Hepatitis C virus infection and genotypes in Southern Israel and the Gaza Strip.

The Gaza Strip borders the southern part of Israel and Egypt. There is a remarkable difference in the prevalence of antibodies to hepatitis C virus (HCV) between Israel (0.5%) and Egypt (10%). A few thousand inhabitants cross the borders daily from the Gaza Strip to both countries. The objectives of this study were to investigate the prevalence of HCV infection in the Gaza Strip, an area that was not studied before, and to study HCV transmission in the Gaza Strip by characterizing the genotypes of HCV in Southern Israel and the Gaza Strip and comparing them with those found in Egypt. HCV prevalence in the Gaza Strip was found to be 2.2%, relatively higher than in Israel but lower than in Egypt. The most common genotypes found were type 1 b in Southern Israel and type 4 in the Gaza Strip, corresponding to the most prevalent genotype in Egypt. Similarity between type 4 isolates from the Gaza Strip and Egypt was illustrated further by sequence analysis of the HCV 5' noncoding region (NCR).

Targeted disruption of the mouse Caspase 8 gene ablates cell death induction by the TNF receptors, Fas/Apo1, and DR3 and is lethal prenatally.

Homozygous targeted disruption of the mouse Caspase 8 (Casp8) gene was found to be lethal in utero. The Caspase 8 null embryos exhibited impaired heart muscle development and congested accumulation of erythrocytes. Recovery of hematopoietic colony-forming cells from the embryos was very low. In fibroblast strains derived from these embryos, the TNF receptors, Fas/Apo1, and DR3 were able to activate the Jun N-terminal kinase and to trigger IkappaB alpha phosphorylation and degradation. They failed, however, to induce cell death, while doing so effectively in wild-type fibroblasts. These findings indicate that Caspase 8 plays a necessary and nonredundant role in death induction by several receptors of the TNF/NGF family and serves a vital role in embryonal development.

Increased soluble tumor necrosis factor receptor expression and release by human immunodeficiency virus type 1 infection.

High levels of circulating soluble tumor necrosis factor receptors (sTNF-R) are associated with HIV-1 infection and disease. To understand better this association, we have investigated p55 and p75 TNF-R expression on peripheral blood mononuclear cell (PBMC) subsets and in the promonocytic cell line U937, with or without HIV infection. Using flow cytometry and monoclonal antibodies both to sTNF-R and to PBMC subsets, TNF-R were found to be expressed mostly by monocytes and in decreasing amounts and intensity in the following order: CD14+ cells > CD8+ cells > CD4+ cells. Expression of TNF-R was higher on cells obtained from HIV-infected than from noninfected subjects, and expression of p75 sTNF-R was much higher than that of p55 sTNF-R. Studying the U937 cells revealed that over 80% of the cells expressed both sTNF-R, but with greater fluorescence intensity in the HIV-1 chronically infected cells (U-937-IIIB). Treatment of the cells with PMA caused an accelerated release into the medium of both sTNF-R, with a sharp decline in their cell surface expression. Basal levels of mRNA transcripts for p75 TNF-R were higher in the U-937-IIIB cells than in the uninfected cells, but p55 TNF-R mRNA was expressed only in the HIV-1-infected cells. These findings show that HIV-1 infection is accompanied by predominant elevation of p75 TNF-R surface expression on monocytes and CD8+ lymphocytes, and results in both increased message and expression of these receptors in monocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning, sequencing and partial functional characterization of the 5' region of the human p75 tumor necrosis factor receptor-encoding gene (TNF-R).

A 1887-bp region at the 5' flank of the human p75 tumor necrosis factor receptor (p75 TNF-R)-encoding gene was found to be active in driving expression of the luc (luciferase-encoding) reporter gene, suggesting that it contains the promoter for the receptor. Rather unexpectedly, a 1827-bp region at the 3' end of the first intron of the p75 TNF-R gene also displayed promoter activity. This activity may be artefactual, reflecting only the presence of an enhancer in this region; yet it also raises the possibility that p75 TNF-R is controlled by more than one promoter and that it encodes various forms of the receptor, or even other proteins. We present here the nucleotide sequences of the 5' flanking and intron regions. Possible implications for the transcriptional regulation of the p75 TNF-R gene are discussed.

Cloning and partial characterization of the promoter for the human p55 tumor necrosis factor (TNF) receptor.

