M atrix metalloproteinases (MMPs) encompasses a family of secreted, calcium dependent, zinc containing endopeptidases. These enzymes are responsible for tissue remodelling and are capable of degrading all kinds of extracellular matrix proteins including collagens, matrix glycoproteins, gelatin, elastins, and proteoglycans. MMPs are involved in regulation of cell behaviours like cellular differentiation, proliferation, angiogenesis, invasion, and apoptosis. Thus, these enzymes have multi-factorial roles during tumor progression: promoting establishment, tumor cell exfoliation, invasion and angiogenesis. The degradation of extracellular matrix components is crucial for tumor cell invasion and MMPs, especially MMP2 (72 kDa gelatinase A) and MMP9 (92 kDa gelatinase B), play critical role in degradation of gelatin and type IV collagen, the two major constituents of extracellular matrix. Short CommunicationAbstract | Matrix Metalloproteinases (MMP) 2 & 9 are overexpressed in wide variety of cancers and play important roles in tumour cell proliferation, invasion and metastasis. Tissue and serum levels of MMP2 and MMP9 correlate with disease prognosis. Real-time PCR is emerging as an alternative or supplementary technique to immunohistochemistry (IHC) for sensitive detection of tumour markers in tissues. Therefore, in this study real-time PCR assays were standardized for detecting over-expression of MMP2 and MMP9 in canine mammary tumour (CMT). Primers designed for real-time PCR analysis of MMP2 & MMP9 were specific as indicated by amplification plots and meltcurve analysis of amplicons. These amplicons showed sharply defined melting curves with single peaks at expected melting points. The over-expression of MMP2 and MMP9 was examined in 12 CMT tissues by real-time PCR analysis. Over-expression of MMP2 & MMP9 was observed in 58.3(7/12) and 41.6 % (5/12) cases of CMT respectively. Expression levels of MMP2 were 2.23±0.10 to 29.80±3.5 fold higher and for MMP2, 1.5±0.56 to 22.67±2.35 fold higher in cases of CMT than normal mammary gland biopsy. Approximately 33.3 % (4/12) of CMT tissues showed co-expression of both MMP2 and MMP9. Further studies are required to correlate MMP2 and MMP9 expression levels with CMT prognosis.

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The present study reports the determination of optimum thawing temperature and holding period of the Mareks Disease (MD) cell associated vaccine after reconstitution by plaque assay. The highest titre of MD HVT+SB-1 bivalent vaccine was obtained at the thawing temperature of 35°C for 45 sec followed by 26°C for 45 sec, 20°C for 60 sec and 40°C at 45 sec; when the vaccine was held on ice after reconstitution. The difference in virus titre among 30, 90 and 120 min holding period at 26°C and 35°C thawing for 45 sec was significant (P≤0.001). However the variation in titre between 30 and 90 min holding period was not significant for thawing temperatures of 20°C and 40°C (P>0.05). No plaques were seen when the bivalent HVT+SB-1 vaccine was thawed at 45°C for 45 sec. The titre obtained at 40°C was extremely low after a holding period of 120 min. After thawing at 35°C for 45 sec and when the reconstituted vaccine was held at RT, there was a drastic reduction in virus titre by 120 min. The study revealed that thawing at 20°C and 40°C for 45 sec would severely lower the titre of HVT+SB-1 vaccine. The complete loss of virus titre was seen at thawing temperature of 45°C for 45 sec. The study reasserts the importance of maintaining the cold chain, appropriate thawing temperature and holding period after reconstitution of HVT+SB-1, a cell associated MD vaccine in maintaining the requisite PFU/dose of vaccine.

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