Daniel Laubitz

Interests

Teaching

NGS techniques, Microbiome analysis, Whole Genome and Whole Exome Sequencing in diagnosis of rare genetic diseases

Research

Microbiome in human health and disease

Courses

Scholarly Contributions

Journals/Publications

• Jandova, J., Schiro, G., Duca, F. A., Laubitz, D., & Wondrak, G. T. (2024). Exposure to chlorinated drinking water alters the murine fecal microbiota. The Science of the total environment, 914, 169933.
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  An abundant body of scientific studies and regulatory guidelines substantiates antimicrobial efficacy of freshwater chlorination ensuring drinking water safety in large populations worldwide. In contrast to the purposeful use of chlorination ensuring antimicrobial safety of drinking water, only a limited body of research has addressed the molecular impact of chlorinated drinking water exposure on the gut microbiota. Here, for the first time, we have examined the differential effects of drinking water regimens stratified by chlorination agent [inorganic (HOCl) versus chloramine (TCIC)] on the C57BL/6J murine fecal microbiota. To this end, we exposed C57BL/6J mice to chlorinated drinking water regimens followed by fecal bacterial microbiota analysis at the end of the three-week feeding period employing 16S rRNA sequencing. α-diversity was strongly reduced when comparing chlorinated versus control drinking water groups and community dissimilarities (β-diversity) were significant between groups even when comparing HOCl and TCIC. We detected significant differences in fecal bacterial composition as a function of drinking water chlorination observable at the phylum and genus levels. Differential abundance analysis of select amplicon sequence variants (ASVs) revealed changes as a function of chlorination exposure [up: Lactobacillus ASV1; Akkermansia muciniphila ASV7; Clostridium ss1 ASV10; down: Ileibacterium valens ASV5; Desulfovibrio ASV11; Lachnospiraceae UCG-006 ASV15]. Given the established complexity of murine and human gastrointestinal microbiota and their role in health and disease, the translational relevance of the chlorination-induced changes documented by us for the first time in the fecal murine microbiota remains to be explored.
• Sheikh, I. A., Bianchi-Smak, J., Laubitz, D., Schiro, G., Midura-Kiela, M. T., Besselsen, D. G., Vedantam, G., Jarmakiewicz, S., Filip, R., Ghishan, F. K., Gao, N., & Kiela, P. R. (2024). Transplant of microbiota from Crohn's disease patients to germ-free mice results in colitis. Gut microbes, 16(1), 2333483.
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  Although the role of the intestinal microbiota in the pathogenesis of inflammatory bowel disease (IBD) is beyond debate, attempts to verify the causative role of IBD-associated dysbiosis have been limited to reports of promoting the disease in genetically susceptible mice or in chemically induced colitis. We aimed to further test the host response to fecal microbiome transplantation (FMT) from Crohn's disease patients on mucosal homeostasis in ex-germ-free (xGF) mice. We characterized and transferred fecal microbiota from healthy patients and patients with defined Crohn's ileocolitis (CD_L3) to germ-free mice and analyzed the resulting microbial and mucosal homeostasis by 16S profiling, shotgun metagenomics, histology, immunofluorescence (IF) and RNAseq analysis. We observed a markedly reduced engraftment of CD_L3 microbiome compared to healthy control microbiota. FMT from CD_L3 patients did not lead to ileitis but resulted in colitis with features consistent with CD: a discontinued pattern of colitis, more proximal colonic localization, enlarged isolated lymphoid follicles and/or tertiary lymphoid organ neogenesis, and a transcriptomic pattern consistent with epithelial reprograming and promotion of the Paneth cell-like signature in the proximal colon and immune dysregulation characteristic of CD. The observed inflammatory response was associated with persistently increased abundance of , , and and unexpected growth of toxigenic , which was below the detection level in the community used for inoculation. Our study provides the first evidence that the transfer of a dysbiotic community from CD patients can lead to spontaneous inflammatory changes in the colon of xGF mice and identifies a signature microbial community capable of promoting colonization of pathogenic and conditionally pathogenic bacteria.
• Demergasso, C., Neilson, J. W., Tebes-Cayo, C., Véliz, R., Ayma, D., Laubitz, D., Barberán, A., Chong-Díaz, G., & Maier, R. M. (2023). Corrigendum: Hyperarid soil microbial community response to simulated rainfall. Frontiers in microbiology, 14, 1327998.
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  [This corrects the article DOI: 10.3389/fmicb.2023.1202266.].
• Demergasso, C., Neilson, J. W., Tebes-Cayo, C., Véliz, R., Ayma, D., Laubitz, D., Barberán, A., Chong-Díaz, G., & Maier, R. M. (2023). Hyperarid soil microbial community response to simulated rainfall. Frontiers in microbiology, 14, 1202266.
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  The exceptionally long and protracted aridity in the Atacama Desert (AD), Chile, provides an extreme, terrestrial ecosystem that is ideal for studying microbial community dynamics under hyperarid conditions. Our aim was to characterize the temporal response of hyperarid soil AD microbial communities to simulated rainfall (5% g water/g dry soil for 4 weeks) without nutrient amendment. We conducted replicated microcosm experiments with surface soils from two previously well-characterized AD hyperarid locations near Yungay at 1242 and 1609 masl (YUN1242 and YUN1609) with distinct microbial community compositions and average soil relative humidity levels of 21 and 17%, respectively. The bacterial and archaeal response to soil wetting was evaluated by 16S rRNA gene qPCR, and amplicon sequencing. Initial YUN1242 bacterial and archaeal 16S rRNA gene copy numbers were significantly higher than for YUN1609. Over the next 4 weeks, qPCR results showed significant increases in viable bacterial abundance, whereas archaeal abundance decreased. Both communities were dominated by 10 prokaryotic phyla (Actinobacteriota, Proteobacteria, Chloroflexota, Gemmatimonadota, Firmicutes, Bacteroidota, Planctomycetota, Nitrospirota, Cyanobacteriota, and Crenarchaeota) but there were significant site differences in the relative abundances of Gemmatimonadota and Chloroflexota, and specific actinobacterial orders. The response to simulated rainfall was distinct for the two communities. The actinobacterial taxa in the YUN1242 community showed rapid changes while the same taxa in the YUN1609 community remained relatively stable until day 30. Analysis of inferred function of the YUN1242 microbiome response implied an increase in the relative abundance of known spore-forming taxa with the capacity for mixotrophy at the expense of more oligotrophic taxa, whereas the YUN1609 community retained a stable profile of oligotrophic, facultative chemolithoautotrophic and mixotrophic taxa. These results indicate that bacterial communities in extreme hyperarid soils have the capacity for growth in response to simulated rainfall; however, historic variations in long-term hyperaridity exposure produce communities with distinct putative metabolic capacities.
• Martinez, T. M., Wachsmuth, H. R., Meyer, R. K., Weninger, S. N., Lane, A. I., Kangath, A., Schiro, G., Laubitz, D., Stern, J. H., & Duca, F. A. (2023). Differential effects of plant-based flours on metabolic homeostasis and the gut microbiota in high-fat fed rats. Nutrition & metabolism, 20(1), 44.
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  The gut microbiome is a salient contributor to the development of obesity, and diet is the greatest modifier of the gut microbiome, which highlights the need to better understand how specific diets alter the gut microbiota to impact metabolic disease. Increased dietary fiber intake shifts the gut microbiome and improves energy and glucose homeostasis. Dietary fibers are found in various plant-based flours which vary in fiber composition. However, the comparative efficacy of specific plant-based flours to improve energy homeostasis and the mechanism by which this occurs is not well characterized.
• Weninger, S. N., Ding, A., Browne, E. N., Frost, M. L., Schiro, G., Laubitz, D., & Duca, F. A. (2023). Longitudinal Characterization of the Gut Microbiota in the Diabetic ZDSD Rat Model and Therapeutic Potential of Oligofructose. Metabolites, 13(5).
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  The complex development of type 2 diabetes (T2D) creates challenges for studying the progression and treatment of the disease in animal models. A newly developed rat model of diabetes, the Zucker Diabetic Sprague Dawley (ZDSD) rat, closely parallels the progression of T2D in humans. Here, we examine the progression of T2D and associated changes in the gut microbiota in male ZDSD rats and test whether the model can be used to examine the efficacy of potential therapeutics such as prebiotics, specifically oligofructose, that target the gut microbiota. Bodyweight, adiposity, and fed/fasting blood glucose and insulin were recorded over the course of the study. Glucose and insulin tolerance tests were performed, and feces collected at 8, 16, and 24 weeks of age for short-chain fatty acids and microbiota analysis using 16s rRNA gene sequencing. At the end of 24 weeks of age, half of the rats were supplemented with 10% oligofructose and tests were repeated. We observed a transition from healthy/nondiabetic to prediabetic and overtly diabetic states, via worsened insulin and glucose tolerance and significant increases in fed/fasted glucose, followed by a significant decrease in circulating insulin. Acetate and propionate levels were significantly increased in the overt diabetic state compared to healthy and prediabetic. Microbiota analysis demonstrated alterations in the gut microbiota with shifts in alpha and beta diversity as well as alterations in specific bacterial genera in healthy compared to prediabetic and diabetic states. Oligofructose treatment improved glucose tolerance and shifted the cecal microbiota of the ZDSD rats during late-stage diabetes. These findings underscore the translational potential of ZDSD rats as a model of T2D and highlight potential gut bacteria that could impact the development of the disease or serve as a biomarker for T2D. Additionally, oligofructose treatment was able to moderately improve glucose homeostasis.
