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PubMed | National Institute of Pathology ICMR, JLNM Hospital and AIIMS
Type: Journal Article | Journal: Tumori | Year: 2016

Hepatosplenic T-cell lymphoma (HSTL) is a rare extranodal and systemic lymphoma derived from cytotoxic T cells usually of T cell receptor type. It is characterized by primary extranodal disease with typical sinusoidal infiltration of liver, spleen, and bone marrow by medium-sized lymphoid cells.A 29-year-old man, with no significant prior medical history, presented with fever and massive splenomegaly. A diagnosis of HSTL was established by histologic examination and immunohistochemistry. Staging workup demonstrated bone marrow involvement by lymphoma. In addition, the patient was found to have hepatitis B infection. The association of these 2 entities has been described rarely.Hepatosplenic T-cell lymphoma is a distinct T cell lymphoma associated with an aggressive clinical course, a poor response to conventional treatment, and an exceedingly high mortality rate. An association of HSTL with hepatitis B as seen in the present case is exceedingly rare, with few cases reported in the literature.


Chugh R.M.,National Institute of Pathology ICMR | Chaturvedi M.,National Institute of Pathology ICMR | Yerneni L.K.,National Institute of Pathology ICMR
PLoS ONE | Year: 2015

Growth arrested Swiss mouse embryonic 3T3 cells are used as feeders to support the growth of epidermal keratinocytes and several other target cells. The 3T3 cells have been extensively subcultured owing to their popularity and wide distribution in the world and, as a consequence selective inclusion of variants is a strong possibility in them. Inadvertently selected variants expressing innate resistance to mitomycin C may continue to proliferate even after treatment with such growth arresting agents. The failure of growth arrest can lead to a serious risk of proliferative feeder contamination in target cell cultures. In this study, we passaged Swiss 3T3 cells (CCL-92, ATCC) by different seeding densities and incubation periods. We tested the resultant cultures for differences in anchorage-independent growth, resumption of proliferation after mitomycin C treatment and occurrence of proliferative feeder contaminants in an epidermal keratinocyte co-culture system. The study revealed subculture dependent differential responses. The cultures of a particular subculture procedure displayed unique cell size distribution and disintegrated completely in 6 weeks following mitomycin C treatment, but their repeated subculture resulted in feeder regrowth as late as 11 weeks after the growth arrest. In contrast, mitomycin C failed to inhibit cell proliferation in cultures of the other subculture schemes and also in a clone that was established from a transformation focus of super-confluent culture. The resultant proliferative feeder cells contaminated the keratinocyte cultures. The anchorage-independent growth appeared in late passages as compared with the expression of mitomycin C resistance in earlier passages. The feeder regrowth was prevented by identifying a safe subculture protocol that discouraged the inclusion of resistant variants. We advocate routine anchorage-independent growth assay and absolute confirmation of feeder disintegration to qualify feeder batches and caution on the use of fetal bovine serum. © 2015 Chugh et al.


Selvapandiyan A.,Institute of Molecular Medicine | Dey R.,CBER | Gannavaram S.,CBER | Solanki S.,Institute of Molecular Medicine | And 3 more authors.
Vaccine | Year: 2014

Visceral leishmaniasis (VL) is fatal if not treated and is prevalent widely in the tropical and sub-tropical regions of world. VL is caused by the protozoan parasite Leishmania donovani or Leishmania infantum. Although several second generation vaccines have been licensed to protect dogs against VL, there are no effective vaccines against human VL [1]. Since people cured of leishmaniasis develop lifelong protection, development of live attenuated Leishmania parasites as vaccines, which can have controlled infection, may be a close surrogate to leishmanization. This can be achieved by deletion of genes involved in the regulation of growth and/or virulence of the parasite. Such mutant parasites generally do not revert to virulence in animal models even under conditions of induced immune suppression due to complete deletion of the essential gene(s). In the Leishmania life cycle, the intracellular amastigote form is the virulent form and causes disease in the mammalian hosts. We developed centrin gene deleted L. donovani parasites that displayed attenuated growth only in the amastigote stage and were found safe and efficacious against virulent challenge in the experimental animal models. Thus, targeting genes differentially expressed in the amastigote stage would potentially attenuate only the amastigote stage and hence controlled infectivity may be effective in developing immunity. This review lays out the strategies for attenuation of the growth of the amastigote form of Leishmania for use as live vaccine against leishmaniasis, with a focus on visceral leishmaniasis. © 2014 Elsevier Ltd.


