Shigella flexneri Infection in Caenorhabditis elegans: Cytopathological Examination and Identification of Host Responses

The Gram-negative bacterium Shigella flexneri is the causative agent of shigellosis, a diarrhoeal disease also known as bacillary dysentery. S. flexneri infects the colonic and rectal epithelia of its primate host and induces a cascade of inflammatory responses that culminates in the destruction of the host intestinal lining. Molecular characterization of host-pathogen interactions in this infection has been challenging due to the host specificity of S. flexneri strains, as it strictly infects humans and non-human primates. Recent studies have shown that S. flexneri infects the soil dwelling nematode Caenorhabditis elegans, however, the interactions between S. flexneri and C. elegans at the cellular level and the cause of nematode death are unknown. Here we attempt to gain insight into the complex host-pathogen interactions between S. flexneri and C. elegans. Using transmission electron microscopy, we show that live S. flexneri cells accumulate in the nematode intestinal lumen, produce outer membrane vesicles and invade nematode intestinal cells. Using two-dimensional differential in-gel electrophoresis we identified host proteins that are differentially expressed in response to S. flexneri infection. Four of the identified genes, aco-1, cct-2, daf-19 and hsp-60, were knocked down using RNAi and ACO-1, CCT-2 and DAF-19, which were identified as up-regulated in response to S. flexneri infection, were found to be involved in the infection process. aco-1 RNAi worms were more resistant to S. flexneri infection, suggesting S. flexneri-mediated disruption of host iron homeostasis. cct-2 and daf-19 RNAi worms were more susceptible to infection, suggesting that these genes are induced as a protective mechanism by C. elegans. These observations further our understanding of the processes involved in S. flexneri infection of C. elegans, which is immensely beneficial to the routine use of this new in vivo model to study S. flexneri pathogenesis.     

In Vitro,In Silico and In Vivo Studies of Ursolic Acid as an Anti-Filarial Agent

As part of our drug discovery program for anti-filarial agents from Indian medicinal plants, leaves of Eucalyptus tereticornis were chemically investigated, which resulted in the isolation and characterization of an anti-filarial agent, ursolic acid (UA) as a major constituent. Antifilarial activity of UA against the human lymphatic filarial parasite Brugia malayi using in vitro and in vivo assays, and in silico docking search on glutathione-s-transferase (GST) parasitic enzyme were carried out. The UA was lethal to microfilariae (mf; LC₁₀₀: 50; IC₅₀: 8.84 µM) and female adult worms (LC₁₀₀: 100; IC50: 35.36 µM) as observed by motility assay; it exerted 86% inhibition in MTT reduction potential of the adult parasites. The selectivity index (SI) of UA for the parasites was found safe. This was supported by the molecular docking studies, which showed adequate docking (LibDock) scores for UA (−8.6) with respect to the standard antifilarial drugs, ivermectin (IVM −8.4) and diethylcarbamazine (DEC-C −4.6) on glutathione-s-transferase enzyme. Further, in silico pharmacokinetic and drug-likeness studies showed that UA possesses drug-like properties. Furthermore, UA was evaluated in vivo in B. malayi-M. coucha model (natural infection), which showed 54% macrofilaricidal activity, 56% female worm sterility and almost unchanged microfilaraemia maintained throughout observation period with no adverse effect on the host. Thus, in conclusion in vitro, in silico and in vivo results indicate that UA is a promising, inexpensive, widely available natural lead, which can be designed and developed into a macrofilaricidal drug. To the best of our knowledge this is the first ever report on the anti-filarial potential of UA from E. tereticornis, which is in full agreement with the Thomson Reuter''s ‘Metadrug’ tool screening predictions.     

Preventing Slips and Falls through Leisure-Time Physical Activity: Findings from a Study of Limited-Service Restaurants

Background/Objective

Physical activity has been shown to be beneficial at improving health in some medical conditions and in preventing injury. Epidemiologic studies suggest that physical activity is one factor associated with a decreased risk for slips and falls in the older (≥65 years) adult population. While the risk of slips and falls is generally lower in younger than in older adults; little is known of the relative contribution of physical activity in preventing slips and falls in younger adults. We examined whether engagement in leisure-time physical activity (LTPA) was protective of slips and falls among a younger/middle-aged (≤50 years old) working population.

Methods

475 workers from 36 limited-service restaurants in six states in the U.S. were recruited to participate in a prospective cohort study of workplace slipping. Information on LTPA was collected at the time of enrollment. Participants reported their slip experience and work hours weekly for up to 12 weeks. We investigated the association between the rate of slipping and the rate of major slipping (i.e., slips that resulted in a fall and/or injury) and LTPA for workers 50 years of age and younger (n = 433, range 18–50 years old) using a multivariable negative binomial generalized estimating equation model.

