Transgenic cyclooxygenase-2 expression and high salt enhanced susceptibility to chemical-induced gastric cancer development in mice.

Cyclooxoygenase (COX)-2 overexpression is involved in gastric carcinogenesis. While high-salt intake is a known risk factor for gastric cancer development, we determined the effects of high salt on gastric chemical carcinogenesis in COX-2 transgenic (TG) mice. COX-2 TG mice were developed in C57/BL6 strain using the full-length human cox-2 complementary DNA construct. Six-week-old COX-2 TG and wild-type (WT) littermates were randomly allocated to receive alternate week of N-methyl-N-nitrosourea (MNU, 240 p.p.m.) in drinking water or control for 10 weeks. Two groups of mice were further treated with 10% NaCl during the initial 10 weeks. All mice were killed at the end of week 50. Both forced COX-2 overexpression and high-salt intake significantly increased the frequency of gastric cancer development in mice as compared with WT littermates treated with MNU alone. However, no additive effect was observed on the combination of high salt and COX-2 expression. We further showed that MNU and high-salt treatment increased chronic inflammatory infiltrates and induced prostaglandin E(2) (PGE(2)) production in the non-cancerous stomach. Whereas high-salt treatment markedly increased the expression of inflammatory cytokines (tumor necrosis factor-alpha, interferon-gamma, interleukin (IL)-1 beta and IL-6) in the gastric mucosa, COX-2 overexpression significantly altered the cell kinetics in the MNU-induced gastric cancer model. In conclusion, both high salt and COX-2 overexpression promote chemical-induced gastric carcinogenesis, possibly related to chronic inflammation, induction of PGE(2), disruption of cell kinetics and induction of inflammatory cytokines.

BRE is an antiapoptotic protein in vivo and overexpressed in human hepatocellular carcinoma.

BRE binds to the cytoplasmic domains of tumor necrosis factor receptor-1 and Fas, and in cell lines can attenuate death receptor-initiated apoptosis by inhibiting t-BID-induced activation of the mitochondrial apoptotic pathway. Overexpression of BRE by transfection can also attenuate intrinsic apoptosis and promote growth of the transfected Lewis lung carcinoma line in mice. There is, however, a complete lack of in vivo data about the protein. Here, we report that by using our BRE-specific monoclonal antibody on the immunohistochemistry of 123 specimens of human hepatocellular carcinoma (HCC), significant differences in BRE expression levels between the paired tumoral and non-tumoral regions (P<2.2e-16) were found. Marked overexpression of BRE was detected in majority of the tumors, whereas most non-tumoral regions expressed the same low level of the protein as in normal livers. To investigate whether BRE overexpression could promote cell survival in vivo, liver-specific transgenic BRE mice were generated and found to be significantly resistant to Fas-mediated lethal hepatic apoptosis. The transgenic model also revealed post-transcriptional regulation of Bre level in the liver, which was not observed in HCC and non-HCC cell lines. Indeed, all cell lines analysed express high levels of BRE. In conclusion, BRE is antiapoptotic in vivo, and may promote tumorigenesis when overexpressed.

In vivo functional characterization of the SARS-Coronavirus 3a protein in Drosophila.

The Severe Acute Respiratory Syndrome-Coronavirus (SARS-CoV) 3a locus encodes a 274 a.a. novel protein, and its expression has been confirmed in SARS patients. To study functional roles of 3a, we established a transgenic fly model for the SARS-CoV 3a gene. Misexpression of 3a in Drosophila caused a dominant rough eye phenotype. Using a specific monoclonal antibody, we demonstrated that the 3a protein displayed a punctate cytoplasmic localization in Drosophila as in SARS-CoV-infected cells. We provide genetic evidence to support that 3a is functionally related to clathrin-mediated endocytosis. We further found that 3a misexpression induces apoptosis, which could be modulated by cellular cytochrome c levels and caspase activity. From a forward genetic screen, 78 dominant 3a modifying loci were recovered and the identity of these modifiers revealed that the severity of the 3a-induced rough eye phenotype depends on multiple cellular processes including gene transcriptional regulation.

