Systems analysis of the transcriptional response of human ileocecal epithelial cells to Clostridium difficile toxins and effects on cell cycle control

<p>Abstract</p> <p>Background</p> <p>Toxins A and B (TcdA and TcdB) are <it>Clostridium difficile</it>'s principal virulence factors, yet the pathways by which they lead to inflammation and severe diarrhea remain unclear. Also, the relative role of either toxin during infection and the differences in their effects across cell lines is still poorly understood. To better understand their effects in a susceptible cell line, we analyzed the transciptome-wide gene expression response of human ileocecal epithelial cells (HCT-8) after 2, 6, and 24 hr of toxin exposure.</p> <p>Results</p> <p>We show that toxins elicit very similar changes in the gene expression of HCT-8 cells, with the TcdB response occurring sooner. The high similarity suggests differences between toxins are due to events beyond transcription of a single cell-type and that their relative potencies during infection may depend on differential effects across cell types within the intestine. We next performed an enrichment analysis to determine biological functions associated with changes in transcription. Differentially expressed genes were associated with response to external stimuli and apoptotic mechanisms and, at 24 hr, were predominately associated with cell-cycle control and DNA replication. To validate our systems approach, we subsequently verified a novel G₁/S and known G₂/M cell-cycle block and increased apoptosis as predicted from our enrichment analysis.</p> <p>Conclusions</p> <p>This study shows a successful example of a workflow deriving novel biological insight from transcriptome-wide gene expression. Importantly, we do not find any significant difference between TcdA and TcdB besides potency or kinetics. The role of each toxin in the inhibition of cell growth and proliferation, an important function of cells in the intestinal epithelium, is characterized.</p

Adenosine receptors differentially mediate enteric glial cell death induced by Clostridioides difficile Toxins A and B

Increased risk of intestinal dysfunction has been reported in patients after Clostridioides difficile infection (CDI). Enteric glial cells (EGCs), a component of the enteric nervous system (ENS), contribute to gut homeostasis. Previous studies showed that adenosine receptors, A2A and A2B, modulate inflammation during CDI. However, it is unknown how these receptors can modulate the EGC response to the C. difficile toxins (TcdA and TcdB). We investigated the effects of these toxins on the expression of adenosine receptors in EGCs and the role of these receptors on toxin-induced EGC death. Rat EGCs line were incubated with TcdA or TcdB alone or in combination with adenosine analogues 1h prior to toxins challenge. After incubation, EGCs were collected to evaluate gene expression (adenosine receptors and proinflammatory markers) and cell death. In vivo, WT, A2A, and A2B KO mice were infected with C. difficile, euthanized on day 3 post-infection, and cecum tissue was processed. TcdA and TcdB increased A2A and A3 transcripts, as well as decreased A2B. A2A agonist, but not A2A antagonist, decreased apoptosis induced by TcdA and TcdB in EGCs. A2B blocker, but not A2B agonist, diminished apoptosis in EGCs challenged with both toxins. A3 agonist, but not A3 blocker, reduced apoptosis in EGCs challenged with TcdA and TcdB. Inhibition of protein kinase A (PKA) and CREB, both involved in the main signaling pathway driven by activation of adenosine receptors, decreased EGC apoptosis induced by both toxins. A2A agonist and A2B antagonist decreased S100B upregulation induced by C. difficile toxins in EGCs. In vivo, infected A2B KO mice, but not A2A, exhibited a decrease in cell death, including EGCs and enteric neuron loss, compared to infected WT mice, reduced intestinal damage and decreased IL-6 and S100B levels in cecum. Our findings indicate that upregulation of A2A and A3 and downregulation of A2B in EGCs and downregulation of A2B in intestinal tissues elicit a protective response against C. difficile toxins. Adenosine receptors appear to play a regulatory role in EGCs death and proinflammatory response induced by TcdA and TcdB, and thus may be potential targets of intervention to prevent post-CDI intestinal dysmotility

Cryptosporidium Priming Is More Effective than Vaccine for Protection against Cryptosporidiosis in a Murine Protein Malnutrition Model