Results

The rate of major slips among workers who engaged in moderate (Adjusted Rate Ratio (RR)  = 0.65; 95% Confidence Interval (CI)  =  [0.18–2.44]) and vigorous (RR = 0.64; 95%CI  =  [0.18–2.26]) LTPA, while non-significant, were approximately one-third lower than the rate of major slips among less active workers.

Conclusion

While not statistically significant, the results suggest a potential association between engagement in moderate and vigorous LTPA and the rate of major slips in younger adults. Additional studies that examine the role of occupational and non-occupational physical activity on the risk of slips, trips and falls among younger and middle aged adults appear warranted.     

Post Kala-Azar Dermal Leishmaniasis following Treatment with 20 mg/kg Liposomal Amphotericin B (Ambisome) for Primary Visceral Leishmaniasis in Bihar,India

Background

The skin disorder Post Kala-Azar Dermal Leishmaniasis (PKDL) occurs in up to 10% of patients treated for visceral leishmaniasis (VL) in India. The pathogenesis of PKDL is not yet fully understood. Cases have been reported in India following therapy with most available treatments, but rarely in those treated with liposomal amphotericin B (Ambisome). Between July 2007 and August 2012 with the support of the Rajendra Memorial Research Institute (RMRI), Médecins Sans Frontières (MSF) supported a VL treatment programme in Bihar, India—an area highly endemic for Leishmania donovani—in which 8749 patients received 20 mg/kg intravenous Ambisome as first-line treatment. This study describes the characteristics of patients who returned to the MSF supported treatment programme with PKDL.

Methods and Principal Findings

Over a 5-year period, Ambisome was administered to 8749 patients with laboratory-confirmed VL (clinical signs, rK39 positive, with/without parasite confirmation) in four intravenous doses of 5 mg/kg to a total of 20 mg/kg, with a high initial-cure rate (99.3%) and low default rate (0.3%). All patients received health education highlighting the possibility and symptoms of developing PKDL, and advice to return to the MSF programme if these symptoms developed. This is an observational retrospective cohort study of the programme outcomes. Of the 8311 patients completing treatment for their first episode of VL, 24 (0.3%) returned passively to the programme complaining of symptoms subsequently confirmed as PKDL, diagnosed from clinical history, appearance consistent with PKDL, and slit-skin smear examination. Of the 24 patients, 89% had macular lesions, with a median time (interquartile range) to development of 1.2 (0.8–2.2) years following treatment. Comparison of the demographic and clinical characteristics of the VL patients treated with Ambisome who later developed PKDL, with those of the remaining cohort did not identify any significant risk factors for PKDL. However, the time to developing PKDL was significantly shorter with Ambisome than in a subset of patients presenting to the programme with PKDL following previous sodium stibogluconate treatment for VL.

Conclusions

In this large cohort of patients with VL in Bihar who were treated with 20 mg/kg Ambisome, PKDL following treatment appears to be infrequent with no predictive risk factors. The shorter median time to developing symptoms of PKDL compared with that after conventional VL treatments should be taken into account when counseling patients treated with regimens including Ambisome.     

Risk Factors for Visceral Leishmaniasis Relapse in Immunocompetent Patients following Treatment with 20 mg/kg Liposomal Amphotericin B (Ambisome) in Bihar,India

Background

A proportion of all immunocompetent patients treated for visceral leishmaniasis (VL) are known to relapse; however, the risk factors for relapse are not well understood. With the support of the Rajendra Memorial Research Institute (RMRI), Médecins Sans Frontières (MSF) implemented a program in Bihar, India, using intravenous liposomal amphotericin B (Ambisome) as a first-line treatment for VL. The aim of this study was to identify risk factors for VL relapse by examining the characteristics of immunocompetent patients who relapsed following this regimen.

Methods and Principal Findings

This is an observational retrospective cohort study of all VL patients treated by the MSF program from July 2007 to August 2012. Intravenous Ambisome was administered to 8749 patients with VL in four doses of 5 mg/kg (for a total dose of 20 mg/kg) over 4–10 days, depending on the severity of disease. Out of 8588 patients not known to be HIV-positive, 8537 (99.4%) were discharged as initial cures, 24 (0.3%) defaulted, and 27 (0.3%) died during or immediately after treatment. In total, 1.4% (n = 119) of the initial cured patients re-attended the programme with parasitologically confirmed VL relapse, with a median time to relapse of 10.1 months. Male sex, age <5 years and ≥45 years, a decrease in spleen size at time of discharge of ≤0.5 cm/day, and a shorter duration of symptoms prior to seeking treatment were significantly associated with relapse. Spleen size at admission, hemoglobin level, nutritional status, and previous history of relapse were not associated with relapse.