Comparative proteomic analysis identifies protein disulfide isomerase and peroxiredoxin 1 as new players involved in embryonic interdigital cell death.

In this study, we used comparative proteomics to identify proteins that were involved in the regulation of interdigital cell death. The protein profiles of embryonic day (E) 12.5 and 13.5 mouse hindlimb interdigital tissues were compared to identify proteins that were differentially expressed. The interdigital cells are irreversibly committed to programmed cell death (PCD) at E13.5, whereas they are developmentally plastic at E12.5. We established that protein disulfide isomerase (PDI) expression was up-regulated at E13.5, while peroxiredoxin 1 (Prdx1) expression was down-regulated at this time point. Semiquantitative reverse transcriptase-polymerase chain reaction and Western blot analyses confirmed the data obtained from the two-dimensional electrophoresis gels. Furthermore, we were able to up-regulate PDI expression by manipulating the E12.5 interdigital tissues to die during culture, although this up-regulation was not possible when cell survival was promoted. In addition, we could inhibit interdigital cell death and expression of proapoptotic genes (Bmp-4 and Bambi) by treating interdigital tissues with PDI antibodies and bacitracin (a PDI enzyme inhibitor). These findings suggested that PDI was involved in the activation and maintenance of interdigital cell death. Conversely, we determined that Prdx1 expression was maintained when interdigital cultures were manipulated to survive but down-regulated when the cultures were permitted to die. The result suggested that Prdx1 was involved in maintaining interdigital cell survival. However, we were unable to induce interdigital cell death by means of RNA interference-mediated silencing of Prdx1 expression, indicating that Prdx1 down-regulation is not sufficient for PCD to occur. Proteomic analysis of the Prdx1 knock-down cells revealed that the level of NF-kappaB inhibitor epsilon (IkappaBepsilon) was dramatically reduced. Furthermore, we found an increase in NFkappaB activation and reactive oxygen species (ROS) levels in the cytoplasm as a result of Prdx1 knockdown. We also found that silencing Prdx1 made the interdigital cells more susceptible to ROS-induced cell death. Taken together, our study identifies two new players in interdigital cell death and highlights that PCD is regulated by a delicate balance of proapoptotic and survival-promoting activities.

Expression of human BRE in multiple isoforms.

BRE, a putative stress-modulating gene, found able to down-regulate TNF-alpha-induced NF-kappaB activation upon overexpression, is now shown in human cells expressed as multiple mRNA isoforms. A total of six isoforms are produced by alternative splicing predominantly at either end of the gene. Predicted from the cDNA sequences of these isoforms, three of them (alpha(a), alpha(b), and alpha(c)) code for BRE of different C-terminus, and the other three (beta(a), beta(b), and beta(c)) may possibly be the nonfunctional counterparts. All human cells examined coexpress all the predominant splice variants, albeit at different ratios. Comparing with normal cells, immortalized human cell lines uniformly express higher levels of BRE. Interestingly, peripheral blood monocytes responded to LPS by down-regulating the expression of all the BRE isoforms, which was however less obvious in the cell line counterpart, THP-1. Isoform alpha(a), which codes for the canonical BRE with a C-terminal peroxisomal targeting sequence, is the most abundant transcript. We propose that the function of BRE and its isoforms is to regulate peroxisomal activities.

Molecular mimicry: anti-DNA antibodies may arise inadvertently as a response to antibodies generated to microorganisms.

The origin of anti-DNA antibodies remains speculative. We argue that some of these antibodies may arise inadvertently in nature during the course of a normal immune response due to their induction by antibodies which bear structures (mimotopes) that mimic DNA. These antibodies are not necessarily DNA specific but, like the T15 idiotype (id)-positive antibodies which bind to phosphorylcholine, are produced normally to some environmental or microbial antigen. Such a mimotope was found in a T15(+) antibody at the highly specific region encoded principally by the D gene, DFL16.1. This mimotope was also found in human antibodies that are encoded by DXP'1, the human counterpart of DFL16.1 and which is used commonly in anti-DNA antibodies. The mimotope is closely related to the epitope responsible for the T15 id and appears to be cryptic or normally hidden in the native protein. The existence of such a common, conserved sequence raises questions about how easily anti-DNA antibodies can be generated in nature and what purpose these proteins may serve. Molecular mimicry with regard to autoimmunity must thus be viewed as existing not necessarily between the infectious agent and self-antigens, but also between the antibodies induced by the organism and the self-antigens.

