Hitesh N. Pawar

Over-expression of gene encoding heat shock protein 70 from Mycobacterium tuberculosis and its evaluation as vaccine adjuvant

BACKGROUND Heat shock proteins (Hsps) are evolutionary ancient and highly conserved molecular chaperons found in prokaryotes as well as eukaryotes. Hsp70 is a predominant member of Hsp family. Microbial Hsp70s (mHsp70s) have acquired special significance in immunity since they have been shown to be potent activators of the innate immune system and generate specific immune responses against tumours and infectious agents. OBJECTIVES The present study was aimed to clone express and purify recombinant Hsp70 from the Mycobacterium tuberculosis and characterise it immunologically. The study also aimed at determining the potential of recombinant M. tuberculosis heat shock protein (rMTB-Hsp70) as adjuvant or antigen carrier. MATERIALS AND METHODS Cloning of M. tuberculosis heat shock protein (MTB-Hsp70) amplicon was carried out using the pGEMT-Easy vector although for expression, pProExHTb prokaryotic expression vector was used. Purification of recombinant Hsp70 was carried out by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. For immunological characterization and determining the adjuvant effect of MTB-Hsp70, BALB/c mice were used. The data obtained was statistically analysed. RESULTS Hsp70 gene was cloned, sequenced and the sequence data were submitted to National Center for Biotechnology Information (NCBI). Recombinant MTB-Hsp70 was successfully over-expressed using the prokaryotic expression system and purified to homogeneity. The protein was found to be immunodominant. Significant adjuvant effect was produced by the rMTB-Hsp70 when inoculated with recombinant outer membrane protein 31; however, effect was less than the conventionally used the Freunds adjuvant. CONCLUSION Protocol standardised can be followed for bulk production of rHsp70 in a cost-effective manner. Significant adjuvant effect was produced by rMTB-Hsp70; however, the effect was than Freunds adjuvant. Further, studies need to be carried out to explore its applicability as carrier of antigen.

Effect of Year, Season and Parity on Milk Production Traits in Murrah Buffaloes

The objective of this study was to find out the effects of year, season and parity on milk production traits i.e. total lactation milk yield (TLMY), 305 day milk yield (305d MY) and average fat percentage etc. in Murrah buffaloes under organized herd. Records of 515 Murrah buffaloes maintained at Guru Angad Dev Veterinary and Animal Sciences University (GADVASU), Ludhiana, Punjab (India) during the period of 2004-2008 were used. Average TLMY, 305d MY and fat percentage were recorded to be 2229.87± 93.7 kg, 2147.6 ± 87.06 kg and 7.12 ±0.11%. The TLMY was found to be significantly affected by season (P≤0.05) but not by year and parity. The highest milk yield was obtained in buffaloes calving in winter followed by rainy and summer. Milk yield of buffaloes in winter was significantly higher than that of animals in summer (P≤0.05). The TLMY increased over the years with highest milk yield in the year 2006 (2345.1±99.32kg). Similar results were obtained for 305d MY, where only the season was found significant (P≤0.05). The average fat percentage was significantly (P≤0.05) affected by year and season. Milk fat percentage of buffaloes calved in winter was significantly (P≤0.05) higher than that of calved in summer. Similarly milk fat percentage varied significantly among the parities with no consistent increase over the advancement of the parities. In this study season was found to have a significant effect on 305MY and fat % but not on the total lactation milk yield.

Effect of Heat Stress on Milk Production and Composition in Murrah Buffaloes

Temperature humidity index (THI) is widely used to assess the effect of temperature and relative humidity on performance in animals. In summer the THI was between 74 – 89 with average value of 81.18. in winter months THI ranged between 49 -70 with the average of 60. The results showed a significant effect of heat stress on daily milk yield and milk composition. In the present study the daily milk yield decreases from 4.46 to 3.65kg, heat stress reduced milk yield by 18.2%. There was a significant effect of heat stress on milk composition. Heat stress significantly reduced milk fat content from 8.3% during the winter to 7.19% during the summer. Milk protein percentage significantly decreased as a result of summer heat stress (3.08 vs.2.9 %, respectively for the winter and summer). In the present study the SNF decreases from 9.08 to 9.05 %, heat stress reduced SNF % as the THI value went from ≤ 74 to ≥ 83 in summer. Results showed that milk production is a function of THI. The negative slope of regression line indicates that milk production fat%, protein% and SNF% decreases as THI increases. This regression indicates that in general for each point increase in THI value. There was decrease in milk yield of 0.028kg per buffalo per day. Heat stress environments have been associated with depression in milk fat%, protein% and SNF%. There was decrease in milk fat of 0.046% per buffalo per day. There was also decrease in milk protein of 0.00014 % per buffalo per day. The decrease in milk SNF of 0.0047 % per buffalo per day.

