BACKGROUND: Ulcerative colitis (UC) is a type of inflammatory bowel disease associated with persistent inflammation. Animal studies proved the efficacy of metformin in UC.
OBJECTIVE: To investigate the potential role of metformin and its protective pathways in patients with UC.
METHODS: This is a randomized, controlled, and double-blinded clinical trial that included 60 participants with mild to moderate UC and was divided randomly into two groups (n = 30). For 6 months, the mesalamine group received 1 g of mesalamine three times daily (t.i.d.). For six months, the metformin group received mesalamine 1 g t.i.d. and metformin 500 mg twice daily. A gastroenterologist evaluated patients at baseline and 6 months after starting the treatment in order to measure serum levels of zonulin, sphingosine 1 phosphate (S1P), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α). Biopsies from the colon were used to measure gene expression of zonula occuldin-1 (ZO-1), signal transducer and activator of factor-3 (STAT-3), and intracellular adhesion molecule-1 (ICAM-1). The numeric pain rating scale (NRS) and partial Mayo score were also assessed for each patient.
RESULTS: When compared to the mesalamine group, the metformin group demonstrated a statistical decrease in serum IL-6, zonulin, TNF-α, SIP, gene expression of ICAM-1 and STAT-3, and a significant increase in colonic ZO-1 when compared to the mesalamine group. The metformin group also showed a significant decrease in NRS and partial Mayo score index in comparison with the mesalamine group.
CONCLUSIONS: Metformin may be a promising additional therapy for UC patients. Trial registration identifier: NCT05553704.

BACKGROUND: A significant number of patients with axial spondyloarthritis (axSpA) do not respond to biological therapy. Therefore, we decided to investigate the specificity of this group of patients and, in particular, whether haptoglobin (Hp), its polymorphism and zonulin, in addition to other clinical features, are predictors of poor response to biological treatment.
METHODS: 48 patients with axSpA who were unsuccessfully treated with standard drugs were converted to biological treatment, and from this time on, a 12-week follow-up was started to assess the failure of biological treatment (Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) decrease < 2 points). Predictors of treatment failure were identified using logistic regression analysis.
RESULTS: 21% of subjects had biological treatment failure. Patients who had a higher zonulin level, a history of frequent infections, were older, had inflammatory bowel disease (IBD), had a lower Hp level at the time of inclusion in biological therapy showed an increased risk of treatment failure.
CONCLUSIONS: The results of the study support the hypothesis that the effectiveness of biological treatment of axSpA is limited by changed microbiota and intestinal epithelial barrier dysfunction, as an increased risk of biological treatment failure was observed in patients who were older, had higher zonulin level, IBD and repeated courses of antibiotics due to frequent infections. Therefore, starting biological treatment should be followed by reducing intestinal permeability and regulating the disturbed gut microbiome.

Tumor subunit vaccines have great potential in personalized cancer immunotherapy. They are usually administered with adjuvant owing to their low immunogenicity. Cholera toxin (CT) is a biological adjuvant with diverse biological functions and a long history of use. Our earlier study revealed that a CT-like chimeric protein co-delivered with murine granulocyte-macrophage colony stimulating factor (mGM-CSF) and prostate cancer antigen epitope could co-stimulate dendritic cells (DCs) and enhance cross presentation of tumor epitope. To further study the molecular mechanism of CT-like chimeric protein in cross presentation, major histocompatibility complex class I (MHC I)-restricted epitope 257-264 of ovalbumin (OVAT) was used as a model antigen peptide in this study. Recombinant A subunit and pentameric B subunit of CT protein were respectively genetically constructed and purified. Then both assembled into AB5 chimeric protein in vitro. Three different chimeric biomacromolecules containing mGM-CSF and OVAT were constructed according to the different fusion sites and whether the endoplasmic reticulum (ER) retention sequence was included. It was found that A2 domain and B subunit of CT were both available for loading epitopes and retaining GM1 affinity. The binding activity of GM1 was positively correlated with antigen endocytosis. Once internalized, DCs became mature and cross-presented antigen. KDEL helped the whole molecule to be retained in the ER, and this improved the cross presentation of antigen on MHC I molecules. In conclusion, hexameric CT-like chimeric protein with dual effects of GM1 affinity and ER retention sequence were potential in improvement of cross presentation. The results laid a foundation for designing personalized tumor vaccine based on CT-like chimeric protein molecular structure.

