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BACKGROUND: Suicidal thoughts and behaviors (STBs) are prevalent within the Lesbian, Gay, Bisexual, Transgender, and Queers (LGBTQ) community, often exacerbated by challenges in accessing care and the perceived stigma and discrimination tied to disclosing ones identity. Digital health interventions that offer psychosocial self-help present a promising platform to reach individuals at risk of STBs, especially those who may not engage with conventional health services. This review aimed to assess the role of digital-based intervention in reducing STBs among LGBTQ individuals. METHODS: We conducted a systematic literature search from three databases, PsycINFO, PubMed, and CINHAL, from 1st Jan 1990 to 31st December 2023. The review encompassed studies investigating the feasibility, acceptability, and impact of digital interventions on STBs, employing randomized control trials (RCTs), pseudo-RCTs, observational pre-posttest designs, and qualitative studies. Potential bias was evaluated using the McGill Mixed Methods Appraisal Tool (MMAT). RESULTS: Five non-overlapping studies were included, reporting data from 777 participants. The studies featured diverse types of digital interventions, including videos, online writing, and mobile applications. The studies included three RCTs, and two qualitative studies. Across most of these studies, notable enhancements or reductions in the proportion of participants reporting STBs were observed post-intervention, alongside improvements in help-seeking intentions. The findings underscored that the applications used in the studies were engaging, acceptable, and deemed feasible in effectively addressing suicide prevention among the LGBTQ community. CONCLUSION: Overall, digital interventions were found to be feasible and acceptable in suicide prevention among LGBTQ communities, demonstrating preliminary efficacy in increasing help-seeking behavior when experiencing suicidal thoughts and in reducing STBs. Therefore, advocating for widespread promotion and dissemination of digital health interventions is crucial, particularly in low- and middle-income countries (LMICs) with limited access to health services and heightened barriers to obtaining such services. Further research using fully powered RCT is imperative to assess the efficacy of these interventions.

Clostridium perfringens enterotoxin (CPE) is thought to cause lethal enterotoxemia when absorbed from the intestinal lumen into the circulation. CPE action sequentially involves receptor-binding, oligomerization into a prepore, and pore formation. To explore the mechanistic basis by which CPE alters permeability, this study tested the permeability effects of several recombinant CPE (rCPE) species: rCPE and rCPEC186A (which form pores), rC-CPE and rCPED48A (which bind to receptors but cannot oligomerize), rCPEC186A/F91C (which binds and oligomerizes without pore formation), and rCPEY306A/L315A (which has poor receptor-binding ability). On Caco-2 cells, i) only rCPE and rCPEC186A were cytotoxic; ii) rCPE and rCPEC186A affected transepithelial resistance (TEER) and 4 kDa fluorescent dextran (FD4) transit more quickly than binding-capable, but noncytotoxic, rCPE variants; whereas iii) rCPEY306A/L315A did not affect TEER or FD4 transit. Using mouse intestinal loops, rCPE (but not noncytotoxic rC-CPE, rCPED48A or rCPEY306A/L315A) was lethal and caused intestinal histologic damage within 4 h. After 2 h of treatment, rCPE was more strongly absorbed into the serum than those noncytotoxic rCPE species but by 4 h rC-CPE and rCPED48A became absorbed similarly as rCPE, while rCPEY306A/L315A absorption remained low. This increased rC-CPE and rCPED48A absorption from 2 to 4 h did not involve a general intestinal permeability increase because Evans Blue absorption from the intestines did not increase between 2 and 4 h of treatment with rC-CPE or rCPED48A. Collectively, these results indicate that CPE receptor binding is sufficient to slowly affect permeability, but CPE-induced cytotoxicity is necessary for rapid permeability changes and lethality. IMPORTANCE Clostridium perfringens enterotoxin (CPE) causes lethal enterotoxemia when absorbed from the intestines into the bloodstream. Testing recombinant CPE (rCPE) or rCPE variants impaired for various specific steps in CPE action showed that full CPE-induced cytotoxicity causes rapid Caco-2 monolayer permeability alterations, as well as enterotoxemic lethality and rapid CPE absorption in mouse small intestinal loops. However, receptor binding-capable, but noncytotoxic, rCPE variants did cause slow-developing in vitro and in vivo permeability effects. Absorption of binding-capable, noncytotoxic rCPE variants from the intestines did not correlate with general intestinal permeability alterations, suggesting that CPE binding can induce its own uptake. These findings highlight the importance of binding and, especially, cytotoxicity for CPE absorption during enterotoxemia and may assist development of permeability-altering rCPE variants for translational purposes.

