Stable Dual miR-143 and miR-506 Upregulation Inhibits Proliferation and Cell Cycle Progression.

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.

Reproducibility and Relative Validity of a Dietary Screener Adapted for Use among Pregnant Women in Dhulikhel, Nepal.

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. AC1 indicated good to excellent (≥ 0.6) reproducibility for the majority (85%) of food groups across trimesters. There was moderate to excellent validity (AC1 ≥ 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.

Would people living with epilepsy benefit from palliative care?

Palliative care (PC) is an approach to the care of persons living with serious illness and their families that focuses on improving quality of life and reducing suffering by addressing complex medical symptoms, psychosocial needs, spiritual well-being, and advance care planning. While PC has traditionally been associated with hospice care for persons with cancer, there is now recognition that PC is relevant to many noncancer diagnoses, including neurologic illness, and at multiple points along the illness journey, not just end of life. Despite the recent growth of the field of neuropalliative care there has been scant attention paid to the relevance of PC principles in epilepsy or the potential for PC approaches to improve outcomes for persons living with epilepsy and their families. We believe this has been a significant oversight and that PC may provide a useful framework for addressing the many sources of suffering facing persons living with epilepsy, for engaging patients and families in challenging conversations, and to focus efforts to improve models of care for this population. In this manuscript we review areas of significant unmet needs where a PC approach may improve patient and family-centered outcomes, including complex symptom management, goals of care, advance care planning, psychosocial support for patient and family and spiritual well-being. When relevant we highlight areas where epilepsy patients may have unique PC needs compared to other patient populations and conclude with suggestions for future research, clinical, and educational efforts.

Facilitators and barriers to healthy eating in a worksite cafeteria: a qualitative study.

BACKGROUND: Worksite-based nutrition interventions can serve as access points to facilitate healthy eating and translate existing knowledge of cardiometabolic disease prevention. We explored perceptions, facilitators, and barriers for healthy eating in a cafeteria at a large worksite in Mexico City. METHODS: We conducted an exploratory qualitative study in a large department store in Mexico City with ~ 1500 employees. We conducted eight focus group discussions (FGD) with 63 employees stratified by job category (sales, maintenance, shipping, restaurant, cafeteria, administrative staff, and sales managers). Employees were invited to participate in the FGD if they were at the store at the day and time of the FGD for their job type. FGDs were audio-recorded, transcribed verbatim and analyzed using the thematic method. This process involved the researches´ familiarizing themselves with the data, generating initial codes, searching for themes, reviewing the themes, defining and naming themes, and then interpreting the data. RESULTS: Employees defined healthy eating as eating foods that are fresh, diverse, and prepared hygienically. The most commonly reported facilitators of healthy eating at the worksite were availability of affordable healthy food options and employees' high health awareness. Major barriers to healthy eating included unavailability of healthy foods, unpleasant taste of food, and preference for fatty foods and meat. For lower-wage workers, affordability was a major concern. Other barriers included lack of time to eat work and long working hours. CONCLUSION: A broad range of factors affect healthy eating at the cafeteria, some related to nutrition and some related to the employees type of job. Availability of healthy, hygienic, and tasty food at an affordable price could lead to healthier food choices in the worksite cafeteria. These strategies, along with work schedules that allow sufficient time for healthy eating, may help improve dietary behaviors and health of employees.

Patient satisfaction and its socio-demographic correlates in a tertiary public hospital in Nepal: a cross-sectional study.

BACKGROUND: Patient satisfaction is one proxy indicator of the health care quality; however, enhancing patient satisfaction in low-income settings is very challenging due to the inadequacy of resources as well as low health literacy among patients. In this study, we assess patient satisfaction and its correlates in a tertiary public hospital in Nepal. METHODS: We conducted a cross sectional study at outpatient department of Bhaktapur Hospital of Nepal. To recruit participants for the study, we applied a systematic random sampling method. Our study used a validated Patient Satisfaction Questionnaire III (PSQ-III) developed by RAND Corporation including various contextual socio-demographic characteristics. We calculated mean score and percentages of satisfaction across seven dimensions of patient satisfaction. To determine the association between various dimensions of patient satisfaction and socio-demographic characteristics of the patient, we used a multi-ordinal logistic regression. RESULTS: Among 204 patients, we observed a wide variation in patient satisfaction across seven dimensions. About 39% of patients were satisfied in the dimension of general satisfaction, 92% in interpersonal manner, and 45% in accessibility and convenience. Sociodemographic factors such as age (AOR: 6.42; CI: 1.30-35.05), gender (AOR: 2.81; CI: 1.41-5.74), and ethnicity (AOR: 0.26; CI: 0.08-0.77) were associated with general satisfaction of the patients. Other sociodemographic variables such as education, occupation, and religion were associated with a majority of the dimensions of patient satisfaction (p < 0.05). Age was found to be the strongest predictor of patient satisfaction in five out of seven dimensions. CONCLUSIONS: We concluded that patient satisfaction varies across different dimensions. Therefore, targeted interventions that direct to improve the dimensions of patient satisfaction where the proportion of satisfaction is low are needed. Similar studies should be conducted regularly at different levels of health facilities across the country to capture a wider picture of patient satisfaction at various levels.