Cryptosporidium is a major cause of severe diarrhea, especially in malnourished children. Using a murine model of C. parvum oocyst challenge that recapitulates clinical features of severe cryptosporidiosis during malnutrition, we interrogated the effect of protein malnutrition (PM) on primary and secondary responses to C. parvum challenge, and tested the differential ability of mucosal priming strategies to overcome the PM-induced susceptibility. We determined that while PM fundamentally alters systemic and mucosal primary immune responses to Cryptosporidium, priming with C. parvum (106 oocysts) provides robust protective immunity against re-challenge despite ongoing PM. C. parvum priming restores mucosal Th1-type effectors (CD3+CD8+CD103+ T-cells) and cytokines (IFNγ, and IL12p40) that otherwise decrease with ongoing PM. Vaccination strategies with Cryptosporidium antigens expressed in the S. Typhi vector 908htr, however, do not enhance Th1-type responses to C. parvum challenge during PM, even though vaccination strongly boosts immunity in challenged fully nourished hosts. Remote non-specific exposures to the attenuated S. Typhi vector alone or the TLR9 agonist CpG ODN-1668 can partially attenuate C. parvum severity during PM, but neither as effectively as viable C. parvum priming. We conclude that although PM interferes with basal and vaccine-boosted immune responses to C. parvum, sustained reductions in disease severity are possible through mucosal activators of host defenses, and specifically C. parvum priming can elicit impressively robust Th1-type protective immunity despite ongoing protein malnutrition. These findings add insight into potential correlates of Cryptosporidium immunity and future vaccine strategies in malnourished children

Evaluation of HIV protease and nucleoside reverse transcriptase inhibitors on proliferation, necrosis, apoptosis in intestinal epithelial cells and electrolyte and water transport and epithelial barrier function in mice

<p>Abstract</p> <p>Background</p> <p>Protease inhibitors (PI's) and reverse transcriptase drugs are important components of highly active antiretroviral therapy (HAART) for treating human acquired immunodeficiency syndrome (AIDS). Long-term clinical therapeutic efficacy and treatment compliance of these agents have been limited by undesirable side-effects, such as diarrhea. This study aims to investigate the effects of selected antiretroviral agents on intestinal histopathology and function <it>in vivo </it>and on cell proliferation and death <it>in vitro</it>.</p> <p>Methods</p> <p>Selected antiretroviral drugs were given orally over 7 days, to Swiss mice, as follows: 100 mg/kg of nelfinavir (NFV), indinavir (IDV), didanosine (DDI) or 50 mg/kg of zidovudine (AZT). Intestinal permeability measured by lactulose and mannitol assays; net water and electrolyte transport, in perfused intestinal segments; and small intestinal morphology and cell apoptosis were assessed in treated and control mice. <it>In vitro </it>cell proliferation was evaluated using the WST-1 reagent and apoptosis and necrosis by flow cytometry analysis.</p> <p>Results</p> <p>NFV, IDV, AZT and DDI caused significant reductions in duodenal and in jejunal villus length (p < 0.05). IDV and AZT increased crypt depth in the duodenum and AZT increased crypt depth in the jejunum. NFV, AZT and DDI significantly decreased ileal crypt depth. All selected antiretroviral drugs significantly increased net water secretion and electrolyte secretion, except for DDI, which did not alter water or chloride secretion. Additionally, only NFV significantly increased mannitol and lactulose absorption. NFV and IDV caused a significant reduction in cell proliferation <it>in vitro </it>at both 24 h and 48 h. DDI and AZT did not alter cell proliferation. There was a significant increase in apoptosis rates in IEC-6 cells after 24 h with 70 ug/mL of NFV (control: 4.7% vs NFV: 22%) while IDV, AZT and DDI did not show any significant changes in apoptosis compared to the control group. In jejunal sections, IDV and NFV significantly increased the number of TUNEL positive cells.</p> <p>Conclusion</p> <p>The PI's, NFV and IDV, increased cell apoptosis <it>in vivo</it>, water and electrolyte secretion and intestinal permeability and decreased villus length and cell proliferation. NFV was the only drug tested that increased cell apoptosis <it>in vitro</it>. The nucleoside reverse transcriptase inhibitors, AZT and DDI, did not affect cell apoptosis or proliferation. These findings may partly explain the intestinal side-effects associated with PI's.</p

Effects of adenosine A2A receptor activation and alanyl-glutamine in Clostridium difficile toxin-induced ileitis in rabbits and cecitis in mice