Cholera is an exceptionally aggressive infectious disease characterized by the potential to induce acute, copious, watery diarrhea of considerable severity and renal inflammation. Diabetic nephropathy is a serious complication of diabetes mellitus that can lead to kidney failure through inflammation; thus, anti-inflammatory agents are promising therapies for diabetic nephropathy. Previous studies have shown that the essential oil of Zanthoxylum myriacanthum var. pubescens Huang, Maqian essential oil (MQEO), exhibits potent antibacterial, anti-inflammatory, and renoprotective activities in diabetic mice and has emerged as a potential therapeutic drug for the treatment of diabetic nephropathy complications. Therefore, the present study was carried out to screen the potential inhibition of cholera toxin and the diabetic renoprotective activity of MQEO through computational approaches. Twelve chemical constituents derived from MQEO were docked with cholera toxin and the target proteins involved in diabetic nephropathy, namely, TXNIP, Nrf2, and DPP IV, and, subsequently, the predictions of molecular dynamic simulations, the drug-likeness properties, and the ADMET properties were performed. α-terpineol showed high binding affinities toward the cholera toxin protein. For TXNIP, among all the chemical constituents, α-phellandrene and p-cymene showed strong binding affinities with the TXNIP protein and displayed relatively stable flexibility at the hinge regions of the protein, favorable physicochemical properties in the absence of hepatotoxicity, and low cytotoxicity. For Nrf2, α-terpineol exhibited the highest binding affinity and formed a very stable complex with Nrf2, which displayed high pharmacokinetic properties. All compounds had low free-binding energies when docked with the DPP IV protein, which suggests potent biological activity. In conclusion, based on a computational approach, our findings reveal that MQEO constituents have inhibitory activity against cholera toxin and are promising therapeutic agents for suppressing diabetic inflammation and for the treatment of diabetic nephropathy complications.

Immune responses that target sialidase occur following natural cholera and have been associated with protection against cholera. Sialidase is a neuraminidase that facilitates the binding of cholera toxin (CT) to intestinal epithelial cells. Despite this, little is known about age-related sialidase-specific immune responses and the impact of nutritional status and co-infection on sialidase-specific immunity.
We enrolled 50 culture-confirmed Vibrio cholerae O1 cholera cases presenting to the icddr,b Dhaka hospital with moderate to severe dehydration. We evaluated antibody responses out to 18 months (day 540) following cholera. We assessed immune responses targeting sialidase, lipopolysaccharide (LPS), cholera toxin B subunit (CtxB), and vibriocidal responses. We also explored the association of sialidase-specific immune responses to nutritional parameters and parasitic co-infection of cases.
This longitudinal cohort study showed age-dependent differences in anti-sialidase immune response after natural cholera infection. Adult patients developed plasma anti-sialidase IgA and IgG responses after acute infection (P<0.05), which gradually decreased from day 30 on. In children, no significant anti-sialidase IgA, IgM, and IgG response was seen with the exception of a late IgG response at study day 540 (p=0.05 compared to adults). There was a correlation between anti-sialidase IgA with vibriocidal titers, as well as anti-sialidase IgA and IgG with anti-LPS and anti-CtxB antibody responses in adult patients, whereas in children, a significant positive correlation was seen only between anti-sialidase IgA and CtxB IgA responses. Stunted children showed significantly lower anti-sialidase IgA, IgG, and IgM antibody responses and higher LPS IgG and IgM antibody responses than healthy children. The anti-sialidase IgA and IgG responses were significantly higher in cases with concomitant parasitic infection.
Our data suggest that cholera patients develop age-distinct systemic and mucosal immune responses against sialidase. The stunted children have a lower anti-sialidase antibody response which may be associated with gut enteropathy and the neuraminidase plays an important role in augmented immune response in cholera patients infected with parasites.

