Neem Leaf Glycoprotein Activates CD8+ T Cells to Promote Therapeutic Anti-Tumor Immunity Inhibiting the Growth of Mouse Sarcoma

In spite of sufficient data on Neem Leaf Glycoprotein (NLGP) as a prophylactic vaccine, little knowledge currently exists to support the use of NLGP as a therapeutic vaccine. Treatment of mice bearing established sarcomas with NLGP (25 µg/mice/week subcutaneously for 4 weeks) resulted in tumor regression or dormancy (Tumor free/Regressor, 13/24 (NLGP), 4/24 (PBS)). Evaluation of CD8⁺ T cell status in blood, spleen, TDLN, VDLN and tumor revealed increase in cellular number. Elevated expression of CD69, CD44 and Ki67 on CD8⁺ T cells revealed their state of activation and proliferation by NLGP. Depletion of CD8⁺ T cells in mice at the time of NLGP treatment resulted in partial termination of tumor regression. An expansion of CXCR3⁺ and CCR5⁺ T cells was observed in the TDLN and tumor, along with their corresponding ligands. NLGP treatment enhances type 1 polarized T-bet expressing T cells with downregulation of GATA3. Treg cell population was almost unchanged. However, T∶Treg ratios significantly increased with NLGP. Enhanced secretion/expression of IFNγ was noted after NLGP therapy. In vitro culture of T cells with IL-2 and sarcoma antigen resulted in significant enhancement in cytotoxic efficacy. Consistently higher expression of CD107a was also observed in CD8⁺ T cells from tumors. Reinoculation of sarcoma cells in tumor regressed NLGP-treated mice maintained tumor free status in majority. This is correlated with the increment of CD44^hiCD62L^hi central memory T cells. Collectively, these findings support a paradigm in which NLGP dynamically orchestrates the activation, expansion, and recruitment of CD8⁺ T cells into established tumors to operate significant tumor cell lysis.     

Molecular alterations in oral carcinogenesis: significant risk predictors in malignant transformation and tumor progression

In this study an attempt was made to establish the significance of a battery of molecular alterations and thereby identify risk predictors in oral carcinogenesis. For this purpose, EGFR, Stat3, H-ras, c-myc, p53, cyclin D1, p16, Rb, Ki-67 and Bcl-2 were localized immunohistochemically in normal mucosa (n=12), hyperplasia (n=35), dysplasia (n=25), early stage carcinoma (n=65) and advanced stage carcinoma (n=70). Deregulation occurred at an early stage and the number of alterations increased with disease progression. Using multivariate logistic regression analysis, the significant risk predictor for hyperplasia from normal mucosa was Ki-67 (OR=5.75, p=0.021); the significant risk predictors for dysplasia from hyperplasia were EGFR (OR=12.96, p=0.002), Stat3 (OR=17.16, p=0.0001), p16 (OR=5.50, p=0.039) and c-myc (OR=5.99, p=0.052); the significant risk predictors for early stage carcinoma from dysplasia were p53 (OR=6.63, p=0.0001) and Rb (OR=3.81, p=0.056); and the significant risk predictors for further progression were EGFR (OR=5.50, p=0.0001), Stat3 (OR=4.49, p=0.0001), H-ras (OR=4.05, p=0.001) and c-myc (OR=2.99, p=0.015). Cyclin D1 holds a key position linking upstream signaling pathways to cell cycle regulation. Gene products of the mitogenic signaling pathway play an equally significant role as cell cycle regulatory proteins in the hyperplasia-dysplasia-early-advanced-carcinoma sequence and together may provide a reference panel of markers for use in defining premalignant lesions and predicting the risk of malignant transformation and tumor progression.     