<p>Abstract</p> <p>Background</p> <p>Severe <it>Clostridium difficile </it>toxin-induced enteritis is characterized by exuberant intestinal tissue inflammation, epithelial disruption and diarrhea. Adenosine, through its action on the adenosine A_2Areceptor, prevents neutrophillic adhesion and oxidative burst and inhibits inflammatory cytokine production. Alanyl-glutamine enhances intestinal mucosal repair and decreases apoptosis of enterocytes. This study investigates the protection from enteritis by combination therapy with ATL 370, an adenosine A_2Areceptor agonist, and alanyl-glutamine in a rabbit and murine intestinal loop models of <it>C. difficile </it>toxin A-induced epithelial injury.</p> <p>Methods</p> <p>Toxin A with or without alanyl-glutamine was administered intraluminally to rabbit ileal or murine cecal loops. Animals were also given either PBS or ATL 370 parenterally. Ileal tissues were examined for secretion, histopathology, apoptosis, Cxcl1/KC and IL-10.</p> <p>Results</p> <p>ATL 370 decreased ileal secretion and histopathologic changes in loops treated with Toxin A. These effects were reversed by the A_2Areceptor antagonist, SCH 58261, in a dose-dependent manner. The combination of ATL 370 and alanyl-glutamine significantly further decreased ileal secretion, mucosal injury and apoptosis more than loops treated with either drug alone. ATL 370 and alanyl-glutamine also decreased intestinal tissue KC and IL-10.</p> <p>Conclusions</p> <p>Combination therapy with an adenosine A_2Areceptor agonist and alanyl-glutamine is effective in reversing <it>C. difficile </it>toxin A-induced epithelial injury, inflammation, secretion and apoptosis in animals and has therapeutic potential for the management of <it>C. difficile </it>infection.</p

Tracking Inhibitory Alterations during Interstrain <i>Clostridium difficile</i> Interactions by Monitoring Cell Envelope Capacitance

Global threats arising from the increasing use of antibiotics coupled with the high recurrence rates of <i>Clostridium difficil</i>e (<i>C. difficile</i>) infections (CDI) after standard antibiotic treatments highlight the role of commensal probiotic microorganisms, including nontoxigenic <i>C. difficile</i> (NTCD) strains in preventing CDI due to highly toxigenic <i>C. difficile</i> (HTCD) strains. However, optimization of the inhibitory permutations due to commensal interactions in the microbiota requires probes capable of monitoring phenotypic alterations to <i>C. difficile</i> cells. Herein, by monitoring the field screening behavior of the <i>C. difficile</i> cell envelope with respect to cytoplasmic polarization, we demonstrate that inhibition of the host-cell colonization ability of HTCD due to the S-layer alterations occurring after its co-culture with NTCD can be quantitatively tracked on the basis of the capacitance of the cell envelope of co-cultured HTCD. Furthermore, it is shown that effective inhibition requires the dynamic contact of HTCD cells with freshly secreted extracellular factors from NTCD because contact with the cell-free supernatant causes only mild inhibition. We envision a rapid method for screening the inhibitory permutations to arrest <i>C. difficile</i> colonization by routinely probing alterations in the HTCD dielectrophoretic frequency response due to variations in the capacitance of its cell envelope

Tracking Inhibitory Alterations during Interstrain <i>Clostridium difficile</i> Interactions by Monitoring Cell Envelope Capacitance

Global threats arising from the increasing use of antibiotics coupled with the high recurrence rates of <i>Clostridium difficil</i>e (<i>C. difficile</i>) infections (CDI) after standard antibiotic treatments highlight the role of commensal probiotic microorganisms, including nontoxigenic <i>C. difficile</i> (NTCD) strains in preventing CDI due to highly toxigenic <i>C. difficile</i> (HTCD) strains. However, optimization of the inhibitory permutations due to commensal interactions in the microbiota requires probes capable of monitoring phenotypic alterations to <i>C. difficile</i> cells. Herein, by monitoring the field screening behavior of the <i>C. difficile</i> cell envelope with respect to cytoplasmic polarization, we demonstrate that inhibition of the host-cell colonization ability of HTCD due to the S-layer alterations occurring after its co-culture with NTCD can be quantitatively tracked on the basis of the capacitance of the cell envelope of co-cultured HTCD. Furthermore, it is shown that effective inhibition requires the dynamic contact of HTCD cells with freshly secreted extracellular factors from NTCD because contact with the cell-free supernatant causes only mild inhibition. We envision a rapid method for screening the inhibitory permutations to arrest <i>C. difficile</i> colonization by routinely probing alterations in the HTCD dielectrophoretic frequency response due to variations in the capacitance of its cell envelope