BackgroundThe co-existence of undernutrition and overnutrition is a global public health threat. We aim to report the burden of both nutritional deficiency (Protein-Energy Malnutrition) and overweight (high Body Mass Index) in Nepal over a decade (2010-2019) and observe the changes through trend charts.MethodsWe did a secondary data analysis using the Institute for Health Metrics and Evaluation (IHME)'s Global Burden of Disease (GBD) database to download age-standardized data on Protein Energy Malnutrition (PEM) and high Body Mass Index (BMI). We presented the trend of death, Disability Adjusted Life Years (DALYs), Years of Life Lost (YLL), and Years Lost due to Disability (YLD) of PEM and high BMI in Nepal from 2010 to 2019 and also compared data for 2019 among South Asian countries.ResultsBetween 2010 and 2019, in Nepal, the Disability Adjusted Life Years (DALYs) due to PEM were declining while high BMI was in increasing trend. Sex-specific trends revealed that females had higher DALYs for PEM than males. In contrast, males had higher DALYs for high BMI than females. In 2019, Nepal had the highest death rate for PEM (5.22 per 100,000 populations) than any other South Asian country. The burden of PEM in terms of DALY was higher in under-five children (912 per 100,000 populations) and elderly above 80 years old (808.9 per 100,000 populations), while the population aged 65-69 years had the highest burden of high BMI (5893 per 100,000 populations). In the last decade, the DALYs for risk factors contributing to PEM such as child growth failure (stunting and wasting), unsafe water, sanitation and handwashing, and sub-optimal breastfeeding have declined in Nepal. On the contrary, the DALYs for risk factors contributing to high BMI, such as a diet high in sugar-sweetened beverages, a diet high in trans fatty acid, and low physical activity, have increased. This could be a possible explanation for the increasing trend of high BMI and decreasing trend of PEM.ConclusionRapidly growing prevalence of high BMI and the persistent existence of undernutrition indicate the double burden of malnutrition in Nepal. Public health initiatives should be planned to address this problem.

Clostridium perfringens is an extremely versatile pathogen of humans and livestock, causing wound infections like gas gangrene (clostridial myonecrosis), enteritis/enterocolitis (including one of the most common human food-borne illnesses), and enterotoxemia (where toxins produced in the intestine are absorbed and damage distant organs such as the brain). The virulence of this Gram-positive, spore-forming, anaerobe is largely attributable to its copious toxin production; the diverse actions and roles in infection of these toxins are now becoming established. Most C. perfringens toxin genes are encoded on conjugative plasmids, including the pCW3-like and the recently discovered pCP13-like plasmid families. Production of C. perfringens toxins is highly regulated via processes involving two-component regulatory systems, quorum sensing and/or sporulation-related alternative sigma factors. Non-toxin factors, such as degradative enzymes like sialidases, are also now being implicated in the pathogenicity of this bacterium. These factors can promote toxin action in vitro and, perhaps in vivo, and also enhance C. perfringens intestinal colonization, e.g. NanI sialidase increases C. perfringens adherence to intestinal tissue and generates nutrients for its growth, at least in vitro. The possible virulence contributions of many other factors, such as adhesins, the capsule and biofilms, largely await future study.

Clostridium perfringens is an extremely versatile pathogen of humans and livestock, causing wound infections like gas gangrene (clostridial myonecrosis), enteritis/enterocolitis (including one of the most common human food-borne illnesses), and enterotoxemia (where toxins produced in the intestine are absorbed and damage distant organs such as the brain). The virulence of this Gram-positive, spore-forming, anaerobe is largely attributable to its copious toxin production; the diverse actions and roles in infection of these toxins are now becoming established. Most C. perfringens toxin genes are encoded on conjugative plasmids, including the pCW3-like and the recently discovered pCP13-like plasmid families. Production of C. perfringens toxins is highly regulated via processes involving two-component regulatory systems, quorum sensing and/or sporulation-related alternative sigma factors. Non-toxin factors, such as degradative enzymes like sialidases, are also now being implicated in the pathogenicity of this bacterium. These factors can promote toxin action in vitro and, perhaps in vivo, and also enhance C. perfringens intestinal colonization, e.g. NanI sialidase increases C. perfringens adherence to intestinal tissue and generates nutrients for its growth, at least in vitro. The possible virulence contributions of many other factors, such as adhesins, the capsule and biofilms, largely await future study.

