third vaccine dose of BNT162b2 ( NCT04936997 ); primary outcomes were immune responses, with a secondary outcome of safety. At 1 week after a third immunization, 16 participants demonstrated a median threefold increase in neutralizing antibody responses, but no improvement was observed in T cell responses. Adverse events were mild. These results suggest that a third dose of BNT162b2 is safe, improves humoral immunity against SARS-CoV-2 and could be immunologically beneficial for patients with cancer on active chemotherapy.
• Billheimer, D., Gerner, E. W., McLaren, C. E., & LaFleur, B. (2014). Combined benefit of prediction and treatment: a criterion for evaluating clinical prediction models. Cancer informatics, 13(Suppl 2), 93-103.
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  Clinical treatment decisions rely on prognostic evaluation of a patient's future health outcomes. Thus, predictive models under different treatment options are key factors for making good decisions. While many criteria exist for judging the statistical quality of a prediction model, few are available to measure its clinical utility. As a consequence, we may find that the addition of a clinical covariate or biomarker improves the statistical quality of the model, but has little effect on its clinical usefulness. We focus on the setting where a treatment decision may reduce a patient's risk of a poor outcome, but also comes at a cost; this may be monetary, inconvenience, or the potential side effects. This setting is exemplified by cancer chemoprevention, or the use of statins to reduce the risk of cardiovascular disease. We propose a novel approach to assessing a prediction model using a formal decision analytic framework. We combine the predictive model's ability to discriminate good from poor outcome with the net benefit afforded by treatment. In this framework, reduced risk is balanced against the cost of treatment. The relative cost-benefit of treatment provides a useful index to assist patient decisions. This index also identifies the relevant clinical risk regions where predictive improvement is needed. Our approach is illustrated using data from a colorectal adenoma chemoprevention trial.
• Paz, E. A., LaFleur, B., & Gerner, E. W. (2014). Polyamines are oncometabolites that regulate the LIN28/let-7 pathway in colorectal cancer cells. Molecular carcinogenesis, 53 Suppl 1, E96-106.
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  Polyamine metabolism is a highly coordinated process that is essential for normal development and neoplastic growth in mammals. Although polyamine metabolism is a validated pathway for prevention of carcinogenesis, the mechanisms by which polyamines elicit their tumorigenic effects are poorly understood. In this study, we investigated the role of polyamine metabolism in colon cancer by screening a non-coding RNA (ncRNA) platform to identify polyamine responsive signaling nodes. We report that multiple non-coding RNAs are altered by polyamine depletion including induction of microRNA (miRNA) let-7i, a member of the tumor suppressive let-7 family. The let-7 family targets several RNAs for translational repression, including the growth-associated transcription factor HMGA2 and is negatively regulated by the pluripotency factor LIN28. Depletion of polyamines using difluoromethylornithine (DFMO) or genetic knockdown of the polyamine-modified eukaryotic translation initiation factor 5A isoforms 1 and 2 (eIF5A1/2) resulted in robust reduction of both HMGA2 and LIN28. Locked nucleic acid (LNA) oligonucleotides targeting the seed region of the let-7 family rescued the expression of HMGA2, but not LIN28, in both DFMO-treated and eIF5A1/2 knockdown cultures. Our findings suggest that polyamines are oncometabolites that influence specific aspects of tumorigenesis by regulating pluripotency associated factors, such as LIN28, via an eIF5A-dependent but let-7-independent mechanism while the expression of proliferation-related genes regulated by let-7, such as HMGA2, is mediated through microRNA mediated repression. Therefore, manipulating polyamine metabolism may be a novel method of targeting the LIN28/let-7 pathway in specific disease states.
• Chen, H., Hwang, H., McKee, L. A., Perez, J. N., Regan, J. A., Constantopoulos, E., Lafleur, B., & Konhilas, J. P. (2013). Temporal and morphological impact of pressure overload in transgenic FHC mice. Frontiers in physiology, 4, 205.
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  Although familial hypertrophic cardiomyopathy (FHC) is characterized as cardiac disease in the absence of overt stressors, disease penetrance, and pathological progression largely depend on modifying factors. Accordingly, pressure overload by transverse aortic constriction (TAC) was induced in 2-month-old, male mice with and without a FHC (R403Q) mutation in α-myosin heavy chain. A significantly greater number of FHC mice (n = 8) than wild-type (WT) mice (n = 5) died during the 9-week study period. TAC induced a significant increase in cardiac mass whether measured at 2 or 9 weeks post-TAC in both WT and FHC mice, albeit to a different extent. However, the temporal and morphological trajectory of ventricular remodeling was impacted by the FHC transgene. Both WT and FHC hearts responded to TAC with an early (2 weeks post-TAC) and significant augmentation of the relative wall thickness (RWT) indicative of concentric hypertrophy. By 9 weeks post-TAC, RWT decreased in WT hearts (eccentric hypertrophy) but remained elevated in FHC hearts. WT hearts following TAC demonstrated enhanced cardiac function as measured by the end-systolic pressure-volume relationship, pre-load recruitable stroke work (PRSW), and myocardial relaxation indicative of compensatory hypertrophy. Similarly, TAC induced differential histological and cellular remodeling; TAC reduced expression of the sarcoplasmic reticulum Ca(2+)-ATPase (2a) (SERCA2a; 2 and 9 weeks) and phospholamban (PLN; 2 weeks) but increased PLN phosphorylation (2 weeks) and β-myosin heavy chain (β-MyHC; 9 weeks) in WT hearts. FHC-TAC hearts showed increased β-MyHC (2 and 9 weeks) and a late (9 weeks) decrease in PLN expression concomitant with a significant increase in PLN phosphorylation. We conclude that FHC hearts respond to TAC induced pressure overload with increased premature death, severe concentric hypertrophy, and a differential ability to undergo morphological, functional, or cellular remodeling compared to WT hearts.
• Nokes, B. T., Cunliffe, H. E., Lafleur, B., Mount, D. W., Livingston, R. B., Futscher, B. W., & Lang, J. E. (2013). In Vitro Assessment of the Inflammatory Breast Cancer Cell Line SUM 149: Discovery of 2 Single Nucleotide Polymorphisms in the RNase L Gene. Journal of Cancer, 4(2), 104-16.
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  Inflammatory breast cancer (IBC) is a rare, highly aggressive form of breast cancer. The mechanism of IBC carcinogenesis remains unknown. We sought to evaluate potential genetic risk factors for IBC and whether or not the IBC cell lines SUM149 and SUM190 demonstrated evidence of viral infection.
• O'Neal, R. L., Nam, K. T., LaFleur, B. J., Barlow, B., Nozaki, K., Lee, H. J., Kim, W. H., Yang, H. K., Shi, C., Maitra, A., Montgomery, E., Washington, M. K., El Rifai, W., Drapkin, R. I., & Goldenring, J. R. (2013). Human epididymis protein 4 is up-regulated in gastric and pancreatic adenocarcinomas. Human pathology, 44(5), 734-42.
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  Upper gastrointestinal neoplasia in the esophagus, stomach, and pancreas is associated with the formation of preneoplastic metaplasias. We have previously reported the up-regulation of human epididymis protein 4 (HE4) in all metaplasias in the stomach of humans and mice. We have now sought to evaluate the expression of HE4 in metaplasias/preneoplastic precursors and cancers of the human stomach, pancreas, and esophagus. Tissue microarrays for gastric cancers, pancreatic cancers, and esophageal adenocarcinoma were stained with antibodies against HE4. Immunostaining was quantified by digital imaging, and the results were evaluated to assess the expression in metaplasias, the expression in cancer pathological subtypes, and the effects of expression on survival in patients with cancer. In patients with gastric cancer from Korea, HE4 was detected in 74% of intestinal and 90% of diffuse cancers, whereas in a gastric cancer cohort from Johns Hopkins, HE4 was detected in 74% of intestinal-type and 92% of diffuse cancers. Nevertheless, in both cohorts, there was no impact of HE4 expression on overall survival. In the esophagus, we observed the expression of HE4 in scattered endocrine cells within Barrett esophagus samples, but Barrett columnar metaplasias and HE4 were detected in only 2% of esophageal adenocarcinomas. Finally, in the pancreas, HE4 expression was not observed in pancreatic intraepithelial neoplasia lesions, but 46.8% of pancreatic adenocarcinomas expressed HE4. Still, we did not observe any influence of HE4 expression on survival. The results suggest that HE4 is up-regulated during gastric and pancreatic carcinogenesis.
• Weis, V. G., Sousa, J. F., LaFleur, B. J., Nam, K. T., Weis, J. A., Finke, P. E., Ameen, N. A., Fox, J. G., & Goldenring, J. R. (2013). Heterogeneity in mouse spasmolytic polypeptide-expressing metaplasia lineages identifies markers of metaplastic progression. Gut, 62(9), 1270-9.
