5-Fluorouracil (5-FU) is an anticancer agent whose main side effects include intestinal mucositis associated with intestinal motility alterations maybe due to an effect on the enteric nervous system ...(ENS), but the underlying mechanism remains unclear. In this report, we used an animal model to investigate the participation of the S100B/RAGE/NFκB pathway in intestinal mucositis and enteric neurotoxicity caused by 5-FU (450 mg/kg, IP, single dose). 5-FU induced intestinal damage observed by shortened villi, loss of crypt architecture and intense inflammatory cell infiltrate as well as increased GFAP and S100B co-expression and decreased HuC/D protein expression in the small intestine. Furthermore, 5-FU increased RAGE and NFκB NLS immunostaining in enteric neurons, associated with a significant increase in the nitrite/nitrate, IL-6 and TNF-α levels, iNOS expression and MDA accumulation in the small intestine. We provide evidence that 5-FU induces reactive gliosis and reduction of enteric neurons in a S100B/RAGE/NFκB-dependent manner, since pentamidine, a S100B inhibitor, prevented 5-FU-induced neuronal loss, enteric glia activation, intestinal inflammation, oxidative stress and histological injury.
Enteropathogenic
(EPEC) are recognized as one of the leading bacterial causes of infantile diarrhea worldwide. Weaned C57BL/6 mice pretreated with antibiotics were challenged orally with wild-type ...EPEC or
mutant (lacking type 3 secretion system) to determine colonization, inflammatory responses and clinical outcomes during infection. Antibiotic disruption of intestinal microbiota enabled efficient colonization by wild-type EPEC resulting in growth impairment and diarrhea. Increase in inflammatory biomarkers, chemokines, cellular recruitment and pro-inflammatory cytokines were observed in intestinal tissues. Metabolomic changes were also observed in EPEC infected mice with changes in tricarboxylic acid (TCA) cycle intermediates, increased creatine excretion and shifts in gut microbial metabolite levels. In addition, by 7 days after infection, although weights were recovering, EPEC-infected mice had increased intestinal permeability and decreased colonic claudin-1 levels. The
mutant colonized the mice with no weight loss or increased inflammatory biomarkers, showing the importance of the T3SS in EPEC virulence in this model. In conclusion, a murine infection model treated with antibiotics has been developed to mimic clinical outcomes seen in children with EPEC infection and to examine potential roles of selected virulence traits. This model can help in further understanding mechanisms involved in the pathogenesis of EPEC infections and potential outcomes and thus assist in the development of potential preventive or therapeutic interventions.
Clostridioides difficile infection (CDI) is the fifth leading cause of death from nonmalignant gastrointestinal disease in the United States. The contribution of resistance to C. difficile-active ...antibiotics to the outcomes of CDI is unclear. We evaluated the antimicrobial susceptibility of C. difficile isolates in a U.S. hospital and determined associations of clinical variables and binary toxin positivity with antibiotic resistance. C. difficile spores were cultured from fecal specimens of adult patients with CDI for genotyping and antimicrobial susceptibility assay (for clindamycin CLI, fidaxomicin FDX, metronidazole MTZ, moxifloxacin MXF, tigecycline TGC, and vancomycin VAN). Electronic medical records were reviewed for clinical data extraction. Ninety-seven of 130 (75%) fecal samples grew toxigenic C. difficile in culture. Most of the isolates were
(80.4%), and 18.6% and 1% were
and
, respectively. Susceptibility to VAN, MTZ, FDX, TGC, MXF, and CLI was 96%, 94%, 100%, 100%, 8%, and 79%, respectively. Six isolates, all
positive and belonging to the 027 ribotype, were resistant to VAN and/or MTZ. Higher MICs were found in isolates with a mutation in the VAN-related resistance gene
, but not
. In addition,
isolates exhibited higher MICs of VAN, MTZ, TGC, CLI, and MXF compared to
strains. Patients with greater intestinal inflammation or severe disease were more likely to be infected with
strains. Decreased susceptibility to antibiotics is not directly associated with either severe or recurrent CDI. However, antimicrobial susceptibility of C. difficile is decreased in strains positive for the binary toxin gene.
S-nitrosoglutathione (GSNO) is a nitric oxide (NO) donor, which exerts antioxidant, anti-inflammatory, and microbicidal actions. Intragingival application of GSNO was already shown to decrease ...alveolar bone loss, inflammation and oxidative stress in an experimental periodontal disease (EPD) model. In the present study, we evaluated the potential therapeutic effect of topical applications of hydroxypropylmethylcellulose (HPMC)/GSNO solutions on EPD in Wistar rats. EPD was induced by placing a sterilized nylon (3.0) thread ligature around the cervix of the second left upper molar of the animals, which received topical applications of a HPMC solutions containing GSNO 2 or 10 mM or vehicle (HPMC solution), 1 h prior to the placement of the ligature and then twice daily until sacrifice on day 11. Treatment with HPMC/GSNO 10 mM solution significantly reduced alveolar bone loss, oxidative stress and TNF-α e IL-1β levels in the surrounding gingival tissue, and led to a decreased transcription of RANK and TNF-α genes and elevated bone alkaline phosphatase, compared to the HPMC group. In conclusion, topical application of HPMC/GSNO solution is a potential treatment to reduce inflammation and bone loss in periodontal disease.