Differential Expression of Th1- and Th2- Type Cytokines in Peripheral Blood Mononuclear Cells of Murrah Buffalo (Bubalus Bubalis) on TLR2 Induction by B. Subtilis Peptidoglycan

Peripheral blood mononuclear cells (PBMCs) discriminate microbial pathogens and induce T-cell responses of appropriate effector phenotype accordingly. Toll-like receptors (TLRs), in part, mediate this microbial recognition and differentiation while the development of T-cell effector functions critically depends on the release of Th1- or Th2- type cytokines. In the present study, buffalo PBMCs were stimulated under in vitro culture conditions by Bacillus subtilis cell wall petidoglycan, a TLR2 ligand, in a dose-and time- dependent manner. The expression of TLR2 as well as the subsequent differential induction of the Th1 and Th2 type cytokines was measured. Stimulation was analyzed across five doses of peptidoglycan (10 μ/ml, 20 μg/ml, 30 μg/ml, 40 μg/ml and 50 μg/ml) for 3 h, 12 h, 24 h and 36 h incubation periods. We observed the induction of TLR2 expression in a dose- and time-dependent manner and the peptidoglycan induced tolerance beyond 30 μg/ml dose at all incubation periods. The correlation between peptidoglycan stimulation and TLR2 induction was found positive at all doses and for all incubation periods. Increased production of all the cytokines was observed at low doses for 3 h incubation, but the expression of IL-4 was relatively higher than IL-12 at the higher antigen doses, indicating tailoring towards Th2 response. At 12 h incubation, there was a pronounced decrease in IL-4 and IL-10 expression relative to IL-12 in a dose- dependent manner, indicating skewing to Th1 polarization. The expression of IL-12 was highest for all doses across all the incubation intervals at 24 h incubation, indicating Th1 polarization. The relative expression of TNF-α and IFN-γ was also higher while that of IL-4 and IL-10 showed a decrease. For 36 h incubation, at low doses, relative increase in the expression of IL-4 and IL-10 was observed which decreased at higher doses, as did the expression of all other cytokines. The exhaustion of cytokine production at 36 h indicated that PBMCs became refractory to further stimulation. It can be concluded from this study that the cytokine response to sPGN initially was of Th2 type which skews, more pronouncedly, to Th1 type with time till the cells become refractory to further stimulation.

Differential expression of apoptosis-associated genes in canine mammary tumors

Abstract B-cell lymphoma 2 (Bcl-2) and heat shock protein (HSP) families are implicated in various processes of carcinogenesis, owing to their role in regulation of apoptosis and cell cycle, respectively. mRNA expression of Bcl-2, myeloid cell leukemia 1 (Mcl-1), B-cell lymphoma-extra-large (Bcl-xl), Bcl2-associated X (Bax), HSP70 and HSP90-β genes were studied in 30 clinical canine mammary tumors (CMTs). Histological ‘type’ and ‘grade’ were assigned to CMTs and expression was evaluated by SYBR-Green real-time PCR assay. Overall, the tumors exhibited the maximum expression of Bcl-2 amongst the Bcl-2 family members. Sarcoma and carcinosarcoma showed relatively higher expression of Mcl-1, whereas Bcl-2 was over-expressed in carcinoma. In relation to the cancer grades, Bcl-2/Bax ratio tend to increase as the tumor differentiation progressed from well to poorly differentiated. HSP90-β exhibited significantly high expression in carcinoma, carcinosarcoma and all grades of CMTs were suggestive of their elemental role in tumor progression. In conclusion, this study underpins the conjecture that Bcl-2, Mcl-1 and HSP90-β can be used as potential targets of inhibition in future mammary tumor therapeutics.