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  Spasmolytic polypeptide-expressing metaplasia (SPEM) develops as a preneoplastic lesion in the stomachs of mice and humans after parietal cell loss. To identify the commonalities and differences between phenotypic SPEM lineages, SPEM were studied from three different mouse models of parietal cell loss: with chronic inflammation with Helicobacter felis infection; with acute inflammation with L635 treatment; and without inflammation following DMP-777 treatment.
• Chandramouli, A., Onyeagucha, B. C., Mercado-Pimentel, M. E., Stankova, L., Shahin, N. A., LaFleur, B. J., Heimark, R. L., Bhattacharyya, A. K., & Nelson, M. A. (2012). MicroRNA-101 (miR-101) post-transcriptionally regulates the expression of EP4 receptor in colon cancers. Cancer biology & therapy, 13(3), 175-83.
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  Expression of the PGE2 receptor, EP4, is up-regulated during colorectal carcinogenesis. However the mechanism leading to deregulation of the EP4 receptor is not known. The present study was conducted to investigate the regulation of EP4 receptor by miRNAs.
• Sousa, J. F., Ham, A. J., Whitwell, C., Nam, K. T., Lee, H. J., Yang, H. K., Kim, W. H., Zhang, B., Li, M., LaFleur, B., Liebler, D. C., & Goldenring, J. R. (2012). Proteomic profiling of paraffin-embedded samples identifies metaplasia-specific and early-stage gastric cancer biomarkers. The American journal of pathology, 181(5), 1560-72.
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  Early diagnosis and curative resection are the predominant factors associated with increased survival in patients with gastric cancer. However, most gastric cancer cases are still diagnosed at later stages. Since most pathologic specimens are archived as FFPE samples, the ability to use them to generate expression profiles can greatly improve cancer biomarker discovery. We sought to uncover new biomarkers for stomach preneoplastic metaplasias and neoplastic lesions by generating proteome profiles using FFPE samples. We combined peptide isoelectric focusing and liquid chromatography-tandem mass spectrometry analysis to generate proteomic profiles from FFPE samples of intestinal-type gastric cancer, metaplasia, and normal mucosa. The expression patterns of selected proteins were analyzed by immunostaining first in single tissue sections from normal stomach, metaplasia, and gastric cancer and later in larger tissue array cohorts. We detected 60 proteins up-regulated and 87 proteins down-regulated during the progression from normal mucosa to metaplasia to gastric cancer. Two of the up-regulated proteins, LTF and DMBT1, were validated as specific markers for spasmolytic polypeptide-expressing metaplasia and intestinal metaplasia, respectively. In cancers, significantly lower levels of DMBT1 or LTF correlated with more advanced disease and worse prognosis. Thus, proteomic profiling using FFPE samples has led to the identification of two novel markers for stomach metaplasias and gastric cancer prognosis.
• Blaschke, A. J., Allison, M. A., Meyers, L., Rogatcheva, M., Heyrend, C., Mallin, B., Carter, M., Lafleur, B., Barney, T., Poritz, M. A., Daly, J. A., & Byington, C. L. (2011). Non-invasive sample collection for respiratory virus testing by multiplex PCR. Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology, 52(3), 210-4.
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  Identifying respiratory pathogens within populations is difficult because invasive sample collection, such as with nasopharyngeal aspirate (NPA), is generally required. PCR technology could allow for non-invasive sampling methods.
• Chan, E., Lafleur, B., Rothenberg, M. L., Merchant, N., Lockhart, A. C., Trivedi, B., Chung, C. H., Coffey, R. J., & Berlin, J. D. (2011). Dual blockade of the EGFR and COX-2 pathways: a phase II trial of cetuximab and celecoxib in patients with chemotherapy refractory metastatic colorectal cancer. American journal of clinical oncology, 34(6), 581-6.
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  The epidermal growth factor receptor (EGFR) and cyclooxygenase-2 (COX-2) pathways play key and often complementary roles in the pathogenesis of colorectal cancer (CRC). This study explores the clinical and biological effects of combined blockade of these pathways.
• Lafleur, B., Lee, W., Billhiemer, D., Lockhart, C., Liu, J., & Merchant, N. (2011). Statistical methods for assays with limits of detection: Serum bile acid as a differentiator between patients with normal colons, adenomas, and colorectal cancer. Journal of carcinogenesis, 10, 12.
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  In analytic chemistry a detection limit (DL) is the lowest measurable amount of an analyte that can be distinguished from a blank; many biomedical measurement technologies exhibit this property. From a statistical perspective, these data present inferential challenges because instead of precise measures, one only has information that the value is somewhere between 0 and the DL (below detection limit, BDL). Substitution of BDL values, with 0 or the DL can lead to biased parameter estimates and a loss of statistical power. Statistical methods that make adjustments when dealing with these types of data, often called left-censored data, are available in many commercial statistical packages. Despite this availability, the use of these methods is still not widespread in biomedical literature. We have reviewed the statistical approaches of dealing with BDL values, and used simulations to examine the performance of the commonly used substitution methods and the most widely available statistical methods. We have illustrated these methods using a study undertaken at the Vanderbilt-Ingram Cancer Center, to examine the serum bile acid levels in patients with colorectal cancer and adenoma. We have found that the modern methods for BDL values identify disease-related differences that are often missed, with statistically naive approaches.
• Moore, J. C., Thompson, K., Lafleur, B., Book, L. S., Jackson, W. D., O'Gorman, M. A., Black, R. E., Downey, E., Johnson, D. G., Matlak, M. E., Meyers, R. L., Scaife, E., & Guthery, S. L. (2011). Clinical variables as prognostic tools in pediatric-onset ulcerative colitis: a retrospective cohort study. Inflammatory bowel diseases, 17(1), 15-21.
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  Clinical variables may identify a subset of patients with pediatric-onset ulcerative colitis (UC) (≤18 years at diagnosis) at risk for adverse outcomes. We postulated that routinely measured clinical variables measured at diagnosis would predict colectomy in patients with pediatric-onset UC.
• Thorne, C. A., Lafleur, B., Lewis, M., Hanson, A. J., Jernigan, K. K., Weaver, D. C., Huppert, K. A., Chen, T. W., Wichaidit, C., Cselenyi, C. S., Tahinci, E., Meyers, K. C., Waskow, E., Orton, D., Salic, A., Lee, L. A., Robbins, D. J., Huppert, S. S., & Lee, E. (2011). A biochemical screen for identification of small-molecule regulators of the Wnt pathway using Xenopus egg extracts. Journal of biomolecular screening, 16(9), 995-1006.
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  Misregulation of the Wnt pathway has been shown to be responsible for a variety of human diseases, most notably cancers. Screens for inhibitors of this pathway have been performed almost exclusively using cultured mammalian cells or with purified proteins. We have previously developed a biochemical assay using Xenopus egg extracts to recapitulate key cytoplasmic events in the Wnt pathway. Using this biochemical system, we show that a recombinant form of the Wnt coreceptor, LRP6, regulates the stability of two key components of the Wnt pathway (β-catenin and Axin) in opposing fashion. We have now fused β-catenin and Axin to firefly and Renilla luciferase, respectively, and demonstrate that the fusion proteins behave similarly as their wild-type counterparts. Using this dual luciferase readout, we adapted the Xenopus extracts system for high-throughput screening. Results from these screens demonstrate signal distribution curves that reflect the complexity of the library screened. Of several compounds identified as cytoplasmic modulators of the Wnt pathway, one was further validated as a bona fide inhibitor of the Wnt pathway in cultured mammalian cells and Xenopus embryos. We show that other embryonic pathways may be amendable to screening for inhibitors/modulators in Xenopus egg extracts.
• Campbell, K. A., Cook, L. J., LaFleur, B. J., & Keenan, H. T. (2010). Household, family, and child risk factors after an investigation for suspected child maltreatment: a missed opportunity for prevention. Archives of pediatrics & adolescent medicine, 164(10), 943-9.
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  To determine whether a Child Protection Services investigation for suspected child maltreatment is associated with subsequent improvements in household, caregiver, and child risk factors.
• Lee, H. J., Nam, K. T., Park, H. S., Kim, M. A., Lafleur, B. J., Aburatani, H., Yang, H. K., Kim, W. H., & Goldenring, J. R. (2010). Gene expression profiling of metaplastic lineages identifies CDH17 as a prognostic marker in early stage gastric cancer. Gastroenterology, 139(1), 213-25.e3.
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  Intestinal metaplasia (IM) and spasmolytic polypeptide-expressing metaplasia (SPEM) are precursors to gastric carcinogenesis. We sought to identify molecular biomarkers of gastric metaplasias and gastric cancer by gene expression profiling of metaplastic lesions from patients.