Tracking Inhibitory Alterations during Interstrain <i>Clostridium difficile</i> Interactions by Monitoring Cell Envelope Capacitance

Global threats arising from the increasing use of antibiotics coupled with the high recurrence rates of <i>Clostridium difficil</i>e (<i>C. difficile</i>) infections (CDI) after standard antibiotic treatments highlight the role of commensal probiotic microorganisms, including nontoxigenic <i>C. difficile</i> (NTCD) strains in preventing CDI due to highly toxigenic <i>C. difficile</i> (HTCD) strains. However, optimization of the inhibitory permutations due to commensal interactions in the microbiota requires probes capable of monitoring phenotypic alterations to <i>C. difficile</i> cells. Herein, by monitoring the field screening behavior of the <i>C. difficile</i> cell envelope with respect to cytoplasmic polarization, we demonstrate that inhibition of the host-cell colonization ability of HTCD due to the S-layer alterations occurring after its co-culture with NTCD can be quantitatively tracked on the basis of the capacitance of the cell envelope of co-cultured HTCD. Furthermore, it is shown that effective inhibition requires the dynamic contact of HTCD cells with freshly secreted extracellular factors from NTCD because contact with the cell-free supernatant causes only mild inhibition. We envision a rapid method for screening the inhibitory permutations to arrest <i>C. difficile</i> colonization by routinely probing alterations in the HTCD dielectrophoretic frequency response due to variations in the capacitance of its cell envelope

Dielectrophoretic Monitoring and Interstrain Separation of Intact <i>Clostridium difficile</i> Based on Their S(Surface)-Layers

<i>Clostridium difficile</i> (<i>C. difficile</i>) infection (CDI) rates have exhibited a steady rise worldwide over the last two decades and the infection poses a global threat due to the emergence of antibiotic resistant strains. Interstrain antagonistic interactions across the host microbiome form an important strategy for controlling the emergence of CDI. The current diagnosis method for CDI, based on immunoassays for toxins produced by pathogenic <i>C. difficile</i> strains, is limited by false negatives due to rapid toxin degradation. Furthermore, simultaneous monitoring of nontoxigenic <i>C. difficile</i> strains is not possible, due to absence of these toxins, thereby limiting its application toward the control of CDI through optimizing antagonistic interstrain interactions. Herein, we demonstrate that morphological differences within the cell wall of particular <i>C. difficile</i> strains with differing S-layer proteins can induce systematic variations in their electrophysiology, due alterations in cell wall capacitance. As a result, dielectrophoretic frequency analysis can enable the independent fingerprinting and label-free separation of intact microbials of each strain type from mixed <i>C. difficile</i> samples. The sensitivity of this contact-less electrophysiological method is benchmarked against the immunoassay and microbial growth rate methods for detecting alterations within both, toxigenic and nontoxigenic <i>C. difficile</i> strains after vancomycin treatment. This microfluidic diagnostic platform can assist in the development of therapies for arresting clostridial infections by enabling the isolation of individual strains, optimization of antibiotic treatments and the monitoring of microbiomes

Primary Cutaneous Cryptococcosis Treated with Debridement and Fluconazole Monotherapy in an Immunosuppressed Patient: A Case Report and Review of the Literature

Cryptococcus neoformans is an opportunistic yeast present in the environment. Practitioners are familiar with the presentation and management of the most common manifestation of cryptococcal infection, meningoencephalitis, in patients with AIDS or other conditions of immunocompromise. There is less awareness, however, of uncommon presentations where experience rather than evidence guides therapy. We report a case of primary cutaneous cryptococcosis (PCC) in a patient who had been immunosuppressed by chronic high-dose corticosteroid for the treatment of severe asthma. This case highlights the importance of early recognition of aggressive cellulitis that fails standard empiric antibiotic treatment in an immunocompromised patient. It also demonstrates successful treatment of PCC with a multispecialty approach including local debridement and fluconazole monotherapy