• Laubitz, D., Gurney, M. A., Midura-Kiela, M., Clutter, C., Besselsen, D. G., Chen, H., Ghishan, F. K., & Kiela, P. R. (2022). Decreased NHE3 expression in colon cancer is associated with DNA damage, increased inflammation and tumor growth. Scientific reports, 12(1), 14725.
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  Dysregulation of intra- and extracellular pH in cancer contributes to extracellular matrix remodeling, favors cell migration, proliferation, and metastasis. Although the primary attention has been focused on the role of the ubiquitous Na/H exchanger isoform NHE1, the role of NHE3, the predominant apical isoform in colonic surface epithelium in the pathogenesis of colon cancer has not been investigated. Here, we show that NHE3 mRNA expression is significantly reduced in colorectal cancer patients and that low NHE3 expression is associated with poorer survival. Deletion of NHE3 in Apc mice evaluated at 15 weeks of age (significant mortality was observed beyond this time) led to lower body weights, increased mucosal inflammation, increased colonic tumor numbers, evidence of enhanced DNA damage in tumor surface epithelium, and to significant alteration in the gut microbiota. In the absence of the inflammatory and microbial pressors, ca. 70% knockdown of NHE3 expression in SK-CO15 cells led to reduced intracellular pH, elevated apical pH, dramatic differences in their transcriptomic profile, increased susceptibility to DNA damage, increased proliferation, decreased apoptosis and reduced adhesion to extracellular matrix proteins. Our findings suggest that loss of NHE3 in the surface epithelium of colonic tumors has profound consequences for cancer progression and behavior.
• Mashaqi, S., Laubitz, D., Morales, E. J., De Armond, R., Alameddin, H., Ghishan, F. K., Kiela, P. R., & Parthasarathy, S. (2022). Interactive Effect of Combined Intermittent and Sustained Hypoxia and High-Fat Diet on the Colonic Mucosal Microbiome and Host Gene Expression in Mice. Nature and science of sleep, 14, 1623-1639.
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  Gut dysbiosis can cause cardiometabolic disease. Gut dysbiosis can be independently caused by high-fat diet (HFD) and intermittent hypoxia (IH; characterizing obstructive sleep apnea), but the interactive effect of combined intermittent and sustained hypoxia (IH+SH) (characterizing obesity hypoventilation syndrome) and HFD on gut dysbiosis is unclear. We aimed to investigate the interactive effect of a combination of IH and SH and HFD on proximal colonic microbiota and colonic gene expression pattern.
• Meyer, R. K., Lane, A. I., Weninger, S. N., Martinez, T. M., Kangath, A., Laubitz, D., & Duca, F. A. (2022). Oligofructose restores postprandial short-chain fatty acid levels during high-fat feeding. Obesity (Silver Spring, Md.), 30(7), 1442-1452.
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  Obesity is associated with consumption of a Western diet low in dietary fiber, while prebiotics reduce body weight. Fiber induces short-chain fatty acid (SCFA) production, and SCFA administration is beneficial to host metabolic homeostasis. However, the role of endogenous SCFA signaling in the development of obesity is contentious. Therefore, the primary objective of this study is to evaluate the postprandial time course of SCFA production and uptake in healthy (chow-fed), Western diet-fed (high-fat diet [HFD]) obese, and oligofructose-treated HFD-fed (HFD + OFS) rats.
• Detman, A., Laubitz, D., Chojnacka, A., Kiela, P. R., Salamon, A., Barberán, A., Chen, Y., Yang, F., Błaszczyk, M. K., & Sikora, A. (2021). Dynamics of dark fermentation microbial communities in the light of lactate and butyrate production. Microbiome, 9(1), 158.
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  This study focuses on the processes occurring during the acidogenic step of anaerobic digestion, especially resulting from nutritional interactions between dark fermentation (DF) bacteria and lactic acid bacteria (LAB). Previously, we have confirmed that DF microbial communities (MCs) that fed on molasses are able to convert lactate and acetate to butyrate. The aims of the study were to recognize the biodiversity of DF-MCs able and unable to convert lactate and acetate to butyrate and to define the conditions for the transformation.
• Laubitz, D., Typpo, K., Midura-Kiela, M., Brown, C., Barberán, A., Ghishan, F. K., & Kiela, P. R. (2021). Dynamics of Gut Microbiota Recovery after Antibiotic Exposure in Young and Old Mice (A Pilot Study). Microorganisms, 9(3).
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  Antibiotics have improved survival from previously deadly infectious diseases. Antibiotics alter the microbial composition of the gut microbiota, and these changes are associated with diminished innate immunity and decline in cognitive function in older adults. The composition of the human microbiota changes with age over the human lifespan. In this pilot study, we sought to identify if age is associated with differential recovery of the microbiota after antibiotic exposure. Using 16S rRNA gene sequencing, we compared recovery of the gut microbiota after the 10-day broad-spectrum antibiotic treatment in wild-type C57BL/six young and older mice. Immediately after antibiotic cessation, as expected, the number of ASVs, representing taxonomic richness, in both young and older mice significantly declined from the baseline. Mice were followed up to 6 months after cessation of the single 10-day antibiotic regimen. The Bray-Curtis index recovered within 20 days after antibiotic cessation in young mice, whereas in older mice the microbiota did not fully recover during the 6-months of follow-up. , , _NK4A136_group became dominant in older mice, whereas in young mice, the bacteria were more evenly distributed, with only one dominant genus of From 45 genera that became extinct after antibiotic treatment in young mice, 31 (68.9%) did not recover by the end of the study. In older mice, from 36 extinct genera, 27 (75%) did not recover. The majority of the genera that became extinct and never recovered belonged to phylum and family. In our study, age was a factor associated with the long-term recovery of the gut microbiota after the 10-day antibiotic treatment.
• Bernardazzi, C., Xu, H., Tong, H., Laubitz, D., Figliuolo da Paz, V., Curiel, L., & Ghishan, F. K. (2020). An indisputable role of NHE8 in mucosal protection. American journal of physiology. Gastrointestinal and liver physiology, 319(4), G421-G431.
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  The loss of the intestinal Na/H exchanger isoform 8 (NHE8) results in an ulcerative colitis-like condition with reduction of mucin production and dysbiosis, indicating that NHE8 plays an important role in intestinal mucosal protection. The aim of this study was to investigate the potential rebalance of the altered microbiota community of NHE8-deficient mice via fecal microbiota transplantation (FMT) and feeding probiotic VSL#3. We also aimed to stimulate mucin production by sodium butyrate administration via enema. Data from 16S rRNA sequencing showed that loss of NHE8 contributes to colonic microbial dysbiosis with reduction of butyrate-producing bacteria. FMT increased bacterial adhesion in the colon in NHE8 knockout (NHE8KO) mice. Periodic-acid Schiff reagent (PAS) stain and quantitative PCR showed no changes in mucin production during FMT. In mice treated with the probiotic VSL#3, a reduction of and segmented filamentous bacteria (SFB) in NHE8KO mouse colon was detected and an increase in goblet cell theca was observed. In NHE8KO mice receiving sodium butyrate (NaB), 1 mM NaB stimulated Muc2 expression without changing goblet cell theca, but 10 mM NaB induced a significant reduction of goblet cell theca without altering Muc2 expression. Furthermore, 5 mM and 10 mM NaB-treated HT29-MTX cells displayed increased apoptosis, while 0.5 mM NaB stimulated Muc2 gene expression. These data showed that loss of NHE8 leads to dysbiosis with reduction of butyrate-producing bacteria and FMT and VSL#3 failed to rebalance the microbiota in NHE8KO mice. Therefore, FMT, VSL#3, and NaB are not able to restore mucin production in the absence of NHE8 in the intestine. Loss of Na/H exchanger isoform 8 (NHE8), a Slc9 family of exchanger that contributes to sodium uptake, cell volume regulation, and intracellular pH homeostasis, resulted in dysbiosis with reduction of butyrate-producing bacteria and decrease of Muc2 production in the intestine in mice. Introducing fecal microbiota transplantation (FMT) and VSL#3 in NHE8 knockout (NHE8KO) mice failed to rebalance the microbiota in these mice. Furthermore, administration of FMT, VSL#3, and sodium butyrate was unable to restore mucin production in the absence of NHE8 in the intestine.