A human and a mouse anti-idiotypic antibody specific for human T14(+) anti-DNA antibodies reconstructed by phage display.

Little is known about human anti-idiotypic antibodies. Phage display methodology was used to reconstruct these antibodies from lupus patients, which recognize a subset (T14(+)) of anti-DNA antibodies. Antigen-specific B cells were isolated from the blood using a peptide based on a complementarity determining region (V(H)CDR3) of the prototypic T14(+) antibody. cDNA fragments of the V(H) and V(L) genes prepared from the cells were expressed as phage displayed single chain Fv (scFv) fragments using the pCANTAB-5E phagemid vector. From a reactive clone obtained, the Ig genes used were identified to be V(H)3, D5-D3, J(H)4b, V(kappa)I and J(kappa)2. The heavy chain was highly mutated, especially in CDR3, which bears mutations mostly of the replacement type; this region is also unusual in being extremely long due to a D-D fusion. In contrast, a mouse hybridoma antibody, made to the same T14(+) peptide and transformed as a scFv fragment, uses a short V(H)CDR3 comprising five amino acids, three of which are tyrosines. Tyrosines may be important for antigen binding because two of these also exist in the human V(H)CDR3. The light chains of both antibodies may also contribute to the specificity of the protein, because their V(L) segments, including the CDRs, are highly homologous to each other.

Strand bias in Ig somatic hypermutation is determined by signal sequence within the variable region.

Ig genes undergo hypermutation with a nucleotide preference of A over T for mutation on the coding strand. As only with concomitant strand bias can such nucleotide bias be observed, Ig gene hypermutation is generally accepted as a strand-specific process, for which the mechanistic basis remains unknown. It has previously been shown that different non-Ig sequences replacing the LVJ region of an Ig transgene to various extents are targeted for hypermutation with similar mutation frequencies. However, the nucleotide bias characteristic of Ig hypermutation was not found in two of the three such sequences studied. To test whether it is the DNA sequences of the non-Ig substrates that determine the pattern of nucleotide bias in hypermutation or whether the LVJ sequence may contain element(s) that confer strand bias, we have added back all the replaced LVJ sequences to one of the transgenes, L(kappa)-Vgpt*, that expresses no strand bias in hypermutation and studied the outcome. The results show that the gpt sequence in the presence of the complete LVJ sequence hypermutates differently from the same sequence in L(kappa)-Vgpt* where 84% of the LVJ was replaced. The main difference is the resumption of strand bias characteristic of Ig hypermutation. Thus, whether or not a substrate sequence manifests strand bias in hypermutation is not inherently determined by the substrate DNA sequence. This indicates the presence of special element(s) within the LVJ that confer strand bias.

Phage-displayed La/SS-B antigen as a diagnostic reagent.

