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Biomedical subjects

J Brock

Publications and source records attributed to J Brock.

At least 19 recordsLinked to original sources
PubMed2001

Investigation of systemic lupus erythematosus in Nogales, Arizona.

In 1996, a citizens group in Nogales, Arizona, reported to the Arizona Department of Health their concerns about a possible excess prevalence of systemic lupus erythematosus (SLE) due to exposure to environmental contamination in the area. The authors conducted a two-phase study in which the objectives of phase I were to identify potential SLE cases and to determine the prevalence of SLE and the objectives of phase II were to identify potential risk factors associated with the development of SLE and to evaluate the possible association between SLE and environmental exposure to pesticides and inorganic compounds. Participants included 20 confirmed cases and 36 controls. The authors found the prevalence of SLE to be 103 cases per 100,000 population (95 percent confidence interval: 56, 149), two to seven times higher than the prevalence in the US population. They detected elevated levels of 1,1-dichloro-2,2-bis-(p-chorophenyl)ethylene and organophosphate metabolites among cases and controls. In both, levels were higher than the reference mean for the US population. The authors found no statistical association between elevated levels of pesticides and disease status. Their results show that the prevalence of SLE in Nogales is higher than the reported prevalence in the US population and that both cases and controls had past exposure to chlorinated pesticides and have ongoing exposure to organophosphates.

Arizona↗
PubMed2001

The B-chain of mistletoe lectin I efficiently stimulates calcium signaling in human Jurkat T-cells.

Mistletoe lectin I (ML I), a heterodimeric disulfide-linked type II ribosome inactivating protein, exhibits immunomodulatory potency in stimulating the cytokine release in vitro and in vivo. However, data concerning early activation events in T-cells induced by ML I and its A and B chain preceding cytokine secretion and the receptors involved are of limited availability. Here we show by flow cytometric measurements that human T-lymphoblastoid Jurkat cells express surface glycoprotein receptors for ML I. One of which is shown to be the CD2 antigen involved in a variety of T-cell signaling events. The lectin induces in Jurkat T-cells an increase of the cytosolic calcium concentration ([Ca(2+)](i)) consisting of both, the transient release of Ca(2+) from internal stores and a sustained influx of extracellular Ca(2+). Studies with isolated A- and B-chains provided evidence that the lectin-induced increase in [Ca(2+)](i) is mediated by ML IB. The ML I and ML IB stimulated cellular calcium responses are inhibited by saccharidic competitors. In transiently transfected E6.1 cells ML IB stimulated the expression of the luciferase reporter construct pNFAT-TA-Luc that is activated through the nuclear factor of activated T-cells (NFAT). The ML IB stimulated expression of the reporter luciferase (Luc) is completely inhibited by cyclosporin A (0.2 microM) and by FK 506 at 0.05 microM. Pretreatment of Jurkat E6.1 cells with 1-deoxymannojirimycin (dMJ), an inhibitor of cis-Golgi alpha-mannosidase I, strongly reduced cell binding of ML IB-FITC and the ML IB induced calcium response. Benzyl-alpha-GalNAc, an inhibitor of O-linked glycosylation, has slightly decreasing effects in ML IB-FITC binding and was without effects on the lectin stimulated increase in [Ca(2+)](i). Inhibition of the lectin induced calcium responses by cholera toxin and by inhibitors of protein kinases as well as the absence of calcium responses in CD3- and CD45- Jurkat T-cell clones suggest that ML IB has the potency to induce early T-cell activation events.

Adjuvants, Immunologic↗
PubMed2001

Activation of the transcription factor NF-kappaB by the erythropoietin receptor: structural requirements and biological significance.