PubMed | Gb Pant Hospital and National Institute of Pathology ICMR
Type: Journal Article | Journal: Indian heart journal | Year: 2016

The regulatory T cell (Treg) is essential for prevention of autoimmunity. In a preliminary study, we showed significant deficiency of Tregs (CD4CD25 T cells) in rheumatic heart disease (RHD) patients (an autoimmune disease), but the markers used could not reliably differentiate Treg from nonregulatory conventional T cells (Tcon). The study aim was to reassess the level of circulatory Tregs by using more specific markers.70 adults of RHD and 35 controls were studied. Patients were subdivided according to the extent of left-sided valvular involvement. 35 patients with significant mitral-valve disease only were enrolled in the univalvular group while 35 patents with significant involvement of both mitral and aortic-valves in the multivalvular group. Circulating Treg cell level was determined by flow-cytometry.Level of Tregs (CD4+CD25(med-high)CD127(low) Foxp3(high)) in CD4+ T lymphocyte was significantly lower in RHD patients compared to controls (median 0.6% versus 3.2%; p=0.001) with no significant difference in Tcon cells (p=0.94). Within the study group Treg count was significantly lower in patients with multivalvular-disease only (median 0.1% versus 3.2%; p=0.001) with no significant difference in Treg cell count between the univalvular group and control (median 1.9% versus 3.2%, p=0.10).There is significant deficiency of circulating Tregs in patients of chronic RHD and the deficiency is greater in patients with multivalvular than univalvular involvement.


PubMed | National Institute of Pathology ICMR and Jamia Hamdard University
Type: | Journal: Journal of pharmacological and toxicological methods | Year: 2016

Growth-arrested feeder cells following Mitomycin C treatment are instrumental in stem cell culture allowing development of regenerative strategies and alternatives to animal testing in drug discovery. The concentration of Mitomycin C and feeder cell type was described to affect feeder performance but the criticality of feeder cell exposure density was not addressed. We hypothesize that the exposure cell density influences the effectiveness of Mitomycin C in an arithmetic manner.Three different exposure cell densities of Swiss 3T3 fibroblasts were treated with a range of Mitomycin C concentrations for 2h. The cells were replaced and the viable cells counted on 3, 6, 9, 12 and 20days. The cell extinctions were compared with doses per cell which were derived by dividing the product of concentration and volume of Mitomycin C solution with exposure cell number.The periodic post-treatment feeder cell extinctions were not just dependent on Mitomycin C concentration but also on dose per cell. Analysis of linearity between viable cell number and Mitomycin C dose per cell derived from the concentrations of 3 to 10g/ml revealed four distinct categories of growth-arrest. Confluent cultures exposed to low concentration showed growth-arrest failure.The in vitro cell density titration can facilitate prediction of a compounds operational in vivo dosing. For containing the growth arrest failure, an arithmetic volume derivation strategy is proposed by fixing the exposure density to a safe limit. The feeder extinction characteristics are critical for streamlining the stem cell based pharmacological and toxicological assays.


PubMed | National Institute of Pathology ICMR
Type: Journal Article | Journal: The Indian journal of medical research | Year: 2016

