Anti-Proliferative Role of Recombinant Lethal Toxin of Bacillus Anthracis on Primary Mammary Ductal Carcinoma Cells Revealing Its Therapeutic Potential

Bacillus anthracis secretes three secretary proteins; lethal factor (LF), protective antigen (PA) and edema factor (EF). The LF has ability to check proliferation of mammary tumors, chiefly depending on mitogen activated protein kinase (MAPK) signaling pathway. Evaluation of therapeutic potential of recombinant LF (rLF), recombinant PA (rPA) and lethal toxin (rLF + rPA = LeTx) on the primary mammary ductal carcinoma cells revealed significant (p \u3c 0.01) reduction in proliferation of tumor cells with mean inhibition indices of 28.0 ±1.37% and 19.6 ± 1.47% respectively. However, treatment with rPA alone had no significant anti-proliferative effect as evident by low mean inhibition index of 3.4 ± 3.87%. The higher inhibition index observed for rLF alone as compared to LeTx is contrary to the existing knowledge on LF, which explains the requirement of PA dependent endocytosis for its enzymatic activity. Therefore, the plausible existence of PA independent mode of action of LF including direct receptor mediated endocytosis or modulation of signal transduction cascade via unknown means is hypothesized. In silico protein docking analysis of other cellular receptors for any plausibility to play the role of receptor for LF revealed c-Met receptor showing strongest affinity for LF (H bond = 19; Free energy = -773.96), followed by nerve growth factor receptor (NGFR) and human epidermal growth factor receptor (HER)-1. The study summarizes the use of rLF or LeTx as therapeutic molecule against primary mammary ductal carcinoma cells and also the c-Met as potential alternative receptor for LF to mediate and modulate PA independent signal transduction

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Not AvailableUnderstanding pathogenesis during progressive stages of infection by Mycobacterium avium subsp. paratuberculosis (MAP) and finding suitable methods for its diagnosis are key to the control of Johne's disease in animals. Paratuberculosis was experimentally produced in 20 crossbred lambs by oral administration of MAP to study the sequential development of lesions between 10 and 330 days postinfection and to assess commonly used diagnostic methods such as bacterial culture, lymphocyte stimulation test (LST), and enzyme-linked immunosorbent assay (ELISA) during progressive stages of infection. Histologic lesions were classified into four grades from grade 1 (least severe) to grade 4 (most severe) on the basis of location of granulomatous lesions in different regions and layers of intestines, their association with intestinal lymphoid tissues, pattern and distribution of lesions, types of cellular infiltration, and presence of acid-fast bacilli. It is evident that infection first establishes in lymphoid tissues of the small intestine, possibly at multiple sites, producing segmental lesions and from there spreads to lamina propria and local lymph nodes. Wide variability in the histologic lesions in relation to postinfection periods and initial tropism of MAP to the intestinal lymphoid tissues (Peyer's patches) suggests a differential susceptibility of young animals, possibly because of compositional phenotypic variation of Peyer's patches influencing subsequent course of infection. Histopathology was found to be a better indicator of paratuberculous infection than bacteriology in sheep. The LST (reflecting the cellular immune response) and ELISA (reflecting the humoral immune response) had overall sensitivities of 65% (11 of 17) and 42% (8 of 19), respectively, in sheep with different types of pathology but when employed together could detect about 88% of infected animals.Not Availabl

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Not AvailableThe aim of this study was to develop an indirect ELISA using the helicase domain of bovine viral diarrhoea virus (BVDV) NS3 protein instead of full-length NS3 protein for detection of BVDV and BDV antibodies in sheep and goats and its validation by comparing its sensitivity and specificity with virus neutralization test (VNT) as the reference test. The purified 50 kDa recombinant NS3 protein was used as the coating antigen in the ELISA. The optimal concentration of antigen was 320 ng/well at a serum dilution of 1:20 and the optimal positive cut-off optical density value was 0.40 based on test results of 418 VNT negative sheep and goat sera samples. When 569 serum samples from sheep (463) and goats (106) were tested, the ELISA showed a sensitivity of 91.71% and specificity of 94.59% with BVDV VNT. A good correlation (93.67%) was observed between the two tests. It showed a sensitivity of 85% and specificity of 86.6% with VNT in detecting BDV antibody positive or negative samples. This study demonstrates the efficacy of truncated recombinant NS3 antigen based ELISA for seroepidemiological study of pestivirus infection in sheep and goats.Not Availabl