• Thompson, P. A., Wertheim, B. C., Zell, J. A., Chen, W. P., McLaren, C. E., LaFleur, B. J., Meyskens, F. L., & Gerner, E. W. (2010). Levels of rectal mucosal polyamines and prostaglandin E2 predict ability of DFMO and sulindac to prevent colorectal adenoma. Gastroenterology, 139(3), 797-805, 805.e1.
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  Combination of polyamine and prostaglandin E2 (PGE2)-synthesis inhibitors reduced the risk of colorectal adenoma (CRA) by 70% in patients who received polypectomies. We studied effects of the combination of difluoromethylornithine (DFMO) and sulindac on biomarkers and investigated factors that modify their efficacy.
• Thorne, C. A., Hanson, A. J., Schneider, J., Tahinci, E., Orton, D., Cselenyi, C. S., Jernigan, K. K., Meyers, K. C., Hang, B. I., Waterson, A. G., Kim, K., Melancon, B., Ghidu, V. P., Sulikowski, G. A., LaFleur, B., Salic, A., Lee, L. A., Miller, D. M., & Lee, E. (2010). Small-molecule inhibition of Wnt signaling through activation of casein kinase 1α. Nature chemical biology, 6(11), 829-36.
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  Wnt/β-catenin signaling is critically involved in metazoan development, stem cell maintenance and human disease. Using Xenopus laevis egg extract to screen for compounds that both stabilize Axin and promote β-catenin turnover, we identified an FDA-approved drug, pyrvinium, as a potent inhibitor of Wnt signaling (EC(50) of ∼10 nM). We show pyrvinium binds all casein kinase 1 (CK1) family members in vitro at low nanomolar concentrations and pyrvinium selectively potentiates casein kinase 1α (CK1α) kinase activity. CK1α knockdown abrogates the effects of pyrvinium on the Wnt pathway. In addition to its effects on Axin and β-catenin levels, pyrvinium promotes degradation of Pygopus, a Wnt transcriptional component. Pyrvinium treatment of colon cancer cells with mutation of the gene for adenomatous polyposis coli (APC) or β-catenin inhibits both Wnt signaling and proliferation. Our findings reveal allosteric activation of CK1α as an effective mechanism to inhibit Wnt signaling and highlight a new strategy for targeted therapeutics directed against the Wnt pathway.
• Blaschke, A. J., Korgenski, E. K., Daly, J. A., LaFleur, B., Pavia, A. T., & Byington, C. L. (2009). Extended-spectrum beta-lactamase-producing pathogens in a children's hospital: a 5-year experience. American journal of infection control, 37(6), 435-41.
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  Pediatric infection with bacteria producing extended-spectrum beta-lactamases (ESBLs) has not been well described. We sought to determine the proportion of isolates producing ESBLs and the incidence of infection or colonization with these organisms in our tertiary care pediatric facility over 5 years. In addition, we sought to evaluate the characteristics of children affected.
• Guess, C. M., Lafleur, B. J., Weidow, B. L., & Quaranta, V. (2009). A decreased ratio of laminin-332 beta3 to gamma2 subunit mRNA is associated with poor prognosis in colon cancer. Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology, 18(5), 1584-90.
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  Laminin-332 (Ln-332) is a heterotrimeric glycoprotein (alpha3beta3gamma2) unique to epithelial cells with crucial roles in signaling, adhesion, and migration. Altered localization or expression levels of Ln-332, particularly its gamma2 subunit, are of prognostic value in a variety of cancers. However, the lack of standardized methodology and the limited quantification of previous study results have left unanswered questions, including the role of gamma2 transcript variants and whether differential expression of this chain represents dysregulation of the whole heterotrimer. Herein, we test the hypothesis that mRNA changes in one or more Ln-332 encoding genes can be used to distinguish between early- and advanced-stage cancer specimens and shed light on mechanistic questions raised by previous studies. Statistical analyses of human microarray data from the publicly available expression project in Oncology (expO) dataset, including examination of the distributions of Ln-332 subunit mRNA levels, identified a significant decrease in the Ln-332 beta3:gamma2 mRNA ratio between normal (n = 10) and early-stage colon cancer (n = 29) specimens. The beta3:gamma2 ratio was further decreased in metastatic colon cancer (n = 41) compared with early-stage samples. Our findings raise the possibility that Ln-332 gamma2 may be a therapeutic target against metastatic colon cancer because a lowered beta3:gamma2 ratio would reduce expression of heterotrimeric Ln-332 and increase monomeric gamma2 secretion. Further, standardized, quantitative methods for patient prognosis and therapeutic choice could be developed based upon the Ln-332 mRNA changes we uncovered.
• LaFleur, B. J., & Greevy, R. A. (2009). Introduction to permutation and resampling-based hypothesis tests. Journal of clinical child and adolescent psychology : the official journal for the Society of Clinical Child and Adolescent Psychology, American Psychological Association, Division 53, 38(2), 286-94.
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  A resampling-based method of inference -- permutation tests -- is often used when distributional assumptions are questionable or unmet. Not only are these methods useful for obvious departures from parametric assumptions (e.g., normality) and small sample sizes, but they are also more robust than their parametric counterparts in the presences of outliers and missing data, problems that are often found in clinical child and adolescent psychology research. These methods are increasingly found in statistical software programs, making their use more feasible. In this article, we use an application-based approach to provide a brief tutorial on permutation testing. We present some historical perspectives, describe how the tests are formulated, and provide examples of common and specific situations under which the methods are most useful. Finally, we demonstrate the utility of these methods to clinical and adolescent psychology by examining four recent articles employing these methods.
• Simon, T. D., Hall, M., Riva-Cambrin, J., Albert, J. E., Jeffries, H. E., Lafleur, B., Dean, J. M., Kestle, J. R., & , H. C. (2009). Infection rates following initial cerebrospinal fluid shunt placement across pediatric hospitals in the United States. Clinical article. Journal of neurosurgery. Pediatrics, 4(2), 156-65.
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  Reported rates of CSF shunt infection vary widely across studies. The study objective was to determine the CSF shunt infection rates after initial shunt placement at multiple US pediatric hospitals. The authors hypothesized that infection rates between hospitals would vary widely even after adjustment for patient, hospital, and surgeon factors.
• Williams, J. V., Weitkamp, J. H., Blum, D. L., LaFleur, B. J., & Crowe, J. E. (2009). The human neonatal B cell response to respiratory syncytial virus uses a biased antibody variable gene repertoire that lacks somatic mutations. Molecular immunology, 47(2-3), 407-14.
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  The human Ab repertoire exhibits restrictions during fetal life characterized by biases of variable gene segment usage and lack of junctional diversity. We tested the hypotheses that Ab repertoire restriction persists in the early postnatal period and contributes to the observed poor quality of specific Ab responses made by neonates to viruses and vaccines. We analyzed the molecular determinants of B cell responses in humans to respiratory syncytial virus (RSV). Analysis of the variable gene segment usage of adult RSV-specific B cells revealed a repertoire profile in these cells similar to that seen in randomly selected B cells, which was V(H)3-dominant. Four gene segments (V(H)3-23, V(H)3-30, V(H)3-33 and V(H)4-04) accounted for almost half of the V(H) genes used. In contrast, very young infant RSV-specific antibodies exhibited a biased repertoire characterized by comparable use of the V(H)1, V(H)3, and V(H)4 families, and less common use of the four immunodominant gene segments. Infants and children older than three months used an antibody repertoire similar to that of adults. Mutational analysis revealed that the antibody variable genes of infants under three months of age also possessed significantly fewer somatic mutations in both framework and complementarity-determining region (CDR) regions than those of adults, even in a child with recurrent RSV infection. These data suggest that neonates use a biased antibody gene repertoire that is less V(H)3-focused and that possesses a dramatically lower frequency of somatic mutations. These biased features of the RSV-specific repertoire likely contribute to the poor functional Ab response in very young infants.
• Zeft, A., Hollister, R., LaFleur, B., Sampath, P., Soep, J., McNally, B., Kunkel, G., Schlesinger, M., & Bohnsack, J. (2009). Anakinra for systemic juvenile arthritis: the Rocky Mountain experience. Journal of clinical rheumatology : practical reports on rheumatic & musculoskeletal diseases, 15(4), 161-4.
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  Poor outcomes in systemic juvenile arthritis have been associated with persistent thrombocytosis, increased sedimentation rates, anemia, polyarthritis, and prolonged steroid use. Off-label treatment with recombinant interleukin-1 receptor antagonist therapy (anakinra) has become more common since reports of its association with reduced systemic symptoms and arthritis scores, improved laboratory parameters of inflammation, and decreased corticosteroid requirements.