The transcription factor nuclear factor kappa B (NF-kappaB) has been implicated in the regulation of genes mainly involved in inflammation and immune response. We analysed the role of NF-kappaB in signalling pathways induced by the hematopoietic growth factor erythropoietin (EPO). Our data, obtained by electrophoretic mobility shift assays (EMSA) and reporter gene assays, show that the intracellular domain of the EPO receptor (EPOR) transmits signals leading to the activation of NF-kappaB. Studies employing an inhibitor specific for the EPOR-associated tyrosine kinase JAK2 suggest that JAK2-dependent pathways are not involved. The induction of an NF-kappaB-triggered reporter gene construct was inhibited by cotransfection of dominant negative forms of the src kinase Lyn, but not by dominant negative JAK2. Using epidermal growth factor (EGF)/EPOR hybrids containing mutant forms of the EPOR intracellular domain, we were able to further define the critical structures for the induction of NF-kappaB. The data show that although the activity of JAK2 seems to be dispensable, its association to the receptor, as well as the phosphorylation of membrane proximal tyrosine residues, are essential. Furthermore, the functional analysis of different receptor forms revealed a correlation of the abilities to induce NF-kappaB activity and to generate antiapoptotic signals.

Animals↗
PubMed2000

High concentration of soluble HLA-DR in the synovial fluid: generation and significance in "rheumatoid-like" inflammatory joint diseases.

In the search for its role in inflammatory joint diseases, soluble HLA-DR (sHLA-DR) was quantitated in 72 synovial fluids (SF) by a newly established immunoenzyme assay. Unlike other soluble receptors which accumulated only moderately (sCD25, sCD4) or negligibly (sHLA class I, sCD8) in the SF, SF sHLA-DR levels exceeded serum levels by up to 3 orders of magnitude and varied disease dependently from "control" values (traumatic synovitis and osteoarthritis: 9.9 +/- 6.1 ng/ml). Clear-cut different SF sHLA-DR values in HLA-DR-associated "rheumatoid-like" (136.5 +/- 130.0 ng/ml) vs HLA-B27-associated "spondylarthropathy-like" arthritic forms (28.4 +/- 29.1 ng/ml) were most significant comparing oligoarticular juvenile chronic arthritis type I (147.6 +/- 112.6 ng/ml) and type II (3.3 +/- 1.1 ng/ml), thus offering a new classification marker. Also ex vivo, large amounts of sHLA-DR were released spontaneously by SF mononuclear cells and found to be related to the T-cell activation state. SF sHLA-DR may be shed in large complexes or micelles, as it eluted mainly at >450 kDa on gel filtration. Western blotting revealed that the majority of SF sHLA-DR consisted of full-length alpha- and beta-chains. Minor fractions of smaller sized antigens seemed to be generated by proteolytic cleavage rather than by alternative splicing, since only minute amounts of HLA-DRB mRNA lacking the transmembrane exon could be amplified by RT-PCR. Distinct forms of high-dose sHLA-DR, able to provoke rather than to suppress T-cell responses, are discussed as contributing to some HLA-DR disease association.

Adolescent↗
PubMed2000

Immune response after adenoviral gene transfer in syngeneic heart transplants: effects of anti-CD4 monoclonal antibody therapy.

BACKGROUND: E1-deleted adenoviral vectors are frequently used for in vivo gene therapy. However, gene expression after adenovirus-(ad) mediated gene transfer is known to be transient due to the generation of an immune response against virus-infected cells. In this study, we asked whether an anti-CD4 mAb (RIB 5/2) treatment may improve the gene transfer into rat cardiac grafts. METHODS: We injected recombinant ad-constructs encoding for Escherichia coli beta-gal into syngeneic rat heart transplants via the proximal aorta. One-half of the recipients of genetically modified grafts received the anti-CD4 mAb RIB 5/2, whereas the other half received no monoclonal antibody treatment. Genetically unmodified isografts without any treatment of the recipients were used as additional controls. At different time points hearts were harvested and analyzed for reporter gene expression, intragraft cellular infiltration, and cytokine gene expression (quantitative "real time" reverse transcriptase polymerase chain reaction). Serum samples were analyzed for anti-ad-Ig using enzyme-linked-immunosorbent-assay. RESULTS: In control animals the beta-gal reporter gene expression slowly increased until day 7 and then declined. The immunohistological and reverse transcriptase polymerase chain reaction intragraft analyses revealed a strong inflammatory response (cellular infiltration, cytokine expression) in ad-transfected grafts that may explain the delayed expression and fast down-regulation of the transgene. Treatment with RIB 5/2 mAb resulted in a faster and prolonged reporter gene expression, reduced graft infiltration, reduced anti-ad-Ig titers and less interferon-gamma up-regulation. CONCLUSIONS: Our results indicate that modulation of the anti-ad immune response using a nondepleting anti-CD4 mAb may increase the efficiency of ad-vectors for gene therapy in the transplant setting.

