Ravi Kumar Gandham

Canine parvovirus NS1 induced apoptosis involves mitochondria, accumulation of reactive oxygen species and activation of caspases

The non-structural protein (NS1) of parvoviruses plays an important role in viral replication and is thought to be responsible for inducing cell death. However, the detailed mechanism and the pathways involved in canine parvovirus type 2 NS1 (CPV2.NS1) induced apoptosis are not yet known. In the present study, we report that expression of CPV2.NS1 in HeLa cells arrests cells in G1 phase of the cell cycle and the apoptosis is mitochondria mediated as indicated by mitochondrial depolarization, release of cytochrome-c and activation of caspase 9. Treatment of cells with caspase 9 inhibitor Z-LEHD-FMK reduced the induction of apoptosis significantly. We also report that expression of CPV2.NS1 causes accumulation of reactive oxygen species (ROS) and treatment with an antioxidant reduces the ROS levels and the extent of apoptosis. Our results provide an insight into the mechanism of CPV2.NS1 induced apoptosis, which might prove valuable in developing NS1 protein as an oncolytic agent.

Isolation of Coxiella burnetii from bovines with history of reproductive disorders in India and phylogenetic inference based on the partial sequencing of IS1111 element

In the present study, a total of 158 blood samples from 148 bovines and 10 dogs having a history of reproductive disorders were screened for Coxiella burnetii by trans-PCR method. In case of bovines, 6.08% (9/148) blood samples comprised of 4.54% (4/88) cattle and 8.33% (5/60) buffaloes turned out to be positive for C. burnetii DNA while all the samples from dogs (10) were found negative. Of the 9 PCR-positive bovine blood samples, the organism could be isolated only from 3 cases of buffaloes by chick embryo inoculation method. Further, to predict the homology and genetic diversity, the recovered C. burnetii isolates designated as Y1, Y3 and Y7 were partially sequenced for IS1111 gene. On phylogenetic analysis, Y3 and Y7 isolates clustered to a common node away from Y1 isolate. This study may enlighten the nature of circulating C. burnetii isolates in different parts of the world. To the best of our knowledge, this appears to be the first report describing phylogenic analysis of C. burnetii isolates based on IS1111 gene sequence.

Viral genes as oncolytic agents for cancer therapy.

Many viruses have the ability to modulate the apoptosis, and to accomplish it; viruses encode proteins which specifically interact with the cellular signaling pathways. While some viruses encode proteins, which inhibit the apoptosis or death of the infected cells, there are viruses whose encoded proteins can kill the infected cells by multiple mechanisms, including apoptosis. A particular class of these viruses has specific gene(s) in their genomes which, upon ectopic expression, can kill the tumor cells selectively without affecting the normal cells. These genes and their encoded products have demonstrated great potential to be developed as novel anticancer therapeutic agents which can specifically target and kill the cancer cells leaving the normal cells unharmed. In this review, we will discuss about the viral genes having specific cancer cell killing properties, what is known about their functioning, signaling pathways and their therapeutic applications as anticancer agents.

Comparative Evaluation of Different Test Combinations for Diagnosis of Mycobacterium avium Subspecies paratuberculosis Infecting Dairy Herds in India

A total of 355 cows were sampled (serum, n = 315; faeces, n = 355; milk, n = 209) from dairy farms located in the Punjab state of India. Faeces and serum/milk samples were screened by acid fast staining and “indigenous ELISA,” respectively. IS900 PCR was used to screen faeces and milk samples. Bio-load of MAP in dairy cows was 36.9, 15.6, 16.3, and 14.4%, using microscopy, serum ELISA, milk ELISA and milk PCR, respectively. Estimated kappa values between different test combinations: serum and milk ELISA, faecal microscopy and faecal PCR, milk ELISA and milk PCR, faecal PCR and serum ELISA were 0.325, 0.241, 0.682, and 0.677, respectively. Estimation of the relative sensitivity and specificity of different tests in the present study indicated that “serum ELISA” and “milk ELISA” were good screening tests, add “milk PCR” was “confirmatory test” for MAP infection. Combination of milk ELISA with milk PCR may be adopted as a model strategy for screening and diagnosis of JD in lactating/dairy cattle herds in Indian conditions.

