Helicobacter pylori has been associated with gastritis, peptic ulcer, and gastric adenocarcinoma. We report the nucleotide sequence and expression of an immunodominant antigen of H. pylori and the immune response to the antigen during disease. The antigen, named CagA (cytotoxin-associated gene A), is a hydrophilic, surface-exposed protein of 128 kDa produced by most clinical isolates. The size of the cagA gene and its protein varies in different strains by a mechanism that involves duplication of regions within the gene. Clinical isolates that do not produce the antigen do not have the gene and are unable to produce an active vacuolating cytotoxin. An ELISA to detect the immune response against a recombinant fragment of this protein detects 75.3% of patients with gastroduodenal diseass and 100% of patients with duodenal ulcer (P < 0.0005), suggesting that only bacteria harboring this protein are associated with disease.

Helicobacter pylori is a human pathogen that has been associated with gastritis, peptic ulcer and gastric carcinoma. The role of the direct action of H. pylori virulence factors and of the induction of autoreactive immunity in the development of chronic gastritis has not been clarified yet. Here we report the cloning and molecular characterization of a gene of H. pylori coding for a protein of 58 kDa, recognized by sera of patients affected by H. pylori-induced gastroduodenal diseases. This antigen is present in all the H. pylori strains tested and it belongs to the Hsp60 family of heat-shock proteins, with high homology with other bacterial and eukaryotic proteins of the same family. This class of homologous proteins has been implicated in the induction of autoimmune disorders in different systems. The presence in infected patients of anti-H. pylori Hsp60 antibodies, potentially cross-reactivity between human Hsp60 and a rabbit antiserum against H. pylori Hsp60 suggest that a role of this protein in gastroduodenal diseases is possible.

Activation of T‐cells infected by HIV‐1 results in activation of long terminal repeal (LTR)‐dependent viral transcription and ultimately the production of infectious virus. Although fulI T‐cell activation requires a complex series of intracellular signals, including protein kinase C activation, calcium mobilisation, and less‐well defined lymphokine‐induced signals, the HIV‐1 LTR can be activated by subsets of these signals. We have studied the interaction of these signals in the human lymphoma line, Jurkat, in activation of the HIV‐1 LTR. The HIV promoter was induced by IL‐1 and phorbol ester activation of PKC but not by a calcium ionophore. The constitutively active form of Ha‐ras could replace phorbol ester stimulation of the HIV promoter and of a synthetic promoter containing NFκB binding sites.

We have isolated from a genomic library a murine recombinant clone containing the gene coding for interleukin-1 beta m-RNA. A 7000 b.p. DNA fragment has been sequenced. Sequences homologous with human IL-1 beta cDNA have been found distributed within 7 exons. The translation of these sequences allows the prediction of a protein 269 aminoacids long. Hybridization of P388D1 RNA from cells stimulated with phorbol myristic acetate with a genomic DNA probe shows the existence of a 1.6 Kb murine IL-1 beta mRNA which is absent in the unstimulated cells. The comparative analysis between the murine IL-1 beta and the human IL-1 alpha genes shows extreme conservation of the aminoacids at the exon junctions. This observation together with the similarity in number and size of the exons suggests that these genes have diverged from a common ancestor.

We report the immunization protocol used to produce high frequency of specific hybridomas secreting defined antibodies against soluble proteins. The two immunization schemes used consist in 1300 and 115 micrograms of protein distributed over a period of two weeks. The specific efficiency (SE) of positive clones recovered in eleven different fusion experiments ranged between 0.52 and 0.78. These values are referred to hybridomas secreting monoclonal antibodies (MAbs) against soluble proteins such as human chorionic somatomammotropin (hCS), human growth hormone (hGH), and human prostatic acid phosphatase (hPAP). Similar high SE were also recovered by immunizing mice with nonsoluble antigens such as intermediate filaments of cytoskeleton (IF).

Interleukin-1 (IL-1) is known to synergize with phorbol esters in the induction of interleukin-2 (IL-2) expression in T-lymphoid leukemia cells and proliferation of mouse thymocytes. We used a plasmid construct containing the bacterial gene for chloramphenicol acetyltransferase under the control of the human IL-2 promoter to study the nature of this synergism in the murine thymoma cell line EL4. Although IL-1 induction of the IL-2 promoter in these cells required costimulus with phorbol myristate acetate, the signal induced by IL-1 was qualitatively different. We provide evidence to support the hypothesis that the phorbol ester signal is mediated by protein kinase C, and we show that the IL-1 signal is not. That IL-1 and phorbol myristate acetate represent different stimuli was shown by their response to protein kinase C inhibitors, capacity to synergize with increased intracellular free calcium, and requirement for protein synthesis. In addition we show that pretreatment with IL-1 can prime EL4 cells to subsequent activation by concentrations of phorbol esters not normally sufficient to induce IL-2 expression. Pretreated cells remained primed for at least 40 h after removal of the IL-1. Neither phorbol myristate acetate nor a calcium ionophore was capable of preactivating EL4 cells.

No abstract

NIH 3T3 fibroblasts were transfected with the chloramphenicol-acetyltransferase (CAT) gene under the control of the SV40 early promoter, which can be stimulated by IL-1. CAT activity in cell lysates and PGE2 release in the supernatants were measured in control and stimulated cell cultures in parallel. Human IL-1 beta (180 pM) and human rTNF-alpha (3 nM) significantly stimulated both CAT activity and PGE2 release. The combined incubation of the two cytokines resulted in a synergistic effect on PGE2 release. The addition of AA (30 microM) greatly stimulated PGE2 release without affecting CAT activity. Similarly, drugs interfering with AA metabolism were without effect on CAT activity although profoundly reducing PGE2 release. Forskolin (0.1 microM) did not modify either parameter. The glucocorticoid fluocinolone (20 nM) was able to decrease both parameters. Protein kinase inhibitors H7 (5-50 microM) and sphingosine (50 microM) inhibited only IL-1-induced CAT activity, whereas H8 (5-50 microM) and HA1004 (50 microM) were ineffective on both parameters. PMA (0.5 microM) and R59 022, a diacylglycerol kinase inhibitor (10 microM), did not modify either control or IL-1-induced CAT activity. IL-1-stimulated PGE2 release was potentiated by PMA, although this effect was not inhibited by H7. The data suggest that: 1) in NIH 3T3 cells the activation of AA metabolism by IL-1 is not involved in IL-1-induced gene expression; 2) protein kinase C activity is required but not sufficient for IL-1-induced gene expression; and 3) PMA may stimulate AA metabolism by a mechanism in part independent of protein kinase activity.