Molecular biology & medicine最新文献

Evidence from epidemiological and histopathologic studies in humans with autoimmune type 1 (insulin-dependent) diabetes suggests that beta-cell destruction within the islets of Langerhans progresses through a number of stages. In this review we draw on recent experimental evidence in an attempt to define the molecular pathology of these stages. Stage 1 is postulated to be initiated by modification of the beta cell by virus, chemical or other factors, leading to the production of interferon-alpha, hyperexpression of major histocompatibility complex (MHC) class I molecules and induction of MHC class II molecules. Experiments in transgenic mice suggest that overexpression of MHC molecules is in itself detrimental to beta-cell function. Shedding of antigen(s) from dying beta cells in combination with hyperexpression of MHC molecules may be a powerful immunogenic stimulus. Stage 2 commences with infiltration of the islets by immuno-inflammatory cells (termed insulitis). It is proposed that production of cytokines from the infiltrating cells induces "phenotypic switching" in beta cells, with further upregulation of MHC molecules and the induction of intracellular adhesion molecule-1 expression and interleukin-6 production. Together, these properties are seen as a prerequisite for the presentation of autoantigen by beta cells to adherent T lymphocytes and autoimmune activation. The final stage encompasses autoimmune-mediated destruction of the beta cells by the targeted delivery of cytotoxic cytokines and other mediators.

Transgenic mice represent a versatile experimental approach for understanding the pathways by which the immune system regulates responsiveness to self-antigens, thereby establishing self-tolerance. The introduction of immunoglobulin and T cell receptor genes with specificity for self-antigens into the germline of mice has enabled the fate of self-reactive lymphocyte precursors to be followed in vivo. The influence of both developmentally regulated, and tissue-specific gene expression on tolerance to self-antigens has been addressed using transgenic mice expressing novel self-antigens under the transcriptional control of heterologous gene promoters. The generation of transgenic mice expressing structurally altered forms of self-antigens has allowed the role of antigen-processing in the induction of tolerance to be examined. Significantly, while these studies have confirmed the classically derived principles of immunological self-tolerance, they also point to the existence of pathways, as yet undefined, by which tolerance to self-antigens may be implemented and maintained.

The sexual difference in the incidence of autoimmune diseases has remained an enigma for many years. In the examination of the induction of autoimmunity in transgenic mice, evidence has been obtained further implicating the lymphokine interferon-gamma in the etiology of autoimmunity. Sex steroid regulation of the production of this molecule, as well as other cytokines, may help explain the gender-specific differences in the immune system, including autoimmunity.

Reversal of autoimmune disease with monoclonal antibodies to polymorphic determinants associated with class II gene products of the major histocompatibility complex (MHC) and to T-cell receptor variable region segments has been demonstrated in animal models. Recent studies have shown that it is also possible to use mutant peptides to block recognition of self-antigen associated with MHC by T-cells that mediate autoimmune disease. These mutant peptides have been used to prevent the model autoimmune condition experimental allergic encephalomyelitis. The possibility of extending these approaches to human disease is discussed.

Studies of banding induced by restriction enzymes may provide insight into banding mechanisms and chromosome structure. We examined whether or not the sizes of chromosome-specific alphoid DNA fragments created by digestion with various restriction enzymes relate to the presence or absence of C-like bands produced by these same enzymes. We sized alphoid DNA fragments from five different chromosomes, digested with each of six different restriction enzymes. There was no obvious correlation between the length of alphoid restriction fragments at specific human centromeric regions and the production of C-like bands. We used the enzyme AluI and traditional staining (CBG) techniques to band centromeres with conformational alterations. These included dicentric chromosomes, chromosomes from a patient with Roberts syndrome, and 5-azacytidine-treated prometaphase chromosomes. In all cases bands produced by AluI resembled CBG banding. We found that markedly decondensed portions of centromeric regions induced by 5-azacytidine did not band. Our studies demonstrate that restriction endonuclease C-like banding is not strictly related to the presence of restriction sites in alphoid DNA, and the condensed chromatin conformation at the centromeric region may play a role in banding.

