A retrospective analysis of 1019 cases of tuberculous cervical lymphadenitis in a rural setup in 20 years.

BACKGROUND: This article is to review cervical lymphadenitis due to tuberculosis (TB), their presentation, their aetiology, the methods used to diagnose them, the treatment modalities offered and the response to treatment. METHODS: 1019 patients were diagnosed and treated for TB of the lymph nodes of the neck from 1st November 2001 to 31st August 2020 at a tertiary ENT hospital, Nadiad, Gujarat, India. Study consisted about 61% males and 39% females with the mean age being 37.3 years. RESULT: Commonest factor or habit among those diagnosed for tuberculous cervical lymphadenitis was consumption of unpasteurized milk. HIV and diabetes were the most common co-morbid conditions found with this disease. Swelling in the neck was most common clinical feature followed by loss of weight, formation of abscess, fever and fistula. Rifampicin resistance was found in 1.5% of patients among those tested for the same. CONCLUSION: The most commonly affected site for extra pulmonary TB is posterior triangle of neck than the anterior triangle. Patients with HIV and diabetes are at higher risk for the same. Testing for drug susceptibility has to be done due to increased resistant of drugs for extra pulmonary TB. GeneXpert and histopathological examination are important for its confirmation.

CD4+25+ regulatory T cells limit Th1-autoimmunity by inducing IL-10 producing T cells following human lung transplantation.

Chronic human lung allograft rejection is manifested by bronchiolitis obliterans syndrome (BOS). BOS has a multifactorial etiology. Previous studies have indicated that both cellular and humoral alloimmunity play a significant role in the pathogenesis of BOS. Recently, autoimmunity has also been demonstrated to contribute to lung allograft rejection in animal models. However, the significance of autoimmunity in BOS remains unknown. In this report, we investigated the role of naturally occurring CD4(+)CD25(+) regulatory T cells (T-regs) in modulating cellular autoimmunity to collagen type V (col-V), a 'sequestered' yet immunogenic self-protein present in the lung tissue, following lung transplantation (LT). We demonstrated that col-V reactive CD4(+) T cells could be detected in the peripheral blood of lung transplant recipients. There was a predominance of IL-10 producing T cells (T(IL-10)) reactive to col-V with significantly lower levels of IFN-gamma and IL-2 producing T cells (Th1 cells). The col-V specific T(IL-10) cells suppressed the proliferation and expansion of col-V specific Th1 cells by IL-10-dependent and contact-independent pathways. The T(IL-10) cells were distinct but their development was dependent on the presence of T-regs. Furthermore, during chronic lung allograft rejection there was a significant decline of T(IL-10) cells with concomitant expansion of col-V-specific IFN-gammaproducing Th1 cells.

Expression of ZmMET1, a gene encoding a DNA methyltransferase from maize, is associated not only with DNA replication in actively proliferating cells, but also with altered DNA methylation status in cold-stressed quiescent cells.

A cDNA fragment encoding part of a DNA methyltransferase was isolated from maize. The putative amino acid sequence identically matched that deduced from a genomic sequence in the database (accession no. AF063403), and the corresponding gene was designated as ZmMET1. Bacterially expressed ZmMET1 actively methylated DNA in vitro. Transcripts of ZmMET1 could be shown to exclusively accumulate in actively proliferating cells of the meristems of mesocotyls and root apices, suggesting ZmMET1 expression to be associated with DNA replication. This was confirmed by simultaneous decrease of transcripts of ZmMET1 and histone H3, a marker for DNA replication, in seedlings exposed to wounding, desiccation and salinity, all of which suppress cell division. Cold stress also depressed both transcripts in root tissues. In contrast, however, accumulation of ZmMET1 transcripts in shoot mesocotyls was not affected by cold stress, whereas those for H3 sharply decreased. Such a differential accumulation of ZmMET1 transcripts was consistent with ZmMET1 protein levels as revealed by western blotting. Expression of ZmMET1 is thus coexistent, but not completely dependent on DNA replication. Southern hybridization analysis with a methylation-sensitive restriction enzyme revealed that cold treatment induced demethylation of DNA in the Ac/Ds transposon region, but not in other genes, and that such demethylation primarily occurred in roots. These results suggested that the methylation level was decreased selectively by cold treatment, and that ZmMET1 may, at least partly, prevent such demethylation.

