Functional consequences of neuromyelitis optica-IgG astrocyte interactions on blood-brain barrier permeability and granulocyte recruitment

Autoantibody neuromyelitis optica-IgG (NMO-IgG) recognizing aquaporin-4 (AQP4) is implicated as playing a central role in the physiopathology of NMO. The aim of this in vitro-based study was to characterize functional consequences of interaction between NMO-IgG and cells of the neurovascular unit (astrocytes and brain endothelium) that would provide insight into recognized features of NMO, namely altered blood-brain barrier (BBB) permeability and granulocyte recruitment. We used sera from NMO and longitudinally extensive transverse myelitis cases shown to bind in a characteristic perivascular pattern to primate cerebellar slices. Using flow cytometry, we found that sera from NMO-IgG-positive patients reacted with CNS-derived human fetal astrocytes, whereas sera from multiple sclerosis patients did not. We demonstrated that NMO-IgG binding to astrocytes alters aquaporin-4 polarized expression and increases permeability of a human BBB endothelium/astrocyte barrier. We further demonstrated that NMO-IgG binding to human fetal astrocytes can result in NK cell degranulation, astrocyte killing by Ab-dependent cellular cytotoxicity and complement-dependent granulocyte attraction through the BBB model. Our study highlights important functional roles for NMO-IgG that could account for pathological lesions and BBB dysfunction observed in NMO.     

Collagen Promotes Higher Adhesion,Survival and Proliferation of Mesenchymal Stem Cells

Mesenchymal stem cells (MSC) can differentiate into several cell types and are desirable candidates for cell therapy and tissue engineering. However, due to poor cell survival, proliferation and differentiation in the patient, the therapy outcomes have not been satisfactory. Although several studies have been done to understand the conditions that promote proliferation, differentiation and migration of MSC in vitro and in vivo, still there is no clear understanding on the effect of non-cellular bio molecules. Of the many factors that influence the cell behavior, the immediate cell microenvironment plays a major role. In this context, we studied the effect of extracellular matrix (ECM) proteins in controlling cell survival, proliferation, migration and directed MSC differentiation. We found that collagen promoted cell proliferation, cell survival under stress and promoted high cell adhesion to the cell culture surface. Increased osteogenic differentiation accompanied by high active RHOA (Ras homology gene family member A) levels was exhibited by MSC cultured on collagen. In conclusion, our study shows that collagen will be a suitable matrix for large scale production of MSC with high survival rate and to obtain high osteogenic differentiation for therapy.     

Hemozoin formation in malaria: a two-step process involving histidine-rich proteins and lipids

Major blood stage antimalarial drugs like chloroquine and artemisinin target the heme detoxification process of the malaria parasite. Hemozoin formation reactions in vitro using the Plasmodium falciparum histidine-rich protein-2 (Pfhrp-2), lipids, and auto-catalysis are slow and could not explain the speed of detoxification needed for parasite survival. Here, we show that malarial hemozoin formation is a coordinated two component process involving both lipids and histidine-rich proteins. Hemozoin formation efficiency in vitro is 1-2% with Pfhrp-2 and 0.25-0.5% with lipids. We added lipids after 9h in a 12h Pfhrp-2 mediated reaction that resulted in sixfold increase in hemozoin formation. However, a lipid mediated reaction in which Pfhrp-2 was added after 9h produced only twofold increase in hemozoin production compared to the reaction with Pfhrp-2 alone. Synthetic peptides corresponding to the Pfhrp-2 heme binding sequences, based on repeats of AHHAAD, neither alone nor in combination with lipids were able to generate hemozoin in vitro. These results indicate that hemozoin formation in malaria parasite involves both the lipids and the scaffolding proteins. Histidine-rich proteins might facilitate hemozoin formation by binding with a large number of heme molecules, and facilitating the dimer formation involving iron-carboxylate bond between two heme molecules, and lipids may then subsequently assist the mechanism of long chain formation, held together by hydrogen bonds or through extensive networking of hydrogen bonds.     