• Halasa, N. B., O'Shea, A., Shi, J. R., LaFleur, B. J., & Edwards, K. M. (2008). Poor immune responses to a birth dose of diphtheria, tetanus, and acellular pertussis vaccine. The Journal of pediatrics, 153(3), 327-32.
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  To evaluate the safety and immunogenicity of an additional birth dose of diphtheria, tetanus, and acellular pertussis vaccine (DTaP).
• Lockhart, A. C., Harris, E., Lafleur, B. J., Merchant, N. B., Washington, M. K., Resnick, M. B., Yeatman, T. J., & Lee, W. (2008). Organic anion transporting polypeptide 1B3 (OATP1B3) is overexpressed in colorectal tumors and is a predictor of clinical outcome. Clinical and experimental gastroenterology, 1, 1-7.
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  OATP1B3 is an organic anion transporting polypeptide (OATP) that functions as a multispecific transporter in the normal liver. We examined the expression and clinical significance of OATP1B3 in colon cancers in tissue microarrays.
• Manning, H. C., Merchant, N. B., Foutch, A. C., Virostko, J. M., Wyatt, S. K., Shah, C., McKinley, E. T., Xie, J., Mutic, N. J., Washington, M. K., LaFleur, B., Tantawy, M. N., Peterson, T. E., Ansari, M. S., Baldwin, R. M., Rothenberg, M. L., Bornhop, D. J., Gore, J. C., & Coffey, R. J. (2008). Molecular imaging of therapeutic response to epidermal growth factor receptor blockade in colorectal cancer. Clinical cancer research : an official journal of the American Association for Cancer Research, 14(22), 7413-22.
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  To evaluate noninvasive molecular imaging methods as correlative biomarkers of therapeutic efficacy of cetuximab in human colorectal cancer cell line xenografts grown in athymic nude mice. The correlation between molecular imaging and immunohistochemical analysis to quantify epidermal growth factor (EGF) binding, apoptosis, and proliferation was evaluated in treated and untreated tumor-bearing cohorts.
• McCawley, L. J., Wright, J., LaFleur, B. J., Crawford, H. C., & Matrisian, L. M. (2008). Keratinocyte expression of MMP3 enhances differentiation and prevents tumor establishment. The American journal of pathology, 173(5), 1528-39.
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  Matrix metalloproteinase (MMP)-3 is induced by multiple cell types in the skin during processes involved in both normal and pathological tissue remodeling. We previously demonstrated that MMP3-null animals have an increased sensitivity to the development of squamous cell carcinoma, suggesting that overall, MMP3 has a protective role in squamous cell carcinoma. However, not all cellular responses affected by a loss of MMP3 are tumor-protective, and tumor expression of MMP3 is co-incident with an invasive tumor phenotype. Transgenic mice were generated with MMP3 targeted to keratinocytes to examine the biological role of tumor-produced MMP3. Overexpression of MMP3 reduced tumor multiplicity in response to chemically induced squamous cell carcinoma. Vascular density was increased with MMP3 overexpression; however, other cellular processes, including tumor growth and leukocyte infiltration, were unaffected. In accordance with the change in tumor multiplicity, SP-1 murine papilloma cell lines that were generated to stably express MMP3 lost the capacity to establish palpable tumors following orthotopic injection into immunocompromised mice. Analysis of epidermal biopsies taken at 1 to 2 weeks postinjection revealed that these MMP3-expressing Sp-1 lines had reduced levels of proliferation and pronounced differentiation. These same cells demonstrated an increased ability to differentiate in vitro, an effect that was inhibited by broad-spectrum MMP and selective MMP3 inhibition. These studies suggest that keratinocyte expression of MMP3 promotes cellular differentiation, impeding tumor establishment during tumorigenesis.
• Merchant, N. B., Voskresensky, I., Rogers, C. M., Lafleur, B., Dempsey, P. J., Graves-Deal, R., Revetta, F., Foutch, A. C., Rothenberg, M. L., Washington, M. K., & Coffey, R. J. (2008). TACE/ADAM-17: a component of the epidermal growth factor receptor axis and a promising therapeutic target in colorectal cancer. Clinical cancer research : an official journal of the American Association for Cancer Research, 14(4), 1182-91.
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  Activation of the epidermal growth factor receptor (EGFR) requires cell surface cleavage of EGFR ligands, uptake of soluble ligand by the receptor, and initiation of EGFR tyrosine kinase activity. We define these collective events as the EGFR axis. Transforming growth factor-alpha (TGF-alpha) and amphiregulin are two EGFR ligands that are delivered preferentially to the basolateral surface of polarized epithelial cells where the EGFR resides. TACE/ADAM-17 (tumor necrosis factor-alpha converting enzyme/a disintegrin and metalloprotease) has been implicated in ectodomain cleavage of TGF-alpha and amphiregulin.
• Nozaki, K., Ogawa, M., Williams, J. A., Lafleur, B. J., Ng, V., Drapkin, R. I., Mills, J. C., Konieczny, S. F., Nomura, S., & Goldenring, J. R. (2008). A molecular signature of gastric metaplasia arising in response to acute parietal cell loss. Gastroenterology, 134(2), 511-22.
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  Loss of gastric parietal cells is a critical precursor to gastric metaplasia and neoplasia. However, the origin of metaplasia remains obscure. Acute parietal cell loss in gastrin-deficient mice treated with DMP-777 leads to the rapid emergence of spasmolytic polypeptide/trefoil factor family 2 (TFF2)-expressing metaplasia (SPEM) from the bases of fundic glands. We now sought to characterize more definitively the pathway for emergence of SPEM.
• Rauth, T. P., Eckhauser, A. W., LaFleur, B. J., Melvin, W. V., & Holzman, M. D. (2008). Intraband pressure measurements describe a pattern of weight loss for patients with adjustable gastric bands. Journal of the American College of Surgeons, 206(5), 926-32; discussion 932-4.
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  Individuals with adjustable gastric bands experience plateaus in weight loss. Patients commonly attribute this to a "loosening" of their band with time. We sought to elucidate a physiologic mechanism for this pattern in patient behavior and describe the feasibility of a pressure-based adjustment algorithm for adjustable gastric bands.
• Sinnamon, M. J., Carter, K. J., Sims, L. P., Lafleur, B., Fingleton, B., & Matrisian, L. M. (2008). A protective role of mast cells in intestinal tumorigenesis. Carcinogenesis, 29(4), 880-6.
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  Mast cells have been observed in numerous types of tumors; however, their role in carcinogenesis remains poorly understood. The majority of epidemiological evidence suggests a negative association between the presence of mast cells and tumor progression in breast, lung and colonic neoplasms. Intestinal adenomas in the multiple intestinal neoplasia (Min, APC(Min/+)) mouse displayed increased numbers of mast cells and increased abundance of mast cell-associated proteinases as determined by transcriptional profiling with the Hu/Mu ProtIn microarray. To examine the role of mast cells in intestinal tumorigenesis, a mutant mouse line deficient in mast cells, Sash mice (c-kit(W-sh/W-sh)), was crossed with the Min mouse, a genetic model of intestinal neoplasia. The resulting mast cell-deficient Min-Sash mice developed 50% more adenomas than littermate controls and the tumors were 33% larger in Min-Sash mice. Mast cell deficiency did not affect tumor cell proliferation; however, apoptosis was significantly inhibited in mast cell-deficient mice. Mast cells have been shown to act as critical upstream regulators of numerous inflammatory cells. Neutrophil, macrophage and T cell populations were similar between Min and Min-Sash mice; however, eosinophils were significantly less abundant in tumors obtained from Min-Sash animals. These results indicate a protective, antitumor role of mast cells in a genetic model of early-stage intestinal tumorigenesis.
• Sosman, J. A., Carrillo, C., Urba, W. J., Flaherty, L., Atkins, M. B., Clark, J. I., Dutcher, J., Margolin, K. A., Mier, J., Gollob, J., Kirkwood, J. M., Panka, D. J., Crosby, N. A., O'Boyle, K., LaFleur, B., & Ernstoff, M. S. (2008). Three phase II cytokine working group trials of gp100 (210M) peptide plus high-dose interleukin-2 in patients with HLA-A2-positive advanced melanoma. Journal of clinical oncology : official journal of the American Society of Clinical Oncology, 26(14), 2292-8.
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  High-dose interleukin-2 (IL-2) induces responses in 15% to 20% of patients with advanced melanoma; 5% to 8% are durable complete responses (CRs). The HLA-A2-restricted, modified gp100 peptide (210M) induces T-cell immunity in vivo and has little antitumor activity but, combined with high-dose IL-2, reportedly has a 42% (13 of 31 patients) response rate (RR). We evaluated 210M with one of three different IL-2 schedules to determine whether a basis exists for a phase III trial.