• Detman, A., Laubitz, D., Chojnacka, A., Wiktorowska-Sowa, E., Piotrowski, J., Salamon, A., Kaźmierczak, W., Błaszczyk, M. K., Barberan, A., Chen, Y., Łupikasza, E., Yang, F., & Sikora, A. (2020). Dynamics and Complexity of Dark Fermentation Microbial Communities Producing Hydrogen From Sugar Beet Molasses in Continuously Operating Packed Bed Reactors. Frontiers in microbiology, 11, 612344.
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  This study describes the dynamics and complexity of microbial communities producing hydrogen-rich fermentation gas from sugar-beet molasses in five packed-bed reactors (PBRs). The bioreactors constitute a part of a system producing hydrogen from the by-products of the sugar-beet industry that has been operating continuously in one of the Polish sugar factories. PBRs with different working volumes, packing materials, construction and inocula were tested. This study focused on analysis (based on 16S rRNA profiling and shotgun metagenomics sequencing) of the microbial communities selected in the PBRs under the conditions of high (>100 cm/g COD of molasses) and low (
• Jamwal, D. R., Laubitz, D., Harrison, C. A., Figliuolo da Paz, V., Cox, C. M., Wong, R., Midura-Kiela, M., Gurney, M. A., Besselsen, D. G., Setty, P., Lybarger, L., Bhattacharya, D., Wilson, J. M., Ghishan, F. K., & Kiela, P. R. (2020). Intestinal Epithelial Expression of MHCII Determines Severity of Chemical, T-Cell-Induced, and Infectious Colitis in Mice. Gastroenterology, 159(4), 1342-1356.e6.
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  Intestinal epithelial cells (IECs) provide a barrier that separates the mucosal immune system from the luminal microbiota. IECs constitutively express low levels of major histocompatibility complex (MHC) class II proteins, which are upregulated upon exposure to interferon gamma. We investigated the effects of deleting MHCII proteins specifically in mice with infectious, dextran sodium sulfate (DSS)-, and T-cell-induced colitis.
• Yu, S., Balasubramanian, I., Laubitz, D., Tong, K., Bandyopadhyay, S., Lin, X., Flores, J., Singh, R., Liu, Y., Macazana, C., Zhao, Y., Béguet-Crespel, F., Patil, K., Midura-Kiela, M. T., Wang, D., Yap, G. S., Ferraris, R. P., Wei, Z., Bonder, E. M., , Häggblom, M. M., et al. (2020). Paneth Cell-Derived Lysozyme Defines the Composition of Mucolytic Microbiota and the Inflammatory Tone of the Intestine. Immunity, 53(2), 398-416.e8.
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  Paneth cells are the primary source of C-type lysozyme, a β-1,4-N-acetylmuramoylhydrolase that enzymatically processes bacterial cell walls. Paneth cells are normally present in human cecum and ascending colon, but are rarely found in descending colon and rectum; Paneth cell metaplasia in this region and aberrant lysozyme production are hallmarks of inflammatory bowel disease (IBD) pathology. Here, we examined the impact of aberrant lysozyme production in colonic inflammation. Targeted disruption of Paneth cell lysozyme (Lyz1) protected mice from experimental colitis. Lyz1-deficiency diminished intestinal immune responses to bacterial molecular patterns and resulted in the expansion of lysozyme-sensitive mucolytic bacteria, including Ruminococcus gnavus, a Crohn's disease-associated pathobiont. Ectopic lysozyme production in colonic epithelium suppressed lysozyme-sensitive bacteria and exacerbated colitis. Transfer of R. gnavus into Lyz1 hosts elicited a type 2 immune response, causing epithelial reprograming and enhanced anti-colitogenic capacity. In contrast, in lysozyme-intact hosts, processed R. gnavus drove pro-inflammatory responses. Thus, Paneth cell lysozyme balances intestinal anti- and pro-inflammatory responses, with implications for IBD.
• Zhang, X., Bandyopadhyay, S., Araujo, L. P., Tong, K., Flores, J., Laubitz, D., Zhao, Y., Yap, G., Wang, J., Zou, Q., Ferraris, R., Zhang, L., Hu, W., Bonder, E. M., Kiela, P. R., Coffey, R., Verzi, M. P., Ivanov, I. I., & Gao, N. (2020). Elevating EGFR-MAPK program by a nonconventional Cdc42 enhances intestinal epithelial survival and regeneration. JCI insight, 5(16).
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  The regulatory mechanisms enabling the intestinal epithelium to maintain a high degree of regenerative capacity during mucosal injury remain unclear. Ex vivo survival and clonogenicity of intestinal stem cells (ISCs) strictly required growth response mediated by cell division control 42 (Cdc42) and Cdc42-deficient enteroids to undergo rapid apoptosis. Mechanistically, Cdc42 engaging with EGFR was required for EGF-stimulated, receptor-mediated endocytosis and sufficient to promote MAPK signaling. Proteomics and kinase analysis revealed that a physiologically, but nonconventionally, spliced Cdc42 variant 2 (V2) exhibited stronger MAPK-activating capability. Human CDC42-V2 is transcriptionally elevated in some colon tumor tissues. Accordingly, mice engineered to overexpress Cdc42-V2 in intestinal epithelium showed elevated MAPK signaling, enhanced regeneration, and reduced mucosal damage in response to irradiation. Overproducing Cdc42-V2 specifically in mouse ISCs enhanced intestinal regeneration following injury. Thus, the intrinsic Cdc42-MAPK program is required for intestinal epithelial regeneration, and elevating this signaling cascade is capable of initiating protection from genotoxic injury.
• Harrison, C. A., Laubitz, D., Midura-Kiela, M. T., Jamwal, D. R., Besselsen, D. G., Ghishan, F. K., & Kiela, P. R. (2019). Sexual Dimorphism in the Response to Broad-spectrum Antibiotics During T Cell-mediated Colitis. Journal of Crohn's & colitis, 13(1), 115-126.
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  Broad-spectrum antibiotics [Abx], including combination therapy with ciprofloxacin and metronidazole, are often prescribed during the treatment of inflammatory bowel disease [IBD] to alleviate symptoms, but with varying success. In this pilot study, we studied the effects of Abx on the course of experimental colitis, with a particular focus on sex as a determinant of the microbial and inflammatory responses.
• Kim, A. S., Willis, A. L., Laubitz, D., Sharma, S., Song, B. H., Chiu, A. G., Le, C. H., & Chang, E. H. (2019). The effect of maxillary sinus antrostomy size on the sinus microbiome. International forum of allergy & rhinology, 9(1), 30-38.
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  The optimal maxillary antrostomy size to surgically treat sinusitis is not well known. In this study, we examined clinical metrics of disease severity and symptom scores, measured secreted inflammatory markers, and characterized the sinus microbiome to determine if there were significant differences in outcome between different maxillary ostial sizes.
• Harrison, C. A., Laubitz, D., Ohland, C. L., Midura-Kiela, M. T., Patil, K., Besselsen, D. G., Jamwal, D. R., Jobin, C., Ghishan, F. K., & Kiela, P. R. (2018). Microbial dysbiosis associated with impaired intestinal Na/H exchange accelerates and exacerbates colitis in ex-germ free mice. Mucosal immunology, 11(5), 1329-1341.
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  Intestinal epithelial Na/H exchange facilitated by the apical NHE3 (Slc9a3) is a highly regulated process inhibited by intestinal pathogens and in inflammatory bowel diseases. NHE3 mice develop spontaneous, bacterially mediated colitis, and IBD-like dysbiosis. Disruption of epithelial Na/H exchange in IBD may thus represent a host response contributing to the altered gut microbial ecology, and may play a pivotal role in modulating the severity of inflammation in a microbiome-dependent manner. To test whether microbiome fostered in an NHE3-deficient environment is able to drive mucosal immune responses affecting the onset or severity of colitis, we performed a series of cohousing experiments and fecal microbiome transplants into germ-free Rag-deficient or IL-10 mice. We determined that in the settings where the microbiome of NHE3-deficient mice was stably engrafted in the recipient host, it was able accelerate the onset and amplify severity of experimental colitis. NHE3-deficiency was characterized by the reduction in pH-sensitive butyrate-producing Firmicutes families Lachnospiraceae and Ruminococcaceae (Clostridia clusters IV and XIVa), with an expansion of inflammation-associated Bacteroidaceae. We conclude that the microbiome fostered by impaired epithelial Na/H exchange enhances the onset and severity of colitis through disruption of the gut microbial ecology.
• Gurney, M. A., Laubitz, D., Ghishan, F. K., & Kiela, P. R. (2017). Pathophysiology of Intestinal Na/H exchange. Cellular and molecular gastroenterology and hepatology, 3(1), 27-40.