Clostridium perfringens epsilon toxin H149A mutant as a platform for receptor binding studies

Clostridium perfringens epsilon toxin (Etx) is a pore‐forming toxin responsible for a severe and rapidly fatal enterotoxemia of ruminants. The toxin is classified as a category B bioterrorism agent by the U.S. Government Centres for Disease Control and Prevention (CDC), making work with recombinant toxin difficult. To reduce the hazard posed by work with recombinant Etx, we have used a variant of Etx that contains a H149A mutation (Etx‐H149A), previously reported to have reduced, but not abolished, toxicity. The three‐dimensional structure of H149A prototoxin shows that the H149A mutation in domain III does not affect organisation of the putative receptor binding loops in domain I of the toxin. Surface exposed tyrosine residues in domain I of Etx‐H149A (Y16, Y20, Y29, Y30, Y36 and Y196) were mutated to alanine and mutants Y30A and Y196A showed significantly reduced binding to MDCK.2 cells relative to Etx‐H149A that correlated with their reduced cytotoxic activity. Thus, our study confirms the role of surface exposed tyrosine residues in domain I of Etx in binding to MDCK cells and the suitability of Etx‐H149A for further receptor binding studies. In contrast, binding of all of the tyrosine mutants to ACHN cells was similar to that of Etx‐H149A, suggesting that Etx can recognise different cell surface receptors. In support of this, the crystal structure of Etx‐H149A identified a glycan (β‐octyl‐glucoside) binding site in domain III of Etx‐H149A, which may be a second receptor binding site. These findings have important implications for developing strategies designed to neutralise toxin activity.     

Cardiovascular disease after Escherichia coli O157:H7 gastroenteritis

Background:

Escherichia coli O157:H7 is one cause of acute bacterial gastroenteritis, which can be devastating in outbreak situations. We studied the risk of cardiovascular disease following such an outbreak in Walkerton, Ontario, in May 2000.

Methods:

In this community-based cohort study, we linked data from the Walkerton Health Study (2002–2008) to Ontario’s large healthcare databases. We included 4 groups of adults: 3 groups of Walkerton participants (153 with severe gastroenteritis, 414 with mild gastroenteritis, 331 with no gastroenteritis) and a group of 11 263 residents from the surrounding communities that were unaffected by the outbreak. The primary outcome was a composite of death or first major cardiovascular event (admission to hospital for acute myocardial infarction, stroke or congestive heart failure, or evidence of associated procedures). The secondary outcome was first major cardiovascular event censored for death. Adults were followed for an average of 7.4 years.

Results:

During the study period, 1174 adults (9.7%) died or experienced a major cardiovascular event. Compared with residents of the surrounding communities, the risk of death or cardiovascular event was not elevated among Walkerton participants with severe or mild gastroenteritis (hazard ratio [HR] for severe gastroenteritis 0.74, 95% confidence interval [CI] 0.38–1.43, mild gastroenteritis HR 0.64, 95% CI 0.42–0.98). Compared with Walkerton participants who had no gastroenteritis, risk of death or cardiovascular event was not elevated among participants with severe or mild gastroenteritis.

Interpretation:

There was no increase in the risk of cardiovascular disease in the decade following acute infection during a major E. coli O157:H7 outbreak.Escherichia coli O157:H7 is one cause of acute bacterial gastroenteritis, causing 63 000 infections each year and 12 major outbreaks since 2006 in the United States alone.^1,2 This strain was most recently implicated in the outbreak involving beef from XL Foods (September 2012), with 17 confirmed cases across Canada.³ A similar enterohemorrhagic strain E. coli O104:H4 was responsible for an outbreak in Germany in May 2011, causing 3792 cases of gastroenteritis and 43 deaths.^4,5Most patients fully recover from acute gastroenteritis caused by E. coli. However, such an illness may predispose patients to long-term disease. Shiga toxin is produced by E. coli O157:H7; this toxin damages the microvasculature of the kidneys leading to hypertension^6–13 and directly damages the systemic vasculature.^14–16 Infected people may progress from a state of acute inflammation of the vasculature to subclinical chronic inflammation, which could promote atherosclerosis.^17–20In Walkerton, Ontario, in May 2000, heavy rains transported bovine fecal matter into the town’s well, contaminating the inadequately chlorinated municipal water supply with E. coli O157:H7.²¹ Over 2300 people developed acute gastroenteritis, and 7 people died.²² The unique circumstances of this outbreak provided a rare opportunity to study the natural history following exposure to this pathogen in a single cohort.²³ Other outbreaks have been geographically dispersed, making it difficult to track cases.^24,25In Walkerton, affected individuals were followed annually in a clinic to assess their long-term outcomes (Walkerton Health Study, 2002–2008). We previously reported that adults who experienced acute gastroenteritis during the outbreak had a higher than expected incidence of hypertension, chronic kidney disease and self-reported cardiovascular disease in follow-up.²³ However, 46% of participants were lost to follow-up by the end of the study, and there were limitations associated with the assessment of cardiovascular disease by participant recall. Thus, we conducted an expanded and extended follow-up study, linking the Walkerton study data to Ontario’s health care databases. Our objective was to more accurately determine the 10-year risk of major cardiovascular events after exposure to E. coli O157:H7.     