Adenoviridae↗
PubMed2000

Health complaints related to pesticide stored at a public health clinic.

Employees at a health center in Georgia were concerned that symptoms experienced by some employees were related to pesticide exposure at the center. Malathion and DDT, used for mosquito control from 1969 to 1981, had been stored and handled at the center's first floor. We surveyed 117 (91%) of 129 employees to determine whether reported symptoms were associated with pesticide exposure. We performed environmental sampling for pesticides. We analyzed serum samples for 17 chlorinated pesticides, and urine samples for malathion. We found that 37% of the participants had reported a diagnosis of sinusitis and 24% of bronchitis since working at the health center. Frequently reported symptoms were eye irritation (44%) and headache (68%). DDT and malathion were found at levels of 2.4 and 11%, respectively, in bulk samples from the loading dock of the building. Multivariate analysis of responses to the questionnaire showed that the perception of odors, inadequate air flow, and length of employment were significantly associated with the employees' health complaints. Pesticide concentrations in employees' serum and urine samples were not associated with any health complaint. The health complaints reported by the employees at the health center were precipitated by both environmental and psychological factors. The epidemiology and laboratory components of this study highlight the importance of obtaining biological measurements in episodes of perceived environmental exposure.

Adult↗
PubMed2000

cDNA cloning and functional analysis of a truncated STAT5a protein from autonomously growing FDCP-1 cells.

The transcription factor STAT5 is activated by multiple hematopoietic cytokine receptors and has been implicated in the induction of cellular processes such as differentiation, proliferation and antiapoptotic activities. Here, we report cloning of the cDNA and characterization of a mutant STAT5a protein that is expressed in interleukin-3 (IL-3)-independently growing FDCP-1 cells. Analysis of the cDNA revealed a deletion of both the transactivation and the SH2 domains. Stable expression of the protein in parental IL-3-dependent cells results in elevated DNA binding activity of wild type (WT)-STAT5 in the nucleus, enhanced growth rates and a reduced susceptibility to undergo apoptosis after withdrawal of IL-3. Although the protein is not present in DNA/protein complexes in the nucleus, we observed pronounced effects on IL-3-induced signal transduction. The results suggest competition of the mutant protein with cytosolic mechanisms regulating STAT5 activity. In conclusion, the data support the hypothesis of an involvement of STAT5 in mitogenic and antiapoptotic signaling.

Amino Acid Sequence↗
PubMed2000

Activation of STAT5 during EPO-directed suppression of apoptosis.

The ligand-dependent activation of the JAK/STAT (Januskinase/Signal Transducer and Activator of Transcription) pathway has been implicated in the explanation of cytokine-specific regulation of gene expression. Previous studies have reported conflicting results on the role of the transcription factor STAT5 in erythropoietin (EPO)-induced cellular responses. In this study we focused on the functional importance of STAT5 docking sites in the intracellular EPO receptor (EPOR) domain for the mediation of antiapoptotic activities. We demonstrate that EPO-dependent survival of erythroleukemic cell lines is accompanied by sustained STAT5 DNA-binding activity. The role of single tyrosine residues was dissected by the analysis of myeloid FDCP-1 cells stably expressing mutant EPOR proteins. The data show that receptors having a high potential to mediate antiapoptotic signals also effectively activate STAT5, whereas receptors lacking STAT5 docking sites are diminished in both activities. We conclude that the transcription factor STAT5 is functionally implicated in the EPO-dependent survival of cells.