• Fasig, J. H., Dupont, W. D., Olson, S. J., Lafleur, B. J., & Cates, J. M. (2007). Steroid hormone receptor and COX-2 expression in chordoma. American journal of clinical pathology, 128(3), 375-81.
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  Reports of sex steroid receptor expression in chordoma suggest that these tumors may be responsive to hormone manipulation therapy. Immunohistochemical stains for estrogen receptor (ER)-alpha, ER-beta, progesterone receptor (PR), androgen receptor (AR), and cyclooxygenase 2 (COX-2), were performed on a tissue microarray containing 21 samples of chordoma. Most chordomas expressed COX-2, ER-beta, and AR, whereas ER-alpha and PR stains were negative in all cases. ER-beta expression did not correlate with AR expression (P = .4142; McNemar test). There were no statistically significant correlations between the expression of any of these markers and anatomic location of tumor, patient sex, patient age, or disease-free survival. Chordomas commonly express COX-2, AR, and ER-beta. These findings may have therapeutic implications concerning the use of agents that inhibit or modulate these signaling molecules.
• Leys, C. M., Nomura, S., LaFleur, B. J., Ferrone, S., Kaminishi, M., Montgomery, E., & Goldenring, J. R. (2007). Expression and prognostic significance of prothymosin-alpha and ERp57 in human gastric cancer. Surgery, 141(1), 41-50.
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  Prothymosin-alpha and ERp57 were previously identified as markers for gastric metaplasia in a mouse model of Helicobacter-induced gastric metaplasia and neoplasia. In this paper we assess whether the expression of these putative biomarkers in humans is correlated with gastric metaplasia and adenocarcinoma and clinical outcomes.
• Poehling, K. A., Szilagyi, P. G., Grijalva, C. G., Martin, S. W., LaFleur, B., Mitchel, E., Barth, R. D., Nuorti, J. P., & Griffin, M. R. (2007). Reduction of frequent otitis media and pressure-equalizing tube insertions in children after introduction of pneumococcal conjugate vaccine. Pediatrics, 119(4), 707-15.
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  Streptococcus pneumoniae is an important cause of otitis media in children. In this study we estimated the effect of routine childhood immunization with heptavalent pneumococcal conjugate vaccine on frequent otitis media (3 episodes in 6 months or 4 episodes in 1 year) and pressure-equalizing tube insertions.
• Dunckley, T., Beach, T. G., Ramsey, K. E., Grover, A., Mastroeni, D., Walker, D. G., LaFleur, B. J., Coon, K. D., Brown, K. M., Caselli, R., Kukull, W., Higdon, R., McKeel, D., Morris, J. C., Hulette, C., Schmechel, D., Reiman, E. M., Rogers, J., & Stephan, D. A. (2006). Gene expression correlates of neurofibrillary tangles in Alzheimer's disease. Neurobiology of aging, 27(10), 1359-71.
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  Neurofibrillary tangles (NFT) constitute one of the cardinal histopathological features of Alzheimer's disease (AD). To explore in vivo molecular processes involved in the development of NFTs, we compared gene expression profiles of NFT-bearing entorhinal cortex neurons from 19 AD patients, adjacent non-NFT-bearing entorhinal cortex neurons from the same patients, and non-NFT-bearing entorhinal cortex neurons from 14 non-demented, histopathologically normal controls (ND). Of the differentially expressed genes, 225 showed progressively increased expression (AD NFT neurons > AD non-NFT neurons > ND non-NFT neurons) or progressively decreased expression (AD NFT neurons < AD non-NFT neurons < ND non-NFT neurons), raising the possibility that they may be related to the early stages of NFT formation. Immunohistochemical studies confirmed that many of the implicated proteins are dysregulated and preferentially localized to NFTs, including apolipoprotein J, interleukin-1 receptor-associated kinase 1, tissue inhibitor of metalloproteinase 3, and casein kinase 2, beta. Functional validation studies are underway to determine which candidate genes may be causally related to NFT neuropathology, thus providing therapeutic targets for the treatment of AD.
• Weinberg, A., Wiznia, A. A., Lafleur, B. J., Shah, S., & Levin, M. J. (2006). Cytomegalovirus-specific cell-mediated immunity in HIV-infected children on HAART. AIDS research and human retroviruses, 22(3), 283-8.
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  The objectives of this study were to define the magnitude, time course, and virologic and immunologic correlates of HAART-associated reconstitution of cytomegalovirus (CMV)-specific cell-mediated immunity (CMI) in pediatric HAART recipients. Thirty-five HIV-infected CMV-seropositive subjects < or = 22 years on or about to receive HAART had CMV-CMI measured by responder cell frequency (RCF) and interferon-gamma (IFN-gamma) secretion over 3 years. RCF was detected in 33, 52, 38, and 28% before HAART and at years 1, 2, and > or = 3, respectively. Corresponding percentages for IFN-gamma were 100, 85, 100, and 38%. Neither RCF nor IFN-gamma was significantly associated with CD4% before or after HAART initiation. Lower HIV replication was associated with a higher proportion of subjects with positive RCF, but not IFN-gamma. There were no clinical CMV manifestations during the study. HIV-infected children did not demonstrate a significant increase in CMV-CMI with longer HAART duration, which suggests that CMV immunereconstitution involves more complex immunologic and virologic interactions than previously anticipated.
• Guthrie, S. O., Lynn, C., Lafleur, B. J., Donn, S. M., & Walsh, W. F. (2005). A crossover analysis of mandatory minute ventilation compared to synchronized intermittent mandatory ventilation in neonates. Journal of perinatology : official journal of the California Perinatal Association, 25(10), 643-6.
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  Mandatory minute ventilation (MMV) is a novel ventilator mode that combines synchronized intermittent mandatory ventilation (SIMV) breaths with pressure-supported spontaneous breaths to maintain a desired minute volume. The SIMV rate is automatically adjusted to maintain minute ventilation.
• Mintz, M. B., Sowers, R., Brown, K. M., Hilmer, S. C., Mazza, B., Huvos, A. G., Meyers, P. A., Lafleur, B., McDonough, W. S., Henry, M. M., Ramsey, K. E., Antonescu, C. R., Chen, W., Healey, J. H., Daluski, A., Berens, M. E., Macdonald, T. J., Gorlick, R., & Stephan, D. A. (2005). An expression signature classifies chemotherapy-resistant pediatric osteosarcoma. Cancer research, 65(5), 1748-54.
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  Osteosarcoma is the most common malignant bone tumor in children. Osteosarcoma patients who respond poorly to chemotherapy are at a higher risk of relapse and adverse outcome. Therefore, it was the aim of this study to identify prognostic factors at the time of diagnosis to characterize the genes predictive of poor survival outcome and to identify potential novel therapeutic targets. Expression profiling of 30 osteosarcoma diagnostic biopsy samples, 15 with inferior necrosis following induction chemotherapy (Huvos I/II) and 15 with superior necrosis following induction chemotherapy (Huvos III/IV), was conducted using Affymetrix U95Av2 oligonucleotide microarrays. One hundred and four genes were found to be statistically significant and highly differentially expressed between Huvos I/II and III/IV patients. Statistically significant genes were validated on a small independent cohort comprised of osteosarcoma xenograft tumor samples. Markers of Huvos I/II response predominantly were gene products involved in extracellular matrix (ECM) microenvironment remodeling and osteoclast differentiation. A striking finding was the significant decrease in osteoprotegerin, an osteoclastogenesis inhibitory factor. Additional genes involved in osteoclastogenesis and bone resorption, which were statistically different, include annexin 2, SMAD, PLA2G2A, and TGFbeta1. ECM remodeling genes include desmoplakin, SPARCL1, biglycan, and PECAM. Gene expression of select genes involved in tumor progression, ECM remodeling, and osteoclastogenesis were validated via quantitative reverse transcription-PCR in an independent cohort. We propose that osteosarcoma tumor-driven changes in the bone microenvironment contribute to the chemotherapy-resistant phenotype and offer testable hypotheses to potentially enhance therapeutic response.
• Rothenberg, M. L., LaFleur, B., Levy, D. E., Washington, M. K., Morgan-Meadows, S. L., Ramanathan, R. K., Berlin, J. D., Benson, A. B., & Coffey, R. J. (2005). Randomized phase II trial of the clinical and biological effects of two dose levels of gefitinib in patients with recurrent colorectal adenocarcinoma. Journal of clinical oncology : official journal of the American Society of Clinical Oncology, 23(36), 9265-74.
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  The clinical objective of this trial was to evaluate gefitinib in patients with metastatic colorectal cancer that had progressed despite prior treatment. Serial tumor biopsies were performed when possible and analyzed for activation of the epidermal growth factor receptor (EGFR) signaling pathway. Serial serum samples were measured for amphiregulin and transforming growth factor-alpha (TGFalpha).