Over the past decades, it has become increasingly clear that many neurodevelopmental disorders can be characterized by aberrations in the neuro-anatomical connectome of intermediary hubs. Yet, despite the advent in unidirectional transsynaptic tracing technologies, we are still lacking an efficient approach to identify individual neurons based on both their precise input and output relations, hampering our ability to elucidate the precise connectome in both the healthy and diseased condition. Here, we bridge this gap by combining anterograde transsynaptic- and retrograde (cATR) tracing in Ai14 reporter mice, using adeno-associated virus serotype 1 expressing Cre and cholera toxin subunit B as the anterograde and retrograde tracer, respectively. We have applied this innovative approach to selectively identify individual neurons in the brainstem that do not only receive input from one or more of the cerebellar nuclei (CN), but also project to the primary motor cortex (M1), the amygdala or the ventral tegmental area (VTA). Cells directly connecting CN to M1 were found mainly in the thalamus, while a large diversity of midbrain and brainstem areas connected the CN to the amygdala or VTA. Our data highlight that cATR allows for specific, yet brain-wide, identification of individual neurons that mediate information from a cerebellar nucleus to the cerebral cortex, amygdala or VTA via a disynaptic pathway. Given that the identified neurons in healthy subjects can be readily quantified, our data also form a solid foundation to make numerical comparisons with mouse mutants suffering from aberrations in their connectome due to a neurodevelopmental disorder.

BACKGROUND: Centella asiatica (L.) Urb or Indian pennywort is a plant of ethnopharmacological relevance, commonly called as Brahmi in South India known for its antimicrobial property in gut and for the treatment of other gut ailments. Natural anti-virulence drugs that disarm pathogens by directly targeting virulence factors or the cell viability and are thus preferred over antibiotics as these drugs impose limited selection pressure for resistance development. In this regard, an in-vitro experimental study was conducted to know the effect of extract of Centella asiatica(L.) Urb. on cholera toxin, gene expression and its vibriocidal effect on five standard strains of Vibrio cholerae; IDH03097 (El Tor variant), N16961 (El Tor), O395 (Classical) as well as five clinical strains (Haitian variant).
OBJECTIVE: To study the effect of extract of Centella asiatica on Vibrio cholerae.
METHODS: Crude extract was prepared from the leaves and stem part of the plant. The vibriocidal concentration was tested at different concentrations of the extract. The amount of cholera toxin released from the strains before and after exposure to the extract of Centella asiatica to Vibrio cholerae was measured using Bead ELISA. ctxA gene expression in the strains before and after exposure to extract of Centella asiatica was measured using quantitative real time PCR. All the above assays were performed with commercially obtained asiaticoside as well.
RESULTS: The vibriocidal activity was tested at the different concentration of the extract, where 1g/mL of crude extract and 12.5mg/mL of asiaticoside was found to be vibriocidal. The amount of cholera toxin released before and after the exposure to extract of C. asiatica was measured using Bead ELISA, showing a reduction of 70%, 89% and 93% toxin produced by classical, El Tor and variant respectively. ctxA gene expression before and after exposure to extract of Centella asiatica as well as asiaticoside was measured using qRT-PCR. We found a decrease in expression of ctxA gene transcription by 6.19 fold in classical strain, 4.29 fold in El Tor, 1.133 fold in variant strains and about 10.13-10.20 fold for the clinical strains of V. cholerae using the extract of C.asiatica while, the reduction with the exposure to the asiaticoside were 2.762 fold in classical strain, 4.809 in El Tor, 24.1 in variant strain and 34.77 - 34.8 for the clinical strains.
CONCLUSIONS: Centella asiatica extract inhibited the CT production in Vibrio cholerae as well as decreased the transcription of ctxA gene expression.