Objectives Here we examined the reproducibility and validity of a dietary screener which was translated and adapted to assess diet quality among pregnant Nepalese women. Methods A pilot cohort of singleton pregnant women (N = 101; age 25.9 ± 4.1 years) was recruited from a tertiary, periurban hospital in Nepal. An adapted Nepali version of the PrimeScreen questionnaire, a brief 21-item dietary screener that assesses weekly consumption of 12 healthy and 9 unhealthy food groups, was administered twice, and a month apart, in both the 2nd and 3rd trimesters. Up to four inconsecutive 24-h dietary recalls (24-HDRs) were completed each trimester and utilized as the reference method for validation. For each trimester, data from multiple 24-HDRs were averaged across days, and items were grouped to match the classification and three weekly consumption categories (0-1, 2-3, or 4 + servings/week) of the 21 food groups represented on the PrimeScreen. Results Gwet's agreement coefficients (AC1) were used to evaluate the reproducibility and validity of the adapted PrimeScreen against the 24-HDRs in both the 2nd and 3rd trimester. AC.sub.1 indicated good to excellent ([greater than or equal to] 0.6) reproducibility for the majority (85%) of food groups across trimesters. There was moderate to excellent validity (AC.sub.1 [greater than or equal to] 0.4) for all food groups except for fruits and vegetables in the 2nd trimester, and green leafy vegetables and eggs in both the 2nd and 3rd trimesters. Conclusions The modified PrimeScreen questionnaire appears to be a reasonably valid and reliable instrument for assessing the dietary intake of most food groups among pregnant women in Nepal., Author(s): Kelly Martin [sup.1] [sup.2] , Krupali Shah [sup.1] , Abha Shrestha [sup.3] , Emily Barrett [sup.4] , Kusum Shrestha [sup.5] , Cuilin Zhang [sup.6] , Archana Shrestha [sup.5] [sup.7] [...]

BACKGROUND:In Nepal, the burden of noncommunicable, chronic diseases is rapidly rising, and disproportionately affecting low and middle-income countries. Integrated interventions are essential in strengthening primary care systems and addressing the burden of multiple comorbidities. A growing body of literature supports the involvement of frontline providers, namely mid-level practitioners and community health workers, in chronic care management. Important operational questions remain, however, around the digital, training, and supervisory structures to support the implementation of effective, affordable, and equitable chronic care management programs. METHODS:A 12-month, population-level, type 2 hybrid effectiveness-implementation study will be conducted in rural Nepal to evaluate an integrated noncommunicable disease care management intervention within Nepal's new municipal governance structure. The intervention will leverage the government's planned roll-out of the World Health Organization's Package of Essential Noncommunicable Disease Interventions (WHO-PEN) program in four municipalities in Nepal, with a study population of 80,000. The intervention will leverage both the WHO-PEN and its cardiovascular disease-specific technical guidelines (HEARTS), and will include three evidence-based components: noncommunicable disease care provision using mid-level practitioners and community health workers; digital clinical decision support tools to ensure delivery of evidence-based care; and training and digitally supported supervision of mid-level practitioners to provide motivational interviewing for modifiable risk factor optimization, with a focus on medication adherence, and tobacco and alcohol use. The study will evaluate effectiveness using a pre-post design with stepped implementation. The primary outcomes will be disease-specific, "at-goal" metrics of chronic care management; secondary outcomes will include alcohol and tobacco consumption levels. DISCUSSION:This is the first population-level, hybrid effectiveness-implementation study of an integrated chronic care management intervention in Nepal. As low and middle-income countries plan for the Sustainable Development Goals and universal health coverage, the results of this pragmatic study will offer insights into policy and programmatic design for noncommunicable disease care management in the future. TRIAL REGISTRATION:ClinicalTrials.gov, NCT04087369. Registered on 12 September 2019.