An 809-bp region at the 5' flank of the human p55 tumor necrosis factor receptor (TNF-R)-encoding gene was found to be active in driving expression of the cat reporter gene, indicating that it contains a functional promoter. Deletion analysis showed that the promoter activity is present in the region between nucleotides (nt) -385 and -207; the sequence upstream from this region (nt -809 to -385) has an inhibitory effect. The promoter for the p55 TNF-R resembles housekeeping-type promoters in that it drives transcription from multiple start points (tsp) and lacks canonical TATA and CAAT box motifs. The cluster of tsp lies in a region which is particularly C+T rich. In this region, upstream from and near to the furthest upstream tsp, two closely located TCC repeat motifs were identified. These motifs also appear twice in the promoter for the epidermal growth factor receptor, where they were shown to be essential for promoter activity. The human p55 TNF-R promoter shows an overall resemblance, yet also some marked dissimilarities, to the recently described promoter for the mouse p55 TNF-R.

Selective up-regulation of the 75-kDa tumor necrosis factor (TNF) receptor and its mRNA by TNF and IL-1.

In cells of the fibroblastoid line SV-80, rapid down-modulation of TNF binding in response to TNF itself, or to IL-1, was followed by a gradual recovery of binding, which occurred even in the continuous presence of the cytokines. Untreated cells carried mainly the 55-kDa receptor species. In cells treated with TNF or IL-1, the 55-kDa TNF-R, although increasing after initial down-modulation, remained lower than before treatment. Expression of the 75-kDa TNF-R species upon treatment with either cytokine was markedly increased. Both TNF and IL-1 also induced a strong increase of the mRNA for the 75-kDa receptors, whereas the amount of mRNA for the 55-kDa receptors decreased. The effects of TNF on cell surface expression of the TNF-R could not be blocked with antibodies to IL-1, nor could the effects of IL-1 be blocked with antibodies to TNF, indicating that each cytokine affects the cell surface expression of the receptors independently of the other. Applying both cytokines together resulted in much stronger increase in expression of the 75-kDa TNF-R than applying each alone. Similar changes in cell surface expression and mRNA levels of the two TNF-R as observed in SV-80 cells were also found in TNF and IL-1-treated human foreskin fibroblasts. It is suggested that these sustained changes in the pattern of receptor expression contribute to the adjustment of the cellular response to TNF when formation of TNF and IL-1 takes place over a prolonged period.

Selective decrease in cell surface expression and mRNA level of the 55-kDa tumor necrosis factor receptor during differentiation of HL-60 cells into macrophage-like but not granulocyte-like cells.

Expression of the two known receptors for TNF was studied in the promyelocytic leukemia cell line HL-60 before and after differentiation of the cells along the granulocyte lineage (induced by incubation with retinoic acid), or along the macrophage lineage (induced by incubation with the phorbol diester, PMA). The extent of inhibition of TNF binding by receptor-specific antisera, as well as the size of the complexes formed after cross-linking TNF to its receptors on intact cells, indicated that both receptor species were expressed on the surface of the undifferentiated HL60 cells. Differentiation into granulocyte-like cells resulted in some increase in TNF binding. The increase was apparently due to enhanced expression of the 75-kDa TNF-R, whereas the amounts of the 55-kDa TNF-R did not change significantly. In contrast, in HL-60 cells induced to differentiate into macrophage-like cells, expression of the 55-kDa TNF-R species was completely abolished. The pattern of TNF-R expression in the differentiated HL-60 cells was similar to that observed in leukocytes isolated from peripheral blood: on granulocytes, there were about equal amounts of both receptor species, whereas on monocytes the 75-kDa receptor was predominant. The loss of 55-kDa receptors during differentiation of HL-60 cells into macrophage-like cells was accompanied by a pronounced decrease in the level of the mRNA for that receptor, suggesting that at least part of the change in TNF-R expression is due to mechanisms that control the amounts of receptor mRNA. Although little is yet known regarding the functional differences between the two receptor species, marked changes in the pattern of their expression, as observed during HL-60 cell differentiation, are likely to alter the kind of response of the cells to TNF and may therefore play an important role in the coordination of TNF effects in the organism.

Cytoplasmic truncation of the p55 tumour necrosis factor (TNF) receptor abolishes signalling, but not induced shedding of the receptor.