Leprosy type 1 reactions (T1R) are acute episodes of immune exacerbation that are a major cause of inflammation and nerve damage. T1R are diagnosed clinically and supported by histopathology. No laboratory marker is currently available that can accurately predict a T1R. Increased plasma and tissue expression of inducible nitric oxide synthase (i-NOS) and chemokine CXCL10 have been demonstrated in T1R. We studied the gene expression and immunoexpression of i-NOS, CXCL10 and its receptor CXCR3 in clinically and histopathologically confirmed patients with T1R and compared with non-reactional leprosy patients to understand which biomarker has better potential in distinguishing reaction from non-reaction.Gene expression of i-NOS, CXCL10 and CXCR3 was studied in 30 skin biopsies obtained from patients with borderline tuberculoid (BT), mid-borderline (BB) and borderline lepromatous (BL) leprosy with and without T1R by real-time PCR. Further validation was done by immunohistochemical expression on 60 borderline leprosy biopsies with and without T1R.Of the 120 patients histopathological evaluation confirmed T1R in 65 (54.2%) patients. CXCR3 gene expression was significantly (P<0.05) higher in BT- and BB-T1R patients compared to those without T1R. The CXCL10 gene expression was significantly higher (P<0.05) in BB leprosy with T1R but the difference was not significant in patients with BT with or without T1R. Immunoexpression for CXCR3 was significant in both BB-T1R and BB (P<0.001) and BT and BT-T1R (P<0.001). Immunoexpression of CXL10 was significant only in differentiating BB from BB-T1R leprosy (P<0.01) and not the BT cases. i-NOS immunoexpression was not useful in differentiating reactional from non-reactional leprosy.Both CXCL10 and CXCR3 appeared to be useful in differentiating T1R reaction in borderline leprosy while CXCR3 alone differentiated BT from BT-T1R. CXCR3 may be a potentially useful immunohistochemical marker to predict an impending T1R.


PubMed | Vardhaman Mahavir Medical College VMMC & Safdarjung Hospital and National Institute of Pathology ICMR
Type: Journal Article | Journal: The journal of maternal-fetal & neonatal medicine : the official journal of the European Association of Perinatal Medicine, the Federation of Asia and Oceania Perinatal Societies, the International Society of Perinatal Obstetricians | Year: 2015

The aim of the study was to characterize the expression of cyclooxygenase-2 (COX-2) and prostaglandin-E2 (PGE2) genes in spontaneously aborted tissues from women infected with Chlamydia trachomatis.A total of 135 spontaneous aborters (Group I) and 45 induced aborters (controls; Group II) attending Obstetrics and Gynaecology Department at Safdarjung hospital (New Delhi, India), were enrolled. Polymerase chain reaction (PCR) assay was performed to detect C. trachomatis DNA in endometrial curettage tissue (ECT). Differential expression of COX-2 and PGE2 receptors at mRNA level was analysed in ECT using reverse transcription PCR and real-time PCR.In total, 14.8% patients were diagnosed as C. trachomatis-positive in Group I whereas all control patients were C. trachomatis-negative. Qualitative expression of COX-2 (p<0.05) and PGE2 (p<0.0001) receptors was found increased in C. trachomatis-positive patients (Group I) in comparison to controls. Quantitative real-time PCR analysis also showed upregulation in transcript levels of both COX-2 (p<0.002) and PGE2 (p<0.0001) receptors in infected patients (Group I) versus Group II. COX-2 and PGE2 expression was higher (p<0.002) in recurrent spontaneous aborters in comparison to sporadic spontaneous aborters.Results suggest that chlamydial infection leads to upregulation of COX-2 in C. trachomatis-positive recurrent spontaneous aborters, which probably mediates increased prostaglandin synthesis.


PubMed | National Institute of Pathology ICMR
Type: Journal Article | Journal: PloS one | Year: 2015