BACKGROUND: The detection of antibodies to La/SS-B, a nuclear RNA-binding protein in mammalian cells, aids in the diagnosis of Sjogren's syndrome and systemic lupus erythematosus (SLE). This is performed conventionally by immunoprecipitation using a crude splenic extract and more recently, by the more sensitive and rapid enzyme-linked immunosorbent assay (ELISA) which uses a purified La/SS-B antigen. The latter antigen is obtained from cellular extracts of the antigen or from bacterial cell lysates containing the recombinant antigen usually by affinity chromatographic method. OBJECTIVE: To produce a La/SS-B antigen for use in ELISA that can be obtained easily and inexpensively without the need for extensive purification (including affinity chromatography). STUDY DESIGN: The antigen was produced as a fusion protein of the minor coat protein of M13 bacteriophage and used in this phage-associated form in an ELISA. La/SS-B cDNA derived from Hep-2 cells was cloned into the phagemid, pCANTAB-5E, and transfected to Escherichia coli. Phage clones selected for the presence of insert both by gene and antigenic analyses were used in the ELISA to detect anti-La/SS-B antibodies from patients with Sjogren's syndrome and SLE. RESULTS: A phage clone was obtained which contained a La/SS-B cDNA fragment truncated at the C-terminal end (after base-pair 631). The phage-displayed antigen derived from this clone was obtained by precipitation of the phage particles from the bacterial culture supernatant with polyethylene glycol. Used in the ELISA, this antigen detected 27 of 28 precipitin-positive sera and was negative for 50 control sera. The soluble (phage-free) form of the antigen was obtained from a nonsuppressor host as a cell lysate which could not be used in this form in an ELISA for antibody detection. It was useable, however, in Western blot analysis which confirmed the reactivity of the recombinant antigen. CONCLUSION: Phage-displayed antigens may be used in place of soluble forms of these antigens in detection assays which have the advantage that they are easy and inexpensive to produce.

Identification of a surrogate TSL-1 antigen of Trichinella spiralis from a phage library of random peptides.

We sought to find a peptide analogue of an important antigen (TSL-1) of Trichinella spiralis which is recognized by the 7C2C5 antibody. A phage library which displays a short (15-mer) randomly-generated peptide at the filament of the minor coat protein of the virion was used for selection by the 7C2C5 antibody. A peptide thus identified, ICDASGLGCWCWSLSP, was found to be a true surrogate since its binding to the antibody could be blocked by the native antigen and, conversely, an antiserum made to the peptide could recognize the native antigen. In addition, the peptide appeared to detect T. spiralis-infected pigs although it was less discriminatory than the native antigen.

Common occurrence of an antiidiotypic antibody that recognizes T14+ anti-DNA antibodies in patients with systemic lupus erythematosus.

OBJECTIVE: To investigate whether antibodies to a T14 anti-DNA antibody can be found in patients with systemic lupus erythematosus (SLE). METHODS: Seventy-six serum samples (37 from patients with SLE) were randomly selected from among sera submitted for routine antinuclear antibody testing. Short, overlapping peptides based on the partial VH (variable region of the heavy chain) sequence of the T14 antibody were synthesized on multipins and screened for reactivity with SLE sera. In addition, selected peptides from T14 and related proteins were synthesized in bulk and screened for reactivity with both SLE and control sera. A monoclonal antibody was generated to determine the prevalence of the T14 idiotype (T14+ Id) in the different study populations. RESULTS: Antibodies were detected by a peptide based on the third complementarity-determining region (CDR3) of the T14 protein in 15 (41%) of 37 patients with SLE or 15 (54%) of 28 who had anti-DNA antibodies, in 3 (9%) of 34 patients without anti-DNA antibodies (9 of whom had SLE), and in 6 (10%) of 57 healthy controls. In SLE sera, the antiidiotypic (anti-Id) responses (IgM and IgG) correlated well with the anti-DNA responses (IgG), and both responses correlated well with the T14+ Id activity in SLE sera. Control peptides based on the 18/2 (16/6+ Id) and S107 proteins detected low antibody activities in SLE sera, attributable to cross-reactivity with the T14 peptide. A peptide based on an unrelated human antibody was not reactive with these sera. CONCLUSION: Anti-Id antibodies directed to T14 VHCDR3 were found commonly in the sera of patients with SLE, and they appeared to be induced by the anti-DNA antibodies present in the sera. Based on these findings, these secondary antibodies may be pathogenic in SLE.

Identification of C-type natriuretic peptide gene transcripts in glial cells.