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  Several members of the family of Na/H exchangers are expressed in the gut, with varying expression patterns and cellular localization. Not only do they participate in the regulation of basic epithelial cell functions, including control of transepithelial Na absorption, intracellular pH (pH ), cell volume, and nutrient absorption, but also in cellular proliferation, migration, and apoptosis. Additionally, they modulate the extracellular milieu in order to facilitate other nutrient absorption and to regulate the intestinal microbial microenvironment. Na/H exchangers are frequent targets of inhibition in gastrointestinal pathologies, either by intrinsic factors (e.g. bile acids, inflammatory mediators) or infectious agents and associated microbial toxins. Based on emerging evidence, disruption of NHE activity via impaired expression or function of respective isoforms may contribute not only to local and systemic electrolyte imbalance, but also to the disease severity via multiple mechanisms. Here, we review the current state of knowledge about the roles Na/H exchangers play in the pathogenesis of disorders of diverse origin and affecting a range of GI tissues.
• Larmonier, C. B., Shehab, K. W., Laubitz, D., Jamwal, D. R., Ghishan, F. K., & Kiela, P. R. (2016). Transcriptional Reprogramming and Resistance to Colonic Mucosal Injury in Poly(ADP-ribose) Polymerase 1 (PARP1)-deficient Mice. The Journal of biological chemistry, 291(17), 8918-30.
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  Poly(ADP-ribose) polymerases (PARPs) synthesize and bind branched polymers of ADP-ribose to acceptor proteins using NAD as a substrate and participate in the control of gene transcription and DNA repair. PARP1, the most abundant isoform, regulates the expression of proinflammatory mediator cytokines, chemokines, and adhesion molecules, and inhibition of PARP1 enzymatic activity reduced or ameliorated autoimmune diseases in several experimental models, including colitis. However, the mechanism(s) underlying the protective effects of PARP1 inhibition in colitis and the cell types in which Parp1 deletion has the most significant impact are unknown. The objective of the current study was to determine the impact of Parp1 deletion on the innate immune response to mucosal injury and on the gut microbiome composition. Parp1 deficiency was evaluated in DSS-induced colitis in WT, Parp1(-/-), Rag2(-/-), and Rag2(-/-)×Parp1(-/-) double knock-out mice. Genome-wide analysis of the colonic transcriptome and fecal 16S amplicon profiling was performed. Compared with WT, we demonstrated that Parp1(-/-) were protected from dextran-sulfate sodium-induced colitis and that this protection was associated with a dramatic transcriptional reprogramming in the colon. PARP1 deficiency was also associated with a modulation of the colonic microbiota (increases relative abundance of Clostridia clusters IV and XIVa) and a concomitant increase in the frequency of mucosal CD4(+)CD25(+) Foxp3(+) regulatory T cells. The protective effects conferred by Parp1 deletion were lost in Rag2(-/-) × Parp1(-/-) mice, highlighting the role of the adaptive immune system for full protection.
• Laubitz, D., Harrison, C. A., Midura-Kiela, M. T., Ramalingam, R., Larmonier, C. B., Chase, J. H., Caporaso, J. G., Besselsen, D. G., Ghishan, F. K., & Kiela, P. R. (2016). Reduced Epithelial Na+/H+ Exchange Drives Gut Microbial Dysbiosis and Promotes Inflammatory Response in T Cell-Mediated Murine Colitis. PloS one, 11(4), e0152044.
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  Inflammatory bowel diseases (IBD) are associated with functional inhibition of epithelial Na+/H+ exchange. In mice, a selective disruption of NHE3 (Slc9a3), a major apical Na+/H+ exchanger, also promotes IBD-like symptoms and gut microbial dysbiosis. We hypothesized that disruption of Na+/H+ exchange is necessary for the development of dysbiosis, which promotes an exacerbated mucosal inflammatory response. Therefore, we performed a temporal analysis of gut microbiota composition, and mucosal immune response to adoptive T cell transfer was evaluated in Rag2-/- and NHE3-/-/Rag2-/- (DKO) mice with and without broad-spectrum antibiotics. Microbiome (16S profiling), colonic histology, T cell and neutrophil infiltration, mucosal inflammatory tone, and epithelial permeability were analyzed. In adoptive T cell transfer colitis model, Slc9a3 status was the most significant determinant of gut microbial community. In DKO mice, NHE3-deficiency and dysbiosis were associated with dramatically accelerated and exacerbated disease, with rapid body weight loss, increased mucosal T cell and neutrophil influx, increased mucosal cytokine expression, increased permeability, and expansion of CD25-FoxP3+ Tregs; this enhanced susceptibility was alleviated by oral broad-spectrum antibiotics. Based on these results and our previous work, we postulate that epithelial electrolyte homeostasis is an important modulator in the progression of colitis, acting through remodeling of the gut microbial community.
• Ehebauer, M. T., Zimmermann, M., Jakobi, A. J., Noens, E. E., Laubitz, D., Cichocki, B., Marrakchi, H., Lanéelle, M. A., Daffé, M., Sachse, C., Dziembowski, A., Sauer, U., & Wilmanns, M. (2015). Characterization of the mycobacterial acyl-CoA carboxylase holo complexes reveals their functional expansion into amino acid catabolism. PLoS pathogens, 11(2), e1004623.
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  Biotin-mediated carboxylation of short-chain fatty acid coenzyme A esters is a key step in lipid biosynthesis that is carried out by multienzyme complexes to extend fatty acids by one methylene group. Pathogenic mycobacteria have an unusually high redundancy of carboxyltransferase genes and biotin carboxylase genes, creating multiple combinations of protein/protein complexes of unknown overall composition and functional readout. By combining pull-down assays with mass spectrometry, we identified nine binary protein/protein interactions and four validated holo acyl-coenzyme A carboxylase complexes. We investigated one of these--the AccD1-AccA1 complex from Mycobacterium tuberculosis with hitherto unknown physiological function. Using genetics, metabolomics and biochemistry we found that this complex is involved in branched amino-acid catabolism with methylcrotonyl coenzyme A as the substrate. We then determined its overall architecture by electron microscopy and found it to be a four-layered dodecameric arrangement that matches the overall dimensions of a distantly related methylcrotonyl coenzyme A holo complex. Our data argue in favor of distinct structural requirements for biotin-mediated γ-carboxylation of α-β unsaturated acid esters and will advance the categorization of acyl-coenzyme A carboxylase complexes. Knowledge about the underlying structural/functional relationships will be crucial to make the target category amenable for future biomedical applications.
• Machová, I., Snašel, J., Zimmermann, M., Laubitz, D., Plocinski, P., Oehlmann, W., Singh, M., Dostál, J., Sauer, U., & Pichová, I. (2014). Mycobacterium tuberculosis phosphoenolpyruvate carboxykinase is regulated by redox mechanisms and interaction with thioredoxin. The Journal of biological chemistry, 289(19), 13066-78.
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  Tuberculosis remains a major health concern worldwide. Eradication of its causative agent, the bacterial pathogen Mycobacterium tuberculosis, is particularly challenging due to a vast reservoir of latent carriers of the disease. Despite the misleading terminology of a so-called dormant state associated with latent infections, the bacteria have to maintain basic metabolic activities. Hypoxic conditions have been widely used as an in vitro system to study this dormancy. Such studies identified a rearrangement of central carbon metabolism to exploit fermentative processes caused by the lack of oxygen. Phosphoenolpyruvate carboxykinase (Pck; EC 4.1.1.32) is the enzyme at the center of these metabolic rearrangements. Although Pck is associated with gluconeogenesis under standard growth conditions, the enzyme can catalyze the reverse reaction, supporting anaplerosis of the tricarboxylic acid cycle, under conditions leading to slowed or stopped bacterial replication. To study the mechanisms that regulate the switch between two Pck functions, we systematically investigated factors influencing the gluconeogenic and anaplerotic reaction kinetics. We demonstrate that a reducing environment, as found under hypoxia-triggered non-replicating conditions, accelerates the reaction in the anaplerotic direction. Furthermore, we identified proteins that interact with Pck. The interaction between Pck and the reduced form of mycobacterial thioredoxin, gene expression of which is increased under hypoxic conditions, also increased the Pck anaplerotic activity. We thus propose that a reducing environment and the protein-protein interaction with thioredoxin in particular enable the Pck anaplerotic function under fermentative growth conditions.
• Płociński, P., Laubitz, D., Cysewski, D., Stoduś, K., Kowalska, K., & Dziembowski, A. (2014). Identification of protein partners in mycobacteria using a single-step affinity purification method. PloS one, 9(3), e91380.
