Role of antimicrobial peptides in combating shigellosis and in antibiotic-associated diarrhea

Antimicrobial peptides (AMPs) constitute front-line components of innate immunity in multicellular organisms. AMPs are able to kill a wide range of pathogens and exhibit additional important functions such as chemotaxis, angiogenesis and wound healing. These peptides are constitutively expressed in immune and/or epithelial cells. However, their expression can also be induced or suppressed by different stimuli in a cell and tissue specific manner. In this thesis, the functional relevance of cathelicidins and defensins, two major families of AMPs in mammals, was studied in the context of enteric infectious diseases. Induction of these AMPs was explored in Shigella infection. The effect of antibiotics on the expression of AMPs in colonic epithelial cells was also investigated and the implication of this effect on Clostridium difficile associated diarrhea (CDAD) was assessed. In a rabbit model of shigellosis, cathelicidin CAP-18 was downregulated in the large intestinal epithelia. Oral treatment with sodium butyrate (NaB) counteracted this downregulation and led to the conversion of the proform of CAP-18 into its active form in the stool (paper I). These findings correlated with reduced Shigella load in the intestinal lumen and clinical as well as histopathological recovery. Association between CAP-18 induction and reduction of bacterial load was supported by partial blocking of the antimicrobial activity in stool extract of a butyrate treated rabbit with CAP-18 specific antibody along with in vitro shigellacidal activity of CAP-18 peptide. In patients with shigellosis, administration of NaB enema as adjunct to antibiotic therapy led to an early improvement of rectal histopathology along with early reduction of inflammatory cells and proinflammatory cytokines in the stool compared to placebo treated patients (paper II). NaB treatment also resulted in enhanced expression of the human cathelicidin LL-37 in the rectal epithelia and sustained secretion of LL-37 in the stool. Since all patients were treated with antibiotics, attenuation of Shigella load in stool and clinical symptoms occurred simultaneously in both group of patients. Sodium-4-phenyl-butyrate (PB), an analogue of butyrate, was demonstrated to exhibit a similar therapeutic efficiency as NaB in the rabbit model of shigellosis (paper III). Downregulation of CAP-18 expression was also observed in the epithelia of lung and trachea. This suppression could render the respiratory tract susceptible to secondary infections during shigellosis. After oral treatment with PB or NaB, CAP-18 reappeared in the lung epithelia, which might strengthen the immunity of the respiratory tract against opportunistic respiratory pathogens. Ciprofloxacin and clindamycin were found to suppress butyrate-induced expression of LL-37 in the colonic epithelial cell line, HT-29 (paper IV). In vivo inhibitory effect of ciprofloxacin was observed on CAP-18 expression in the rectal epithelia of Shigellainfected rabbits treated with NaB as well as of healthy rabbits. Induction of genes encoding HBD-3 and additional AMPs by NaB were also inhibited by ciprofloxacin. In vitro antibacterial activity of LL-37 against ciprofloxacin-resistant C. difficile indicates that the reduction of AMPs after antibiotic treatment may allow the overgrowth of C. difficile in the gut. In conclusion, induction of AMPs is a promising therapeutic strategy against shigellosis. Suppression of AMP expression by antibiotics may contribute to CDAD, which is classically known to occur through disruption of the intestinal microbiota after antibiotic treatment

Efficacy of sodium butyrate adjunct therapy in shigellosis: a randomized, double-blind, placebo-controlled clinical trial

BACKGROUND: Treatment of shigellosis in rabbits with butyrate reduces clinical severity and counteracts the downregulation of cathelicidin (CAP-18) in the large intestinal epithelia. Here, we aimed to evaluate whether butyrate can be used as an adjunct to antibiotics in the treatment of shigellosis in patients. METHODS: A randomized, double-blind, placebo-controlled, parallel-group designed clinical trial was conducted. Eighty adult patients with shigellosis were randomized to either the Intervention group (butyrate, n = 40) or the Placebo group (normal saline, n = 40). The Intervention group was given an enema containing sodium butyrate (80 mM), twice daily for 3 days, while the Placebo group received the same dose of normal saline. The primary endpoint of the trial was to assess the efficacy of butyrate in improving clinical, endoscopic and histological features of shigellosis. The secondary endpoint was to study the effect of butyrate on the induction of antimicrobial peptides in the rectum. Clinical outcomes were assessed and concentrations of antimicrobial peptides (LL-37, human beta defensin1 [HBD-1] and human beta defensin 3 [HBD-3]) and pro-inflammatory cytokines (interleukin-1β [IL-1β] and interleukin-8 [IL-8]) were measured in the stool. Sigmoidoscopic and histopathological analyses, and immunostaining of LL-37 in the rectal mucosa were performed in a subgroup of patients. RESULTS: Compared with placebo, butyrate therapy led to the early reduction of macrophages, pus cells, IL-8 and IL-1β in the stool and improvement in rectal histopathology. Butyrate treatment induced LL-37 expression in the rectal epithelia. Stool concentration of LL-37 remained significantly higher in the Intervention group on days 4 and 7. CONCLUSION: Adjunct therapy with butyrate during shigellosis led to early reduction of inflammation and enhanced LL-37 expression in the rectal epithelia with prolonged release of LL-37 in the stool. TRIAL REGISTRATION: ClinicalTrials.gov, NCT00800930