HN Protein of Newcastle Disease Virus Induces Apoptosis Through SAPK/JNK Pathway.

Many viral proteins are responsible for causing induction of apoptosis in the target cells. Hemagglutinin neuraminidase (HN), a multifunctional protein of Newcastle disease virus (NDV), is one of such proteins. The present study was undertaken to determine the apoptotic potential of the HN gene in cultured human cervical cancer cell line (HeLa cell) and to elucidate the molecular mechanisms involved. The results of the study indicate that HN protein causes apoptosis in HeLa cells, as observed by the translocation of Phosphatidylserine, activation of caspases, cleavage of poly (ADP-ribose) polymerase (PARP), and DNA fragmentation. Further, we report that expression of HN protein upregulates the SAPK/JNK pathway leading to transactivation of c-Jun which in turn activates apoptosis signaling. The results of our study provide an insight into the mechanism through which HN induces apoptosis.

Canine parvovirus NS1 protein exhibits anti-tumor activity in a mouse mammary tumor model.

Many viral proteins have the ability to kill tumor cells specifically without harming the normal cells. These proteins, on ectopic expression, cause lysis or induction of apoptosis in the target tumor cells. Parvovirus NS1 is one of such proteins, which is known to kill high proliferating tumor cells. In the present study, we assessed the apoptosis inducing ability of canine parvovirus type 2 NS1 protein (CPV2.NS1) in vitro in 4T1 cells, and found it to cause significant cell death due to induction of apoptosis through intrinsic or mitochondrial pathway. Further, we also evaluated the oncolytic activity of CPV2.NS1 protein in a mouse mammary tumor model. The results suggested that CPV2.NS1 was able to inhibit the growth of 4T1 induced mouse mammary tumor as indicated by significantly reduced tumor volume, mitotic, AgNOR and PCNA indices. Further, inhibition of tumor growth was found to be because of induction of apoptosis in the tumor cells, which was evident by a significant increase in the number of TUNEL positive cells. Further, CPV2.NS1 was also able to stimulate the immune cells against the tumor antigens as indicated by the increased CD4+ and CD8+ counts in the blood of CVP2.NS1 treated mice. Further optimization of the delivery of NS1 protein and use of an adjuvant may further enhance its anti-tumor activity.

Combined administration of the apoptin gene and poly (I:C) induces potent anti-tumor immune response and inhibits growth of mouse mammary tumors.

BACKGROUND Many viral proteins exhibit selective cytotoxicity for tumor cells without affecting the normal diploid cells. The apoptin protein of chicken infectious anemia virus is one of such proteins, which has been shown to kill tumor cells specifically. However, an effective cancer treatment strategy also requires assistance from the immune system. Recently, poly (I:C) has been shown to be an effective cancer vaccine adjuvant. AIM In this study, we assessed the anti-tumor potential of apoptin gene transfer alone and in combination with poly (I:C) in a 4T1 mouse mammary tumor model. METHODS 4T1 cells were used to induce mammary tumor in Balb/c mice. Mice bearing tumors were divided into 6 groups, and each group received six intratumoral injections during a period of one month. After the last immunization, the animals were sacrificed, and peripheral blood, spleen, lungs, liver, heart, kidney and tumor tissues were collected for immunological, molecular and pathological analysis. RESULTS We report that intratumoral administration of apoptin plasmid along with poly (I:C) not only significantly inhibited the growth of mammary tumor, but also induced a potent anti-tumor immune response as indicated by the increase in blood CD4+, CD8+ cells and infiltration of immune cells in the tumor tissue. Further, blood serum analysis of the cytokines revealed increased secretion of Th1 cytokines (IFN-γ and IL-2). CONCLUSIONS The results of our study demonstrate that the inclusion of poly (I:C) significantly enhanced the anti-tumor activity of apoptin mainly by inducing a potent anti-tumor immune response. Therefore, we report the use of apoptin and poly (I:C) combination as a novel and powerful strategy for cancer immunotherapy.