Animals↗
PubMed2000

Ischemia/reperfusion injury-mediated down-regulation of adenovirus-mediated gene expression in a rat heart transplantation model is inhibited by co-application of a TNFRp55-Ig chimeric construct.

E1-deleted adenoviral vectors are efficient vectors for somatic gene therapy. Recently, we have shown that intratracheal application of an adenoviral reporter construct leads to significant reporter gene expression in rat lungs within 24 h. In contrast, reporter gene expression in syngeneic rat heart transplants after adenovirus-mediated gene transfer was delayed. Since the adenovirus cannot replicate, down-regulation of the hCMV-IE promoter controlled reporter gene expression in initially infected cells by cytokines, which are released as a result of ischemia/reperfusion injury, might be involved. In order to investigate the role of proinflammatory cytokines, eg TNF-alpha in affecting hCMV-IE promoter-driven reporter gene expression, transient blockade of TNF-alpha was achieved by local co-application of an Ad-construct encoding for a soluble TNFRp55-Ig chimeric molecule in a syngeneic rat heart transplantation model. Co-application of the reporter construct together with the TNFRp55-Ig chimeric molecule significantly increased the early reporter gene expression after transplantation. Moreover, infiltration of inflammatory cells (T cells, macrophages, NK cells) and production of TNF-alpha in the transplant was markedly reduced. Our results indicate that: (1) proinflammatory cytokines are involved in down-regulation of reporter gene expression in ischemia/reperfusion injured tissues; and (2) inhibition of TNF-alpha might be a useful tool to increase early gene expression in gene therapy protocols, particularly in transplantation. Gene Therapy (2000) 7, 1238-1243.

Adenoviridae↗
PubMed2000

Determination of cytokine mRNA-expression in term human placenta of patients with gestational hypertension, intrauterine growth retardation and gestational diabetes mellitus using polymerase chain reaction.

OBJECTIVE: Our objective was to test the hypothesis, that pregnancy-related diseases are going along with changes in cytokine mRNA-expression at the placental site, either as a part of a pathological process or in connection with regulatory mechanisms induced by disturbances at the feto-maternal interface resulting from previous pathological changes--in the sense of counterregulation. MATERIAL AND METHODS: The cytokines chosen for this investigation are known to 1.) be expressed in the human placental tissue, 2.) to be involved in immunological processes and 3.) the regulation of growth and differentiation processes of different cell types of the placenta or decidua, 4.) to play a role in the angiogenesis at the feto-placental interface and 5.) to be involved in pathological processes in other human diseases. 32 samples derived from term human placentas were examined for messenger RNA levels of interleukin 1 alpha (II-1 alpha), tumor necrosis factor-alpha (TNF-alpha), platelet derived growth factor-A chain (PDGF-A), platelet derived growth factor-B chain (PDGF-B), and platelet derived growth factor receptor (PDGF-R) using a semiquantitative reverse transcriptase (RT) polymerase chain reaction (PCR) protocol. To calibrate samples in our procedure, beta-actin mRNA (messenger ribonucleic acid) known as a "house keeping" gene was proven to be constantly expressed. The sample-groups consisted of normal pregnancies (n = 8), gestational hypertension (GH, n = 7), intrauterine growth retardation (IUGR, n = 6), gestational diabetes mellitus (GDM, n = 5), and gemini (n = 3 x 2). RESULTS: Throughout the 32 samples, a significant correlation between PDGF-A and PDGF-R expression, PDGF-A and TNF-alpha expression was stated (p = 0.007). Compared with the pattern of expression in normal placentas, placentas of growth retarded pregnancies had higher Il-1 alpha mRNA (p = 0.016), PDGF-A (p = 0.029) and PDGF-B (p = 0.001) levels. The samples of the gestational hypertension group and placentas of patients with gestational diabetes displayed a significantly stronger PDGF-R mRNA signal (p = 0.0029 and p = 0.008). CONCLUSIONS: Though these marked differences in cytokine mRNA levels between clinical groups were statistically proven, clear correlation of these differences with clinical data was not found.

