Ban-Hock Toh

GM-CSF Deficiency Reduces Macrophage PPAR-γ Expression and Aggravates Atherosclerosis in ApoE-Deficient Mice

Objective—Granulocyte-macrophage colony-stimulating factor (GM-CSF) is expressed in atherosclerotic lesions but its significance for lesion development is unknown. Consequently, we investigated the significance of GM-CSF expression for development of atherosclerotic lesions in apolipoprotein E-deficient (apoE−/−) mice. Methods and Results—We generated apoE−/− mice deficient in GM-CSF (apoE−/−.GM-CSF−/− mice), fed them a high-fat diet, and compared lesion development with apoE−/− mice. We measured lesion size, macrophage, smooth muscle cell, and collagen accumulation at the aortic sinus, and expression of genes that regulate cholesterol transport and inflammation. No differences in serum cholesterol were found between the 2 groups. Lesion size in hyperlipidemic apoE−/−.GM-CSF−/− increased by 30% (P<0.05), macrophage accumulation doubled, and collagen content reduced by 15% (P<0.05); smooth muscle cell accumulation and vascularity were unaffected. Analysis of PPAR-&ggr;, ABCA1, and CD36 in lesions showed reduced expression (50%, 65%, and 55%, respectively), whereas SR-A doubled. In peritoneal macrophages, PPAR-&ggr; and ABCA1 expression was also reduced by 50% and 70%, respectively, as was cholesterol efflux, by 50%. In lesions, pro-inflammatory MCP-1 and tumor necrosis factor (TNF)-&agr; expression increased 2- and 3.5-fold, respectively, vascular cell adhesion molecule (VCAM)-1 expression enhanced and interleukin (IL)-1 receptor antagonist reduced by 50%. Conclusions—GM-CSF deficiency increases atherosclerosis under hypercholesterolemic conditions, indicating antiatherogenic role for GM-CSF. We suggest this protective role is mediated by PPAR-&ggr; and ABCA1, molecules that affect cholesterol homeostasis and inflammation.

The glycolytic enzyme enolase is present in sperm tail and displays nucleotide-dependent association with microtubules

We examined the expression and localisation of enolase (2-phospho-D-glycerate hydrolase) in differentiating rat spermatogenic cells. We found that enolase is most abundant in mature spermatozoa and in residual cytoplasmic bodies detached from elongating spermatids with little to no enolase detected in meiotic primary spermatocytes and round spermatids. We localised enolase mostly to the tail of mature spermatozoa by immunoblotting and by immunofluorescence. RT-PCR analysis of differentiating spermatogenic cells detected only the alpha isoform of enolase. As several glycolytic enzymes are known to associate with microtubules prepared from brain, we investigated the association of enolase with brain and testis microtubules. We found that only a small fraction of testis and brain-derived cytosolic enolase (4.9% and 11.2%, respectively) co-sediments with microtubules stabilised in the presence of taxol. In the presence of certain nucleotides in excess (3 mM ATP, CTP, GTP and ITP) the association of enolase with microtubules was disrupted, however, this was not the case for UTP. This observation is consistent with the finding that in the presence of 0.5 mM AMP-PNP, a nonhydrolysable analogue of ATP, there is an increased association of enolase with microtubules. We propose that the nucleotide-dependent association of enolase with microtubules regulates enzyme activity by linking energy production to utilisation.

TGF-BETA AND CANCER: IS SMAD3 A REPRESSOR OF HTERT GENE?

Transforming growth factor β (TGF-β) carries out tumor suppressor activity in epithelial and lymphoid cells, whereas telomerase is required for most cancers. Although the molecular mechanisms by which TGF-β acts as a tumor suppressor are yet to be fully established, a link between TGFb and its tumor suppressor activity by telomerase has been suggested. Recently, we have noted a novel mode of action for TGF-β through which human telomerase reverse transcriptase (hTERT) gene is repressed in immortal and neoplastic cells, confirming that one of the mechanisms underlying TGF-β suppression of tumor growth may be through inhibiting hTERT gene transcription. Moreover, the inhibition of hTERT gene by TGF-β suggests a cis action of the TGF-β signaling molecule Smad3 on hTERT promoter directly. This article examines our current understanding and investigation of TGF-β regulation of telomerase activity, and presents a model in which Smad3 participates in regulating hTERT gene transcription by acting as a repressor directly. Engineering the interface between Smad3 and hTERT gene may lead to a new strategy to inhibit telomerase activity in cancer.

Gene Therapy Strategies Towards Immune Tolerance to Treat the Autoimmune Diseases

Autoimmune diseases such as type 1 diabetes and multiple sclerosis pose a significant health burden on our society. As a whole, autoimmune diseases affect approximately 6% of the population and are the third largest disease burden after heart disease and cancer. Such pathologic manifestations arise by way of damaging reactions of B-cell derived antibodies and/or T-cells to self-antigens and are triggered by genetic and environmental factors. Currently there is no known cure, with treatment restricted to toxic, long-term immunosuppressive regimes, replacement therapy and in intractable cases, transplantation of autologous or allogeneic haematopoietic stem cells. In experimental models of autoimmunity, gene therapeutic approaches have demonstrated promise in treating the autoimmune diseases. These include delivery of anti-inflammatory cytokines and exploitation of regulatory T cells. However, none of these approaches provide lasting, long-term benefit. We hypothesise that therapeutically transduced haematopoietic stem cells followed by transplantation is an alternative strategy to establish permanent immune tolerance that can not only prevent autoimmunity but also cure these diseases. Our approach is focused on directing autoimmune disease-specific autoantigen expression in the thymus by genetic manipulation of haematopoietic stem cells to establish molecular chimeras. Our hypothesis originates from experimental studies with a mouse model of experimental autoimmune gastritis (EAG) and more recently with the non-obese diabetic (NOD) mouse model for type 1 diabetes (T1D).