Cholera remains a major global public health threat and continuous emergence of new Vibrio cholerae strains is of major concern. We conducted a molecular epidemiological study to detect virulence markers and antimicrobial resistance patterns of V. cholerae isolates obtained from the 2012-2015 cholera outbreaks in Ghana. Archived clinical isolates obtained from the 2012, 2014 and 2015 cholera outbreaks in Ghana were revived by culture and subjected to microscopy, biochemical identification, serotyping, antibiotic susceptibility testing, molecular detection of distinct virulence factors and Multi-Locus Variable-Number of Tandem-Repeat Analysis (MLVA). Of 277 isolates analysed, 168 (60.6%) were confirmed to be V. cholerae and 109 (39.4%) isolates constituted other bacteria (Escherichia coli, Aeromonas sobria, Pseudomonas aeruginosa, Enterobacter cloacae and Enterococci faecalis). Serotyping the V. cholerae isolates identified 151 (89.9%) as Ogawa, 3 (1.8%) as Inaba and 14 (8.3%) as non-O1/O139 serogroup. The O1 serogroup isolates (154/168, 91.7%) carried the cholera toxin ctxB gene as detected by PCR. Additional virulence genes detected include zot, tcpA, ace, rtxC, toxR, rtxA, tcpP, hlyA and tagA. The most common and rare virulence factors detected among the isolates were rtxC (165 isolates) and tcpP (50 isolates) respectively. All isolates from 2014 and 2015 were multidrug resistant against the selected antibiotics. MLVA differentiated the isolates into 2 large unique clones A and B, with each predominating in a particular year. Spatial analysis showed clustering of most isolates at Ablekuma sub-district. Identification of several virulence genes among the two different genotypes of V. cholerae isolates and resistance to first- and second-line antibiotics, calls for scaleup of preventive strategies to reduce transmission, and strengthening of public health laboratories for rapid antimicrobial susceptibility testing to guide accurate treatment. Our findings support the current WHO licensed cholera vaccines which include both O1 Inaba and Ogawa serotypes.

The development of neuroprotective drugs has proven to be extremely difficult because of the blood-brain barrier. Intranasal administration is thought to transport the drug from the nasal cavity along the olfactory and trigeminal nerves to the brain, thus bypassing the blood-brain barrier. However, macromolecular protein drugs have low delivery efficiency via this route in general. We hypothesized that an innocuous cholera toxin-like chimeric protein could better enhance the efficiency of protein delivery through the intranasal route. To test this hypothesis, we designed an enhanced green fluorescent protein (EGFP) chimera to evaluate the effect of the cholera toxin (CT) as a carrier for drug delivery into the brain. Then, the EGFP was replaced with epidermal growth factor (EGF) in the chimeric protein, and the therapeutic effect of the new chimeric protein was studied in an LPS-induced neuritis mouse model. The results suggest that the CT-like chimeric protein can bypass the blood-brain barrier and enter the brain in approximately 30 min. This EGF chimeric protein can effectively protect the spatial cognitive ability of and confer anti-anxiety protection to mice. The results indicate that cholera toxin-like chimeric proteins are potential tools for effectively delivering macromodecular drugs into the brain through intranasal administration.

Antibody avidity is an important measure of the quality of vaccine-induced immune responses. Murine and human studies suggest that antibody avidity may be augmented by limiting access to antigen. The primary objective of this study was to evaluate in primed Swedish adults if booster vaccination with fractional doses (1/5th and 1/25th) of a model oral vaccine, the cholera vaccine Dukoral®, results in higher avidity antibody responses compared to boosting with a full vaccine dose. We also evaluated if fractional booster vaccination elicited similar magnitudes of antibody response compared to a full dose, and if the previously observed increase in antibody avidity after booster vaccination 1-2 years later occurred when boosting after a shorter interval. To this end, a randomised, open-label, exploratory Phase-II trial was performed. Swedish adults (n = 44), primed with two full doses of Dukoral®, were randomised into three groups and given a booster dose at either full (n = 14), 1/5th (n = 17) or 1/25th (n = 13) dose four months later. Antibody responses to cholera toxin B-subunit (CTB) were measured in serum and mucosal antibody in lymphocyte secretions (ALS). We found that the 1/5th and 1/25th booster doses had similar abilities as the full dose to induce significantly higher avidity anti-CTB antibody responses in both ALS and serum samples, as compared to after priming vaccination. There was a non-significant trend to lower magnitudes of ALS and serum IgA responses after the 1/5th compared to the full booster dose, and responses after the 1/25th dose were significantly lower. Our findings suggest fractional booster doses of Dukoral® four months after priming result in anti-toxoid mucosal antibody responses with increased antibody avidity compared to after priming vaccinations. ISRCTN registry identifier 11806026.