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  Tuberculosis is a leading cause of death in developing countries. Efforts are being made to both prevent its spread and improve curability rates. Understanding the biology of the bacteria causing the disease, Mycobacterium tuberculosis (M. tuberculosis), is thus vital. We have implemented improved screening methods for protein-protein interactions based on affinity purification followed by high-resolution mass spectrometry. This method can be efficiently applied to both medium- and high-throughput studies aiming to characterize protein-protein interaction networks of tubercle bacilli. Of the 4 tested epitopes FLAG, enhanced green fluorescent protein (eGFP), protein A and haemagglutinin, the eGFP tag was found to be most useful on account of its easily monitored expression and its ability to function as a simultaneous tool for subcellular localization studies. It presents a relatively low background with cost-effective purification. RNA polymerase subunit A (RpoA) was used as a model for investigation of a large protein complex. When used as bait, it co-purified with all remaining RNA polymerase core subunits as well as many accessory proteins. The amount of RpoA strongly correlated with the amount of quantification peptide used as part of the tagging system in this study (SH), making it applicable for semi-quantification studies. Interactions between the components of the RpoA-eGFP protein complex were further confirmed using protein cross-linking. Dynamic changes in the composition of protein complexes under induction of UV damage were observed when UvrA-eGFP expressing cells treated with UV light were used to co-purify UvrA interaction partners.
• Larmonier, C. B., Laubitz, D., Hill, F. M., Shehab, K. W., Lipinski, L., Midura-Kiela, M. T., McFadden, R. M., Ramalingam, R., Hassan, K. A., Golebiewski, M., Besselsen, D. G., Ghishan, F. K., & Kiela, P. R. (2013). Reduced colonic microbial diversity is associated with colitis in NHE3-deficient mice. American journal of physiology. Gastrointestinal and liver physiology, 305(10), G667-77.
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  Chronic inflammation and enteric infections are frequently associated with epithelial Na(+)/H(+) exchange (NHE) inhibition. Alterations in electrolyte transport and in mucosal pH associated with inflammation may represent a key mechanism leading to changes in the intestinal microbial composition. NHE3 expression is essential for the maintenance of the epithelial barrier function. NHE3(-/-) mice develop spontaneous distal chronic colitis and are highly susceptible to dextran sulfate (DSS)-induced mucosal injury. Spontaneous colitis is reduced with broad-spectrum antibiotics treatment, thus highlighting the importance of the microbiota composition in NHE3 deficiency-mediated colitis. We herein characterized the colonic microbiome of wild-type (WT) and NHE3(-/-) mice housed in a conventional environment using 454 pyrosequencing. We demonstrated a significant decrease in the phylogenetic diversity of the luminal and mucosal microbiota of conventional NHE3(-/-) mice compared with WT. Rederivation of NHE3(-/-) mice from conventional to a barrier facility eliminated the signs of colitis and decreased DSS susceptibility. Reintroduction of the conventional microflora into WT and NHE3(-/-) mice from the barrier facility resulted in the restoration of the symptoms initially described in the conventional environment. Interestingly, qPCR analysis of the microbiota composition in mice kept in the barrier facility compared with reconventionalized mice showed a significant reduction of Clostridia classes IV and XIVa. Therefore, the gut microbiome plays a prominent role in the pathogenesis of colitis in NHE3(-/-) mice, and, reciprocally, NHE3 also plays a critical role in shaping the gut microbiota. NHE3 deficiency may be a critical contributor to dysbiosis observed in patients with inflammatory bowel disease.
• Radhakrishnan, V. M., Ramalingam, R., Larmonier, C. B., Thurston, R. D., Laubitz, D., Midura-Kiela, M. T., McFadden, R. M., Kuro-O, M., Kiela, P. R., & Ghishan, F. K. (2013). Post-translational loss of renal TRPV5 calcium channel expression, Ca(2+) wasting, and bone loss in experimental colitis. Gastroenterology, 145(3), 613-24.
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  Dysregulated Ca(2+) homeostasis likely contributes to the etiology of inflammatory bowel disease-associated loss of bone mineral density. Experimental colitis leads to decreased expression of Klotho, a protein that supports renal Ca(2+) reabsorption by stabilizing the transient receptor potential vanilloid 5 (TRPV5) channel on the apical membrane of distal tubule epithelial cells.
• Midura-Kiela, M. T., Radhakrishnan, V. M., Larmonier, C. B., Laubitz, D., Ghishan, F. K., & Kiela, P. R. (2012). Curcumin inhibits interferon-γ signaling in colonic epithelial cells. American journal of physiology. Gastrointestinal and liver physiology, 302(1), G85-96.
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  Curcumin (diferulolylmethane) is an anti-inflammatory phenolic compound found effective in preclinical models of inflammatory bowel diseases (IBD) and in ulcerative colitis patients. Pharmacokinetics of curcumin and its poor systemic bioavailability suggest that it targets preferentially intestinal epithelial cells. The intestinal epithelium, an essential component of the gut innate defense mechanisms, is profoundly affected by IFN-γ, which can disrupt the epithelial barrier function, prevent epithelial cell migration and wound healing, and prime epithelial cells to express major histocompatibility complex class II (MHC-II) molecules and to serve as nonprofessional antigen-presenting cells. In this report we demonstrate that curcumin inhibits IFN-γ signaling in human and mouse colonocytes. Curcumin inhibited IFN-γ-induced gene transcription, including CII-TA, MHC-II genes (HLA-DRα, HLA-DPα1, HLA-DRβ1), and T cell chemokines (CXCL9, 10, and 11). Acutely, curcumin inhibited Stat1 binding to the GAS cis-element, prevented Stat1 nuclear translocation, and reduced Jak1 phosphorylation and phosphorylation of Stat1 at Tyr(701). Longer exposure to curcumin led to endocytic internalization of IFNγRα followed by lysosomal fusion and degradation. In summary, curcumin acts as an IFN-γ signaling inhibitor in colonocytes with biphasic mechanisms of action, a phenomenon that may partially account for the beneficial effects of curcumin in experimental colitis and in human IBD.
• Krześniak, N., Paziewska, A., Rubel, T., Skrzypczak, M., Mikula, M., Dzwonek, A., Goryca, K., Wyrwicz, L. S., Jarosz, D., Laubitz, D., Woszczyński, M., Bielecki, K., & Ostrowski, J. (2011). Gene expression alterations induced by low molecular weight heparin during bowel anastomosis healing in rats. Acta biochimica Polonica, 58(1), 79-87.
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  Colon anastomosis is therapeutically challenging because multiple, usually undetectable factors influence a spectrum of repair mechanisms. We hypothesized that low molecular weight heparins, routinely administered perioperatively, may differentially affect gene expression related to colon healing. Twenty pairs of untreated and enoxaparin-treated rats underwent left-side hemicolectomy with a primary end-to-end anastomosis. Normal colon and anastomotic bowel segments were resected on day 0 and on days 1, 3, 5, and 7 after surgery, respectively. Serial anastomosis transverse cross-sections were evaluated microscopically and by microarray (Rat Genome 230 2.0, Affymetrix). Differentially expressed probe sets were annotated with Gene Ontology. We also examined the influence of enoxaparin on fibroblast proliferation and viability in vitro. Among the 5476 probe sets, we identified differential expression at each healing time point, yielding 79 subcategories. Most indicated genes were involved in wound healing, including multicellular organismal development, locomotory behavior, immune response, cell adhesion, inflammatory response, cell-cell signaling, blood vessel development, and tissue remodeling. Although we found no intensity differences in histological features of healing between enoxaparin-treated and control rats, treatment did induce significant expression changes during early healing. Of these changes, 83 probe sets exhibited at least twofold changes and represented different functional annotations, including inflammatory response, regulation of transcription, regulation of apoptosis, and angiogenesis. Fibroblast culture confirmed an anti-viability effect of enoxaparin. Enoxaparin affects colon wound-related gene expression profiles, but further studies will resolve whether heparin treatment is a risk factor after intestinal surgery, at least in some patients.
• Larmonier, C. B., Laubitz, D., Thurston, R. D., Bucknam, A. L., Hill, F. M., Midura-Kiela, M., Ramalingam, R., Kiela, P. R., & Ghishan, F. K. (2011). NHE3 modulates the severity of colitis in IL-10-deficient mice. American journal of physiology. Gastrointestinal and liver physiology, 300(6), G998-G1009.
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  NHE3, the major intestinal Na(+)/H(+) exchanger, was shown to be downregulated and/or inhibited in patients with inflammatory bowel disease (IBD), a phenomenon believed to contribute to inflammation-associated diarrhea. NHE3(-/-) mice spontaneously develop colitis and demonstrate high susceptibility to dextran sulfate-induced mucosal injury. We investigated the effects of NHE3 deficiency on the development of chronic colitis in an IL-10 knockout (KO) mouse model of Crohn's disease. NHE3(-/-) mice were first backcrossed to 129/SvEv mice for >10 generations, with no apparent changes in their survival or phenotype. These mice were crossed with IL-10(-/-) mice on the same genetic background, and the phenotypes of 10-wk-old wild-type (WT), IL-10(-/-), NHE3(-/-), and IL-10(-/-)/NHE3(-/-) (double-KO) mice were studied. Histological and immunohistochemical examination of the colon established important architectural alterations, including increased neutrophilic and mononuclear cell infiltration in double- compared with single-KO mice. Double-KO mice demonstrated increased colonic expression of neutrophil collagenase matrix metalloproteinase-8 and the chemokines macrophage inflammatory protein-2, CXCL1, CXCL10, and CXCL11. Colonic IFNγ, IL-17, and IL-12/23 p40 protein secretion was significantly increased in double- compared with single-KO mice. IL-10(-/-)/NHE3(-/-) mouse colonic epithelium exhibited increased hallmarks of apoptosis, including a significantly increased number of cleaved caspase-3-positive surface epithelial cells. These results highlight the importance of NHE3 in the maintenance of intestinal barrier integrity and in modulating the inflammatory process in IL-10-deficient mice. Chronic NHE3 inhibition or underexpression observed in IBD may therefore contribute to the pathogenesis of IBD by influencing the extent of the epithelial barrier defect and affect the ultimate degree of inflammation.