• Weitkamp, J. H., Kallewaard, N. L., Bowen, A. L., Lafleur, B. J., Greenberg, H. B., & Crowe, J. E. (2005). VH1-46 is the dominant immunoglobulin heavy chain gene segment in rotavirus-specific memory B cells expressing the intestinal homing receptor alpha4beta7. Journal of immunology (Baltimore, Md. : 1950), 174(6), 3454-60.
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  Memory B cells expressing the intestinal homing marker alpha4beta7 are important for protective immunity against human rotavirus (RV). It is not known whether the B cell repertoire of intestinal homing B cells differs from B cells of the systemic compartment. In this study, we analyzed the RV-specific VH and VL repertoire in human IgD- B cells expressing the intestinal homing marker alpha4beta7. The mean frequency of RV-specific B cells in the systemic compartment of healthy adult subjects was 0.6% (range, 0.2-1.2). The mean frequency of IgD- B cells that were both RV specific and alpha4beta7 was 0.04% (range, 0.01-0.1), and a mean of 10% (range, 1-32) of RV-specific peripheral blood human B cells exhibited an intestinal homing phenotype. We previously demonstrated that VH1-46 is the dominant Ab H chain gene segment in RV-specific systemic B cells from adults and infants. RV-specific systemic IgD- or intestinal homing IgD-/alpha4beta7+ B cells in the current study also used the gene segment VH1-46 at a high frequency, while randomly selected B cells with those phenotypes did not. These data show that VH1-46 is the immunodominant gene segment in human RV-specific effector B cells in both the systemic compartment and in intestinal homing lymphocytes. The mean replacement/silent mutation ratio of systemic compartment IgD- B cells was >2, consistent with a memory phenotype and antigenic selection. Interestingly, RV-specific intestinal homing IgD-/alpha4beta7+ B cells using the VH1-46 gene segment were not mutated, in contrast to systemic RV-specific IgD- B cells.
• Weitkamp, J. H., Lafleur, B. J., Greenberg, H. B., & Crowe, J. E. (2005). Natural evolution of a human virus-specific antibody gene repertoire by somatic hypermutation requires both hotspot-directed and randomly-directed processes. Human immunology, 66(6), 666-76.
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  Somatic hypermutation of antibody genes is mediated by activation-induced cytidine deaminase and targets primarily hotspot motifs. We tested the hypothesis that the antibody variable genes of virus-specific B cells from infants exhibit a decreased frequency of somatic mutations compared with adults. We also sought to determine whether virus-specific B cells exhibit predominantly hotspot or randomly directed processes. We analyzed somatic mutations in rotavirus (RV)-specific B cells from otherwise healthy but recently RV-infected infants or adults in comparison with B cells from healthy volunteers not recently infected. We compared these antibody variable gene sequences with those derived from RV-specific B cells from an adult patient with X-linked hyper-IgM syndrome (XHIM). We found that the overall mutational frequency within the antibody variable region was lowest in RV-specific B cells from RV-infected infants, followed by randomly selected B cells, followed by RV-specific B cells from the patient with XHIM. RV-specific memory B cells from healthy adults exhibited the highest frequency of mutations. Approximately half of mutations in random or RV-specific B cells from adults or infants occurred at the DGYW/WRCH or WA/TW hotspot motifs. These findings suggest that virus-specific antibodies require both hotspot and randomly-directed processes.
• Amiri, K. I., Horton, L. W., LaFleur, B. J., Sosman, J. A., & Richmond, A. (2004). Augmenting chemosensitivity of malignant melanoma tumors via proteasome inhibition: implication for bortezomib (VELCADE, PS-341) as a therapeutic agent for malignant melanoma. Cancer research, 64(14), 4912-8.
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  Melanoma poses a great challenge to patients, oncologists, and biologists because of its nearly universal resistance to chemotherapy. Many studies have shown that nuclear factor kappaB is constitutively activated in melanoma, thereby promoting the proliferation of melanoma cells by inhibiting the apoptotic responses to chemotherapy. Nuclear factor kappaB activity is regulated by phosphorylation and subsequent degradation of inhibitor of nuclear factor kappaB by the ubiquitin-proteasome pathway. In this study, we show that the novel proteasome inhibitor, bortezomib, inhibited the growth of melanoma cells in vitro at a concentration range of 0.1-10 nM and in combination with the chemotherapeutic agent temozolomide, the inhibitory effect on melanoma cell growth was even more prominent. Data from a murine model showed reduced tumor growth when bortezomib was administered to human melanoma tumors. Strikingly, animals receiving bortezomib in combination with temozolomide achieved complete remission of palpable tumors after only 30 days of therapy, lasting >200 days. Our data indicate strongly that bortezomib in combination with chemotherapeutic agents should be studied additionally for the treatment of melanoma.
• Deane, N. G., Lee, H., Hamaamen, J., Ruley, A., Washington, M. K., LaFleur, B., Thorgeirsson, S. S., Price, R., & Beauchamp, R. D. (2004). Enhanced tumor formation in cyclin D1 x transforming growth factor beta1 double transgenic mice with characterization by magnetic resonance imaging. Cancer research, 64(4), 1315-22.
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  Transgenic mice that overexpress cyclin D1 protein in the liver develop liver carcinomas with high penetrance. Transforming growth factor beta (TGF-beta) serves as either an epithelial cell growth inhibitor or a tumor promoter, depending on the cellular context. We interbred LFABP-cyclin D1 and Alb-TGF-beta1 transgenic mice to produce cyclin D1/TGF-beta1 double transgenic mice and followed the development of liver tumors over time, characterizing cellular and molecular changes, tumor incidence, tumor burden, and tumor physiology noninvasively by magnetic resonance imaging. Compared with age-matched LFABP-cyclin D1 single transgenic littermates, cyclin D1/TGF-beta1 mice exhibited a significant increase in tumor incidence. Tumor multiplicity, tumor burden, and tumor heterogeneity were higher in cyclin D1/TGF-beta1 mice compared with single transgenic littermates. Characteristics of cyclin D1/TGF-beta1 livers correlated with a marked induction of the peripheral periductal oval cell/stem cell compartment of the liver. A number of cancerous lesions from cyclin D1/TGF-beta1 mice exhibited unique features such as ductal plate malformations and hemorrhagic nodules. Some lesions were contiguous with the severely diseased background liver and, in some cases, replaced the normal architecture of the entire organ. Cyclin D1/TGF-beta1 lesions, in particular, were associated with malignant features such as areas of vascular invasion by hepatocytes and heterogeneous hyperintensity of signal on T2-weighted magnetic resonance imaging. These findings demonstrate that TGF-beta1 promotes stem cell activation and tumor progression in the context of cyclin D1 overexpression in the liver.
• Jones, T. F., Pavlin, B. I., LaFleur, B. J., Ingram, L. A., & Schaffner, W. (2004). Restaurant inspection scores and foodborne disease. Emerging infectious diseases, 10(4), 688-92.
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  Restaurants in the United States are regularly inspected by health departments, but few data exist regarding the effect of restaurant inspections on food safety. We examined statewide inspection records from January 1993 through April 2000. Data were available from 167,574 restaurant inspections. From 1993 to 2000, mean scores rose steadily from 80.2 to 83.8. Mean inspection scores of individual inspectors were 69-92. None of the 12 most commonly cited violations were critical food safety hazards. Establishments scoring
• Mitchell, S. A., Brown, K. M., Henry, M. M., Mintz, M., Catchpoole, D., LaFleur, B., & Stephan, D. A. (2004). Inter-platform comparability of microarrays in acute lymphoblastic leukemia. BMC genomics, 5, 71.
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  Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy and has been the poster-child for improved therapeutics in cancer, with life time disease-free survival (LTDFS) rates improving from 80% today. There are numerous known genetic prognostic variables in ALL, which include T cell ALL, the hyperdiploid karyotype and the translocations: t(12;21)[TEL-AML1], t(4;11)[MLL-AF4], t(9;22)[BCR-ABL], and t(1;19)[E2A-PBX]. ALL has been studied at the molecular level through expression profiling resulting in un-validated expression correlates of these prognostic indices. To date, the great wealth of expression data, which has been generated in disparate institutions, representing an extremely large cohort of samples has not been combined to validate any of these analyses. The majority of this data has been generated on the Affymetrix platform, potentially making data integration and validation on independent sample sets a possibility. Unfortunately, because the array platform has been evolving over the past several years the arrays themselves have different probe sets, making direct comparisons difficult. To test the comparability between different array platforms, we have accumulated all Affymetrix ALL array data that is available in the public domain, as well as two sets of cDNA array data. In addition, we have supplemented this data pool by profiling additional diagnostic pediatric ALL samples in our lab. Lists of genes that are differentially expressed in the six major subclasses of ALL have previously been reported in the literature as possible predictors of the subclass.