Health system gaps in cardiovascular disease prevention and management in Nepal.

BACKGROUND: Cardiovascular diseases (CVDs) are the leading cause of deaths and disability in Nepal. Health systems can improve CVD health outcomes even in resource-limited settings by directing efforts to meet critical system gaps. This study aimed to identify Nepal's health systems gaps to prevent and manage CVDs. METHODS: We formed a task force composed of the government and non-government representatives and assessed health system performance across six building blocks: governance, service delivery, human resources, medical products, information system, and financing in terms of equity, access, coverage, efficiency, quality, safety and sustainability. We reviewed 125 national health policies, plans, strategies, guidelines, reports and websites and conducted 52 key informant interviews. We grouped notes from desk review and transcripts' codes into equity, access, coverage, efficiency, quality, safety and sustainability of the health system. RESULTS: National health insurance covers less than 10% of the population; and more than 50% of the health spending is out of pocket. The efficiency of CVDs prevention and management programs in Nepal is affected by the shortage of human resources, weak monitoring and supervision, and inadequate engagement of stakeholders. There are policies and strategies in place to ensure quality of care, however their implementation and supervision is weak. The total budget on health has been increasing over the past five years. However, the funding on CVDs is negligible. CONCLUSION: Governments at the federal, provincial and local levels should prioritize CVDs care and partner with non-government organizations to improve preventive and curative CVDs services.

Varicose veins and its risk factors among nurses at Dhulikhel hospital: a cross sectional study.

BACKGROUND: Women in nursing professions are at high risk for developing varicose veins as it requires physical work and prolonged standing. The aim of the study is to estimate the current prevalence of varicose veins among nurses at Dhulikhel Hospital and assess its risk factors. METHODS: A cross sectional study was carried out among 181 female nurses from different clinical settings of Dhulikhel Hospital. A structured questionnaire was administered to gather the demographic, work related and medical history information. The participants underwent Doppler ultrasound for varicose veins confirmation. Varicose veins was defined as Doppler finding of reflux or vein diameter equal or greater than 5 mm. RESULTS: A total of 181 nurses participated in this study and 83 (46%) had varicose veins. The mean standing time was 4.28 (0.8) hours /day, mean sitting time was 1.28 (0.6) hours/day, mean walking time was 2.37 (0.8) hours/day. In the adjusted model the odds of having varicose veins was 27 times greater with every 1 hour increase in standing time per day (adjusted OR: 27.44; 95% CI 4.09-180.77; p-value <0.00). CONCLUSIONS: Varicose veins was prevalent among nurses' at Dhulikhel Hospital. Prolonged standing was found to be a significant factor for varicose veins.

Potential Therapeutic Effects of Mepacrine against Clostridium perfringens Enterotoxin in a Mouse Model of Enterotoxemia.

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.

Nepal Pioneer Worksite Intervention Study to lower cardio-metabolic risk factors: design and protocol.

BACKGROUND: To increase cardiovascular disease prevention efforts, worksite interventions can promote healthy food choices, facilitate health education, increase physical activity and provide social support. This pioneer study will measure the effectiveness of a cafeteria and a behavioral intervention on cardio-metabolic risk in a worksite in Nepal. METHODS: The Nepal Pioneer Worksite Intervention Study is a two-step intervention study conducted in Dhulikhel Hospital in eastern Nepal. In the first step, we will assess the effectiveness of a 6-month cafeteria intervention on cardio-metabolic risk using a pre-post design. In the second step, we will conduct a 6-month, open-masked, two-arm randomized trial by allocating half of the participants to an individual behavioral intervention based on the 'diabetes prevention program' for the prevention of cardio-metabolic risk. We will recruit 366 full time employees with elevated blood pressure, fasting blood sugar, or glycosylated haemoglobin (HbA1c). At baseline, we will measure their demographic variables, lifestyle factors, anthropometry, fasting blood sugar, HbA1c,and lipid profiles. We will measure cardio-metabolic outcomes at 6 months, 12 months, and 18 months. At 12 months, we will compare the proportion of participants who have attained two or more cardio-metabolic risk factor reduction goals (HbA1c decrease ≥0.5%; systolic blood pressure decrease ≥5 mmHg; or triglycerides decrease ≥10 mg/dL) during the cafeteria intervention period and the control period using generalized estimating equations. At 18 months, we will compare the proportion from the 'cafeteria only arm' to the 'cafeteria and behavior arm' for the same outcome using a chi-square test. DISCUSSION: This pioneer study will estimate the effect of environmental-level changes on lowering cardio-metabolic risks; and added benefit of an individual-level dietary intervention. If the study demonstrates a significant effect, a scaled up approach could produce an important reduction in cardiovascular disease burden through environmental and individual level prevention programs in Nepal and similar worksites worldwide. TRIAL REGISTRATION: The trial was retrospectively registered on clincaltrials.gov (Identification Member: NCT03447340 ; Date of Registration: February 27, 2018).