• Larmonier, C. B., Midura-Kiela, M. T., Ramalingam, R., Laubitz, D., Janikashvili, N., Larmonier, N., Ghishan, F. K., & Kiela, P. R. (2011). Modulation of neutrophil motility by curcumin: implications for inflammatory bowel disease. Inflammatory bowel diseases, 17(2), 503-15.
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  Neutrophils (PMN) are the first cells recruited at the site of inflammation. They play a key role in the innate immune response by recognizing, ingesting, and eliminating pathogens and participate in the orientation of the adaptive immune responses. However, in inflammatory bowel disease (IBD) transepithelial neutrophil migration leads to an impaired epithelial barrier function, perpetuation of inflammation, and tissue destruction via oxidative and proteolytic damage. Curcumin (diferulolylmethane) displays a protective role in mouse models of IBD and in human ulcerative colitis, a phenomenon consistently accompanied by a reduced mucosal neutrophil infiltration.
• Thurston, R. D., Larmonier, C. B., Majewski, P. M., Ramalingam, R., Midura-Kiela, M., Laubitz, D., Vandewalle, A., Besselsen, D. G., Mühlbauer, M., Jobin, C., Kiela, P. R., & Ghishan, F. K. (2010). Tumor necrosis factor and interferon-gamma down-regulate Klotho in mice with colitis. Gastroenterology, 138(4), 1384-94, 1394.e1-2.
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  Klotho (KL) is an anti-inflammatory protein that protects the endothelium from nitric oxide (NO)-induced dysfunction, reduces the expression of endothelial adhesion molecules, and potentially regulates T-cell functions. KL deficiency leads to premature senescence and impaired Ca2+/Pi homeostasis, which can lead to inflammatory bowel disease (IBD)-associated osteopenia/osteoporosis. We investigated the changes in renal expression of Kl as a consequence of colitis.
• Guilloteau, P., Zabielski, R., David, J. C., Blum, J. W., Morisset, J. A., Biernat, M., Wolinski, J., Laubitz, D., & Hamon, Y. (2009). Sodium-butyrate as a growth promoter in milk replacer formula for young calves. Journal of dairy science, 92(3), 1038-49.
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  In milk-fed calves, the effects of sodium-butyrate (Na-butyrate) to replace flavomycin on growth performance and some mechanisms involved were studied. Pancreatic and intestinal morphology, digestive enzyme activities, plasma gut regulatory peptide concentrations, and expression of their receptors in the gastrointestinal tract were measured. Gastrointestinal tract defense systems were examined by measuring protein levels of 2 heat-shock proteins (HSP27 and HSP70). The calves were randomly allocated into 2 groups fed the same basic diet with flavomycin as an antimicrobial growth promoter or with Na-butyrate (3 g/kg of dry matter). Sodium-butyrate disappeared quickly in the upper gut and was not found in circulating blood. Supplementation with Na-butyrate enhanced growth rate and improved feed conversion into body weight gain compared with the flavomycin group. Supplementation with Na-butyrate was likely associated with an improvement in efficacy of the gastrointestinal tract digestive capacities expressed by enhanced production of digestive enzymes and increased absorptive capacities in the upper small intestine. The effects could have been controlled by insulin-like growth factor-1 but probably not by any of the cholecystokinin/gastrin peptide family. Concentrations of HSP27 and HSP70 were increased in stomach and colon of calves receiving Na-butyrate, thereby assuring protection of cells with intensive metabolism (chaperone function). In conclusion, beneficial effects of Na-butyrate on maturation of gastrointestinal functions were shown in milk-fed calves and may be applied to young mammals of other species.
• Kiela, P. R., Laubitz, D., Larmonier, C. B., Midura-Kiela, M. T., Lipko, M. A., Janikashvili, N., Bai, A., Thurston, R., & Ghishan, F. K. (2009). Changes in mucosal homeostasis predispose NHE3 knockout mice to increased susceptibility to DSS-induced epithelial injury. Gastroenterology, 137(3), 965-75, 975.e1-10.
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  NHE3 is a target of inhibition by proinflammatory cytokines and pathogenic bacteria, an event contributing to diarrhea in infectious and idiopathic colitis. In mice, NHE3 deficiency leads to mild diarrhea, increased intestinal expression of interferon (IFN)-gamma, and distal colitis, suggesting its role in epithelial barrier homeostasis. Our aim was to investigate the role of NHE3 in maintaining mucosal integrity.
• Jankowska, A., Laubitz, D., Antushevich, H., Zabielski, R., & Grzesiuk, E. (2008). Competition of Lactobacillus paracasei with Salmonella enterica for adhesion to Caco-2 cells. Journal of biomedicine & biotechnology, 2008, 357964.
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  Competition of commensal and probiotic bacteria with pathogens for adhesion and colonization is one of the important protective mechanisms of gastrointestinal tract. In this study, we examined the ability of Lactobacillus paracasei to inhibit the adhesion of pathogenic Salmonella enterica to human colon adenocarcinoma Caco-2 cells. Caco-2 cells were grown for 6 or 21 days to obtain nondifferentiated or well-differentiated cells, respectively. In adhesion experiments, bacteria were added to the cells for 2 or 4 hours. The number of attached bacteria was expressed as colony-forming units (CFUs), Caco-2 cells were counted in hematocytometer. Both bacterial strains used adhered better to well-differentiated than to nondifferentiated Caco-2 cells, however, the amount of Salmonella adhered to Caco-2 after 2 hours of contact was 12-fold higher in comparison to L. paracasei and almost 27-fold higher after 4 hours of contact. Two types of experiments were done: coincubation (both bacteria were added to Caco-2 cells simultaneously), and preincubation (L. paracasei was incubated with Caco-2 cells first, and then S. enterica was added). In coincubation experiment, the presence of L. paracasei decreased S. enterica adhesion by 4-fold and in preincubation experiment even 7-fold. Generally, Lactobacillus spent culture supernatants (SCSs) acted weaker as inhibitors of Salmonella adhesion in comparison to the whole L. paracasei culture in coincubation experiment. In conclusion, the displacement of pathogens by lactic acid bacteria and its secretions showed here depends on the time of bacteria-epithelial cell contact, and also on the stage of Caco-2 differentiation.
• Jankowska, A., Wrzesinski, M., Laubitz, D., Kazimierczak, W., Skrzypek, H., Bardowski, J., Zabielski, R., & Grzesiuk, E. (2008). Intestinal MMC-related electric fields and pancreatic juice control the adhesion of Gram-positive and Gram-negative bacteria to the gut epithelium--in vitro study. Journal of physiology and pharmacology : an official journal of the Polish Physiological Society, 59(4), 795-810.
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  The adhesion of six different Lactobacillus and Lactococcus and three pathogenic Escherichia and Salmonella strains was studied using Caco-2 cell line. In this in vitro model system the influence of weak electric field (EF) on bacterial adhesion was tested. The EF source was the in vitro reconstruction of spiking potentials recorded in the duodenum of a healthy calf during one myoelectrical migration complex (MMC) cycle. The ability to adhere to Caco-2 cells of bacteria belonging to two groups, Gram-positive lactobacilli and lactococci, and Gram-negative Escherichia and Salmonella differed considerably. The pathogenic bacteria adhered better to well-differentiated Caco-2 cells whereas lactobacilli and lactococci displayed better adhesion to non-differentiated Caco-2 cells. In the presence of MMC-related EF an increased adhesion of Lactobacillus and Lactococcus but not of Salmonella enterica s. Enteritidis and E. coli 269 to Caco-2 cells was observed. Two later strains adhered even less in the presence of EF. The same tendency was found in the presence of pancreatic juice in a cell medium. In conclusion, the myoelectric component of the small intestinal motility, the MMC-related EF, and pancreatic juice may increase the ability of lactic acid bacteria to adhere to GI epithelial cells, creating better environmental conditions for colonization of the intestine and competition with Gram-negative pathogens.
• Larmonier, C. B., Uno, J. K., Lee, K. M., Karrasch, T., Laubitz, D., Thurston, R., Midura-Kiela, M. T., Ghishan, F. K., Sartor, R. B., Jobin, C., & Kiela, P. R. (2008). Limited effects of dietary curcumin on Th-1 driven colitis in IL-10 deficient mice suggest an IL-10-dependent mechanism of protection. American journal of physiology. Gastrointestinal and liver physiology, 295(5), G1079-91.
