The Contribution of Non-Conventional T Cells and NK Cells in the Mycobacterial-Specific IFNγ Response in Bacille Calmette-Guérin (BCG)-Immunized Infants

Background

The Mycobacterium bovis Bacille Calmette-Guérin (BCG) vaccine is given to >120 million infants each year worldwide. Most studies investigating the immune response to BCG have focused on adaptive immunity. However the importance of TCR-gamma/delta (γδ) T cells and NK cells in the mycobacterial-specific immune response is of increasing interest.

Methods

Participants in four age-groups were BCG-immunized. Ten weeks later, in vitro BCG-stimulated blood was analyzed for NK and T cell markers, and intracellular IFNgamma (IFNγ) by flow cytometry. Total functional IFNγ response was calculated using integrated median fluorescence intensity (iMFI).

Results

In infants and children, CD4 and CD4-CD8- (double-negative (DN)) T cells were the main IFNγ-expressing cells representing 43-56% and 27-37% of total CD3+ IFNγ+ T cells respectively. The iMFI was higher in DN T cells compared to CD4 T cells in all age groups, with the greatest differences seen in infants immunized at birth (p=0.002) or 2 months of age (p<0.0001). When NK cells were included in the analysis, they accounted for the majority of total IFNγ-expressing cells and, together with DN Vδ2 γδ T cells, had the highest iMFI in infants immunized at birth or 2 months of age.

Conclusion

In addition to CD4 T cells, NK cells and DN T cells, including Vδ2 γδ T cells, are the key populations producing IFNγ in response to BCG immunization in infants and children. This suggests that innate immunity and unconventional T cells play a greater role in the mycobacterial immune response than previously recognized and should be considered in the design and assessment of novel tuberculosis vaccines.     

Identification of Key Contributory Factors Responsible for Vascular Dysfunction in Idiopathic Recurrent Spontaneous Miscarriage

Poor endometrial perfusion during implantation window is reported to be one of the possible causes of idiopathic recurrent spontaneous miscarriage (IRSM). We have tested the hypothesis that certain angiogenic and vasoactive factors are associated with vascular dysfunction during implantation window in IRSM and, therefore, could play a contributory role in making the endometrium unreceptive in these women. This is a prospective case-controlled study carried out on 66 women with IRSM and age and BMI matched 50 fertile women serving as controls. Endometrial expression of pro-inflammatory (IL-1β, TNF-α, IFN-γ, TGF-β1), anti-inflammatory (IL-4, -10), angiogenesis-associated cytokines (IL-2, -6, -8), angiogenic and vasoactive factors including prostaglandin E2 (PGE2), vascular endothelial growth factor (VEGF), endothelial nitric oxide synthase (eNOS), nitric oxide (NO) and adrenomedullin (ADM) were measured during implantation window by ELISA. Subendometrial blood flow (SEBF) was assessed by color Doppler ultrasonography. Multivariate analysis was used to identify the significant factor(s) responsible for vascular dysfunction in IRSM women during window of implantation and further correlated with vascular dysfunction. Endometrial expression of pro-inflammatory cytokines and PGE2 were up-regulated and anti-inflammatory and angiogenesis-associated cytokines down-regulated in IRSM women as compared with controls. Further, the angiogenic and vasoactive factors including VEGF, eNOS, NO and ADM were found to be down-regulated and SEBF grossly affected in these women. Multivariate analysis identified IL-10, followed by VEGF and eNOS as the major factors contributing towards vascular dysfunction in IRSM women. Moreover, these factors strongly correlated with blood flow impairment. This study provides an understanding that IL-10, VEGF and eNOS are the principal key components having a contributory role in endometrial vascular dysfunction in women with IRSM. Down-regulation of these factors is also associated with impaired endometrial perfusion which possibly makes the endometrium unreceptive that may eventually cause early pregnancy loss.     

Microbiota Dynamics in Patients Treated with Fecal Microbiota Transplantation for Recurrent Clostridium difficile Infection

Clostridium difficile causes antibiotic-associated diarrhea and pseudomembraneous colitis and is responsible for a large and increasing fraction of hospital-acquired infections. Fecal microbiota transplantation (FMT) is an alternate treatment option for recurrent C. difficile infection (RCDI) refractory to antibiotic therapy. It has recently been discussed favorably in the clinical and scientific communities and is receiving increasing public attention. However, short- and long-term health consequences of FMT remain a concern, as the effects of the transplanted microbiota on the patient remain unknown. To shed light on microbial events associated with RCDI and treatment by FMT, we performed fecal microbiota analysis by 16S rRNA gene amplicon pyrosequencing of 14 pairs of healthy donors and RCDI patients treated successfully by FMT. Post-FMT patient and healthy donor samples collected up to one year after FMT were studied longitudinally, including one post-FMT patient with antibiotic-associated relapse three months after FMT. This analysis allowed us not only to confirm prior reports that RCDI is associated with reduced diversity and compositional changes in the fecal microbiota, but also to characterize previously undocumented post-FMT microbiota dynamics. Members of the Streptococcaceae, Enterococcaceae, or Enterobacteriaceae were significantly increased and putative butyrate producers, such as Lachnospiraceae and Ruminococcaceae were significantly reduced in samples from RCDI patients before FMT as compared to post-FMT patient and healthy donor samples. RCDI patient samples showed more case-specific variations than post-FMT patient and healthy donor samples. However, none of the bacterial groups were invariably associated with RCDI or successful treatment by FMT. Overall microbiota compositions in post-FMT patients, specifically abundances of the above-mentioned Firmicutes, continued to change for at least 16 weeks after FMT, suggesting that full microbiota recovery from RCDI may take much longer than expected based on the disappearance of diarrheal symptoms immediately after FMT.     