Diversity and metabolic potential of culturable root-associated bacteria from Origanum vulgare in sub-Himalayan region

Study of rhizospheric bacteria from important plants is very essential, as they are known to influence plant growth and productivity, and also produce industrially important metabolites. Origanum vulgare is a perennial medicinal aromatic plant rich in phenolic antioxidants. Present study investigates the diversity of culturable root-associated bacteria from this plant in Palampur, India, which constitutes a unique ecosystem due to high rain fall, wide temperature fluctuations and acidic soil. Both root endophytes and rhizospheric soil bacteria were isolated, which resulted in a total of 120 morphologically different isolates. They were found to group into 21 phylotypes based on restriction fragment length polymorphism analysis. Growth medium composition had significant effect on the diversity of the isolated bacterial populations. The isolates were characterized for various metabolic, plant growth promoting (PGP) and other biotechnologically useful activities, based on which they were clustered into groups by principal component analysis. Majority of the isolates belonged to ??-Proteobacteria and Firmicutes. Pseudomonas and Stenotrophomonas were the most dominant species and together constituted 27.5?% of the total isolates. Many isolates, especially ??-Proteobacteria, showed very high PGP activities. Few isolates exhibited very high antioxidant activity, which may find potential applications in food and health industries. Firmicutes were catabolically the most versatile group and produced several hydrolytic enzymes. To the best of our knowledge, it is the first study describing rhizospheric microbial community of O. vulgare.     

Internal virus polarization model for virus retention by the Ultipor® VF Grade DV20 membrane

Several recent studies have reported a decline in virus retention during virus challenge filtration experiments, although the mechanism(s) governing this phenomenon for different filters remains uncertain. Experiments were performed to evaluate the retention of PP7 and PR772 bacteriophage through Ultipor VF Grade DV20 virus filters during constant pressure filtration. While the larger PR772 phage was fully retained under all conditions, a 2‐log decline in retention of the small PP7 phage was observed at high throughputs, even under conditions where there was no decline in filtrate flux. In addition, prefouling the membrane with an immunoglobulin G solution had no effect on phage retention. An internal polarization model was developed to describe the decline in phage retention arising from the accumulation of phage in the upper (reservoir) layer within the filter which increases the challenge to the lower (rejection) layer. Independent support for this internal polarization phenomenon was provided by confocal microscopy of fluorescently labeled phage within the membrane. The model was in good agreement with phage retention data over a wide range of phage titers, confirming that virus retention is throughput dependent and supporting current recommendations for virus retention validation studies. These results provide important insights into the factors governing virus retention by membrane filters and their dependence on the underlying structure of the virus filter membrane. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:856–863, 2014     

Burden of Complicated Malaria in a Densely Forested Bastar Region of Chhattisgarh State (Central India)

Background

A prospective study on severe and complicated malaria was undertaken in the tribal dominated area of Bastar division, Chhattisgarh (CG), Central India, with an objective to understand the clinical epidemiology of complicated malaria in patients attending at a referral hospital.