Actins↗
PubMed2000

Maternal seafood diet, methylmercury exposure, and neonatal neurologic function.

OBJECTIVE: To determine whether neonatal neurologic function is adversely affected by seafood contaminants from maternal diet during pregnancy. STUDY DESIGN: One hundred eighty-two singleton term births were evaluated in the Faeroe Islands, where marine food includes pilot whale. Maternal serum, hair, and milk and umbilical cord blood were analyzed for contaminants. Levels of essential fatty acids, selenium, and thyroid hormones were determined in cord blood. Each infant's neurologic optimality score was determined at 2 weeks of age adjusted for gestational age, and predictors were assessed by regression analysis. RESULTS: Exposures to methylmercury and polychlorinated biphenyls were increased in relation to maternal seafood intake, as were omega3 fatty acid concentrations in cord serum. Thyroid function was normal. After adjustment for confounders, a 10-fold increase of the cord-blood mercury concentration was associated with a decreased neurologic optimality score of 2.0 (P =. 03). This effect corresponds to a decrease in gestational age of about 3 weeks. Other indicators of the seafood diet had no effect on this outcome. CONCLUSIONS: Prenatal exposure to methylmercury from contaminated seafood was associated with an increased risk of neurodevelopmental deficit. Thus in this North Atlantic population, methylmercury constituted an important neurologic risk factor, although effects of other seafood components were not detectable.

Adult↗
PubMed2000

Involvement of CD2 and CD3 in galectin-1 induced signaling in human Jurkat T-cells.

Galectin-1 (gal-1) a member of the mammalian beta-galactoside-binding proteins recognizes preferentially Galbeta1-4GlcNAc sequences of oligosaccharides associated with several cell surface glycoconjugates. In the present work, gal-1 has been identified to be a ligand for the CD3-complex as well as for CD2 as detected by affinity chromatography of Jurkat T-cell lysates on gal-1 agarose and by binding of the biotinylated lectin to CD3 and CD2 immunoprecipitates on blots. In CD45(+)Jurkat E6.1 cells, the lectin stimulates a sustained increase in the intracytoplasmic calcium concentration ([Ca(2+)](i)) consisting of both the release of calcium from intracellular stores and the calcium influx from the extracellular space. This effect of gal-1 on [Ca(2+)](i)is completely inhibited by lactose at 10 mM and was absent in CD45(-)Jurkat J45.01 cells. Preincubation of Jurkat E6.1 cells with cholera toxin or with the protein tyrosine kinase inhibitor herbimycin A reduced the gal-1 induced calcium response whereas the increase in [Ca(2+)](i)stimulated by CD2 or CD3 monoclonal antibodies (mAbs) was completely inhibited. Depolarization of E6.1 cells in a high-potassium buffer, a standard method to activate voltage-operated calcium channels, was without effect on [Ca(2+)](i). Membrane depolarization with gramicidin or by a high-potassium buffer was without effects on the lectin-mediated calcium release from intracellular stores but inhibited the gal-1 induced receptor-operated calcium influx. In Jurkat E6.1 cells the lectin stimulates the transient generation of inositol-1,4,5-trisphosphate and the tyrosine phosphorylation of phospholipase Cgamma1. The results suggest that the ligation of CD2 and CD3 by gal-1 induces early events in T-cell activation comparable with that elicited by CD2 or CD3 mAbs.

Antibodies, Monoclonal↗
PubMed2000

Induction of erythroid proliferation and differentiation by a trophoblast-specific cytokine involves activation of the JAK/STAT pathway.