Conclusions

This is the largest cohort of VL patients treated with Ambisome worldwide. The risk factors for relapse included male sex, age <5 and ≥45 years, a smaller decrease in splenomegaly at discharge, and a shorter duration of symptoms prior to seeking treatment. The majority of relapses in this cohort occurred 6–12 months following treatment, suggesting that a 1-year follow-up is appropriate in future studies.     

HSP70 Domain II of Mycobacterium tuberculosis Modulates Immune Response and Protective Potential of F1 and LcrV Antigens of Yersinia pestis in a Mouse Model

No ideal vaccine exists to control plague, a deadly dangerous disease caused by Yersinia pestis. In this context, we cloned, expressed and purified recombinant F1, LcrV antigens of Y. pestis and heat shock protein70 (HSP70) domain II of M. tuberculosis in E. coli. To evaluate the protective potential of each purified protein alone or in combination, Balb/C mice were immunized. Humoral and cell mediated immune responses were evaluated. Immunized animals were challenged with 100 LD₅₀ of Y. pestis via intra-peritoneal route. Vaccine candidates i.e., F1 and LcrV generated highly significant titres of anti-F1 and anti-LcrV IgG antibodies. A significant difference was noticed in the expression level of IL-2, IFN-γ and TNF-α in splenocytes of immunized animals. Significantly increased percentages of CD4+ and CD8+ T cells producing IFN-γ in spleen of vaccinated animals were observed in comparison to control group by flow cytometric analysis. We investigated whether the F1, LcrV and HSP70(II) antigens alone or in combination can effectively protect immunized animals from any histopathological changes. Signs of histopathological lesions noticed in lung, liver, kidney and spleen of immunized animals on 3^rd day post challenge whereas no lesions in animals that survived to day 20 post-infection were observed. Immunohistochemistry showed bacteria in lung, liver, spleen and kidney on 3^rd day post-infection whereas no bacteria was observed on day 20 post-infection in surviving animals in LcrV, LcrV+HSP70(II), F1+LcrV, and F1+LcrV+HSP70(II) vaccinated groups. A significant difference was observed in the expression of IL-2, IFN-γ, TNF-α, and CD4+/CD8+ T cells secreting IFN-γ in the F1+LcrV+HSP70(II) vaccinated group in comparison to the F1+LcrV vaccinated group. Three combinations that included LcrV+HSP70(II), F1+LcrV or F1+LcrV+HSP70(II) provided 100% protection, whereas LcrV alone provided only 75% protection. These findings suggest that HSP70(II) of M. tuberculosis can be a potent immunomodulator for F1 and LcrV containing vaccine candidates against plague.     

Geographic Separation of Domestic and Wild Strains of Toxoplasma gondii in French Guiana Correlates with a Monomorphic Version of Chromosome1a

Background

Previous studies have stressed the genetic divergence and high pathogenicity of strains of T. gondii from French Guiana. Although strains from coastal, human adapted environments (so called anthropized) resemble those found in other regions of the Caribbean, strains collected from inland jungle environment are genetically quite diverse. To better understand the composition of these distinct strain types, we undertook a more in depth analysis of T. gondii strains from French Guiana including profiling of chromosome 1a (Chr1a), which is often shared as a single monomorphic haplotype among lineages that are otherwise genetically distinct.

Methodology/Principal Findings

Comparison of intron sequences from selectively neutral genes indicated that anthropized strains were most closely related to clonal type III strains from North America, although wider RFLP analysis revealed that they are natural hybrids. In contrast, strains isolated from the jungle were genetically very diverse. Remarkably, nearly all anthropized strains contained the monomorphic version of Chr1a while wild stains were extremely divergent. The presence of the monomorphic Chr1a strongly correlated with greater transmission in domestic cats, although there were several exceptions, indicating that other factors also contribute. Anthropized strains also varied in their virulence in laboratory mice, and this pattern could not be explained by the simple combination of previously identified virulence factors, indicating that other genetic determinants influence pathogenicity.

Conclusions/Significance

Our studies underscore the marked genetic separation of anthropized and wild strains of T. gondii in French Guiana and provide additional evidence that the presence of Chr1a is associated with successful expansion of widely different lineages within diverse geographic areas. The predominance of Chr1a among strains in the anthropized environment suggests that it may confer an advantage for transmission in this environment, and thus potentially contribute to the spread of pathogenecity determinants.