Methods

Blood smears, collected from the general medicine and pediatric wards of a government tertiary health care facility located in Jagdalpur, CG, were microscopically examined for malaria parasite from July 2010 to December 2013. The Plasmodium falciparum positive malaria cases who met enrollment criteria and provided written informed consent were enrolled under different malaria categories following WHO guidelines. PCR was performed to reconfirm the presence of P.falciparum mono infection among enrolled cases. Univariate and multivariate logistic regression analysis was done to identify different risk factors using STATA 11.0.

Results

A total of 40,924 cases were screened for malaria. The prevalence of malaria and P.falciparum associated complicated malaria (severe and cerebral both) in the hospital was 6% and 0.81%, respectively. P.falciparum malaria prevalence, severity and associated mortality in this region peaked at the age of>4–5 years and declined with increasing age. P.falciparum malaria was significantly more prevalent in children than adults (P<0.00001). Among adults, males had significantly more P.falciparum malaria than females (P<0.00001). Case fatality rate due to cerebral malaria and severe malaria was, respectively, 32% and 9% among PCR confirmed mono P.falciparum cases. Coma was the only independent predictor of mortality in multivariate regression analysis. Mortality was significantly associated with multi-organ complication score (P = 0.0003).

Conclusion

This study has revealed that the pattern of morbidity and mortality in this part of India is very different from earlier reported studies from India. We find that the peak morbidity and mortality in younger children regardless of seasonality. This suggests that this age group needs special care for control and clinical management.     

Functional and phylogenetic diversity of root-associated bacteria of Ajuga bracteosa in Kangra valley

Present study investigates the cultivable diversity of root-associated bacteria from a medicinal plant Ajuga bracteosa in the Kangra valley, in order to determine their plant growth promoting (PGP) and biotechnological potential. The plant was found to exhibit a positive rhizosphere effect of 1.3-1.5. A total of 123 morphologically different bacteria were isolated from the rhizospheric soil and roots of the plant. Medium composition was found to have significant effect on the composition of isolated bacterial populations. Majority of the rhizospheric soil isolates belonged to α- and γ-Proteobacteria, with Pseudomonas constituting the most dominant species. Endophytic bacterial community, on other hand, consisted almost exclusively of Firmicutes. Majority of the isolates showed PGP activity by producing siderophores and indole acetic acid. Several isolates were found to exhibit very high antioxidant activity in the culture medium. A significant proportion of isolates also demonstrated other ecologically important activities like phosphate solubilization, nitrogen fixation, and production of hydrolytic enzymes including amylase, protease, lipase, chitinase, cellulase, pectinase and phosphatase. Firmicutes were found to be metabolically the most versatile group and performed multiple enzyme activities. This is the first systematic study of culturable bacterial community from the rhizosphere of A. bracteosa, particularly in the Kangra valley region.     

Antibacterial and ulcer healing effects of organoselenium compounds in naproxen induced and Helicobacter pylori infected Wistar rat model

Aim of the present study was to evaluate in vitro toxicity and in vivo antibacterial, anti-inflammatory, antiulcer, and antioxidant activities of two organoselenium compounds, selenocystine (SeCys) and ebselen (Ebs). The study was conducted in experimentally induced ulcers in rodent model infected with Helicobacter pylori (H. pylori). In vitro toxicological studies on normal spleenic lymphocytes revealed that SeCys and Ebs were non-toxic to the cells even at 100 μM concentration. Antibacterial activity was observed at 500 μg/mL concentration of either of the compounds against H. pylori. In vivo studies after treatment with SeCys and Ebs (500 μg/kg/day) resulted in significant reduction in ROS production and inhibition of lipid peroxidation in gastric tissue. The antioxidant and anti-inflammatory activities of both the compounds were also confirmed by their ability to lower GSH reduction, to induce the expression of antioxidant genes such as GPx-4, and MnSOD and to suppress inflammatory genes namely COX-2, TNF-α and TGF-β. In addition, the immunomodulatory activity of both the compounds was evident by enhance of the CD4 levels and maintenance of the IgG, IL-6 and IL-10 levels. Persistent treatment (500 μg/kg, for 28 days) with both the compounds showed considerable (p < 0.05) ulcer healing property supporting its role in gastro protection. In conclusion, the results of our study suggest that both SeCys and Ebs possess broad spectrum of activities without any potential toxicity.