Co-administration of IL-1+IL-6+TNF-α with Mycobacterium tuberculosis infected macrophages vaccine induces better protective T cell memory than BCG

BCG has been administered globally for more than 75 years, yet tuberculosis (TB) continues to kill more than 2 million people annually. Further, BCG protects childhood TB but is quite inefficient in adults. This indicates that BCG fails to induce long-term protection. Hence there is a need to explore alternative vaccination strategies that can stimulate enduring T cell memory response. Dendritic cell based vaccination has attained extensive popularity following their success in various malignancies. In our previous study, we have established a novel and unique vaccination strategy against Mycobacterium tuberculosis (M. tb) and Salmonella typhimurium by utilizing infected macrophages (IM). In short-term experiments (30 days), substantial degree of protection was observed. However, remarkable difference was not observed in long-term studies (240 days) due to failure of the vaccine to generate long-lasting memory T cells. Hence, in the present study we employed T cell memory augmenting cytokines IL-1+IL-6+TNF-α and IL-7+IL-15 for the induction of the enhancement of long-term protection by the vaccine. We co-administered the M. tb infected macrophages vaccine with IL-1+IL-6+TNF-α (IM-1.6.α) and IL-7+IL-15 (IM-7.15). The mice were then rested for a reasonably large period (240 days) to study the bona fide T cell memory response before exposing them to aerosolized M. tb. IM-1.6.α but not IM-7.15 significantly improved memory T cell response against M. tb, as evidenced by recall responses of memory T cells, expansion of both central as well as effector memory CD4 and CD8 T cell pools, elicitation of mainly Th1 memory response, reduction in the mycobacterial load and alleviated lung pathology. Importantly, the protection induced by IM-1.6.α was significantly better than BCG. Thus, this study demonstrates that not only antigen-pulsed DCs can be successfully employed as vaccines against cancer and infectious diseases but also macrophages infected with M. tb can be utilized with great efficacy especially in protection against TB.     

Rescuing melanoma epitope-specific cytolytic T lymphocytes from activation-induced cell death, by SP600125, an inhibitor of JNK: implications in cancer immunotherapy

Activation-induced cell death (AICD) as well as programmed cell death (PCD) serve to control the expansion of activated T cells to limit untoward side effects of continued effector responses by T cells and to maintain homeostasis. AICD of T cells in tumor immunotherapy can be counterproductive particularly if the activated T cells undergo apoptotic death after the very first secondary encounter of the specific epitope. We examined the extent to which tumor epitope-specific CTLs that are activated and expanded in an in vitro-matured dendritic cell-based primary stimulation protocol undergo AICD following their first secondary encounter of the cognate epitope. Using the MART-1(27-35) epitope as a prototype vaccine epitope, we also examined whether these CTLs could be rescued from AICD. Our results demonstrate that a substantial fraction of MART-1(27-35) epitope-specific primary CTLs undergo AICD upon the very first secondary encounter of the cognate epitope. The AICD in these CTLs is neither caspase dependent nor is it triggered by the extrinsic death signaling pathways (Fas, TNFR, etc.). These CTLs, interestingly, could be rescued from AICD by the JNK inhibitor, SP600125. We also found that SP600125 interferes with their IFN-gamma response but does not block their cytolytic function. The rescued CTLs, however, regain their capacity to synthesize IFN-gamma if continued in culture without the inhibitor. These observations have implications in tumor immunotherapy and in further studies for regulation of AICD in CTLs.     

A neural network approach for the prediction of in vitro culture parameters for maximum biomass yields in hairy root cultures