Role of virulence plasmid of Aeromonas hydrophila in the pathogenesis of ulcerative disease syndrome in Clarias batrachus

Pathogenic Aeromonas hydrophila (strain VB21), a multiple-drug resistance strain contains a plasmid of about 21 kb. After curing of plasmid, the isolates became sensitive to antimicrobials, to which they were earlier resistant. The cured bacteria exhibited significant alterations in their surface structure, growth profile and virulence properties, and failed to cause ulcerative disease syndrome (UDS) when injected into the Indian catfish Clarias batrachus. Routine biochemical studies revealed that the plasmid curing did not alter the biochemical properties of the bacteria. After transformation of the plasmid into cured A. hydrophila the bacterium regained its virulence properties and induced all the characteristic symptoms of UDS when injected into fish. Thus, the plasmid plays a pivotal role in the phenotype, growth and virulence of A. hydrophila and pathogenesis of aeromonad UDS.     

Correlates of Treatment Outcomes and Drug Resistance among Pulmonary Tuberculosis Patients Attending Tertiary Care Hospitals of Kolkata,India

Background

Worldwide highest number of new pulmonary tuberculosis (PTB) cases, was reported from India in 2012. Adverse treatment outcomes and emergence of drug resistance further complicated the prevailing scenario owing to increased duration, cost and toxicity associated with the treatment of drug-resistant cases. Hence to reinforce India’s fight against TB, identification of the correlates of adverse treatment outcomes and drug resistance, seemed critical.

Methods

To estimate the associations between diagnostic findings, patient types (based on treatment outcomes), drug resistance and socio-demographic characteristics of PTB patients, a cross-sectional study was conducted in two tertiary-care hospitals in Kolkata between April 2010 and March 2013. Altogether, 350 consenting Mycobacterium tuberculosis sputum-culture positive PTB patients were interviewed about their socio-demographic background, evaluated regarding their X-ray findings (minimal/moderately advanced/far advanced/cavities), sputum-smear positivity, and treatment history/outcomes (new/defaulter/relapse/treatment-failure cases). Multiple-allele-specific polymerase chain reaction (MAS-PCR) was conducted to diagnose drug resistance.

Results

Among all participants, 31.43% were newly diagnosed, while 44%, 15.43% and 9.14% patients fell into the categories of relapsed, defaulters and treatment-failures, respectively. 12.29% were multi-drug-resistant (MDR: resistant to at least isoniazid and rifampicin), 57.71% had non-MDR two-drug resistance and 12% had single-drug resistance. Subjects with higher BMI had lower odds of being a relapse/defaulter/treatment failure case while females were more likely to be defaulters and older age-groups had more relapse. Elderly, females, unmarried, those with low BMI and higher grade of sputum-smear positivity were more likely to have advanced X-ray features. Higher grade of sputum-smear positivity and advanced chest X-ray findings were associated with relapse/treatment-failures. Elderly, unmarried, relapse/defaulter/treatment-failure cases had higher odds and those with higher BMI and moderately/far advanced X-ray findings had lower odds of having MDR/non-MDR two-drug resistant PTB.

Conclusion

Targeted intervention and appropriate counseling are needed urgently to prevent adverse treatment outcomes and development of drug resistance among PTB patients in Kolkata.     

Establishment and characterization of a piscean PCF cell line for toxicity and gene expression studies as in vitro model

A new piscean fibroblastic cell line termed as PCF derived from the caudal fin tissue of dark mahseer, Puntius (Tor) chelynoides was established and characterized in the present study which was found to be suitable for toxicity and gene expression studies as in vitro model. The cell line grew well in Leibovitz's L-15 medium supplemented with 10% fetal bovine serum (FBS). The cells were able to grow at a temperature ranging from 20 to 28 °C with an optimal growth at 24 °C and the cell line have been expanded in culture for more than 70 passages. Authentication of the cell line was carried out using mitochondrial DNA markers (Cytochrome Oxidase subunit I and 16S ribosomal RNA). Presence of vimentin in the cells confirmed the fibroblastic origin of cell line. Significant cytopathic effects were observed upon exposure of PCF cell line to bacterial extracellular products and the study also validated the suitability of cell line in transgenic applications as well as in genotoxicity assessment as an in vitro model.     

Bio-transformation of artemisinin using soil microbe: Direct C-acetoxylation of artemisinin at C-9 by Penicillium simplissimum

Potent antimalarial compound artemisinin, 1 was bio-transformed to C-9 acetoxy artemisinin, 2 using soil microbe Penicillium simplissimum along with C-9 hydroxy derivative 3. The products were characterized using high field NMR and MS–MS data. The absolute stereochemistry of the newly generated chiral centers has been ascertained by COSY and 1D NOESY experiments. This is the first Letter of direct C-acetoxylation of artemisinin using microbial strains.     