Indirect recognition of porcine swine leukocyte Ag class I molecules expressed on islets by human CD4+ T lymphocytes.

Xenotransplantation of porcine islets is considered a viable alternative treatment for type 1 diabetes mellitus. Therefore, we characterized human PBL responding to porcine islets both in vitro by coculture and in vivo using SCID mice reconstituted with human PBLs (HuPBL-SCID) and transplanted with porcine islets. T cell lines generated in vitro and graft-infiltrating T cells obtained from HuPBL-SCID mice were CD4+-proliferated specifically to porcine islets cultured with autologous APC. This proliferation was abrogated by an anti-human class II Ab. These T cell lines also proliferated to purified swine leukocyte Ag (SLA) class I molecules in the presence of self-APC, indicating that the primary xenoantigens recognized are peptides derived from SLA. This CD4+ T cell line lysed porcine islets but not splenocytes. CD4+ T cell clones with Th0, Th1, and Th2 cytokine profiles were isolated. The Th0 and Th1 clones lysed porcine islets, whereas the Th2 clone that secreted a large amount of IL-4 was not lytic. These results demonstrate that human T cells responding to porcine islets are primarily CD4+ and recognize porcine xenoantigens by the indirect Ag pathway presentation. These activated T cells produce cytokines that lyse islets. Furthermore, we demonstrate that the major porcine xenoantigens recognized are SLA class I molecules.

A tobacco NtMET1 cDNA encoding a DNA methyltransferase: molecular characterization and abnormal phenotypes of transgenic tobacco plants.

A cDNA encoding a DNA methyltransferase, with a predicted polypeptide of 1556 amino acid residues containing all motifs conserved in this enzyme family, was isolated from tobacco plants, and the corresponding gene was designated as NtMET1. RNA blot analysis indicated NtMET1 transcripts to accumulate in dividing tissues of tobacco plants, and they could be detected during the S phase in synchronized dividing BY2 cells. In situ hybridization revealed the transcripts to be localized exclusively in actively proliferating tissues around axillary apical meristem. In order to ascertain physiological roles, transgenic tobacco plants that had the antisense construct were made and examined for phenotypes. Methylation levels of genomic DNA from transgenic plants significantly decreased in comparison with wild-type levels, and distinct phenotypic changes including small leaves, short internodes and abnormal flower morphology were noted. Microscopic observation revealed that leaf structure differed between transgenic and wild-type plants. These results suggest that NtMET1 functions during DNA replication, and that DNA methylation plays an important role in plant morphogenesis.

Diverse response of rice and maize genes encoding homologs of WPK4, an SNF1-related protein kinase from wheat, to light, nutrients, low temperature and cytokinins.

The wheat gene WPK4 encodes a 56-kDa protein kinase that belongs to group 3 of the SNF1-related protein kinase family (SnRK3), and is up-regulated by light and cytokinins and down-regulated by sucrose. In order to determine whether or not this particular regulation pattern is general among plant species, we isolated and characterized homologous genes from rice and maize. Two rice genes, OsPK4 and OsPK7, encode proteins comprising 508 and 520 amino acids, and show, respectively, 75% and 76% sequence similarity to WPK4. OsPK4 and OsPK7 proteins produced in Escherichia coli were able to phosphorylate themselves and myelin basic proteins, the reaction requiring magnesium and/or manganese ions. Transcripts of OsPK4 were detected in all tissues tested, and amounts were increased upon illumination, nutrient deprivation and treatment with cytokinins. In contrast, transcripts of OsPK7 were not found in any tissues except in mature leaves at low levels, and did not accumulate under any of the stress conditions examined. A maize gene, ZmPK4, encodes a protein with 518 amino acids that shows 74% similarity to WPK4. Its transcripts were constitutively expressed in all tissues, regardless of light, nutrient and cytokinin status, but were increased upon exposure to low temperature. These results indicate that, despite the sequence similarity between their products, genes for SnRK3 proteins are differentially regulated in response to environmental stimuli.

Characterization of an HLA-A3 restricted human kidney specific T cell clone.