Differential Host Immune Responses to Epidemic and Endemic Strains of Shigella dysenteriae Type 1

Shigella dysenteriae type 1 causes devastating epidemics in developing countries with high case-fatality rates in all age-groups. The aim of the study was to compare host immune responses to epidemic (T2218) and endemic strains of S. dysenteriae type 1. Shigellacidal activity of serum from rabbits immunized with epidemic or endemic strains, S. dysenteriae type 1-infected patients, and healthy adult controls from Shigella endemic and non-endemic regions was measured. Immunogenic cross-reactivity of antibodies against Shigella antigens was evaluated by Western blot analysis. Oxidative burst and phagocytic responses of monocytes and neutrophils to selected S. dysenteriae type 1 strains were assessed by flow cytometry. Rabbit antisera against epidemic strain were less effective in killing heterologous bacteria compared to endemic antisera (p=0.0002). Patients showed an increased serum shigellacidal response after two weeks of onset of diarrhoea compared to the acute stage (3-4 days after onset) against their respective homologous strains; the response against T2218 and heterologous endemic S. dysenteriae type 1 strains was not significant. The serum shigellacidal response against all the S. dysenteriae type 1 strains was similar among healthy controls from endemic and non-endemic regions and was comparable with the acute stage response by patients. Compared to endemic strains of S. dysenteriae type 1, T2218 was significantly resistant to phagocytosis by both monocytes and neutrophils. No obvious differences were obtained in the induction of oxidative burst activity and cathelicidin-mediated killing. Cross-reactivity of antibody against antigens present in the epidemic and endemic strains showed some differences in protein/peptide complexity and intensity by Western blot analysis. In summary, epidemic T2218 strain was more resistant to antibody-mediated defenses, namely phagocytosis and shigellacidal activity, compared to endemic S. dysenteriae type 1 strains. Part of this variation may be attributed to the differential complexity of protein/peptide antigens

Differential Host Immune Responses to Epidemic and Endemic Strains of Shigella dysenteriae Type 1

Shigella dysenteriae type 1 causes devastating epidemics in developing countries with high case-fatality rates in all age-groups. The aim of the study was to compare host immune responses to epidemic (T2218) and endemic strains of S. dysenteriae type 1. Shigellacidal activity of serum from rabbits immunized with epidemic or endemic strains, S. dysenteriae type 1-infected patients, and healthy adult controls from Shigellaendemic and non-endemic regions was measured. Immunogenic cross-reactivity of antibodies against Shigella antigens was evaluated by Western blot analysis. Oxidative burst and phagocytic responses of monocytes and neutrophils to selected S. dysenteriae type 1 strains were assessed by flow cytometry. Rabbit antisera against epidemic strain were less effective in killing heterologous bacteria compared to endemic antisera (p=0.0002). Patients showed an increased serum shigellacidal response after two weeks of onset of diarrhoea compared to the acute stage (3-4 days after onset) against their respective homologous strains; the response against T2218 and heterologous endemic S. dysenteriae type 1 strains was not significant. The serum shigellacidal response against all the S. dysenteriae type 1 strains was similar among healthy controls from endemic and non-endemic regions and was comparable with the acute stage response by patients. Compared to endemic strains of S. dysenteriae type 1, T2218 was significantly resistant to phagocytosis by both monocytes and neutrophils. No obvious differences were obtained in the induction of oxidative burst activity and cathelicidin-mediated killing. Cross-reactivity of antibody against antigens present in the epidemic and endemic strains showed some differences in protein/peptide complexity and intensity by Western blot analysis. In summary, epidemic T2218 strain was more resistant to antibody-mediated defenses, namely phagocytosis and shigellacidal activity, compared to endemic S. dysenteriae type 1 strains. Part of this variation may be attributed to the differential complexity of protein/peptide antigens

High rates of placental inflammation among samples collected by the Multi-Omics for Mothers and Infants consortium.