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  Curcumin (diferulolylmethane) demonstrates profound anti-inflammatory effects in intestinal epithelial cells (IEC) and in immune cells in vitro and exhibits a protective role in rodent models of chemically induced colitis, with its presumed primary mechanism of action via inhibition of NF-kappaB. Although it has been demonstrated effective in reducing relapse rate in ulcerative colitis patients, curcumin's effectiveness in Crohn's disease (CD) or in Th-1/Th-17 mediated immune models of CD has not been evaluated. Therefore, we investigated the effects of dietary curcumin (0.1-1%) on the development of colitis, immune activation, and in vivo NF-kappaB activity in germ-free IL-10(-/-) or IL-10(-/-);NF-kappaB(EGFP) mice colonized with specific pathogen-free microflora. Proximal and distal colon morphology showed a mild protective effect of curcumin only at 0.1%. Colonic IFN-gamma and IL-12/23p40 mRNA expression followed similar pattern ( approximately 50% inhibition at 0.1%). Secretion of IL-12/23p40 and IFN-gamma by colonic explants and mesenteric lymph node cells was elevated in IL-10(-/-) mice and was not decreased by dietary curcumin. Surprisingly, activation of NF-kappaB in IL-10(-/-) mice (phospho-NF-kappaBp65) or in IL-10(-/-);NF-kappaB(EGFP) mice (whole organ or confocal imaging) was not noticeably inhibited by curcumin. Furthermore, we demonstrate that IL-10 and curcumin act synergistically to downregulate NF-kappaB activity in IEC and IL-12/23p40 production by splenocytes and dendritic cells. In conclusion, curcumin demonstrates limited effectiveness on Th-1 mediated colitis in IL-10(-/-) mice, with moderately improved colonic morphology, but with no significant effect on pathogenic T cell responses and in situ NF-kappaB activity. In vitro studies suggest that the protective effects of curcumin are IL-10 dependent.
• Laubitz, D., Larmonier, C. B., Bai, A., Midura-Kiela, M. T., Lipko, M. A., Thurston, R. D., Kiela, P. R., & Ghishan, F. K. (2008). Colonic gene expression profile in NHE3-deficient mice: evidence for spontaneous distal colitis. American journal of physiology. Gastrointestinal and liver physiology, 295(1), G63-G77.
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  Na+/H+ exchanger 3 (NHE3) provides a major route for intestinal Na+ absorption. NHE3 has been considered a target of proinflammatory cytokines and enteropathogenic bacteria, and impaired NHE3 expression and/or activity may be responsible for inflammation-associated diarrhea. However, the possibility of loss of NHE3 function reciprocally affecting gut immune homeostasis has not been investigated. In this report, we describe that NHE3-deficient mice spontaneously develop colitis restricted to distal colonic mucosa. NHE3(-/-) mice housed in a conventional facility exhibited phenotypic features such as mild diarrhea, occasional rectal prolapse, and reduced body weight. Genomewide microarray analysis identified not only a large group of transport genes that potentially represent an adaptive response, but also a considerable number of genes consistent with an inflammatory response. Histological examination demonstrated changes in the distal colon consistent with active inflammation, including crypt hyperplasia with an increased number of 5-bromo-2'-deoxyuridine-positive cells, diffuse neutrophilic infiltrate with concomitant 15-fold increase in matrix metalloproteinase 8 expression, an increased number of pSer276-RelA-positive cells, and a significant decrease in periodic acid-Schiff-positive goblet cells. Real-time PCR demonstrated elevated expression of inducible nitric oxide synthase (38-fold), TNF-alpha (6-fold), macrophage inflammatory protein-2 (48-fold), and IL-18 (3-fold) in the distal colon of NHE3(-/-) mice. NHE3(-/-) mice showed enhanced bacterial adhesion and translocation in the distal colon. Colitis was ameliorated by oral administration of broad-spectrum antibiotics. In conclusion, NHE3 deficiency leads to an exacerbated innate immune response, an observation suggesting a potentially novel role of NHE3 as a modifier gene, which when downregulated during infectious or chronic colitis may modulate the extent and severity of colonic inflammation.
• Kapica, M., Laubitz, D., Puzio, I., Jankowska, A., & Zabielski, R. (2006). The ghrelin pentapeptide inhibits the secretion of pancreatic juice in rats. Journal of physiology and pharmacology : an official journal of the Polish Physiological Society, 57(4), 691-700.
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  Ghrelin, a 28 amino acids polypeptide was recognized as an endogenous ligand for the growth hormone secretagogue receptor. It turned out that the entire sequence of ghrelin is not necessary for performing the above-mentioned functions. It was suggested that 5 residues (Gly-Ser-Ser(n-octanoyl)-Phe, pentaghrelin) constituted functionally active part of the full-length polypeptide. Ghrelin-28 was found to inhibit pancreatic enzyme output in rats, though the effect of pentaghrelin was not studied so far. The study aimed to determine the involvement of pentaghrelin in pancreatic juice secretion in anaesthetized rats. Male Wistar rats (220 +/- 20 g body weight, b. wt.) were anesthetized, the external jugular vein and common biliary-pancreatic duct were cannulated. Pentaghrelin boluses (i.v., 1.2, 12, and 50 nmol kg(-1) b. wt.) were injected every 30 min with or without CCK-8 infusion, duodenal mucosal CCK(1) receptor blockade with tarazepide, vagotomy and capsaicin pretreatment. Pentaghrelin boluses reduced the volume of pancreatic-biliary juice, protein and trypsin outputs both under basal and CCK-8-stimulated conditions in a dose-dependent manner. However, exogenous pentaghrelin failed to affect the pancreatic secretion in rats subjected to vagotomy, capsaicin deactivation of afferents or pretreatment with Tarazepide. In conclusion, pentaghrelin may control exocrine pancreas secretion by affecting duodenal neurohormonal mechanism(s) involving CCK and vagal nerves in rats.
• Laubitz, D., Jankowska, A., Nieminuszczy, J., Wrzesiński, M., Jaworski, A., Romanowicz, K., Matyjek, R., Grzesiuk, E., Zebrowska, T., & Zabielski, R. (2006). Pancreatic secretion differs according to the genotype of growing pigs. Journal of physiology and pharmacology : an official journal of the Polish Physiological Society, 57(4), 677-89.
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  The objective of this study was to investigate the secretion of pancreatic enzymes and antibacterial activity in weaned pigs of three pure breeds, Pietrain, Duroc and Polish synthetic line 990, to look for eventual differences related to the genotype. Six male pigs of each breed, about 24 kg mean body weight, were equipped with chronic pancreatic duct catheters and duodenal cannulas to assess pure pancreatic juice, and jugular vein catheters for blood withdrawal. Pancreatic juice was collected before and after the morning feeding. Protein output and enzyme activities revealed two distinct profiles: strong manifestation of the prandial phase in Pietrain and line 990 pigs, and weak manifestation in Duroc. The antibacterial activity did not follow the enzyme kinetics, and it was the strongest in pancreatic juice from Pietrain pigs. Postprandial insulinaemia was reduced in the order of: line 990>Pietrain>Duroc. A slight (not significant) tendency towards a reduction of leptin after feeding in synthetic line 990 corresponded with elevated secretion of pancreatic enzymes and plasma insulin. The presented results suggest that the prandial secretion of pancreatic juice differs according to genotype, and the differences may be in part related to release of insulin.
• Laubitz, D., Jankowska, A., Sikora, A., Woliński, J., Zabielski, R., & Grzesiuk, E. (2006). Gut myoelectrical activity induces heat shock response in Escherichia coli and Caco-2 cells. Experimental physiology, 91(5), 867-75.