Low-income and middle-income countries are struggling with a growing epidemic of non-communicable diseases. To achieve the Sustainable Development Goals, their healthcare systems need to be strengthened and redesigned. The Starfield 4Cs of primary care-first-contact access, care coordination, comprehensiveness and continuity-offer practical, high-quality design options for non-communicable disease care in low-income and middle-income countries. We describe an integrated non-communicable disease intervention in rural Nepal using the 4C principles. We present 18 months of retrospective assessment of implementation for patients with type II diabetes, hypertension and chronic obstructive pulmonary disease. We assessed feasibility using facility and community follow-up as proxy measures, and assessed effectiveness using singular 'at-goal' metrics for each condition. The median follow-up for diabetes, hypertension and chronic obstructive pulmonary disease was 6, 6 and 7 facility visits, and 10, 10 and 11 community visits, respectively (0.9 monthly patient touch-points). Loss-to-follow-up rates were 16%, 19% and 22%, respectively. The median time between visits was approximately 2 months for facility visits and 1 month for community visits. 'At-goal' status for patients with chronic obstructive pulmonary disease improved from baseline to endline (p=0.01), but not for diabetes or hypertension. This is the first integrated non-communicable disease intervention, based on the 4C principles, in Nepal. Our experience demonstrates high rates of facility and community follow-up, with comparatively low lost-to-follow-up rates. The mixed effectiveness results suggest that while this intervention may be valuable, it may not be sufficient to impact outcomes. To achieve the Sustainable Development Goals, further implementation research is urgently needed to determine how to optimise non-communicable disease interventions.

Clostridium perfringens enterotoxin (CPE) is a pore-forming toxin that causes the symptoms of common bacterial food poisoning and several non-foodborne human gastrointestinal diseases, including antibiotic-associated diarrhea and sporadic diarrhea. In some cases, CPE-mediated disease can be very severe or fatal due to the involvement of enterotoxemia. Therefore, the development of potential therapeutics against CPE action during enterotoxemia is warranted. Mepacrine, an acridine derivative drug with broad-spectrum effects on pores and channels in mammalian membranes, has been used to treat protozoal intestinal infections in human patients. A previous study showed that the presence of mepacrine inhibits CPE-induced pore formation and activity in enterocyte-like Caco-2 cells, reducing the cytotoxicity caused by this toxin in vitro Whether mepacrine is similarly protective against CPE action in vivo has not been tested. When the current study evaluated whether mepacrine protects against CPE-induced death and intestinal damage using a murine ligated intestinal loop model, mepacrine protected mice from the enterotoxemic lethality caused by CPE. This protection was accompanied by a reduction in the severity of intestinal lesions induced by the toxin. Mepacrine did not reduce CPE pore formation in the intestine but inhibited absorption of the toxin into the blood of some mice. Protection from enterotoxemic death correlated with the ability of this drug to reduce CPE-induced hyperpotassemia. These in vivo findings, coupled with previous in vitro studies, support mepacrine as a potential therapeutic against CPE-mediated enterotoxemic disease.

Several enteric clostridial diseases can affect humans and animals. Of these, the enteric infections caused by Clostridium perfringens and Clostridium difficile are amongst the most prevalent and they are reviewed here. C. perfringens type A strains encoding alpha toxin (CPA) are frequently associated with enteric disease of many animal mammalian species, but their role in these diseased mammals remains to be clarified. C. perfringens type B encoding CPA, beta (CPB) and epsilon (ETX) toxins causes necro-hemorrhagic enteritis, mostly in sheep, and these strains have been recently suggested to be involved in multiple sclerosis in humans, although evidence of this involvement is lacking. C. perfringens type C strains encode CPA and CPB and cause necrotizing enteritis in humans and animals, while CPA and ETX producing type D strains of C. perfringens produce enterotoxemia in sheep, goats and cattle, but are not known to cause spontaneous disease in humans. The role of C. perfringens type E in animal or human disease remains poorly defined. The newly revised toxinotype F encodes CPA and enterotoxin (CPE), the latter being responsible for food poisoning in humans, and the less prevalent antibiotic associated and sporadic diarrhea. The role of these strains in animal disease has not been fully described and remains controversial. Another newly created toxinotype, G, encodes CPA and necrotic enteritis toxin B-like (NetB), and is responsible for avian necrotic enteritis, but has not been associated with human disease. C. difficile produces colitis and/or enterocolitis in humans and multiple animal species. The main virulence factors of this microorganism are toxins A, B and an ADP-ribosyltransferase (CDT). Other clostridia causing enteric diseases in humans and/or animals are Clostridium spiroforme, Clostridium piliforme, Clostridium colinum, Clostridium sordellii, Clostridium chauvoei, Clostridium septicum, Clostridium botulinum, Clostridium butyricum and Clostridium neonatale. The zoonotic transmission of some, but not all these clostridsial species, has been demonstrated.