The present study deals with ANN based prediction of culture parameters in terms of inoculum density, pH and volume of growth medium per culture vessel and sucrose content of the growth medium for Glycyrrhiza hairy root cultures. This kind of study could be a model system in exploitation of hairy root cultures for commercial production of pharmaceutical compounds using large bioreactors. The study is aimed to evaluate the efficiency of regression neural network and back propagation neural network for the prediction of optimal culture conditions for maximum hairy root biomass yield. The training data for regression and back propagation networks were primed on the basis of function approximation, where final biomass fresh weight (f_wt) was considered as a function of culture parameters. On this basis the variables in culture conditions were described in the form of equations which are for inoculum density: y=0.02x+0.04, for pH of growth medium: y=x+2.8, for sucrose content in medium: y=9.9464x+(−9.7143) and for culture medium per culture vessel: y=10x. The fresh weight values obtained from training data were considered as target values and further compared with predicted fresh weight values. The empirical data were used as testing data and further compared with values predicted from trained networks. Standard MATLAB inbuilt generalized regression network with radial basis function radbas as transfer function in layer one and purelin in layer two and back propagation having purelin as transfer function in output layer and logsig in hidden layer were used. Although in comparative assessment both the networks were found efficient for prediction of optimal culture conditions for high biomass production, more accuracy in results was seen with regression network.     

MicroRNA-21 Is a Downstream Effector of AKT That Mediates Its Antiapoptotic Effects via Suppression of Fas Ligand

MicroRNA-21 (miR-21) is highly up-regulated during hypertrophic and cancerous cell growth. In contrast, we found that it declines in cardiac myocytes upon exposure to hypoxia. Thus, the objective was to explore its role during hypoxia. We show that miR-21 not only regulates phosphatase and tensin homologue deleted on chromosome 10 (PTEN), but also targets Fas ligand (FasL). During prolonged hypoxia, down-regulation of miR-21 proved necessary and sufficient for enhancing expression of both proteins. We demonstrate here for the first time that miR-21 is positively regulated via an AKT-dependent pathway, which is depressed during prolonged hypoxia. Accordingly, hypoxia-induced down-regulation of miR-21 and up-regulation of FasL and PTEN were reversed by activated AKT and reproduced by a dominant negative mutant, wortmannin, or PTEN. Moreover, the antiapoptotic function of AKT partly required miR-21, which was sufficient for inhibition of caspase-8 activity and mitochondrial damage. In consensus, overexpression of miR-21 in a transgenic mouse heart resulted in suppression of ischemia-induced up-regulation of PTEN and FasL expression, an increase in phospho-AKT, a smaller infarct size, and ameliorated heart failure. Thus, we have identified a unique aspect of the function of AKT by which it inhibits apoptosis through miR-21-dependent suppression of FasL.     

Modified iridoid glycosides as anti-implantation agents: inhibition of cell adhesion as an approach for developing pregnancy interceptive agents

Structural modifications in iridoid glycosides and evaluation of their efficacy on adhering capability (in vitro) of immature hamster uterine epithelial cells to the substratum have been studied. Out of 31, eight compounds in vitro, five compounds in utero and two in vivo showed adhesion/implantation preventing activity, respectively. The results provide an indication for further exploration in the line of development of anti-adhesive agents.     

POLG mutation in a patient with cataracts, early-onset distal muscle weakness and atrophy, ovarian dysgenesis and 3-methylglutaconic aciduria

Mutations in POLG account for one of the most frequent nuclear encoded causes of mitochondrial disorders to date. Individuals harboring POLG mutations exhibit fairly heterogeneous clinical presentations leading to increasing difficulties in classifying these patients into defined clinical phenotypes. This study aims to investigate the molecular basis of a mitochondrial cytopathy in a patient with 3-methylglutaconic aciduria and to expand the clinical phenotype associated with POLG mutations. Clinical, molecular and genetic analyses as well as neurophysiological examinations were carried out for a 23-year-old woman of mixed Caucasian and Latin American ancestry with a history of cataracts diagnosed at age 1 year, she had onset of distal muscle weakness at age 2 years progressing to atrophy and ovarian dysgenesis at puberty. The patient was found to have 3-methylglutaconic acid with normal 3 hydroxyisovaleric acid on urine organic acid analysis. POLG sequencing was done and a heterozygous variant, c.2851T>A (p.Y951N) was found which is predicted to be deleterious. There are limited reports of POLG mutations in individuals with 3-methylglutaconic aciduria. This case report of a young woman with a heterozygous mutation in POLG, presenting with muscle weakness and atrophy at a young age aims to aid clinicians in similar challenging diagnostic situations as well as enhances our understanding of POLG-related disease phenotypes.