Growth arrested Swiss mouse embryonic 3T3 cells are used as feeders to support the growth of epidermal keratinocytes and several other target cells. The 3T3 cells have been extensively subcultured owing to their popularity and wide distribution in the world and, as a consequence selective inclusion of variants is a strong possibility in them. Inadvertently selected variants expressing innate resistance to mitomycin C may continue to proliferate even after treatment with such growth arresting agents. The failure of growth arrest can lead to a serious risk of proliferative feeder contamination in target cell cultures. In this study, we passaged Swiss 3T3 cells (CCL-92, ATCC) by different seeding densities and incubation periods. We tested the resultant cultures for differences in anchorage-independent growth, resumption of proliferation after mitomycin C treatment and occurrence of proliferative feeder contaminants in an epidermal keratinocyte co-culture system. The study revealed subculture dependent differential responses. The cultures of a particular subculture procedure displayed unique cell size distribution and disintegrated completely in 6 weeks following mitomycin C treatment, but their repeated subculture resulted in feeder regrowth as late as 11 weeks after the growth arrest. In contrast, mitomycin C failed to inhibit cell proliferation in cultures of the other subculture schemes and also in a clone that was established from a transformation focus of super-confluent culture. The resultant proliferative feeder cells contaminated the keratinocyte cultures. The anchorage-independent growth appeared in late passages as compared with the expression of mitomycin C resistance in earlier passages. The feeder regrowth was prevented by identifying a safe subculture protocol that discouraged the inclusion of resistant variants. We advocate routine anchorage-independent growth assay and absolute confirmation of feeder disintegration to qualify feeder batches and caution on the use of fetal bovine serum.


PubMed | National Institute of Pathology ICMR and Central Institute of Orthopedics CIO
Type: Journal Article | Journal: International journal of rheumatic diseases | Year: 2016

Reportedly, there is little information on the magnitude of genitourinary-induced reactive arthritis (gReA) from India. Genital infection with Chlamydia trachomatis is a major health problem in India because of its high prevalence; therefore, this study was conducted with the aim to screen ReA/undifferentiated spondyloarthropathy (uSpA) patients (n=20) attending a major city hospital in New Delhi, for investigating the presence of intra-articular chlamydial antigen in knee joints. Patients with rheumatoid arthritis (RA) and osteoarthritis (OA) served as controls (n=20).Synovial fluid samples were screened for chlamydial elementary bodies (EBs) using a commercial kit (MicroTrak C.trachomatis Direct Specimen Test; Trinity Biotech, USA) for performing direct fluorescence assay (DFA).Chlamydia trachomatis EBs were detected in the synovial fluid cell deposits of six patients in Group I, namely, 33.3% (4/12) ReA and 25% (2/8) uSpA. All C.trachomatis positive patients exhibited an oligoarticular clinical picture with knee joint involvement. In the synovial fluid cell deposits of control patients, namely, RA/OA, no chlamydial EBs could be detected.This is the first study reporting the presence of C.trachomatis EBs in the synovial fluid of spondyloarthropathy patients, namely, ReA/uSpA from our country and it can be concluded that the prevalence of C.trachomatis-induced ReA is underestimated. Although our study had limitations in terms of sample size and lower sensitivity of DFA, yet this test can be used as an initial diagnostic tool for screening and patients with positive results may undergo specific tests for validation.


PubMed | Kalyani University and National Institute of Pathology ICMR
Type: Journal Article | Journal: Journal of receptor and signal transduction research | Year: 2016

Computer-aided antibody engineering has been successful in the design of new biologics for disease diagnosis and therapeutic interventions. Interleukin-6 (IL-6), a well-recognized drug target for various autoimmune and inflammatory diseases such as rheumatoid arthritis, multiple sclerosis, and psoriasis, was investigated in silico to design potential lead antibodies. Here, crystal structure of IL-6 along with monoclonal antibody olokizumab was explored to predict antigen-antibody (Ag-Ab)-interacting residues using DiscoTope, Paratome, and PyMOL. Tyr56, Tyr103 in heavy chain and Gly30, Ile31 in light chain of olokizumab were mutated with residues Ser, Thr, Tyr, Trp, and Phe. A set of 899 mutant macromolecules were designed, and binding affinity of these macromolecules to IL-6 was evaluated through Ag-Ab docking (ZDOCK, ClusPro, and Rosetta server), binding free-energy calculations using Molecular Mechanics/Poisson Boltzman Surface Area (MM/PBSA) method, and interaction energy estimation. In comparison to olokizumab, eight newly designed theoretical antibodies demonstrated better result in all assessments. Therefore, these newly designed macromolecules were proposed as potential lead antibodies to serve as a therapeutics option for IL-6-mediated diseases.

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