Previously, we reported the development of a human Aγ-globin gene lentivirus (LV), GbG, which expresses high levels of HbF to correct the sickle cell anemia (SCA) phenotype in the Berkeley SCA mouse model, and then modified the γ-globin gene by substituting glycine at codon 16 with aspartic acid in the Aγ-globin gene to generate GbGM LV. In the present study, we evaluated the long-term safety of human Aγ-globin gene carrying GbGM LV in wild-type mice after primary and secondary transplants of GbGM-modified hematopoietic stem cells (HSC) over 18 months. The safety of the GbGM bone marrow transplant was assessed by monitoring the effects on body weight, hematology, histopathology, malignancy formation, and survival. Mice transplanted with Mock-transduced and spleen focus forming virus (SFFV) γ-retroviral vector (RV)-transduced HSC served as negative and positive controls, respectively. The mean donor-cell engraftment was comparable across Mock, GbGM LV, and SFFV RV groups. There were no significant differences in body weight, clinical signs, immunophenotype, or histopathology in the GbGM-treated mice compared to controls. Four SFFV RV-treated mice, but none of the GbGM-treated mice, developed donor-derived, vector-positive lymphomas as demonstrated by flow cytometry analysis and in situ hybridization. These results highlight the safety of the administration of GbGM LV-modified HSC with long-term follow-up after primary and secondary transplants in mice. This data supported the initiation of phase 1/2 first-in-human SCA clinical trial in the United States. [ABSTRACT FROM AUTHOR]

In humans and livestock, Clostridium perfringens is an important cause of intestinal infections that manifest as enteritis, enterocolitis, or enterotoxemia. This virulence is largely related to the toxin-producing ability of C. perfringens. This article primarily focuses on the C. perfringens type F strains that cause a very common type of human food poisoning and many cases of nonfoodborne human gastrointestinal diseases. The enteric virulence of type F strains is dependent on their ability to produce C. perfringens enterotoxin (CPE). CPE has a unique amino acid sequence but belongs structurally to the aerolysin pore-forming toxin family. The action of CPE begins with binding of the toxin to claudin receptors, followed by oligomerization of the bound toxin into a prepore on the host membrane surface. Each CPE molecule in the prepore then extends a beta-hairpin to form, collectively, a beta-barrel membrane pore that kills cells by increasing calcium influx. The cpe gene is typically encoded on the chromosome of type F food poisoning strains but is encoded by conjugative plasmids in nonfoodborne human gastrointestinal disease type F strains. During disease, CPE is produced when C. perfringens sporulates in the intestines. Beyond type F strains, C. perfringens type C strains producing beta-toxin and type A strains producing a toxin named CPILE or BEC have been associated with human intestinal infections. C. perfringens is also an important cause of enteritis, enterocolitis, and enterotoxemia in livestock and poultry due to intestinal growth and toxin production.