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Not AvailableThe aim of this study was to determine the in vitro permissivity of peripheral blood mononuclear cells (PBMCs) from bovine viral diarrhea virus (BVDV)-immune field cattle to homologous and heterologous BVDVs. PBMCs from seventeen BVDV-naïve and sixteen BVDV-immune animals were infected with noncytopathic BVDV-1 or BVDV-2. The immune status of cattle was indicated by the presence of virus neutralizing antibodies, while viral load of PBMCs was determined by real-time RT-PCR. The results revealed that the PBMCs from naïve or immune animals were permissive to either BVDV-1 or BVDV-2, but the viral load was significantly higher for the naïve than for the immune animals. Furthermore, the load of homologous virus in PBMCs from immune animals was lower than that of heterologous virus. Our results provide evidence that the PBMCs from BVDV-immune cattle in field are susceptible to reinfection with homologous or heterologous BVDV, albeit to a lower extent in the former case.Not Availabl

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Not AvailableThe aim of the presented study was to characterise the development of the cellular part of the immune system in pigs from 1 d to 20 weeks of age. Haematological examination and flow cytometry were used to establish the relative and absolute counts of various leukocyte subsets. During the first 5 months of pig life, a significant age-dependent increase in lymphocyte and granulocyte counts was noted. Moreover, a decrease in relative size of lymphocytes connected with increasing proportion of granulocytes was observed. The absolute size of CD3+ and CD21+ increased approximately two-fold during the analysed period. The absolute number of CD4+CD8- , CD4-CD8+ and CD4+CD8+ cells increased almost twice from birth till the 6th week of age. After this time, only CD4+CD8- subset remained stable, while the number of CD4-CD8+ and CD4+CD8+ subsets gradually increased 1.5- and 2.5-fold from 6 weeks to 5 months of age for CD4-CD8+ and CD4+CD8+ , respectively. In conclusion, changes in the absolute size of lymphocyte subsets are not always consistent with changes in their relative size. Moreover, because there are age-related differences in leukocyte subsets in porcine blood, there is a need to use appropriate age matched control groups, especially in experiments, which include immunophenotyping of lymphocytes. We suggest that immunophenotyping of lymphocytes, especially for diagnostic purposes, should be based on the absolute size rather than on the percentage of lymphocyte subpopulations.Not Availabl

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Not AvailableThe aim of this study was to determine the in vitro permissivity of peripheral blood mononuclear cells (PBMCs) from bovine viral diarrhea virus (BVDV)-immune field cattle to homologous and heterologous BVDVs. PBMCs from seventeen BVDV-naïve and sixteen BVDV-immune animals were infected with noncytopathic BVDV-1 or BVDV-2. The immune status of cattle was indicated by the presence of virus neutralizing antibodies, while viral load of PBMCs was determined by real-time RT-PCR. The results revealed that the PBMCs from naïve or immune animals were permissive to either BVDV-1 or BVDV-2, but the viral load was significantly higher for the naïve than for the immune animals. Furthermore, the load of homologous virus in PBMCs from immune animals was lower than that of heterologous virus. Our results provide evidence that the PBMCs from BVDV-immune cattle in field are susceptible to reinfection with homologous or heterologous BVDV, albeit to a lower extent in the former case. PMID: 24957715Not Availabl

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Not AvailableThe objective of this study was to optimize an immune electron microscopy protocol for detection of Bovine Viral Diarrhoea Virus (BVDV) and to compare its detection limit with that of direct negative staining technique. Indian non-cytopathic BVDV isolate (Ind S1449) infected SFTR cell supernatant with a titre of 105.3 TCID 50 per ml was used. For IEM, various parameters including anti-BVDV polyclonal serum dilution, virus-antibody incubation temperature and time, and centrifugation time were optimized using the undiluted BVDV stock. Incubation with 1: 400 dilution of anti-BVDV polyclonal serum at 37°C for one hour was found to be optimum. To compare the detection limits of both the protocols, serial ten-fold dilutions of the virus stock (undiluted to 10−4 containing 105.3 to 101.3 TCID 50per ml, respectively) were used. Phosphotungstic acid stained grids were viewed under transmission electron microscope. BVDV was visualized as immune-aggregates of varying sizes distributed diffusely across IEM grids. In direct negative stained grids, the virus was detected mostly as individual particles. The detection limit of the IEM protocol was 103.3 TCID 50 per ml which was one hundred-fold more than that of direct negative staining protocol. The present study demonstrates that examination of suspected sample by immune electron microscopic procedures is feasible for BVDV detection and it can be used as routine diagnostic tool, especially for screening of cell culture supernatants infected with suspected clinical specimens. Suitability of this assay for direct screening for identification of persistently infected animals which are of great concern for control programmes, needs to be explored.Not Availabl