Pregnancy is characterized by increased erythropoiesis within maternal and fetal compartments. The placenta has been shown to produce factors that stimulate erythropoiesis but convincing evidence for placental production of erythropoietin (EPO) is still lacking. Prolactin-like protein E (PLP-E) was recently found to stimulate expression of the adult beta major globin gene in mouse erythroleukemia cells. Here we demonstrate that PLP-E transiently expressed in COS-7 cells stimulates proliferation and erythroid differentiation of murine and human erythroid progenitor cell lines. Electrophoretic mobility shift assays were used to show the activation of STAT5 by PLP-E in the human erythroid cell line TF1. Furthermore, we compared the effects of PLP-E on murine myeloid FDCP1 cells which do not express EPO receptors (EPORs) with effects on cells genetically engineered to express functional EPORs. We provide evidence that PLP-E-dependent proliferation and STAT5 activation is independent of the expression of the EPOR. Taken together, these data suggest that PLP-E acts on specific receptors of erythroid-committed murine and human cells by the activation of intracellular signaling pathways promoting cell growth and differentiation.

Animals↗
PubMed1999

Intragraft overexpression of interleukin-4 is neither sufficient nor essential for tolerance induction to cardiac allografts in a high-responder strain combination.

BACKGROUND: Recently we have demonstrated that the nondepleting anti-CD4 monoclonal antibody (mAb) RIB5/2 induces long-term acceptance of kidney and heart allografts in all rat strain combinations tested. Cytokine gene expression studies by reverse transcriptase-polymerase chain reaction revealed a reversed intragraft interleukin (IL)-4/interferon-gamma ratio. Whether IL-4 mediated immune deviation contributes to transplantation tolerance is not clear so far. METHODS: To learn more about the functional relevance of the relative IL-4 up-regulation, IL-4 was overexpressed in rat heart allografts by using ex vivo adenoviral gene transfer. The efficiency of gene transfer was analyzed by reporter gene assays as well by reverse transcriptase-polymerase chain reaction analysis of IL-4 mRNA expression. RESULTS: The intragraft overexpression of IL-4 did not prolong the allograft survival compared with controls. Moreover, neutralization of IL-4 by OX81 mAb did not prevent tolerance induction by RIB5/2 treatment. CONCLUSIONS: Anti-CD4 mAb-induced tolerance is associated with an intragraft type1/type2 shift, however, the up-regulation of IL-4 alone is neither sufficient nor essential to induce tolerance to cardiac allografts in a high-responder strain combination.

Animals↗
PubMed1999

Galectin-1, a natural ligand for the receptor-type protein tyrosine phosphatase CD45.

Galectin-1 binds preferentially to N-acetyllactosamine residues on oligosaccharides associated with several cell surface glycoconjugates. In the present work, placental galectin-1 has been identified to be a natural ligand for the receptor-type protein tyrosine phosphatase CD45. The binding of galectin-1 to CD45 was detected by affinity chromatography of NP 40 solubilized Jurkat T cell membranes on galectin-1 agarose followed by immunoblotting of the galectin-1 agarose bound fraction applying monoclonal antibodies to CD45 isoforms. The PTPase activity of the galectin-1 agarose binding membrane fraction could be inhibited by sodium orthovanadate. Preincubation of Jurkat T cell membrane preparations with galectin-1 decreased the membrane-associated PTPase activity in a concentration-dependent manner. Incubation of Jurkat cells with galectin-1 suppressed the immunoprecipitated PTPase activity of CD45. Galectin-1 stimulates the cell surface expression of phosphatidylserine an early indicator of apoptosis. In CD45+ Jurkat T cells, galectin-1 induces higher levels of phosphatidylserine when compared with CD45- Jurkat cells. These observations indicate that galectin-1-mediated ligation of CD45 is involved in the induction of apoptosis in Jurkat T cells.

Apoptosis↗