• Poehling, K. A., Lafleur, B. J., Szilagyi, P. G., Edwards, K. M., Mitchel, E., Barth, R., Schwartz, B., & Griffin, M. R. (2004). Population-based impact of pneumococcal conjugate vaccine in young children. Pediatrics, 114(3), 755-61.
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  To determine the population impact of pneumococcal conjugate vaccine (PCV) on pneumococcal-related diseases, including pneumonia and otitis media.
• Richard, C., Gao, J., LaFleur, B., Christman, B. W., Anderson, J., Brown, N., & Reese, J. (2004). Patency of the preterm fetal ductus arteriosus is regulated by endothelial nitric oxide synthase and is independent of vasa vasorum in the mouse. American journal of physiology. Regulatory, integrative and comparative physiology, 287(3), R652-60.
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  Patency of the fetal ductus arteriosus (DA) is maintained in an environment of low relative oxygen tension and a preponderance of vasodilating forces. In addition to prostaglandins, nitric oxide (NO), a potent vasodilator in the pulmonary and systemic vasculatures, has been implicated in regulation of the fetal DA. To further define the contribution of NO to DA patency, the expression and function of NO synthase (NOS) isoforms were examined in the mouse DA on days 17-19 of pregnancy and after birth. Our results show that endothelial NOS (eNOS) is the predominant isoform expressed in the mouse DA and is localized in the DA endothelium by in situ hybridization. Despite rapid constriction of the DA after birth, eNOS expression levels were unchanged throughout the fetal and postnatal period. Pharmacological inhibition of prostaglandin vs. NO synthesis in vivo showed that the preterm fetal DA on day 16 is more sensitive to NOS inhibition than the mature fetal DA on day 19, whereas prostaglandin inhibition results in marked DA constriction on day 19 but minimal effects on the day 16 DA. Combined prostaglandin and NO inhibition caused additional DA constriction on day 16. The contribution of vasa vasorum to DA regulation was also examined. Immunoreactive platelet endothelial cell adhesion molecule and lacZ tagged FLK1 localized to DA endothelial cells but revealed the absence of vasa vasorum within the DA wall. Similarly, there was no evidence of vasa vasorum by vascular casting. These studies indicate that eNOS is the primary source of NO in the mouse DA and that vasomotor tone of the preterm fetal mouse DA is regulated by eNOS-derived NO and is potentiated by prostaglandins. In contrast to other species, mechanisms for DA patency and closure appear to be independent of any contribution of the vasa vasorum.
• Talbot, T. R., Ziel, E., Doersam, J. K., LaFleur, B., Tollefson, S., & Edwards, K. M. (2004). Risk of vaccinia transfer to the hands of vaccinated persons after smallpox immunization. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America, 38(4), 536-41.
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  Transmission of vaccinia virus after smallpox vaccination is a concern. We conducted a prospective examination of the protection afforded by vaccination-site bandages in recently vaccinated individuals. After smallpox vaccination, inoculation sites were covered with 2 occlusive dressings. Site assessment and bandage changes occurred every 3-5 days until the site was healed. At each visit, specimens from the vaccination site, outer dressing surface, and contralateral hand were obtained for vaccinia culture. For 148 vaccinated subjects, vaccinia was detected from vaccination lesions of every subject on several occasions. Only 6 (0.65%) of 918 dressing (95% CI, 0.24%-1.4%) and 2 (0.22%) of 926 hand (95% CI, 0.03%-0.78%) specimens tested positive for vaccinia. The mean number of bandage changes was 9.6 (95% CI, 9.17-10.0). Vaccinia autoinoculation did not occur. The rate of vaccinia recovery outside occlusive bandages covering smallpox vaccination sites was remarkably low, suggesting excellent protection against inadvertent transmission.
• Weinberg, A., Wiznia, A. A., LaFleur, B. J., Shah, S., & Levin, M. J. (2004). Varicella-Zoster virus-specific cell-mediated immunity in HIV-infected children receiving highly active antiretroviral therapy. The Journal of infectious diseases, 190(2), 267-70.
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  Herpes zoster (HZ) is a frequent complication of advanced human immunodeficiency virus (HIV) infection. We determined the effect of highly active antiretroviral therapy (HAART) on reconstitution of varicella-zoster virus (VZV)-specific cell-mediated immunity (VZV-CMI) in 56 VZV- and HIV-infected children. VZV-CMI did not change over the course of >/=3 years of observation, despite a reduction in HIV load. VZV-CMI correlated with lower HIV load but not with CD4 cell percentage. The incidence of HZ was unaffected by HAART. None of 5 patients who developed HZ during the study had VZV-CMI before developing HZ. After developing HZ, only the 2 HAART-compliant patients developed VZV-CMI. Thus, VZV-specific immune reconstitution in HIV infection may require antigenic reexposure, in addition to control of HIV replication.
• Wong, E. C., Schreiber, S., Criss, V. R., LaFleur, B., Rais-Bahrami, K., Short, B., & Luban, N. L. (2004). Feasibility of red blood cell transfusion through small bore central venous catheters used in neonates. Pediatric critical care medicine : a journal of the Society of Critical Care Medicine and the World Federation of Pediatric Intensive and Critical Care Societies, 5(1), 69-74.
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  To determine whether packed red blood cell transfusions through small-bore central venous catheters used in critically ill neonates results in significant hemolysis.
• Cutler, N. S., Graves-Deal, R., LaFleur, B. J., Gao, Z., Boman, B. M., Whitehead, R. H., Terry, E., Morrow, J. D., & Coffey, R. J. (2003). Stromal production of prostacyclin confers an antiapoptotic effect to colonic epithelial cells. Cancer research, 63(8), 1748-51.
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  The mechanism whereby cyclooxygenase-2 and its prostaglandin (PG) products are involved in colonic carcinogenesis is not fully understood. Prostacyclin (PGI(2)) is a major PG with antiapoptotic activity and is produced in the gastrointestinal tract. We reported previously that a human colorectal cancer (CRC) cell line, HCA-7, produces significant levels of PGE(2), PGD(2), thromboxane, and PGF(2alpha), but not PGI(2). We now report that human colonic fibroblast cell lines produce significant amounts of PGI(2) and that fibroblast lines derived from normal-appearing colonic mucosa of hereditary nonpolyposis CRC individuals produce 50-fold more PGI(2) than normal fibroblast lines derived from individuals with nonhereditary CRC. Coculture of HCA-7 cells with hereditary nonpolyposis CRC fibroblasts, but not normal fibroblasts, markedly reduced butyrate-induced apoptosis of HCA-7 cells. This antiapoptotic effect was inhibited by the cyclooxygenase-2 inhibitor rofecoxib and was restored by the stable PGI(2) analogue carbaprostacyclin. PGI(2) binds either G protein-coupled cell surface PGI(2) receptor or the nuclear peroxisome proliferator-activated receptor (PPAR) delta. PPAR delta likely mediates this antiapoptotic effect because HCA-7 cells express this receptor, and another PPAR delta agonist, docosahexaenoic acid, mimics the effect. We propose a novel mechanism by which stromal production of PGI(2) promotes survival of colonocytes through PPAR delta activation. This mechanism may have relevance to maintenance of cells in the normal crypt and to clonal expansion of mutant colonocytes during tumorigenesis.
• Fortunato, S. J., LaFleur, B., & Menon, R. (2003). Collagenase-3 (MMP-13) in fetal membranes and amniotic fluid during pregnancy. American journal of reproductive immunology (New York, N.Y. : 1989), 49(2), 120-5.
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  To examine the expression, site of production and a role of collagenase-3 in human fetal membranes and amniotic fluid (AF).
• Khatua, S., Peterson, K. M., Brown, K. M., Lawlor, C., Santi, M. R., LaFleur, B., Dressman, D., Stephan, D. A., & MacDonald, T. J. (2003). Overexpression of the EGFR/FKBP12/HIF-2alpha pathway identified in childhood astrocytomas by angiogenesis gene profiling. Cancer research, 63(8), 1865-70.