C-type natriuretic peptide (CNP), a third member of the natriuretic peptide family, is found throughout the central nervous system (CNS), particularly in those regions involved in neuroendocrine regulation. Astrocytes, which have important physiological roles in normal neuronal functioning, express receptors of CNP. Using reverse transcription-polymerase chain reaction (RT-PCR), followed by hybridization with a digoxigenin-labelled cDNA probe, we have demonstrated the expression of CNP gene transcripts in both cultured mouse astrocytes and rat C6 glioma cells, with the former expressing the gene at a considerably higher level than the latter. Our data raise the possibility that CNP may act in autocrine and/or paracrine fashion in glial cell physiology and neuromodulate communication between glial cells and neurones.

Targeting of non-Ig sequences in place of the V segment by somatic hypermutation.

Affinity maturation of antibodies is characterized by localized hypermutation of the DNA around the V segment. Here we show, using mice containing single or multiple transgene constructs, that an immunoglobulin V kappa segment can be replaced by human beta-globin or prokaryotic neo or gpt genes without affecting the rate of hypermutation; the V gene itself is not necessary for recruiting hypermutation. The ability to target hypermutation to heterologous genes in vivo could find more general applications in biology.

A reporter gene to analyse the hypermutation of immunoglobulin genes.

The affinity maturation of antibodies is driven by somatic hypermutation which is localized to specific segments of the coding genes. The information available on this process derives from studies in vivo. With the intention of developing new approaches, we have constructed a fusion gene between a kappa chain and a selectable neomycin resistance gene, neor. The neor gene, which includes the SV40 small t intron and polyadenylation site, but not the upstream elements nor its first 12 amino acids, is an in-frame substitution of the FR2-CDR3 fragment of a rearranged V kappa OX1-J kappa 5 gene. Expression of neor activity is therefore dependent on the upstream immunoglobulin sequence. A stop codon was placed in the CDR1 region so that only mutants survive treatment with geneticin sulphate (G418). The effectiveness of the system was tested by transfecting the NS0 myeloma cell line and isolating spontaneous mutants. Neomycin-resistant clones arose at an estimated rate of 1 x 10(-8)/cell division, and over 90% were authentic structural mutants. Unlike the somatic hypermutations, the majority arose by in-frame deletions including the stop codon, although up to 30% involved a point mutation. The reporter gene was then modified by substituting all the sequences downstream of the J kappa 5 with others known to be required for full hypermutation in vivo. Different cell lines were transfected and G418-resistant clones analyzed. No significant increase in the rate of reversion or in the generation of point mutations versus deletions was detected, even using conditioned culture medium. In the presence of azacytidine however, a mutant involving multiple events (single base addition and deletion plus two point mutations) was detected. The reporter gene system therefore seems suitable to test culture conditions and modifications of the host cells aimed at the derivation of an in vitro assay of somatic hypermutation.

Structural analysis of a phosphorylcholine-binding antibody which exhibits a unique carrier specificity for Trichinella spiralis.

A phosphorylcholine (PC)-binding IgG (Mab2) antibody produced by a hybridoma derived from a BALB/c mouse which had been immunized against Trichinella spiralis was found to bind to the immunizing antigen (TSC) but not to other PC-associated antigens such as pneumococcal antigen (PNC) and PC-conjugated ovalbumin (PC-OVA). Sequence analysis of the protein revealed the presence of a heavy chain (VH) which was very similar (differing in only four amino acids) to that of the M511 myeloma protein, and a light chain (VL) which was completely identical to that of the M167 myeloma protein. Several M511/M167+ proteins, including the prototypic M511 protein and PC-binding proteins of other families (TEPC 15 and W3207), were examined in their binding to the various PC-associated antigens. These were found to be largely indiscriminate although subtle differences were observed for some antigens with some of the antibodies. A comparison of the VH sequences of Mab2 and these proteins revealed that of the differences seen, the single most important substitution in Mab2 which could contribute to the unique specificity of the molecule is the glycine residue at 49H. None of the other proteins, including other PCV-binding proteins published to-date, which utilize the same VH segment (99 in total), has this substitution.

CD4-positive cells from patients with IgA nephropathy demonstrate increased mRNA of cytokines that induce the IgA switch and differentiation.