Clostridium perfringens enterotoxin (CPE) is responsible for the gastrointestinal symptoms of C. perfringens type A food poisoning and some cases of nonfoodborne gastrointestinal diseases, such as antibiotic-associated diarrhea. In the presence of certain predisposing medical conditions, this toxin can also be absorbed from the intestines to cause enterotoxemic death. CPE action in vivo involves intestinal damage, which begins at the villus tips. The cause of this CPE-induced intestinal damage is unknown, but CPE can induce caspase-3-mediated apoptosis in cultured enterocyte-like Caco-2 cells. Therefore, the current study evaluated whether CPE activates caspase-3 in the intestines and, if so, whether this effect is required for the development of intestinal tissue damage or enterotoxemic lethality. Using a mouse ligated small intestinal loop model, CPE was shown to cause intestinal caspase-3 activation in a dose- and time-dependent manner. Most of this caspase-3 activation occurred in epithelial cells shed from villus tips. However, CPE-induced caspase-3 activation occurred after the onset of tissue damage. Furthermore, inhibition of intestinal caspase-3 activity did not affect the onset of intestinal tissue damage. Similarly, inhibition of intestinal caspase-3 activity did not reduce CPE-induced enterotoxemic lethality in these mice. Collectively, these results demonstrate that caspase-3 activation occurs in the CPE-treated intestine but that this effect is not necessary for the development of CPE-induced intestinal tissue damage or enterotoxemic lethality.

Clostridium perfringens enterotoxin (CPE) has significant medical importance due to its involvement in several common human gastrointestinal diseases. This 35 kDa single polypeptide toxin consists of two domains: a C-terminal domain involved in receptor binding and an N-terminal domain involved in oligomerization, membrane insertion and pore formation. The action of CPE starts with its binding to receptors, which include certain members of the claudin tight junction protein family; bound CPE then forms a series of complexes, one of which is a pore that causes the calcium influx responsible for host cell death. Recent studies have revealed that CPE binding to claudin receptors involves interactions between the C-terminal CPE domain and both the 1st and 2nd extracellular loops (ECL-1 and ECL-2) of claudin receptors. Of particular importance for this binding is the docking of ECL-2 into a pocket present in the C-terminal domain of the toxin. This increased understanding of CPE interactions with claudin receptors is now fostering the development of receptor decoy therapeutics for CPE-mediated gastrointestinal disease, reagents for cancer therapy/diagnoses and enhancers of drug delivery.

Background: Worksite-based health programs have shown positive impacts on employee health and have led to significant improvements in cardiovascular risk factor profiles. We aimed to determine the effect of cafeteria intervention on cardio-metabolic risk factors diet in a worksite setting (Dhulikhel Hospital) in Nepal. Methods: In this one-arm pre-post intervention study, we recruited 277 non-pregnant hospital employees aged 18–60 with prediabetes or pre-hypertension. The study was registered in clinicaltrials.gov (NCT03447340; 2018/02/27). All four cafeterias in the hospital premises received cafeteria intervention encouraging healthy foods and discouraging unhealthy foods for six months. We measured blood pressure, fasting glucose level, glycated hemoglobin, cholesterol in the laboratory, and diet intake (in servings per week) using 24-hour recall before and six months after the intervention. The before and after measures were compared using paired-t tests. Results: After six months of cafeteria intervention, the median consumption of whole grains, mono/polyunsaturated fat, fruits, vegetable and nuts servings per week increased by 2.24(p<0.001), 2.88(p<0.001), 0.84(p<0.001) 2.25(p<0.001) and nuts 0.55 (p<0.001) servings per week respectively. The median consumption of refined grains decreased by 5.07 servings per week (p<0.001). Mean systolic and diastolic blood pressure decreased by 2 mmHg (SE = 0.6; p = 0.003) and 0.1 mmHg (SE = 0.6; p = 0.008), respectively. The low-density lipoprotein (LDL) was significantly reduced by 6 mg/dL (SE = 1.4; p<0.001). Conclusion: Overall, we found a decrease in consumption of refined grains and an increase in consumption of whole grains, unsaturated fats, fruits, and nuts observed a modest reduction in blood pressure and LDL cholesterol following a 6-month cafeteria-based worksite intervention incorporating access to healthy foods. [ABSTRACT FROM AUTHOR]