The mechanistic relationship between the signalling for the TNF effects by the human p55 TNF receptor (hu-p55-TNF-R) and the formation of a soluble form of the receptor, which is inhibitory to these effects, was explored by examining the function of C-terminally truncated mutants of the receptor, expressed in rodent cells. The 'wild-type' receptor signalled for a cytocidal effect when cross-linked with specific antibodies and exhibited spontaneous shedding. Shedding of the receptor was not affected by TNF but was markedly enhanced by 4 beta-phorbol-12-myristate-13-acetate (PMA). Receptor mutants with 53%, 83% and 96% C-terminal deletions could not signal for the cytocidal effect. Furthermore, they were found to associate with the endogenous rodent receptors, interfering with their signalling. Yet even the deletion of 96% of the intracellular domain did not abolish shedding of the receptor in response to PMA. These findings suggest that signalling and shedding of the p55 TNF-R are mechanistically distinct.

The gene for the type II (p75) tumor necrosis factor receptor (TNF-RII) is localized on band 1p36.2-p36.3.

The gene encoding the type II (p75) tumor necrosis factor receptor (TNF-RII) has been localized on human chromosome 1, band 1p36.2 by nonradioactive in situ hybridization. The gene encoding the type I (p55) TNF-R, which is structurally homologous to the type II (p75) TNF-R, has been previously localized on chromosome 12 band 12p13. Thus, despite their probable common ancestry, the genes for the two TNF-Rs are localized on different chromosomes.

Soluble and cell surface receptors for tumor necrosis factor.

Tumor necrosis factor (TNF) initiates its multiple effects on cell function by binding at a high affinity to specific cell surface receptors. Two different molecular species of these receptors, which are expressed differentially in different cells, have been identified. The cDNAs of both receptors have recently been cloned. Antibodies to one of these receptor species (the p55, type I receptor) can trigger a variety of TNF like effects by cross-linking of the receptor molecules. Thus, it is not TNF itself but its receptors that provide the signal for the response to this cytokine. The intracellular domains of the two receptors differ in structure, suggesting that they mediate different activities. Their extracellular domains, however, are structurally related. Both contain cysteine-rich repeats which are homologous to repeated structures found in the extracellular domains of the nerve growth factor receptor and the CDw40 protein. Truncated soluble forms of the two receptors, corresponding to these cysteine-rich repeated structures, have been detected in human urine and were later found to be present also in the serum. The serum levels of those soluble TNF receptors increase dramatically in certain pathological situations. Release of the soluble receptors from the cells seems to occur by proteolytic cleavage of the cell surface forms and appears to be a way of down-regulating the cell response to TNF. Because of their ability to bind TNF, the soluble receptors exert an inhibitory effect on TNF function, and may thus act as physiological attenuators of its activity.

Soluble forms of tumor necrosis factor receptors (TNF-Rs). The cDNA for the type I TNF-R, cloned using amino acid sequence data of its soluble form, encodes both the cell surface and a soluble form of the receptor.

Two proteins which specifically bind tumor necrosis factor (TNF) have recently been isolated from human urine in our laboratory. The two proteins cross-react immunologically with two species of cell surface TNF receptors (TNF-R). Antibodies against one of the two TNF binding proteins (TBPI) were found to have effects characteristic of TNF, including stimulating phosphorylation of specific cellular proteins. Oligonucleotide probes designed on the basis of the NH2-terminal amino acid sequence of TBPI were used to clone the cDNA for the structurally related cell surface type 1 TNF-R. It is notable that although this receptor can signal the phosphorylation of cellular proteins, it appears from its amino acid sequence to be devoid of intrinsic protein kinase activity. The extracellular domain of the receptor is composed of four internal cysteine-rich repeats, homologous to structures repeated four times in the extracellular domains of the nerve growth factor receptor and the B lymphocytes surface antigen CDw40. The amino acid composition and size of the extracellular domain of the type I TNF-R closely resemble those of TBPI. The COOH-terminal amino acid sequence of the four cysteine rich repeats within the extracellular domain of the type I TNF-R matches the COOH-terminal sequence of TBPI. Amino acid sequences in the extracellular domain also fully match other sequences found in TBPI. On the other hand, amino acid sequences in the soluble form of the type II TNF-R (TBPII), while indicating a marked homology of structure, did not suggest any identity between this protein and the extracellular domain of the type I TNF-R. CHO cells transfected with type I TNF-R cDNA produced both cell surface and soluble forms of the receptor. The receptor produced by CHO cells was recognized by several monoclonal antibodies against TBPI, reacting with several distinct epitopes in this molecule. These data suggest that the soluble forms of the TNF-Rs are structurally identical to the extracellular cytokine binding domains of these receptors and are consistent with the notion that the soluble forms are, at least partly, derived from the same transcripts that encode the cell surface receptors.