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  The heat shock response is associated with the intracellular expression of a number of highly conserved heat shock proteins (Hsps). According to their molecular size, Hsps have been divided into several groups, which are strongly conserved and show high homology between the species, e.g., Hsp70, MW 70 kDa (Lindquist & Craig, 1998; Morimoto, 1998; Jolly & Morimoto, 2000; Zylicz et al. 2001). In all organisms the Hsp expression under stress conditions is regulated at transcriptional level, e.g., in humans by the heat shock transcription factor Hsf1 (Morimoto, 1998; Wu, 1995), while in Escherichia coli by replacement of the sigma factor sigma(70) in RNA polymerase by the sigma factor sigma(32) (Gross, 1987). The Hsps allow cell survival under stress conditions by renaturating of denaturated proteins, protecting of stress-labile proteins, preventing protein aggregation (chaperone functions), and by degradation of damaged proteins (protease activities) (Lindquist & Craig, 1988; Morimoto, 1998; Jolly & Morimoto, 2000). They have also many housekeeping functions under non-stressful conditions during the cell cycle, growth, development, and differentiation (Morimoto, 1998). Among a number of plausible inducing factors already studied, extremely low artificial electromagnetic fields have been shown to induce stress response in various cells, such as expression of sigma(32) mRNA (Cairo et al. 1998) and induction of DnaJ and DnaK proteins in Eschericha coli (Chow & Tung, 2000); expression of hsp-16 gene in Caenorhabditis elegans (Miyakawa et al., 2001); induction of heat shock transcription factor Hsf1 and Hsp70, Hsp90 and Hsp27 in human cells (Lin et al. 1997; Lin et al. 1998; Goodman & Blank, 1998; Pipkin et al. 1999). Nevertheless, the role of endogenous electromagnetic fields, i.e., generated by electrically active cells within a body remains controversial. Heat shock proteins (Hsps) protect cells against various environmental and endogenous stressors. Cytoprotection caused by Hsps involves tolerance induced by one agent against other, more severe agents. We have found that exposure of prokaryotic (Escherichia coli) and eukaryotic (Caco-2) cells to an electrical field (EF) connected with a myoelectrical migrating complex (MMC) generated by the small intestine smooth muscle induces the heat shock response. Using Western blot analysis, we have detected an elevated level of sigma factor 32 in E. coli cells exposed to MMC-related EF, and confocal microscopy indicated an increased level of the inducible form of Hsp70 protein in EF-stimulated Caco-2 cells. Additionally, we have found that this induced level of Hsp70 protected the Caco-2 cells against apoptosis caused by camptothecin. Our observations suggest that the myoelectrical activity of the gut may induce heat shock mechanisms in the cells of gut epithelium as well as in gastrointestinal micro-organisms.
• Kotunia, A., Woliński, J., Laubitz, D., Jurkowska, M., Romé, V., Guilloteau, P., & Zabielski, R. (2004). Effect of sodium butyrate on the small intestine development in neonatal piglets fed [correction of feed] by artificial sow. Journal of physiology and pharmacology : an official journal of the Polish Physiological Society, 55 Suppl 2, 59-68.
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  Feeding of neonates with artificial milk formulas delays the maturation of the gastrointestinal mucosa. Na-butyrate has a complex trophic effect on the gastrointestinal epithelium in adults. The present study aimed to determine the effect of milk formula supplementation with Na-butyrate on the gut mucosa in neonatal piglets. Sixteen 3 day old piglets were randomly divided into two groups: control (C, n = 8), and Na-butyrate (B, n = 8). Animals were feed for 7 days with artificial milk formula alone (C) or supplemented with Na-butyrate (B). At the 10(th) day of life the piglets were sacrificed and whole thickness samples of the upper gut were taken for analyses. Administration of Na-butyrate led to significant increase in daily body weight gain as compared to control. In the duodenum, the villi length and mucosa thickness were reduced, however, in the distal jejunum and ileum, the crypt depth, villi length and mucosa thickness were increased in Na-butyrate supplemented piglets as compared to control. Supplementation with Na-butyrate did not affect the intestinal brush border enzyme activities but increased plasma pancreatic polypeptide and cholecystokinin concentrations. These results suggest that supplementation with Na-butyrate may enhance the development of jejunal and ileal mucosa in formula-fed piglets.
• Laubitz, D., Zabielski, R., Woliński, J., Nieminuszczy, J., & Grzesiuk, E. (2003). Physiological and chemical characteristics of antibacterial activity of pancreatic juice. Journal of physiology and pharmacology : an official journal of the Polish Physiological Society, 54(2), 283-90.
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  Attempts were made to find and characterize an antibacterial activity (ABA) factor in porcine pancreatic juice (PJ). Its isolation requires several steps. Since ABA factor was found to be heat resistant, the first step was heating for 30 min at 65 degrees C. Afterwards column chromatography, ethanol precipitation and polyacrylamide gel electrophoresis were involved. Finally, we obtained a pancreatic juice fraction with antibacterial activity against Escherichia coli strain AB1157. In the presence of this fraction the number of living bacterial cells in overnight culture decreased about 10,000 fold and a spot-test gave clearly positive results. The results of analysis suggest that the antibacterial factor is a polypeptide active in a pH range 8.0-8.5, that migrates in polyacrylamide gel electrophoresis as a band under 14,000 Da. Mass spectroscopy analysis of active fraction showed high concentration of porcine pancreatic spasmolytic polypeptide (PSP). In conclusion, a polypeptide controlling bacterial homeostasis has been found in the porcine pancreatic juice.
• Grzesiuk, E., Laubitz, D., Wójcik-Sikora, A., Zabielski, R., & Pierzynowski, S. G. (2001). Influence of intestinal myoelectrical activity on the growth of Escherichia coli. Bioelectromagnetics, 22(6), 449-55.
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  Intestinal bacteria, particularly those adhering to intestinal epithelial cells, are exposed to electric fields and currents generated by the muscular activity of the small intestine. This activity displays a regular pattern known as the myoelectrical migrating complex (MMC). In order to explore the possibility that these endogenous electric fields could affect bacterial growth, a digitised duodenal signal obtained via serosal electrodes from a healthy calf was recorded and then applied via platinum electrodes to Escherichia coli cultures. The culture tubes were placed within a Faraday shield, incubated at 37 degrees C with shaking, and stimulated by the electric current for 5 or 8 h. The growth of E. coli stimulated by the electric current was significantly altered compared to those of non-stimulated controls: after a period of intensive growth, inhibition of cell division was observed. This was not the case when the bacteria with lon mutation were used. Moreover, synchronic bacterial culture could not be achieved in the presence of the MMC-related electric field. These results suggest that the myoelectrical activity of the duodenum, through action on cell membrane, can affect cell division of intestinal bacteria.
• Wójcik-Sikora, A., Laubitz, D., Pierzynowski, S. G., & Grzesiuk, E. (2001). Exposure of Escherichia coli to intestinal myoelectrical activity-related electric field induces resistance against subsequent UV(254 nm) (UVC) irradiation. Mutation research, 496(1-2), 97-104.
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  Survival of Escherichia coli K-12 AB1157 irradiated with UVC (UV(254 nm)) was enhanced after pre-treatment with a low-tension electric field (EF). The EF used was identical to the electrical field generated by the small intestine (myoelectrical migrating complex--MMC), registered in a healthy calf and transmitted into the memory of an EF generator. The EF emitted by the generator was transmitted via electrodes placed in shaken bacterial cultures. The protective effects of the EF on the E. coli survival after exposure to UV were: (i) observed only for the dnaJ(+)dnaK(+) strain, and not for the DeltadnaJdnaK heat shock mutant; (ii) strictly dependent on the temperature at which the bacteria were grown; (iii) most obvious when the bacteria were incubated at 37 degrees C. Moreover, the MMC-related EF and a higher temperature (40 degrees C) show a similar protective effect against UV-irradiation. The results point to the involvement of the heat shock response in the low-tension EF-induced protection of bacterial cells against UVC-irradiation. Additionally, treatment with the MMC-related EF affects total protein contents and their pattern in E. coli cells. The EF-treatment did not show any influence on the level of the argE3(ochre) --> Arg(+) reversions.
• Jakob, S., Mosenthin, R., Zabielski, R., Rippe, C., Winzell, M. S., Gacsalyi, U., Laubitz, D., Grzesiuk, E., & Pierzynowski, S. G. (2000). Fats infused intraduodenally affect the postprandial secretion of the exocrine pancreas and the plasma concentration of cholecystokinin but not of peptide YY in growing pigs. The Journal of nutrition, 130(10), 2450-5.
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  In pigs, the spontaneous secretion of the exocrine pancreas and the release of cholecystokinin (CCK) and peptide YY (PYY) after intraduodenal infusion of fully saturated synthetic fats differing in chain length was studied. Growing pigs (n = 6) were prepared with pancreatic duct catheters, duodenal T-cannulas and catheters placed in the jugular vein. The pigs were fed 2 g/100 g body twice daily. Beginning with the morning feeding, a medium-chain triglyceride (MCT: glycerol tricaprylate), a long-chain triglyceride (LCT: glycerol tristearate) or saline was infused at a rate of 0.1 g/100 g body. Pancreatic juice was collected, beginning 1 h preprandially until 3 h postprandially. Blood samples were obtained 15 min preprandially and 15, 45, 90 and 150 min postprandially. The infusion of MCT evoked a change in the trend of the curve for the volume of secretion of pancreatic juice, lipase and colipase concentrations and outputs. The trend of the curve did not change over time for CCK and PYY. Differences between the trends of the curves for the saline and MCT treatment were observed for volume of secretion, protein output, lipase content and output, trypsin and colipase output. Differences in the trends of the curves between MCT and LCT were obtained for the outputs of protein, lipase and colipase. Plasma CCK levels were lower as a result of the MCT treatment compared with the saline and LCT treatments. The results suggest an immediate, distinguished response of the porcine exocrine pancreas to fats differing in chain length.