The mainstays of lung cancer pathogenesis are cell cycle progression dysregulation, impaired apoptosis, and unregulated cell proliferation. While individual microRNA (miR) targeting or delivering is a promising approach that has been extensively studied, combination of miR targeting can enhance therapeutic efficacy and overcome limitations present in individual miR regulations. We previously reported on the use of a miR-143 and miR-506 combination via transient transfections against lung cancer. In this study, we evaluated the effect of miR-143 and miR-506 under stable deregulations in A549 lung cancer cells. We used lentiviral transductions to either up- or downregulate the two miRs individually or in combination. The cells were sorted and analyzed for miR deregulation via qPCR. We determined the miR deregulations' effects on the cell cycle, cell proliferation, cancer cell morphology, and cell motility. Compared to the individual miR deregulations, the combined miR upregulation demonstrated a miR-expression-dependent G2 cell cycle arrest and a significant increase in the cell doubling time, whereas the miR-143/506 dual downregulation demonstrated increased cellular motility. Furthermore, the individual miR-143 and miR-506 up- and downregulations exhibited cellular responses lacking an apparent miR-expression-dependent response in the respective analyses. Our work here indicates that, unlike the individual miR upregulations, the combinatorial miR treatment remained advantageous, even under prolonged miR upregulation. Finally, our findings demonstrate potential advantages of miR combinations vs. individual miR treatments. [ABSTRACT FROM AUTHOR]

Clostridium perfringens enterotoxin (CPE) action starts when the toxin binds to claudin receptors. Claudins contain two extracellular loop domains, with the second loop (ECL-2) being slightly smaller than the first. CPE has been shown to bind to ECL-2 in receptor claudins. We recently demonstrated that Caco-2 cells (a naturally CPE-sensitive enterocyte-like cell line) can be protected from CPE-induced cytotoxicity by preincubating the enterotoxin with soluble full-length recombinant claudin-4 (rclaudin-4), which is a CPE receptor, but not with recombinant nonreceptor claudins, such as rclaudin-1. The current study evaluated whether a synthetic peptide corresponding to the claudin-4 ECL-2 sequence can similarly inhibit CPE action in vitro and in vivo. Significant protection of Caco-2 cells was also observed using either rclaudin-4 or the claudin-4 ECL-2 peptide in both a preincubation assay and a coincubation assay. This inhibitory effect was specific, since rclaudin-1 and a synthetic peptide based on the claudin-1 ECL-2 offered no protection to Caco-2 cells. However, the claudin-4 ECL-2 peptide was unable to neutralize cytotoxicity if CPE had already bound to Caco-2 cells. When the study was repeated in vivo using a rabbit small intestinal loop assay, preincubation or coincubation of CPE with the claudin-4 ECL-2 peptide significantly and specifically inhibited the development of CPE-induced luminal fluid accumulation and histologic lesions in rabbit small intestinal loops. No similar in vivo protection from CPE was afforded by the claudin-1 ECL-2 peptide. These results suggest that claudin-4 ECL-2 peptides should be further investigated for their potential therapeutic application against CPE-associated disease.

Clostridium perfringens enterotoxin causes the gastrointestinal (GI) symptoms of C. perfringens type A food poisoning and CPE-associated non-food-borne human GI diseases. It is well established that CPE induces fluid accumulation and severe tissue damage in ligated small intestinal loops of rabbits and other animals. However, a previous study had also reported that CPE binds to rabbit colonic cells yet does not significantly affect rabbit colonic loops. To the contrary, the current study determined that treatment with 50 or 100 μg/ml of CPE causes significant histologic lesions and luminal fluid accumulation in rabbit colonic loops. Interestingly, a CPE-neutralizing monoclonal antibody blocked the development of CPE-induced histologic damage but not luminal fluid accumulation in these loops. Similar luminal fluid accumulation, without significant histologic damage, also occurred after treatment of colonic loops with heat-inactivated CPE, antibody alone, or bovine serum albumin (BSA), indicating that increased osmolarity was causing or contributing to fluid accumulation in CPE-treated colonic loops. Comparative studies revealed the similar development of histologic damage and luminal fluid accumulation in both small intestinal loops and colonic loops after as little as a 1-h treatment with 50 μg/ml of CPE. Consistent with the CPE sensitivity of the small intestine and colon, Western blotting detected CPE binding and large-complex formation in both organs. In addition, Western blotting demonstrated the presence of the high-affinity CPE receptors claudin-3 and -4 in both organs of rabbits, consistent with the observed toxin binding. Collectively, these results offer support for the possible involvement of the colon in CPE-mediated GI disease.