Cardio-metabolic disease risk factors among South Asian labour migrants to the Middle East: a scoping review and policy analysis.

This paper aims to explore the burgeoning burden of cardiovascular and metabolic disease (CMD) risk factors among South Asian labor migrants to the Middle East. We conducted a qualitative synthesis of literature using PubMed/Medline and grey literature searches, supplemented by a policy review of policies from the South Asian countries. We found a high burden of cardio-metabolic risk factors among the migrants as well as among the populations in the home and the host countries. For example, two studies reported the prevalence of diabetes mellitus (DM) ranging between 9 and 17% among South Asian migrants. Overweight and obesity were highly prevalent amongst South Asian male migrants; prevalence ranged from 30 to 66% (overweight) and 17-80% (obesity) respectively. The home country population had a significant CMD risk factor burden. Nearly 14 to 40% have three or more risk factors: such as hypertension (17 to 37%), diabetes (3 to 7%), overweight (18 to 41%), and obesity (2 to 15%). The host country also exhibited similar burden of risk factors: hypertension (13 to 38%), diabetes (8 to 17%), overweight (33 to 77%) and obesity (35 to 41%). Only Nepal, Bangladesh and Sri Lanka have some provisions related to screening of CMDs before labor migration. Further, analysis of policy papers showed that none of the reviewed documents had requirements for screening of any specific CMDs, but chronic diseases were used generically, failing to specify specific screening target. Given the high burden of risk factors, migrants' health should become an urgent priority. The lack of specific focus on screening during different stages of labor migration should receive attention. The International Labour Organization and the International Office for Migration, through their country coordination teams should engage local stakeholders to create policies and plans to address this concern. Similarly, there is a need for the host country to become an equal partner in these efforts, as migrant's better cardiometabolic health is in the benefit of both host and home countries.

Prevalence and associated factors of childhood overweight/obesity among primary school children in urban Nepal.

BACKGROUND: Childhood overweight/obesity has become a major public health concern globally because of its adverse health consequences and escalating prevalence. The factors underlying the disease conditions manifested during adulthood commonly originate in childhood. Nepal is going through a transition where under-nutrition co-exists with obesity; however, there is a lack of well-documented information on childhood overweight or obesity in Nepal. This study was carried out to determine the prevalence and associated factors of childhood overweight/obesity among urban primary school children. METHODS: A cross-sectional survey was conducted from May to October of 2017. Behavioral data were collected using a structured self-administered questionnaire with parents of children aged 6-13 years old in grades 1-5 studying in private schools of Lalitpur district in Nepal. Study participants were selected using two-stage cluster random sampling from 10 private schools. Height and weight measurements of 575 children were taken and BMI-for-age-sex was calculated using WHO AnthroPlus. Data were analyzed using SPSS version 21. Associated factors were examined using Chi-square tests followed by multivariate logistic regression analyses. RESULTS: The study found that out of 575 students, 107 (18.6%) were overweight and 41 (7.1%) were obese. Among 328 male children, 62 (19.0%) were overweight and 35 (10.6%) were obese. Likewise, among 247 female children, 45 (18.2%) were overweight and 6 (2.4%) were obese. Male children (aOR = 2.21, 95% CI: 1.38-3.53), children of mothers with a high school (aOR = 3.13, 95% CI: 1.39-7.12) or university level of education (aOR = 3.09, 95% CI: 1.23-7.70) and children of mothers in a professional field (aOR = 1.34, 95% CI: 1.02-4.05) had a greater likelihood of being overweight/obese. Likewise, students consuming energy-dense less nutrient food (aOR = 2.92, 95% CI: 1.66-5.12), lacking active travel to and from school (aOR = 2.38, 95% CI: 1.12-4.79) and those having sedentary behaviors (aOR = 3.01, 95% CI: 1.20-7.29) were likely to be overweight/obese. CONCLUSIONS: More than one-quarter of the children in urban Lalitpur were found to be overweight/obese. High junk food consumption and sedentary activity were found to be significantly associated with childhood overweight/obesity. School health and awareness programs aiming to reduce the intake of energy-dense foods and promote an active lifestyle including active transportation to school among children are imperative. Future studies to objectively measure the type and amount of food intake and physical activity of students are recommended.

