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T M Haqqi

Publications and source records attributed to T M Haqqi.

42 records · Page 3Linked to original sources
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The inducible cytotoxic T-lymphocyte-associated gene transcript CTLA-1 sequence and gene localization to mouse chromosome 14.

Classical phenomenological approaches to the study of the mechanism of T-cell-mediated cytotoxicity have now given way to a search for molecules involved in this function; this is attempted either by subcellular and biochemical fractionation of material from cytotoxic cells, or through the characterization of molecules recognized by cytotoxicity-inhibiting monoclonal antibodies Molecules having a role in cytotoxicity may also be identified by detecting the corresponding messenger RNA transcripts. Such an approach may include, as a first step, the search for transcripts as specific as possible to cytotoxic T cells; only secondarily can their actual relevance to cytotoxicity be investigated. We report here the preparation and systematic screening of a differential complementary DNA bank, in which we detected three distinct messenger RNA transcripts (CTLA-1, CTLA-2 and CTLA-3) present in various cytotoxic T cells but not (or less so) in a range of non-cytotoxic lymphoid cells. We describe the co-inducibility of these transcripts and of cytotoxicity in thymocytes and hybridoma cells, the sequence of CTLA-1 cDNA, its protein homology with serine esterases and the localization of the corresponding gene to mouse chromosome 14.

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Immunization with TCR Vbeta10 peptide reduces the frequency of type-II collagen-specific Th1 type T cells in BUB/BnJ (H-2q) mice.

OBJECTIVE: Collagen induced arthritis (CIA) in mice is mediated by synergistic T cell and humoral immune responses specific for type II collagen (CII). We have previously shown that in arthritic joints of BUB mice (TCR Vbetaa, H-2q) the TCR repertoire is enrichedfor Vbeta10 expressing T cells, and that immunization with a Vbeta10 peptide (Vbeta10p) prevents the phenotypic expression of disease. The objective of the present study was to understand how immunization with a synthetic TCR Vbeta peptide affected the development of the pathogenic CII-specific immune response in BUB mice. METHODS: Arthritic and protected animals were tested for Vbeta10p- and CII-specific cytokine production by a highly specific and sensitive ELISA spot assay, andfor CII-specific antibody production by standard ELISA. In adoptive transfer experiments, Vbeta10p-specific LN cells (INF-gamma producing) were injected into naive mice prior to immunization with type-II collagen/CFA. RESULTS: Immune cells from arthritic animals produced IFN-gamma and IL-2, without IL-4 and IL-5 in response to CII and an immunodominant epitope, A2, derivedfrom CII. Serum from these mice contained anti-CII antibodies of both IgGI and IgG2a subtypes. Our results show for thefirst time that immunization with Vbeta10p resulted in Vbeta10p-specific IFN-gamma and IL-2 production that was restricted to the CD4+ T cell subset. Emergence of this Vbeta10p-specific immune response was associated with a dramatic decrease in the frequency of CII and A2-specific, cytokine producing T cells in arthritis protected mice. Protective immunity was cell mediated and could be adoptively transferred. In contrast, the protective immunization had only a marginal effect on the anti-CII antibody response indicating that the CII specific humoral immune response was not significantly affected. CONCLUSION: Immunization with TCR Vbeta10p leads to expansion of a population of Vbeta10p- specific CD4+ Tcells. This anti-TCR Vbeta10p specific type 1 cytokine producing immune response was protective in adoptive transfer studies and appears to inhibit the expansion of the pathogenic anti-CII cellular immunity. Additionally, the anti-TCR Vbeta10p-specific cellular immune response was mediated by CD4+ T cells and these T cells did not produce IL-4 or IL-5. Thus, our results suggest that protection against CIA in mice immunized with synthetic TCR Vbeta10p was achieved by a specific down-regulation of the CII-specific Thl type cellular immune response and not via immune deviation.

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Induction of CYP1A1 mRNA in rat epidermis and cultured human epidermal keratinocytes by benz(a)anthracene and beta-naphthoflavone.

Cytochrome P-4501A1 (CYP1A1) plays a major role in the bioactivation of procarcinogens in target tissues, including skin. However, the factors controlling CYP1A1 expression in mammalian skin are unknown. Utilizing the reverse transcriptase-linked polymerase chain reaction, we analyzed the effect of treatment with beta-naphthoflavone (beta-NF) and benz(a)anthracene on the expression of CYP1A1 mRNA in rat epidermis and normal human epidermal keratinocytes (NHEK). Inducer treatment of rats and NHEKs resulted in several-fold increases in aryl hydrocarbon hydroxylase activity. Following inducer treatment of adult or neonatal rats, increase in CYP1A1 gene message occurred as compared with that in controls. Higher basal level and inducibility were detected in mature rather than in 4-day-old rats. This induction occurred as early as 4 hr after beta-NF application. Exposure to beta-NF and benz(a)anthracene also resulted in substantial increases in gene message in NHEK. Northern blot analyses complemented the polymerase chain reaction data. These results indicate that CYP1A1 gene expression is increased in mammalian epidermis by inducers of epidermal aryl hydrocarbon hydroxylase activity.

Animals↗
PubMedDate not supplied

Biochemical and molecular characterization of stromelysin synthesized by human osteoarthritic chondrocytes stimulated with recombinant human interleukin-1.

OBJECTIVE: To study the biochemical and molecular characterization of stromelysin synthesized by human chondrocytes derived from osteofemoral heads. METHODS: First passage human chondrocyte cultures were incubated with recombinant human interleukin-1 alpha or recombinant human interleukin-1 beta (10-1000 pg ml-1) for either 24 or 48 hrs. The medium compartment of these cultures was assayed for stromelysin activity. Total cellular RNA was used to determine: (i) the molecular structure of the stromelysin synthesized by these cells; and (ii) whether or not these chondrocytes expressed the Type II procollagen gene (COL2A1). RESULTS: Human osteoarthritic chondrocytes released into the medium on enzyme requiring tryspin activation that possessed Substance P (SP) cleaving activity. SP cleaving activity was completely inhibited by EDTA. Casein zymography showed lysis zones produced by trypsin-activated chondrocyte culture medium that co-migrated with casein lysis zones produced by recombinant human prostromelysin. The majority of SP cleaving activity was eluted from a Zn-Sepharose column with 0.25 M glycine. Enzyme activity eluted from Zn-Sepharose produced casein lysis zones which co-migrated with lysis zones produced by recombinant human prostromelysin. Immunoblotting revealed the presence of prostromelysin (M(r), 55-57 kDa) in the pooled chondrocyte culture media applied to Zn-Sepharose and in the 0.25 M glycine eluate. Trypsin-activation converted prostromelysin to a mature stromelysin form (M(r), 45-47 kDa). Polymerase chain reaction (PCR) amplification of human chondrocyte cDNA demonstrated COL2A1 transcripts. A PCR product of expected size (680 bp) was produced by amplification of chondrocyte cDNA using stromelysin-1 oligonucleotide primers. The cloned and sequenced PCR product showed 100% homology between the chondrocyte stromelysin-1 mRNA-derived cDNA and the stromelysin-1 mRNA-derived cDNA of cultured human synovial, gingival and skin fibroblasts. CONCLUSIONS: By several criteria, human osteoarthritic chondrocytes synthesized stromelysin which was biochemically and antigenically identical, and molecularly homologous with human fibroblast stromelysin-1. These results suggest that a quantitative imbalance between stromelysin-1 and endogenous stromelysin-1 inhibitors rather than the transcription of a new stromelysin gene is the mechanism underlying the increased proteoglycan degradation seen in osteoarthritic cartilage.

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