IgA nephropathy (IgAN) is characterized by raised serum IgA1 and mesangial IgA1 deposits. We have previously shown increased T-cell activation in IgAN. Recently, transforming growth factor-beta (TGF-beta) has been shown to induce IgA isotype switch at a clonal level and interleukin 5 (IL5) promotes differentiation into IgA-bearing B cells. In the present study we have examined the TGF-beta and IL5 mRNA expression by mitogen-activated CD4-positive T cells from patients with IgAN (n = 25), patients with other primary nephritides (CGN) (n = 24), and healthy control subjects (n = 25). The cytokine genes were analysed by reverse transcription (RT)-polymerase chain reaction (PCR) and were semi-quantitated by normalizing the differences occurring during RT and PCR using a housekeeping gene, beta-actin. CD4-positive T cells from IgA nephritic patients expressed a higher level of IL5 mRNA than healthy controls (P < 0.01) and patients with CGN (P < 0.005). CD4-positive T cells from IgA nephritic patients expressed a higher level of TGF-beta mRNA than healthy controls (P < 0.01) but no difference was demonstrated on comparison with CGN patients. Elevated TGF-beta mRNA expression in patients with CGN probably reflects its other important function as a 'sclerogenic' factor involved in the glomerulosclerosis found in these nephritides. Our data suggest that there is increased expression of cytokine genes which induce the IgA isotype switch and differentiation; these immunological abnormalities may be important in the pathogenesis of IgAN.

Circulating leukocyte-endothelial adhesion molecules in IgA nephropathy.

There is accumulating evidence that leukocyte-endothelial adhesion molecules are important in inflammatory injury in being involved in the primary step of entrapment and migration of leukocytes to the site of inflammation. We have used an antigen capture ELISA to measure the levels of circulating intercellular adhesion molecule-1 (cVCAM-1), vascular cell adhesion molecule-1 (cVCAM-1) and E selectin (cE selectin) in the peripheral blood of 33 patients with IgA nephropathy (IgAN) during clinical quiescence and 24 healthy controls. The serum levels of cICAM-1 and E selectin in IgA-nephritic patients were not different from that of healthy controls but the cVCAM-1 level was significantly elevated in IgAN despite a lack of clinical activity (p = 0.008). The differential rise of circulating leukocyte-endothelial adhesion molecules in IgAN probably reflects the origins and nature of these molecules as well as the specific immunological profile of IgAN. There was no correlation between the levels of these three circulating adhesion molecules. When the patients with IgA nephropathy were stratified according to the severity of their glomerular and interstitial lesions, there was an apparent increase in cE selectin and cVCAM-1 associated with increased histopathologic grading. The changes in endothelial adhesion molecules during clinical exacerbation were studied in 10 patients. Coinciding with synpharyngitic macrohematuria, there was a significant rise of cVCAM-1 and cE selectin (p = 0.046 and p = 0.016, respectively) but no similar rise was observed in cICAM-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of cytokine gene expression in CD4+ and CD8+ T cells after activation with phorbol myristate acetate and phytohaemagglutinin.

Cytokines are important mediators involved in the development of effector cells and in the regulation of immune responses. The gene expression of these mediators in T cell subset has yet to be fully elucidated. Using sensitive reverse transcription-polymerase chain reaction (RT-PCR), the kinetics of cytokine gene expression in human CD4+ and CD8+ T cells were examined. CD4+ T cells were more readily activated by phorbol myristate acetate (PMA) and phytohaemagglutinin (PHA) than CD8+ T cells in terms of the IL-2 receptor (IL-2R) mRNA expression. Quantitative differences in cytokine gene expression between CD4+ and CD8+ T cells were confirmed and higher levels of cytokine mRNAs were induced in CD4+ than in CD8+ T cells. Early induction of IL-2 mRNA was observed in both T cell subsets. The demonstration of different kinetics of cytokine gene expression illustrates one of the examples of the complexity of immunoregulation. The differential response of cytokine gene expression in different T cell subsets should be taken into consideration when clinical studies in cytokine production by peripheral blood mononuclear cells are interpreted.