Poly (I:C) enhances the anti-tumor activity of canine parvovirus NS1 protein by inducing a potent anti-tumor immune response

The canine parvovirus NS1 (CPV2.NS1) protein selectively induces apoptosis in the malignant cells. However, for an effective in vivo tumor treatment strategy, an oncolytic agent also needs to induce a potent anti-tumor immune response. In the present study, we used poly (I:C), a TLR3 ligand, as an adjuvant along with CPV2.NS1 to find out if the combination can enhance the oncolytic activity by inducing a potent anti-tumor immune response. The 4T1 mammary carcinoma cells were used to induce mammary tumor in Balb/c mice. The results suggested that poly (I:C), when given along with CPV2.NS1, not only significantly reduced the tumor growth but also augmented the immune response against tumor antigen(s) as indicated by the increase in blood CD4+ and CD8+ counts and infiltration of immune cells in the tumor tissue. Further, blood serum analysis of the cytokines revealed that Th1 cytokines (IFN-γ and IL-2) were significantly upregulated in the treatment group indicating activation of cell-mediated immune response. The present study reports the efficacy of CPV2.NS1 along with poly (I:C) not only in inhibiting the mammary tumor growth but also in generating an active anti-tumor immune response without any visible toxicity. The results of our study may help in developing CPV2.NS1 and poly (I: C) combination as a cancer therapeutic regime to treat various malignancies.

Global gene expression profile of peripheral blood mononuclear cells challenged with Theileria annulata in crossbred and indigenous cattle

Bovine tropical theileriosis is an important haemoprotozoan disease associated with high rates of morbidity and mortality particularly in exotic and crossbred cattle. It is one of the major constraints of the livestock development programmes in India and Southeast Asia. Indigenous cattle (Bos indicus) are reported to be comparatively less affected than exotic and crossbred cattle. However, genetic basis of resistance to tropical theileriosis in indigenous cattle is not well documented. Recent studies incited an idea that differentially expressed genes in exotic and indigenous cattle play significant role in breed specific resistance to tropical theileriosis. The present study was designed to determine the global gene expression profile in peripheral blood mononuclear cells derived from indigenous (Tharparkar) and cross-bred cattle following in vitro infection of T. annulata (Parbhani strain). Two separate microarray experiments were carried out each for cross-bred and Tharparkar cattle. The cross-bred cattle showed 1082 differentially expressed genes (DEGs). Out of total DEGs, 597 genes were down-regulated and 485 were up-regulated. Their fold change varied from 2283.93 to -4816.02. Tharparkar cattle showed 875 differentially expressed genes including 451 down-regulated and 424 up-regulated. The fold change varied from 94.93 to -19.20. A subset of genes was validated by qRT-PCR and results were correlated well with microarray data indicating that microarray results provided an accurate report of transcript level. Functional annotation study of DEGs confirmed their involvement in various pathways including response to oxidative stress, immune system regulation, cell proliferation, cytoskeletal changes, kinases activity and apoptosis. Gene network analysis of these DEGs plays an important role to understand the interaction among genes. It is therefore, hypothesized that the different susceptibility to tropical theileriosis exhibited by indigenous and crossbred cattle is due to breed-specific differences in the dealing of infected cells with other immune cells, which ultimately influence the immune response responded against T. annulata infection.

HN protein of Newcastle disease virus sensitizes HeLa cells to TNF-α-induced apoptosis by downregulating NF-κB expression.

Hemagglutinin neuraminidase (HN) is a membrane protein of Newcastle disease virus (NDV) with the ability to induce apoptosis in many transformed cell lines. TNF-α is a multi-factorial protein that regulates cell survival, differentiation and apoptosis. In a previous study, we reported that HN protein induces apoptosis by downregulating NF-κB expression. Further, we speculated that downregulation of NF-κB expression might sensitize HeLa cells to TNF-α-mediated apoptosis. Therefore, the present study was undertaken to investigate if HN protein could sensitize HeLa cells to TNF-α and to examine the apoptotic potential of the HN protein and TNF-α in combination. The results revealed that the pro-apoptotic effects were more pronounced with the combination of HN and TNF-α than with HN or TNF-α alone, which indicates that the HN protein indeed sensitized the HeLa cells to TNF-α-induced cell death. The results of the study provide a mechanistic insight into the apoptotic action of HN protein along with TNF-α, which could be valuable in treating tumor types that are naturally resistant to TNF-α.