BACKGROUND: The Multi-Omics for Mothers and Infants consortium aims to improve birth outcomes. Preterm birth is a major obstetrical complication globally and causes significant infant and childhood morbidity and mortality. OBJECTIVE: We analyzed placental samples (basal plate, placenta or chorionic villi, and the chorionic plate) collected by the 5 Multi-Omics for Mothers and Infants sites, namely The Alliance for Maternal and Newborn Health Improvement Bangladesh, The Alliance for Maternal and Newborn Health Improvement Pakistan, The Alliance for Maternal and Newborn Health Improvement Tanzania, The Global Alliance to Prevent Prematurity and Stillbirth Bangladesh, and The Global Alliance to Prevent Prematurity and Stillbirth Zambia. The goal was to analyze the morphology and gene expression of samples collected from preterm and uncomplicated term births. STUDY DESIGN: The teams provided biopsies from 166 singleton preterm (<37 weeks gestation) and 175 term (≥37 weeks gestation) deliveries. The samples were fixed in formalin and paraffin embedded. Tissue sections from these samples were stained with hematoxylin and eosin and subjected to morphologic analyses. Other placental biopsies (n=35 preterm, 21 term) were flash frozen, which enabled RNA purification for bulk transcriptomics. RESULTS: The morphologic analyses revealed a surprisingly high rate of inflammation that involved the basal plate, placenta or chorionic villi, and the chorionic plate. The rate of inflammation in chorionic villus samples, likely attributable to chronic villitis, ranged from 25% (Pakistan site) to 60% (Zambia site) of cases. Leukocyte infiltration in this location vs in the basal plate or chorionic plate correlated with preterm birth. Our transcriptomic analyses identified 267 genes that were differentially expressed between placentas from preterm vs those from term births (123 upregulated, 144 downregulated). Mapping the differentially expressed genes onto single-cell RNA sequencing data from human placentas suggested that all the component cell types, either singly or in subsets, contributed to the observed dysregulation. Consistent with the histopathologic findings, gene ontology analyses highlighted the presence of leukocyte infiltration or activation and inflammatory responses in both the fetal and maternal compartments. CONCLUSION: The relationship between placental inflammation and preterm birth is appreciated in developed countries. In this study, we showed that this link also exists in developing geographies. In addition, among the participating sites, we found geographic- and population-based differences in placental inflammation and preterm birth, suggesting the importance of local factors

Seroprevalence of SARS-CoV-2 infection and associated factors among Bangladeshi slum and non-slum dwellers in pre-COVID-19 vaccination era: October 2020 to February 2021

Background Seroprevalence studies have been carried out in many developed and developing countries to evaluate ongoing and past infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Data on this infection in marginalized populations in urban slums are limited, which may offer crucial information to update prevention and mitigation policies and strategies. We aimed to determine the seroprevalence of SARS-CoV-2 infection and factors associated with seropositivity in slum and non-slum communities in two large cities in Bangladesh. Methods A cross-sectional study was carried out among the target population in Dhaka and Chattogram cities between October 2020 and February 2021. Questionnaire-based data, anthropometric and blood pressure measurements and blood were obtained. SARS-CoV-2 serology was assessed by Roche Elecsys® Anti-SARS-CoV-2 immunoassay. Results Among the 3220 participants (2444 adults, ≥18 years; 776 children, 10–17 years), the overall weighted seroprevalence was 67.3% (95% confidence intervals (CI) = 65.2, 69.3) with 71.0% in slum (95% CI = 68.7, 72.2) and 62.2% in non-slum (95% CI = 58.5, 65.8). The weighted seroprevalence was 72.9% in Dhaka and 54.2% in Chattogram. Seroprevalence was positively associated with limited years of formal education (adjusted odds ratio [aOR] = 1.61; 95% CI = 1.43, 1.82), lower income (aOR = 1.23; 95% CI = 1.03, 1.46), overweight (aOR = 1.2835; 95% CI = 1.26, 1.97), diabetes (aOR = 1.67; 95% CI = 1.21, 2.32) and heart disease (aOR = 1.38; 95% CI = 1.03, 1.86). Contrarily, negative associations were found between seropositivity and regular wearing of masks and washing hands, and prior BCG vaccination. About 63% of the population had asymptomatic infection; only 33% slum and 49% non-slum population showed symptomatic infection. Conclusion The estimated seroprevalence of SARS-CoV-2 was more prominent in impoverished informal settlements than in the adjacent middle-income non-slum areas. Additional factors associated with seropositivity included limited education, low income, overweight and pre-existing chronic conditions. Behavioral factors such as regular wearing of masks and washing hands were associated with lower probability of seropositivity.https://doi.org/10.1371/journal.pone.0268093pubpu