Morphological integration in the carnivoran skull

The correlated evolution of traits may be a principal factor in morphological evolution, but it is typically studied in genetic or developmental systems. Most studies examining phenotypic trait correlations, through analysis of morphological integration, consider only few taxa, with limited ability to test hypotheses of the influence of trait integration on morphological variation and diversity. The few comparative studies in less inclusive groups have yielded varying relationships of integration to the key factors of phylogeny and diet. In this paper, I present analyses of cranial morphological integration in 30 species from the mammalian order Carnivora, spanning eight extant families and a wide range of ecological and morphological diversity. Fifty-five cranial landmarks were captured through three-dimensional digitization of 15-22 specimens for each species. Using a node-based phylogenetic distance matrix, a significant correlation was found between similarity in patterns of integration and phylogenetic relatedness within Felidae (cats) and Canidae (dogs), but not within more inclusive clades, when size-related variation was removed. When size was included, significant correlations were found across all Caniformia, Musteloidea, Mustelidae, and Felidae. There was a significant correlation between phylogeny and morphological integration only within the higher-level clade Feliformia (cats, civets, mongooses, and hyaenas) when a branch-length-based phylogenetic distance matrix was analyzed, with and without size. In contrast, diet was significantly correlated with similarity in morphological integration in arctoid carnivorans (bears, raccoons, and weasels), but had no significant relationship with integration in feliforms or canids. These results support the proposition that evolutionary history is correlated with cranial integration across large clades, although in some smaller clades diet also exerts significant influence on the correlated evolution of traits.     

Cranial modularity shifts during mammalian evolution

The mammalian skull has been studied as several separate functional components for decades, but the study of modularity is a more recent, integrative approach toward quantitative examination of independent subsets of highly correlated traits, or modules. Although most studies of modularity focus on developmental and genetic systems, phenotypic modules have been noted in many diverse morphological structures. However, few studies have provided empirical data for comparing modules across higher taxonomic levels, limiting the ability to assess the broader evolutionary significance of modularity. This study uses 18-32 three-dimensional cranial landmarks to analyze phenotypic modularity in 106 mammalian species and demonstrates that cranial modularity is generally conserved in the evolution of therian mammals (marsupials and placentals) but differs between therians and monotremes, the two extant subclasses of Mammalia. Within therians, cluster analyses identify six distinct modules, but only three modules display significant integration in all species. Monotremes display only two highly integrated modules. Specific hypotheses of functional and developmental influences on cranial bones were tested. Theoretical correlation matrices for bones were constructed on the basis of shared function, tissue origin, or mode of ossification, and all three of these models are significantly correlated with observed correlation matrices for the mammalian cranium.     

Aspirin induces apoptosis through the inhibition of proteasome function

Aspirin and other nonsteroidal anti-inflammatory drugs inhibit cell proliferation and induce apoptosis in various cancer cell lines, which is considered to be an important mechanism for their anti-tumor activity and prevention of carcinogenesis. However, the molecular mechanisms through which these compounds induce apoptosis are not well understood. Here we have found that aspirin treatment of the mouse Neuro 2a cells impaired the proteasome function and caused severe mitochondrial abnormalities. Treatment with aspirin lead to a dose- and time-dependent decrease in proteasome activity and an increase in the accumulation of ubiquitylated proteins in the cells, which correlated with its effect on cell death. Aspirin exposure also resulted in an increase in the half-life of pd1EGFP, a model substrate of proteasome, as well as various intracellular substrates like Bax, IkappaB-alpha, p53, and p27(kip1). Aspirin-induced proteasomal malfunction might be responsible, at least in part, for the down-regulation of NF-kappaB activity and neurite outgrowth. Finally, we have shown that aspirin treatment caused changes in the mitochondrial membrane potential, release of cytochrome c from mitochondria, and activation of caspase-9 and -3, which could be because of the proteasomal dysfunction.