BACKGROUND: The tissue specificity of a cytolytic T lymphocyte is determined by the MHC class I bound peptide it recognizes. We have developed an allorestricted human CTL clone, DBS 1.5, that recognizes an epitope found on HLA-A3+ kidney epithelial cells but not on HLA identical B-lymphoblastoid cells. The peptide recognized by this clone has been isolated from HPLC separated, acid eluted peptides from purified HLA class I molecules from HLA-A3+ kidney tissue. This peptide shares no sequence homology with any known protein. METHODS: To confirm the tissue specificity of the HLA-A3 restricted clone and the peptide it recognizes we have transfected the gene for HLA-A3 into a number of tumor cell lines both human and murine not expressing this antigen. The resulting transfected lines, confirmed by immunofluorescent staining, were used as targets to determine if expression of HLA-A3 alone was sufficient to allow recognition and lysis by the HLA-A3 restricted T cell clone. RESULTS: The HLA-A3 restricted T cell clone recognized HLA-A3 when expressed on human kidney epithelial cells and to a lesser extent on human lung epithelium and human epidermal cells. Of the tumor lines transfected with HLA-A3 only the human kidney tumor cell line was lysed at a level equal to the original kidney epithelial cell used to develop the clone. CONCLUSION: These results confirm that this allorestricted human CTL clone is tissue specific recognizing a peptide found in human epithelial tissue that must be presented in the context of HLA-A3 for recognition.

Determination of growth and lysis kinetics in plant cell suspension cultures from the measurement of esterase release.

The death of Medicago sativa L. cells cultivated in a batch culture was investigated by measuring both the appearance of intact dead cells determined on the basis of the trypan blue (TB) dye exclusion, and the release of the cytoplasmic esterase activity into the culture medium upon cell death. Taking into account the strong instability of this released esterase activity, the total dead cell and lysed cell densities have been estimated. A mechanism for cell death and lysis is proposed and the specific rates of cell growth, death and lysis estimated. The specific rate of appearance of TB dead cells was low and essentially constant (0.25 day(-1)) during the first 8 days of the batch culture, and then increased above 1.5 day(-1) after 2 weeks of cultivation. Whereas no lysis occurred during the first seven days, this phenomenon occurred during the second period and accounted for about 20% of the total cell death by the end of the process. Thus, the viability determined by the trypan blue exclusion method appeared to be invalid after 7 days of culture. When lysis of viable cells is taken into consideration, the specific growth rate was significantly increased and growth was shown to continue for a further 8 days. Increased sensitivity of the cells to shear stresses and consequent cell lysis could be the result of a 35% increase in the cell size

A new methodology for plant cell viability assessment using intracellular esterase activity.

A plant cell suspension culture of Alfalfa (Medicago sativa L.) was grown in a bioreactor using a batch procedure. The cytoplasmic esterase activity (EC 3.1) was extracted from the cells and measured during cultivation using fluorescein diacetate as the fluorogenic substrate. This enzymatic activity was conclusively found to be correlated to cell viability assessed with the membrane integrity test using the trypan blue dye. This new viability determination method is convenient, simple and can be reproduced because: (1) the difficult step of counting the cells when using the trypan blue exclusion method is avoided and (2) the esterase activity level per viable cell constituted of numerous enzymes depends on cell viability but is independent of cellular metabolism.

Renal allograft infiltrating lymphocytes: frequency of tissue specific lymphocytes.

Acute rejection, mediated by T lymphocytes recognizing donor MHC class I and II, is a major factor influencing renal transplant survival. To define the specificity of these effector cells we examined cytolytic activity of graft infiltrating T lymphocytes (GIL) from renal biopsies of individuals undergoing acute cellular rejection. The majority of these cells recognized MHC class I on both donor kidney epithelial cells (KCL) and B-lymphoblastoid cells (LCL) suggesting these T cells recognized peptides from various tissues. However, cold target inhibition experiments demonstrated a significant proportion of GIL T cells were tissue specific. We reported previously that kidney specific CTL can be isolated from biopsies of kidney allografts undergoing acute cellular rejection. Here we extend that observation showing we were able to isolate tissue specific CTL from two additional biopsies. Greater than 10% of the clones isolated (4 of 36 and 5 of 37) from these biopsies were CTL recognizing donor KCL but not LCL targets suggesting that peptides, recognized in the context of donor MHC, were tissue specific. Repeated isolation of significant numbers of tissue specific CTL suggests these T cells play a role in allograft rejection and may be important effector cells mediating rejection in HLA matched transplants.

Indirect allorecognition of HLA class I peptides by CD4+ cytolytic T lymphocytes.