Association of Weather and Anthropogenic Factors for Transmission of Japanese Encephalitis in an Endemic Area of India

Weather and anthropogenic factors are important determinants for Japanese encephalitis (JE) transmission. During 2008–2010, an increasing trend of JE was observed in Dibrugarh district of Northeast India. The JE cases were found to be clustered between June to October in each year. Monthly minimum temperature and rainfall were significantly associated with JE transmission at 1 and 2 months lagged. However, the relationship was more prominent at a lag of 1 month than that of two. Regression analysis suggested that rainfall, minimum and maximum temperature, and relative humidity at 6:00 h are significant predictors (P < 0.05) of quarterly occurrence of JE cases. Additional anthropogenic risk factors including the conditions such as pig sty/cattle shed around and lower part of the houses and proximity of rice field to the dwelling houses (P < 0.05) were also found to be predictors for JE occurrence. Meteorological and anthropogenic risk factors can be used to forecast JE outbreaks in Assam which in turn can help the local health authorities to protect communities in JE prone areas.     

Upregulated autophagy protects cardiomyocytes from oxidative stress-induced toxicity

Autophagy is a cellular self-digestion process that mediates protein quality control and serves to protect against neurodegenerative disorders, infections, inflammatory diseases and cancer. Current evidence suggests that autophagy can selectively remove damaged organelles such as the mitochondria. Mitochondria-induced oxidative stress has been shown to play a major role in a wide range of pathologies in several organs, including the heart. Few studies have investigated whether enhanced autophagy can offer protection against mitochondrially-generated oxidative stress. We induced mitochondrial stress in cardiomyocytes using antimycin A (AMA), which increased mitochondrial superoxide generation, decreased mitochondrial membrane potential and depressed cellular respiration. In addition, AMA augmented nuclear DNA oxidation and cell death in cardiomyocytes. Interestingly, although oxidative stress has been proposed to induce autophagy, treatment with AMA did not result in stimulation of autophagy or mitophagy in cardiomyocytes. Our results showed that the MTOR inhibitor rapamycin induced autophagy, promoted mitochondrial clearance and protected cardiomyocytes from the cytotoxic effects of AMA, as assessed by apoptotic marker activation and viability assays in both mouse atrial HL-1 cardiomyocytes and human ventricular AC16 cells. Importantly, rapamycin improved mitochondrial function, as determined by cellular respiration, mitochondrial membrane potential and morphology analysis. Furthermore, autophagy induction by rapamycin suppressed the accumulation of ubiquitinylated proteins induced by AMA. Inhibition of rapamycin-induced autophagy by pharmacological or genetic interventions attenuated the cytoprotective effects of rapamycin against AMA. We propose that rapamycin offers cytoprotection against oxidative stress by a combined approach of removing dysfunctional mitochondria as well as by degrading damaged, ubiquitinated proteins. We conclude that autophagy induction by rapamycin could be utilized as a potential therapeutic strategy against oxidative stress-mediated damage in cardiomyocytes.     

Improved protein A resin for antibody capture in a continuous countercurrent tangential chromatography system

Continuous countercurrent tangential chromatography (CCTC) enables steady-state continuous bioprocessing with low-pressure operation and high productivity. CCTC has been applied to initial capture of monoclonal antibodies (mAb) from clarified cell culture harvest and postcapture polishing of mAb; however, these studies were performed with commercial chromatography resins designed for conventional column chromatography. In this study, a small particle size prototype agarose resin (20–25 µm) with lower cross-linking was co-developed with industrial partner Purolite and tested with CCTC. Due to increased binding capacity and faster kinetics, the resulting CCTC process showed more than a 2X increase in productivity, and a 2X reduction in buffer consumption over commercial protein A resins used in previous CCTC studies, as well as more than a 10X productivity increase versus conventional column operation. Single-pass tangential flow filtration was integrated with the CCTC system, enabling simple control of eluate concentration. A scale-up exercise was conducted to provide a quantitative comparison of CCTC and batch column chromatography. These results clearly demonstrate opportunities for using otherwise unpackable soft small particle size resins with CCTC as the core of a continuous bioprocessing platform.     