Clostridioides difficile is the leading health care-associated pathogen, leading to substantial morbidity and mortality; however, there is no widely accepted model to predict C. difficile infection ...severity. Most currently available models perform poorly or were calibrated to predict outcomes that are not clinically relevant. We sought to validate six of the leading risk models (Age Treatment Leukocyte Albumin Serum Creatinine (ATLAS), C. difficile Disease (CDD), Zar, Hensgens, Shivashankar, and C. difficile Severity Score (CDSS)), guideline severity criteria, and PCR cycle threshold for predicting C. difficile-attributable severe outcomes (inpatient mortality, colectomy/ileostomy, or intensive care due to sepsis). Models were calculated using electronic data available within ±48 h of diagnosis (unavailable laboratory measurements assigned zero points), calibrated using a large retrospective cohort of 3,327 inpatient infections spanning 10 years, and compared using receiver operating characteristic (ROC) and precision-recall curves. ATLAS achieved the highest area under the ROC curve (AuROC) of 0.781, significantly better than the next best performing model (Zar 0.745; 95% confidence interval of AuROC difference 0.0094–0.6222; P = 0.008), and highest area under the precision-recall curve of 0.232. Current IDSA/SHEA severity criteria demonstrated moderate performance (AuROC 0.738) and PCR cycle threshold performed the worst (0.531). The overall predictive value for all models was low, with a maximum positive predictive value of 37.9% (ATLAS cutoff ≥9). No clinical model performed well on external validation, but ATLAS did outperform other models for predicting clinically relevant C. difficile-attributable outcomes at diagnosis. Novel markers should be pursued to augment or replace underperforming clinical-only models.
In the US, Clostridioides difficile (C. difficile) infection (CDI) is the 8th leading cause of hospital readmission and 7th for mortality among all gastrointestinal (GI) disorders. Here, we ...investigated GI dysfunction post-CDI in humans and mice post-acute infection.
From March 2020 to July 2021, we reviewed the clinical records of 67 patients referred to the UVA Complicated C. difficile clinic for fecal microbiota transplantation (FMT) eligibility. C57BL/6 mice were infected with C. difficile and clinical scores were determined daily. Stool samples from mice were collected to measure the shedding of C. difficile and myeloperoxidase (MPO) levels. On day 21 post-infection, Evans's blue and FITC-70kDa methods were performed to evaluate GI motility in mice.
Of the 67 patients evaluated at the C. difficile clinic, 40 patients (59.7%) were confirmed to have CDI, and 22 patients (32.8%) with post-CDI IBS (diarrhea-type, constipation-type, and mixed-type). In infected mice, levels of MPO in stools and clinical score were higher on day 3. On day 21, mice recovered from body weight loss induced by CDI, and fecal MPO was undetectable. The total GI transit time (TGITT) and FITC-70kDa levels on the proximal colon were increased in infected mice (p = 0.002), suggesting a constipation phenotype post-acute phase of CDI. A positive correlation intestinal inflammation on day 3 and TGITT on day 21 was observed.
In conclusion, post-infection intestinal dysfunction occurs in humans and mice post-CDI. Importantly, we have validated in the mouse model that CDI causes abnormal GI transit in the recovery phase of the disease, indicating the potential utility of the model in exploring the underlying mechanisms of post-infectious IBS in humans.
•C. difficile infection cause irritable bowel syndrome post-infection in humans.•Survivors of CDI in mice exhibit gastrointestinal dysfunction post-infection.•Gastrointestinal dysfunction post-CDI correlates positively with severity of disease.•Gastrointestinal dysfunction post-CDI correlates positively with inflammation.
Increased risk of intestinal dysfunction has been reported in patients after
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
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.
, WT, A2A, and A2B KO mice were infected with
, 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
toxins in EGCs.
, 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.
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.