Sickle cell anemia mice develop a unique cardiomyopathy with restrictive physiology.

Cardiopulmonary complications are the leading cause of mortality in sickle cell anemia (SCA). Elevated tricuspid regurgitant jet velocity, pulmonary hypertension, diastolic, and autonomic dysfunction have all been described, but a unifying pathophysiology and mechanism explaining the poor prognosis and propensity to sudden death has been elusive. Herein, SCA mice underwent a longitudinal comprehensive cardiac analysis, combining state-of-the-art cardiac imaging with electrocardiography, histopathology, and molecular analysis to determine the basis of cardiac dysfunction. We show that in SCA mice, anemia-induced hyperdynamic physiology was gradually superimposed with restrictive physiology, characterized by progressive left atrial enlargement and diastolic dysfunction with preserved systolic function. This phenomenon was absent in WT mice with experimentally induced chronic anemia of similar degree and duration. Restrictive physiology was associated with microscopic cardiomyocyte loss and secondary fibrosis detectable as increased extracellular volume by cardiac-MRI. Ultrastructural mitochondrial changes were consistent with severe chronic hypoxia/ischemia and sarcomere diastolic-length was shortened. Transcriptome analysis revealed up-regulation of genes involving angiogenesis, extracellular-matrix, circadian-rhythm, oxidative stress, and hypoxia, whereas ion-channel transport and cardiac conduction were down-regulated. Indeed, progressive corrected QT prolongation, arrhythmias, and ischemic changes were noted in SCA mice before sudden death. Sudden cardiac death is common in humans with restrictive cardiomyopathies and long QT syndromes. Our findings may thus provide a unifying cardiac pathophysiology that explains the reported cardiac abnormalities and sudden death seen in humans with SCA.

Evidence that Clostridium perfringens Enterotoxin-Induced Intestinal Damage and Enterotoxemic Death in Mice Can Occur Independently of Intestinal Caspase-3 Activation.

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.

Comparative pathogenesis of enteric clostridial infections in humans and animals.

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.

Reproducibility and relative validity of food group intake in a food frequency questionnaire developed for Nepalese diet.

We developed a food frequency questionnaire (FFQ) designed to measure the dietary practices of adult Nepalese. The present study examined the validity and reproducibility of the FFQ. To evaluate the reproducibility of the FFQ, 116 subjects completed two 115-item FFQ across a four-month interval. Six 24-h dietary recalls were collected (1 each month) to assess the validity of the FFQ. Seven major food groups and 23 subgroups were clustered from the FFQ based on macronutrient composition. Spearman correlation coefficients evaluating reproducibility for all food groups were greater than 0.5, with the exceptions of oil. The correlations varied from 0.41 (oil) to 0.81 (vegetables). All crude spearman coefficients for validity were greater than 0.5 except for dairy products, pizzas/pastas and sausage/burgers. The FFQ was found to be reliable and valid for ranking the intake of food groups for Nepalese dietary intake.

Moonlighting bacteriophage proteins derepress staphylococcal pathogenicity islands.

Staphylococcal superantigen-carrying pathogenicity islands (SaPIs) are discrete, chromosomally integrated units of approximately 15 kilobases that are induced by helper phages to excise and replicate. SaPI DNA is then efficiently encapsidated in phage-like infectious particles, leading to extremely high frequencies of intra- as well as intergeneric transfer. In the absence of helper phage lytic growth, the island is maintained in a quiescent prophage-like state by a global repressor, Stl, which controls expression of most of the SaPI genes. Here we show that SaPI derepression is effected by a specific, non-essential phage protein that binds to Stl, disrupting the Stl-DNA complex and thereby initiating the excision-replication-packaging cycle of the island. Because SaPIs require phage proteins to be packaged, this strategy assures that SaPIs will be transferred once induced. Several different SaPIs are induced by helper phage 80alpha and, in each case, the SaPI commandeers a different non-essential phage protein for its derepression. The highly specific interactions between different SaPI repressors and helper-phage-encoded antirepressors represent a remarkable evolutionary adaptation involved in pathogenicity island mobilization.

The interaction of Clostridium perfringens enterotoxin with receptor claudins.

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.

A synthetic peptide corresponding to the extracellular loop 2 region of claudin-4 protects against Clostridium perfringens enterotoxin in vitro and in vivo.

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 type A enterotoxin damages the rabbit colon.

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.