The broad-range proteinase inhibitor alpha 1-inhibitor III (alpha 1I3), a member of the complement C3/alpha 2-macroglobulin protein family, is the prototype of a negatively regulated acute phase protein. During an acute inflammatory reaction alpha 1I3 plasma protein and liver mRNA concentrations are decreased three- to fourfold in rats, and in chronic inflammations the protein concentration is reduced between ten- and 20-fold. In search of a cell culture model to study the regulation of the alpha 1I3 gene by mediators of inflammation, five well-established rat hepatoma cell lines were examined. All five lines constitutively expressed the gene, a marker for a highly differentiated hepatic phenotype, although at less than one-tenth the level of its expression in vivo. In the three hepatoma lines FAZA, FTO2B and FAO1, alpha 1I3 mRNA was decreased by treatment with interleukin 6 (IL6) and glucocorticoids. Among these lines untreated FAO1 cells produced the highest constitutive concentrations of alpha 1I3 mRNA and in FAO1 cells alpha 1I3 mRNA concentrations were decreased up to fourfold in a dose-responsive and time-dependent manner after treatment with IL6 alone or with combinations of IL6 and the synthetic glucocorticoid dexamethasone. Thus, IL6 alone was sufficient to negatively regulate alpha 1I3 mRNA levels in hepatoma cells with similar characteristics as occur during an inflammatory response in the liver. A number of other acute phase mRNA species, including alpha 1-acid glycoprotein, T2-kininogen, gamma-fibrinogen and alpha 2-macroglobulin were induced to higher levels by the same hormonal treatments in FAO1 cells. The fourfold reduction of alpha 1I3 mRNA concentrations in FAO1 cells could be reversed by treatment with 1 microM of a water-soluble derivative of forskolin, an activator of the cyclic AMP pathway. Thus, the effect of IL6 on the expression of the alpha 1I3 gene may involve the activation of the cyclic AMP pathway. In contrast, T2 kininogen mRNA levels were not altered by treatment of FAO1 cells with forskolin, suggesting that IL6 may act on this gene through a different mechanism.

Chronic hepatitis B virus (HBV) infection is etiologically related to human hepatocellular carcinoma (HCC). Most HCCs contain integrated HBV DNA in the liver cellular DNA, suggesting that the integration may be involved in carcinogenesis. From a comparison of a single HBV integration site present in a hepatoma with the corresponding unoccupied site in the non-tumourous tissue of the same liver, we have shown that HBV DNA inserted in a putative cellular exon with striking similarity to the DNA-binding domain of the thyroid/steroid hormone receptors. The corresponding cDNA has been isolated (hap gene) and shown to encode the retinoic acid receptor. In the original patient, integration took place so that the first codons of the viral surface protein gene became fused in frame with most of the hap gene. Because retinoic acid is known to regulate the transcription of target genes crucial for cellular growth and differentiation, it is most probable that consequent to the HBV insertion, hap, usually transcribed at a very low level in normal hepatocytes, became inappropriately expressed as an altered chimaeric retinoic acid receptor, thus contributing to the cell transformation. These results strongly support the possibility that HBV may play a direct role in liver carcinogenesis by insertional mutagenesis.

Serum amyloid A (SAA) is one of the major acute-phase proteins in humans and mice. It is synthesized predominantly by the liver and secreted as a major component of the apolipoproteins in the high density lipoprotein particle. While the major physiological function of SAA is unclear, prolonged elevation of plasma SAA levels, as in chronic inflammation, however, results in the pathological condition amyloidosis affecting the liver, kidney and spleen. The expression of SAA mRNA is dramatically elevated in response to infection or systemic inflammation and is due primarily to the increased rate of SAA gene transcription. Studies in vitro and in vivo demonstrated that the expression of SAA genes is regulated by the inflammatory cytokine interleukin-1. Moreover, both the interleukin-1-induced expression and the enhanced liver-specific expression of the SAA gene are controlled by the binding of nuclear proteins to specific DNA sequences upstream from the structural gene.

We have determined the sequence of 250 bases 5' of the transcriptional start site of the apolipoprotein AI gene in a human individual with high serum concentrations of apo AI. One of the alleles contained a G to A substitution at position -75, between the CACAT sequence and the TAAATA box, creating a tandem repeat, CAGGGC-CA*GGGC. The substitution destroys an MspI cutting site, and the polymerase chain reaction and MspI digestion were used to identify the presence of the A or G base. The frequency of the A substitution in 96 healthy men from Bristol was 0.11 and this was increased to 0.25 in men with serum apo AI concentrations greater than 180 mg/dl. Men with the A allele had significantly higher serum concentrations of apo AI, high density lipoprotein (HDL) cholesterol and HDL2 than those with the G allele. In this sample, variation associated with the G to A substitution accounted for 6% and 4.6% of the total variance in apo AI and HDL cholesterol concentrations, respectively. Although there is as yet no functional proof, it is possible that the A substitution may be having a direct positive effect on the rate of apo AI gene transcription and thus be associated with increased apo AI and HDL cholesterol concentrations because of increased production of apo AI protein from the liver or intestine.