T-cell responses to alloantigens can occur either by "direct" recognition of donor MHC molecules, or "indirect" recognition of MHC peptides in association with self-MHC. To evaluate human T cells mediating indirect allorecognition, a CD4+ TCL and clones specific for HLA-A1 or HLA-B8 (residues 60-84) were generated from normal PBLs (A2,29 B62,- DR1,4 DQ3). Most clones were A1 specific (16 out of 17 tested), HLA-DR4 restricted (8 out of 8), and lysed targets pulsed with A1 peptide (16 out of 16). An amino acid substitution at position 86 of the DR4 beta chain (G -> V) abrogated the capacity of CD4+ CTLs to lyse target cells. Chloroquine treatment of A1-pulsed targets reduced their susceptibility to lysis, indicating a requirement for peptide processing. The TCL and clones were stimulated to proliferate by cells bearing intact HLA-A1 when autologous APCs were present, indicating that the epitope contained within the A1 60-84 peptide being recognized is produced when APCs process native HLA-A1. Furthermore, the clones and TCL did not recognize HLA-A1 on target cells carrying this allele plus self-HLA-DR4. These studies suggest a much wider role for CD4+ T cells in allograft immunity.

Cytolytic T lymphocytes from human renal allograft biopsies are tissue specific.

The cytolytic activity of T lymphocytes infiltrating renal allografts from recipients undergoing episodes of acute cellular rejection was studied. These T-cell populations, composed of both CD4+ and CD8+ cells, demonstrated significant cytolytic activity against both donor-derived KCLs and B-LCLs. In five of 21 biopsy-derived lines greater cytolytic activity was measured against donor KCLs compared to donor B-LCLs, suggesting the presence of kidney antigen-specific, MHC-restricted clones. Clones developed by stimulation with donor B-LCLs lysed both donor B-LCLs and KCLs while clones developed on donor KCLs as stimulator cells showed tissue specificity. Three of 26 clones recognized tissue-specific antigens in the context of donor MHC class I antigens lysing donor KCLs but not B-LCLs. These data demonstrate that a subpopulation of T cells recognizing kidney-specific antigens are present in biopsies of renal allograft recipients undergoing acute cellular rejection. This subpopulation of tissue-specific cytotoxic T lymphocytes may prove to contribute significantly to the pathology of allograft rejection.

Cytokine gene transcripts for tumor necrosis factor-alpha, interleukin-2, and interferon-gamma in human pulmonary allografts.

BACKGROUND: Cytokines participate in host responses to allografts, largely through recruiting and activating various regulatory and effector cells. We performed this study to determine the feasibility of using polymerase chain reaction methodology to define the expression of three important cytokines (tumor necrosis factor-alpha, interleukin-2, and interferon-gamma) in human pulmonary allografts. METHODS: Twenty-six graft-derived samples (11 transbronchial biopsy and 8 macrophage and 7 lymphocyte cell pellets isolated from bronchoalveolar lavage) were obtained from 13 lung transplant recipients and treated as follows: extraction of RNA; reverse transcription of RNA to complementary DNA; polymerase chain reaction amplification of cDNA with oligonucleotide primers specific for the three cytokines; gel electrophoresis of the polymerase chain reaction products; and verification of correct cytokine message by Dot blot technique (with specific 32P-labeled oligonucleotide probes). RESULTS: Concomitant pathologic evaluation of biopsy specimens from these 13 recipients showed five diagnostic groups: "normal" (no rejection/infection), n = 2; acute rejection, n = 4; nonspecific inflammation, n = 3; infection, n = 3; and obliterative bronchiolitis, n = 1. Interleukin-2 was expressed predominantly in acute rejection and infection (seven of ten and five of six samples positive, respectively), whereas tumor necrosis factor-alpha was expressed mainly in nonspecific inflammation (four of five samples) and somewhat less in rejection (six of ten). Interferon-gamma was expressed less frequently (in two of six samples with infection, but in none of ten with rejection and none of five with nonspecific inflammation). Serial data from one patient (6 months apart) showed considerable increase in interleukin-2 and interferon-gamma expression as she progressed from normal histologic status to obliterative bronchiolitis. CONCLUSIONS: Cytokine gene transcripts can be determined from minute samples derived directly from pulmonary allografts. Although our data are insufficient to make definitive conclusions, the suggestion of trends of cytokine expression in different posttransplantation pathologic conditions may indicate a useful role for this approach in the clinical evaluation of the lung transplant recipient.