Phenylbutyrate Counteracts Shigella Mediated Downregulation of Cathelicidin in Rabbit Lung and Intestinal Epithelia: A Potential Therapeutic Strategy

BACKGROUND: Cathelicidins and defensins are endogenous antimicrobial peptides (AMPs) that are downregulated in the mucosal epithelia of the large intestine in shigellosis. Oral treatment of Shigella infected rabbits with sodium butyrate (NaB) reduces clinical severity and counteracts the downregulation of cathelicidin (CAP-18) in the large intestinal epithelia. AIMS: To develop novel regimen for treating infectious diseases by inducing innate immunity, we selected sodium 4-phenylbutyrate (PB), a registered drug for a metabolic disorder as a potential therapeutic candidate in a rabbit model of shigellosis. Since acute respiratory infections often cause secondary complications during shigellosis, the systemic effect of PB and NaB on CAP-18 expression in respiratory epithelia was also evaluated. METHODS: The readouts were clinical outcomes, CAP-18 expression in mucosa of colon, rectum, lung and trachea (immunohistochemistry and real-time PCR) and release of the CAP-18 peptide/protein in stool (Western blot). PRINCIPAL FINDINGS: Significant downregulation of CAP-18 expression in the epithelia of rectum and colon, the site of Shigella infection was confirmed. Interestingly, reduced expression of CAP-18 was also noticed in the epithelia of lung and trachea, indicating a systemic effect of the infection. This suggests a causative link to acute respiratory infections during shigellosis. Oral treatment with PB resulted in reduced clinical illness and upregulation of CAP-18 in the epithelium of rectum. Both PB and NaB counteracted the downregulation of CAP-18 in lung epithelium. The drug effect is suggested to be systemic as intravenous administration of NaB could also upregulate CAP-18 in the epithelia of lung, rectum and colon. CONCLUSION: Our results suggest that PB has treatment potential in human shigellosis. Enhancement of CAP-18 in the mucosal epithelia of the respiratory tract by PB or NaB is a novel discovery. This could mediate protection from secondary respiratory infections that frequently are the lethal causes in dysentery

Standardised quantitative assays for anti-SARS-CoV-2 immune response used in vaccine clinical trials by the CEPI Centralized Laboratory Network: a qualification analysis

Background: Accurate quantitation of immune markers is crucial for ensuring reliable assessment of vaccine efficacy against infectious diseases. This study was designed to confirm standardised performance of SARS-CoV-2 assays used to evaluate COVID-19 vaccine candidates at the initial seven laboratories (in North America, Europe, and Asia) of the Coalition for Epidemic Preparedness Innovations (CEPI) Centralized Laboratory Network (CLN). Methods: Three ELISAs (pre-spike protein, receptor binding domain, and nucleocapsid), a microneutralisation assay (MNA), a pseudotyped virus-based neutralisation assay (PNA), and an IFN-γ T-cell ELISpot assay were developed, validated or qualified, and transferred to participating laboratories. Immune responses were measured in ELISA laboratory units (ELU) for ELISA, 50% neuralisation dilution (ND50) for MNA, 50% neutralisation titre (NT50) for PNA, and spot-forming units for the ELISpot assay. Replicate assay results of well characterised panels and controls of blood samples from individuals with or without SARS-CoV-2 infection were evaluated by geometric mean ratios, standard deviation, linear regression, and Spearman correlation analysis for consistency, accuracy, and linearity of quantitative measurements across all laboratories. Findings: High reproducibility of results across all laboratories was demonstrated, with interlaboratory precision of 4·1–7·7% coefficient of variation for all three ELISAs, 3·8–19·5% for PNA, and 17·1–24·1% for MNA, over a linear range of 11–30 760 ELU per mL for the three ELISAs, 14–7876 NT50 per mL for PNA, and 21–25 587 ND50 per mL for MNA. The MNA was also adapted for detection of neutralising antibodies against the major SARS-CoV-2 variants of concern. The results of PNA and MNA (r=0·864) and of ELISA and PNA (r=0·928) were highly correlated. The IFN-γ ELISpot interlaboratory variability was 15·9–49·9% coefficient of variation. Sensitivity and specificity were close to 100% for all assays. Interpretation: The CEPI CLN provides accurate quantitation of anti-SARS-CoV-2 immune response across laboratories to allow direct comparisons of different vaccine formulations in different geographical areas. Lessons learned from this programme will serve as a model for faster responses to future pandemic threats and roll-out of effective vaccines. Funding: CEPI