Epidemiology and prediction of brown leaf rust of mulberry caused by Peridiopsora mori

Brown leaf rust (BLR) caused by Peridiopsora mori is one of the major foliar diseases of mulberry (Morus sp.) in the subtropical hills of eastern India. The disease appeared in first week of August and continued up to September with maximum severity in second and third week of September. The disease symptoms appeared at atmospheric temperature (27.00–20.07°C), relative humidity (92.14–82.43%), rainfall (11.20 cm) and rainy days (7) of the preceding week. Disease severity (>50 PDI) was observed at temperature (26.29–19.29°C), relative humidity (94.14–80.14%), rainfall (4.12 cm) and number of rainy days (2–3 days). Apparent rate of infection was found high at temperature (27.00–19.83°C), relative humidity (94.67–85.00%), rainfall (4.6 cm) and rainy days (2) of the preceding week. The correlation coefficient between disease severity and average meteorological factors of the preceding 7 days revealed that BLR disease severity showed significant negative correlation with minimum temperature. It was also revealed that contribution of maximum and minimum temperature 42.23% and 35.21%, maximum and minimum relative humidity (RH) 11.23% and 10.69% and rainfall and number of rainy days 0.11% and 0.50%, respectively towards development of BLR disease severity. Multiple regression analysis revealed that average of maximum and minimum temperatures and minimum RH of preceding 7 days were found to maximally influence BLR disease severity.     

Signaling role of nitric oxide in the induction of plant defense by exogenous application of abiotic inducers

The objective of this work was to search out the probable molecule behind the activation of broad spectrum resistance during abiotic elicitors such as arachidonic acid, cupric chloride, chitosan, isonicotinic acid and salicylic acid mediated induced systemic resistance (ISR) in Raphanus sativus L. The elicitor compounds were sprayed on the radish leaves of healthy plant and after 24 h incubation a significant increase of β-1,3 glucanase, peroxidase, polyphenol oxidase and phenolics as well as a remarkable increase of nitric oxide (NO), a probable potent defense-signaling molecule in plant, was observed. Furthermore, treatment of the host with NO donor, sodium nitroprusside, also induced the same defense molecules. The results suggests that NO might be the signaling molecule during abiotic elicitor mediated ISR induction in the host system.     

Habitat Preference and Diversity of Anuran in Durgapur, an Industrial City of West Bengal, India

Anuran diversity study was conducted for one year between June 2008 and August 2009 at Durgapur, an industrial city of West Bengal, India. Nine species under four families of the Order Anura were found to occur in Durgapur. They have a wide range of habitat preference like permanent or temporary aquatic bodies, human residential area, forested areas, termite nest, tree hole, under spaces of logs and so forth. Of the nine sp. 8 were confined to Amarabati pond (site-I), 5 were confined to Durgapur Government College campus (site-II) and 7 were confined at Fuljhore (site-III) of Durgapur. Analysis of the collected data on the anuran community of the study area revealed Shannon–Wiener species diversity index minimum (1.312) at site-II and maximum (1.938) at site-III. In contrast Margalef richness index value was minimum (0.627) at site-II and maximum (1.424) at site-III.     

Identification and molecular cytogenetic characterization of a novel complex Y chromosome rearrangement in a boy with disorder of sexual development

Ambiguous genitalia or disorder of the sexual development is a birth defect where the external genitals do not have the typical appearance of either a male or female. Here we report a boy with ambiguous genitalia and short stature. The cytogenetic analysis by G-banding revealed a small Y chromosome and an additional material on the 15p arm. Further, molecular cytogenetic analysis by Fluorescence in situ hybridization (FISH) using whole chromosome paint probes showed the presence of Y sequences on the 15p arm, confirming that it is a Y;15 translocation. Subsequent, FISH with centromere probe Y showed two signals depicting the presence of two centromeres and differing with a balanced translocation. The dicentric nature of the derivative 15 chromosome was confirmed by FISH with both 15 and Y centromeric probes. Further, the delineation of the Y chromosomal DNA was also done by quantitative real time PCR. Additional Y-short tandem repeat typing was performed to find out the extent of deletion on small Y chromosome. Fine mapping was carried out with 8 Y specific BAC clones which helped in defining the breakpoint regions. MLPA was performed to check the presence or absence of subtelomeric regions and SHOX regions on Y. Finally array CGH helped us in confirming the breakpoint regions. In our study we identified and characterized a novel complex Y chromosomal rearrangement with a complete deletion of the Yq region and duplication of the Yp region with one copy being translocated onto the15p arm. This is the first report of novel and unique Y complex rearrangement showing a deletion, duplication and a translocation in the same patient. The possible mechanism of the rearrangement and the phenotype–genotype correlation are discussed.