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  Intense angiogenesis proliferation, a histopathological hallmark distinguishing malignant from benign astrocytoma, is vital for tumor progression. Thus, identifying and targeting specific pathways that promote malignant astrocytoma-induced angiogenesis could have substantial therapeutic benefit. Expression profiling of 13 childhood astrocytomas to determine the expression pattern of 133 angiogenesis-related genes revealed that 44 (33%) genes were differentially expressed (17 were overexpressed, and 27 were underexpressed) between malignant high-grade astrocytomas (HGAs) and benign low-grade astrocytomas. Hierarchical clustering and principal components analysis using only the 133 angiogenesis-related genes distinguished HGA from low-grade astrocytoma in 100% of the samples analyzed, as did unsupervised analyses using the entire set of 9198 expressed genes represented on the array, indicating that the angiogenesis-related genes were reliable markers of pathological grade. A striking new finding was significant overexpression of hypoxia-inducible transcription factor (HIF)-2alpha as well as high-level expression of FK506-binding protein (FKBP) 12 by HGA. Furthermore, 9 of 21 (43%) genes overexpressed by HGA were HIF/FKBP-associated genes. This group included the epidermal growth factor receptor (EGFR), which promotes HIF synthesis, as well as insulin-like growth factor-binding protein 2 (IGFBP2), a target gene of HIF activity. Differential protein expression of HIF-2alpha was validated in an independent group of 16 astrocytomas (P = 0.02). We conclude that the EGFR/FKBP12/HIF-2alpha pathway is important in childhood HGA and represents a potential new therapeutic target.
• Poehling, K. A., Szilagyi, P. G., Edwards, K., Mitchel, E., Barth, R., Hughes, H., Lafleur, B., Schaffer, S. J., Schwartz, B., & Griffin, M. R. (2003). Streptococcus pneumoniae-related illnesses in young children: secular trends and regional variation. The Pediatric infectious disease journal, 22(5), 413-8.
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  Children
• Rieschke, P., LaFleur, B. J., & Janicki, P. K. (2003). Effects of EDTA- and sulfite-containing formulations of propofol on respiratory system resistance after tracheal intubation in smokers. Anesthesiology, 98(2), 323-8.
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  The formulation of sulfite-containing propofol (SCP) has not been thoroughly investigated in patients with the extensive smoking history for the effects on the total respiratory system resistance after tracheal intubation. However adverse effects, including acute asthma and bronchospasm, have been reported with several other parenteral formulations of drugs containing sulfite as preservative. Therefore, the aim of this prospective randomized and double blind study was to investigate the effects of EDTA-containing propofol (ECP) and SCP on total respiratory system resistance (Rrs) in patients with the prolonged smoking history and undergoing propofol-based total intravenous anesthesia with tracheal intubation.
• Slonim, A. D., LaFleur, B. J., Ahmed, W., & Joseph, J. G. (2003). Hospital-reported medical errors in children. Pediatrics, 111(3), 617-21.
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  Medical errors are an important problem for hospitalized adult inpatients. However, medical errors in children remain comparatively understudied, and published research has been relatively limited.
• Talbot, T. R., Bredenberg, H. K., Smith, M., LaFleur, B. J., Boyd, A., & Edwards, K. M. (2003). Focal and generalized folliculitis following smallpox vaccination among vaccinia-naive recipients. JAMA, 289(24), 3290-4.
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  With the reintroduction of smallpox vaccination, detailed contemporary descriptions of adverse reactions to the vaccine are needed to adequately inform the public and clinicians. During a multicenter, randomized controlled trial investigating the efficacy of various dilutions of smallpox vaccine, we observed the appearance of a papulovesicular eruption (focal and generalized) in study volunteers.
• Weitkamp, J. H., Kallewaard, N., Kusuhara, K., Bures, E., Williams, J. V., LaFleur, B., Greenberg, H. B., & Crowe, J. E. (2003). Infant and adult human B cell responses to rotavirus share common immunodominant variable gene repertoires. Journal of immunology (Baltimore, Md. : 1950), 171(9), 4680-8.
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  Ab repertoires exhibit marked restrictions during fetal life characterized by biases of variable gene usage and lack of junctional diversity. We tested the hypothesis that Ab repertoire restriction contributes to the observed poor quality of specific Ab responses made by infants to viral infections. We analyzed the molecular determinants of B cell responses in humans to two Ags of rotavirus (RV), a common and clinically important infection of human infants. We sequenced Ab H and L chain V region genes (V(H) and V(L)) of clones expanded from single B cells responding to RV virus protein 6 or virus protein 7. We found that adults exhibited a distinct bias in use of gene segments in the V(H)1 and V(H)4 families, for example, V(H)1-46, V(H)4-31, and V(H)4-61. This gene segment bias differed markedly from the V(H)3 dominant bias seen in randomly selected adult B cells. Recombinant Abs incorporating any of those three immunodominant V(H) segments bound to RV-infected cells and also to purified RV particles. The RV-specific B cell repertoires of infants aged 2-11 mo and those of adults were highly related when compared by V(H), D, J(H), V(L), and J(L) segment selection, extent of junctional diversity, and mean H chain complementarity determining region 3 length. These data suggest that residual fetal bias of the B cell repertoire is not a limiting determinant of the quality of Ab responses to viruses of infants beyond the neonatal period.
• Felkel, T. O., Smith, A. L., Reichenspurner, H. C., LaFleur, B., Lutz, J. F., Kanter, K. R., Gravanis, M. B., & Johnston, T. S. (2002). Survival and incidence of acute rejection in heart transplant recipients undergoing successful withdrawal from steroid therapy. The Journal of heart and lung transplantation : the official publication of the International Society for Heart Transplantation, 21(5), 530-9.
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  Steroid-free immunosuppression is feasible in selected patients after heart transplantation. Survival and incidence of acute rejection are important parameters to evaluate when weighing risks and benefits of steroid withdrawal.
• Huang, Z. J., LaFleur, B. J., Chamberlain, J. M., Guagliardo, M. F., & Joseph, J. G. (2002). Inpatient childhood asthma treatment: relationship of hospital characteristics to length of stay and cost: analyses of New York State discharge data, 1995. Archives of pediatrics & adolescent medicine, 156(1), 67-72.
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  There is increasing pressure to optimize asthma treatment efficiency. It is possible that hospital characteristics influence such efficiency.
• Jones, T. F., Garman, R. L., LaFleur, B., Stephan, S. J., & Schaffner, W. (2002). Risk factors for tick exposure and suboptimal adherence to preventive recommendations. American journal of preventive medicine, 23(1), 47-50.
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  Tickborne diseases are the most common vectorborne illnesses in the United States. Understanding risk factors for tick bites and adherence to preventive measures are important in preventing morbidity associated with tickborne disease.
• Nixon, R. G., Chang, S. S., Lafleur, B. J., Smith JA, J. A., & Cookson, M. S. (2002). Carcinoma in situ and tumor multifocality predict the risk of prostatic urethral involvement at radical cystectomy in men with transitional cell carcinoma of the bladder. The Journal of urology, 167(2 Pt 1), 502-5.
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  Determining which patients are at risk for prostatic urethral involvement of urothelial carcinoma may alter assessment of the prostatic urethra before radical cystectomy and ultimately influence the choice of urinary diversion. We determined risk factors predictive of prostatic urethral involvement using preoperative bladder tumor characteristics in male patients who underwent radical cystoprostatectomy due to urothelial carcinoma of the bladder.
• Pearl, P. L., LaFleur, B. J., Reigle, S. C., Rich, A. S., Freeman, A. A., McCutchen, C., & Sato, S. (2002). Sawtooth wave density analysis during REM sleep in normal volunteers. Sleep medicine, 3(3), 255-8.
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  Sawtooth waves (STW) are a characteristic EEG feature of REM sleep but their source and function are unknown. We previously reported stereotypical properties of STW at stage REM onset, and alterations in bulbar postpolio syndrome. This study analyzes STW features throughout REM, in order to test the hypothesis that sawtooth wave activity may be predictable and have a consistent relationship across REM periods.
• Siami, F. S., LaFleur, B. J., & Siami, G. A. (2002). Clinafloxacin versus piperacillin/tazobactam in the treatment of severe skin and soft-tissue infections in adults at a Veterans Affairs medical center. Clinical therapeutics, 24(1), 59-72.
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  Severe skin and soft-tissue infections (SSTIs), particularly diabetic foot infections, are a source of considerable morbidity and mortality. Inappropriate antimicrobial therapy may contribute to the increasing emergence of bacterial resistance, as well as to increased health care costs. Thus, there is a continuing search for reasonably safe, well-tolerated, and effective antimicrobial agents that are less susceptible to the development of resistance than older agents.

Poster Presentations

• Patel, S. I., Parthasarathy, S., Miller, J., Youngstedt, S., Grandner, M., Quan, S. F., Patel, I., Woosley, R., Xia, X., LaFleur, B. J., Zareba, W., & Perez, K. (2022). Changes in markers of ventricular repolarization and positive airway pressure therapy: A pilot study. SLEEP.
• Parthasarathy, S., Mashaqi, S., Patel, I., Combs, D. A., Woosley, R., Xia, X., Couderc, J., LaFleur, B. J., Zareba, W., & Patel, S. I. (2021, June 10-13). The Relationship between Sleep Disordered Breathing, Markers of Ventricular Repolarization and Cardiovascular Mortality. SLEEP. Virtual: American Academy of Sleep Medicine.