Cryptosporidiosis is a waterborne protozoal infection that may cause life-threatening diarrhea in undernourished children living in unsanitary environments. The aim of this study is to identify new ...biomarkers that may be related to gut-brain axis dysfunction in children suffering from the malnutrition/infection vicious cycle, necessary for better intervention strategies. Myeloperoxidase (MPO) is a well-known neutrophil-related tissue factor released during enteropathy that could drive gut-derived brain inflammation. We utilized a model of environmental enteropathy in C57BL/6 weanling mice challenged by Cryptosporidium and undernutrition. Mice were fed a 2%-Protein Diet (dPD) for eight days and orally infected with 107-C. parvum oocysts. C. parvum oocyst shedding was assessed from fecal and ileal-extracted genomic DNA by qRT-PCR. Ileal histopathology scores were assessed for intestinal inflammation. Prefrontal cortex samples were snap-frozen for MPO ELISA assay and NF-kb immunostaining. Blood samples were drawn by cardiac puncture after anesthesia and sera were obtained for serum amyloid A (SAA) and MPO analysis. Brain samples were also obtained for Iba-1 prefrontal cortex immunostaining. C. parvum-infected mice showed sustained stool oocyst shedding for six days post-infection and increased fecal MPO and inflammation scores. dPD and cryptosporidiosis led to impaired growth and weight gain. C. parvum-infected dPD mice showed increased serum MPO and serum amyloid A (SAA) levels, markers of systemic inflammation. dPD-infected mice showed greater MPO, NF-kB expression, and Iba-1 immunolabeling in the prefrontal cortex, an important brain region involved in executive function. Our findings suggest MPO as a potential biomarker for intestinal-brain axis dysfunction due to environmental enteropathy.
Clostridioides difficile
(
C. difficile)
produces toxins A (TcdA) and B (TcdB), both associated with intestinal damage and diarrhea. Pannexin-1 (Panx1) channels allows the passage of messenger ...molecules, such as adenosine triphosphate (ATP), which in turn activate the P2X7 receptors (P2X7R) that regulate inflammation and cell death in inflammatory bowel diseases. The aim of this study was to verify the effect of
C. difficile
infection (CDI) in the expression of Panx1 and P2X7R in intestinal tissues of mice, as well as their role in cell death and
IL-6
expression induced by TcdA and TcdB in enteric glial cells (EGCs). Male C57BL/6 mice (8 weeks of age) were infected with
C. difficile
VPI10463, and the control group received only vehicle per gavage. After three days post-infection (p.i.), cecum and colon samples were collected to evaluate the expression of Panx1 by immunohistochemistry.
In vitro
, EGCs (PK060399egfr) were challenged with TcdA or TcdB, in the presence or absence of the Panx1 inhibitor (10Panx trifluoroacetate) or P2X7R antagonist (A438079), and Panx1 and P2X7R expression, caspase-3/7 activity and phosphatidylserine binding to annexin-V, as well as
IL-6
expression were assessed. CDI increased the levels of Panx1 in cecum and colon of mice compared to the control group. Panx1 inhibitor decreased caspase-3/7 activity and phosphatidylserine-annexin-V binding, but not
IL-6
gene expression in TcdA and TcdB-challenged EGCs. P2X7 receptor antagonist accentually reduced caspase-3/7 activity, phosphatidylserine-annexin-V binding, and
IL-6
gene expression in TcdA and TcdB-challenged EGCs. In conclusion, Panx1 is increased during CDI and plays an important role in the effects of
C. difficile
toxins in EGCs, participating in cell death induced by both toxins by promoting caspase-3/7 activation
via
P2X7R, which is also involved in IL-6 expression induced by both toxins.
Objective and design
Oral mucositis (OM) is an intense inflammatory reaction progressing to tissue damage and ulceration. The medicinal uses of Calotropis procera are supported by anti-inflammatory ...capacity. PII-IAA, a highly homogenous cocktail of laticifer proteins (LP) prepared from the latex of C. procera, with recognized pharmacological properties was tested to treat OM.
Materials and subjects
Male Golden Sirius hamsters were used in all treatments.
Treatment
The latex protein samples were injected i.p. (5 mg/Kg) 24 h before mucositis induction (mechanical trauma) and 24 h later.
Methods
Histology, cytokine measurements ELISA, and macroscopic evaluation scores were performed.
Results
PII-IAA eliminated OM, accompanied by total disappearance of myeloperoxidase activity and release of IL-1b, as well as reduced TNF-a. Oxidative stress was relieved by PII-IAA treatment, as revealed by MDA and GSH measurements. PII-IAA also reduced the expression of adhesion molecules (ICAM-1) and Iba-1, two important markers of inflammation, indicating modulatory effects. Histological analyses of the cheek epithelium revealed greater deposition of type I collagen fibers in animals given PII-IAA compared with the control group. This performance was only reached when LPPII was treated with iodoacetamide (IAA), an irreversible inhibitor of proteolytic activity of cysteine proteases. The endogenous proteolytic activity of LPPII induced adverse effects in animals. Candidate proteins involved in the phytomodulatory activity are proposed.
Conclusions
Therapy was successful in treating OM with the laticifer protein fraction, containing peptidases and osmotin, from Calotropis procera. The effective candidate from the latex proteins for therapeutic use is PII-IAA.
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