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Not AvailableBovine viral diarrhea virus (BVDV) is a pestivirus which infects cattle worldwide causing substantial economic losses in cattle farming. BVDV is divided into two recognized species, BVDV-1 and BVDV-2 and one tentative species, BVDV-3. Since, complete genome sequence analysis can provide better insights into molecular epidemiology of BVD, we report here the first complete genome sequence analyses of an Indian BVDV-2 strain isolated from cattle. The full-genome of strain Ind 141353 contains 12285 nucleotides (nt) with a single large open reading frame which codes for 3898 amino acids. Phylogenetic analysis indicated that this strain belongs to the BVDV-2a subtype and has highest (93%) level of genetic identity with the Chinese cattle strain JZ05-1. It was inferred that although introduction from China is possible, introduction of BVDV-2 into Indian and Chinese cattle from a common trade source cannot be ruled out completely. The results in this study extend the spectrum of pestivirus molecular data and provide important insights into BVDV molecular epidemiology.Not Availabl

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Not AvailableThe aim of this study was to develop an improved border disease virus (BDV) specific real time RT-PCR and to evaluate its performance on manually plucked hairs from sheep persistently infected with BDV that may act as a non-invasive alternate sample. The BDV real time RT-PCR assay reported here showed a high analytical sensitivity (100.6 TCID50/ml), specificity (no reactivity with BVDV-1, BVDV-2, HoBi-like pestivirus and CSFV) and reproducibility. When the assay was validated on 210 samples from BDV-infected and uninfected sheep, it showed a 100% diagnostic sensitivity and specificity with virus isolation. Further evaluation of the assay on manually plucked hair follicles from ear (mid-lateral, mid-medial) and tail tip from sheep persistently infected with BDV showed that a minimum of 20 hair follicles need to be tested for correct diagnosis of BDV. The BDV load was comparatively higher in hairs from mid-medial ear than those from other tested locations. Evaluation on other samples from PI sheep demonstrated that the test performance was similar to that of pestivirus generic real-time RT-PCR, but improved than the currently available BDV specific real-time RT-PCR. Although more number of PI animals need to be evaluated, the results of the study showed that manually plucked hairs from mid-medial ear pinna is a suitable alternative sample in real-time RT-PCR for detection of BDV persistently infected sheep. Use of the non-invasive ear hair samples and the improved BDV specific real-time RT-PCR reported here may be useful for BDV surveillance in several sheep rearing countries.Not Availabl

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Not AvailableThe aim of the present study was to understand the replication kinetics of an Indian isolate of highly pathogenic porcine reproductive and respiratory syndrome (PRRS) virus (Ind-297221) in MARC-145 cells infected at different multiplicity of infection (MOI) of 1.0, 0.1, 0.01 and 0.001. PRRSV titre in the infected cell fraction and the culture supernatant harvested at different intervals (12, 36, 48, 72, 96 and 120 h) post infection (hpi) was estimated by immunoperoxidase monolayer assay. Viral RNA copy numbers were quantified by TaqMan RT-PCR. PRRS virus could be detected first in intracellular fraction at 12 hpi in cells infected at 1.0 MOI, whereas in the extracellular fraction, earliest detection was at 36 hpi. Highest PRRSV titre of 1.3 × 105.0 TCID50/mL was achieved in 0.01 and 0.001 MOI groups at 96 hpi. Infection with 0.01 MOI resulted in the maintenance of maximum titre up to 120 hpi. The maximum viral copy numbers observed was 3.15 × 107.0 in 0.1 MOI group at 120 hpi in culture medium. The results of the study showed that MARC-145 cells infected with Indian PRRSV at 0.01 MOI and harvested in 96-120 hpi was found to be optimum for obtaining maximum virus yield and hence can be used for bulk propagation of the virus.Not Availabl