Human CKIαL and CKIαS are encoded by both 2.4- and 4.2-kb transcripts, the longer containing multiple RNA-destablising elements

Casein kinase I (CKI) are a family of conserved second messenger-independent serine/threonine protein kinases found in all eukaryotes. The avian and mammalian CKI alpha isoform has four splice variants differing in the presence or absence of 28 amino acids ((L) insertion) in the catalytic domain and/or 12 amino acids ((S) insertion) in the regulatory domain. Here we report the isolation of cDNAs encoding human CKIalpha(L) and CKIalpha(S). We find human CKIalpha(L) has a preference to phosphorylate phosvitin over casein, with a higher K(m) for casein than phosvitin, the reverse being the case for human CKIalpha(S). Both human CKIalpha(L), and CKIalpha(S) are derived from 4.2-kb mRNA transcripts and 2.4-kb transcripts, the latter probably generated by use of an alternate polyadenylation signal identified in the longer transcripts. The 4. 2-kb transcripts contain six RNA-destabilising AU-rich element (ARE) motifs in the 3-untranslated region (UTR), while the 2.4-kb transcripts contain a single ARE motif. In vitro analysis of CKI alpha 3-UTR RNA sequences suggests that in HeLa cells, the longer 3-UTR transcripts are likely to degrade approximately 13 times faster than the shorter 3-UTR transcripts. This is the first report of a kinase mRNA containing multiple RNA-destabilising AREs in the longer of two mRNA transcripts.

Gene therapy and bone marrow stem-cell transfer to treat autoimmune disease

Current treatment of human autoimmune disease by autologous bone marrow stem-cell transfer is hampered by frequent disease relapses. This is most probably owing to re-emergent self-reactive lymphocytes. Gene therapy combined with bone marrow stem cells has successfully introduced genes lacking in immunodeficiences. Because the bone marrow compartment has a key role in establishing immune tolerance, this combination strategy should offer a rational approach to prevent re-emergent self-reactive lymphocytes by establishing solid, life-long immune tolerance to causative self-antigen. Indeed, we have recently demonstrated the success of this combination approach to prevent and cure an experimental autoimmune disease. We suggest that this combination strategy has the potential for translation to treat human autoimmune diseases in which causative self-antigens are known.

Major immunoreactive domains of human ribosomal P proteins lie N-terminal to a homologous C-22 sequence: application to a novel ELISA for systemic lupus erythematosus

The aim of this study was to identify immunoreactive domains on human ribosomal P0, P1 and P2 proteins, other than the C‐22 peptide, to develop a novel ELISA using a combination of these proteins and to compare this ELISA with one using the C‐22 peptide. Human recombinant P0, P1, P2 and mutant P0 lacking the homologous C‐22 peptide (N‐P0) were produced in bacteria and tested by ELISA and immunoblotting using sera from 48 patients with systemic lupus erythematosus (SLE), 48 with an unrelated inflammatory disorder (Crohns disease) and 47 healthy controls. ELISA with P0, P1 and P2, premixed at equimolar concentrations, gave higher OD readings than each protein tested individually. Eighteen SLE sera tested positive by ELISA with premixed P0, P1, P2 but only 3 tested positive with the C‐22 peptide. Twenty‐two SLE sera reacted positively, as determined by immunoblotting, with 5 different P protein combinations: P1P2, P0P1P2, P1, P0P1, P0 and P1. Only sera reactive with all three P proteins reacted with the C‐22 peptide, with absent or minimal reactivity with N‐P0. Native antigens yielded sensitivity (6/48, 13%) similar to the C‐22 peptide assay. An ELISA with premixed P1 and P2 gave higher OD values than the arithmetic means with P1 or P2. Fifteen SLE patients had antibodies to double stranded (ds)‐DNA, of which 6 also had antibodies to P0P1P2 by ELISA but 12 reactive with P0P1P2 did not have discernable ds‐DNA antibodies. Ribosomal P autoantibodies react mainly with epitopes N‐terminal to a homologous C‐22 peptide. An ELISA with premixed P0, P1 and P2 has 5‐fold greater sensitivity (38%) for SLE than an assay with the conventional C‐22 peptide (7%). The combined sensitivity for SLE for antibodies to P0P1P2 and ds‐DNA is 56%, higher than C‐22 and ds‐DNA, 38%. Only one of the SLE patients had neuropsychiatric lupus.

60 – PARIETAL CELL AND INTRINSIC FACTOR AUTOANTIBODIES

Autoimmune gastritis is the silent pathological lesion that may eventually become clinically manifest as pernicious anaemia after a latency of 20–30 or more years. Autoimmune or type A gastritis is restricted to the parietal cell-containing corpus of the stomach. Circulating autoantibody to gastric parietal cell H/K ATPase, the enzyme responsible for acidification of gastric juice, is a diagnostic marker for autoimmune gastritis. As parietal cell, autoantibody may also be found in relatives of patients with pernicious anaemia and with other autoimmune endocrinopathies such as autoimmune thyroiditis and type 1 diabetes mellitus, it is not diagnostic for pernicious anaemia. The advent of pernicious anaemia is accompanied by the development of autoantibody to intrinsic factor, itself a secretory product of gastric parietal cells. Intrinsic factor autoantibody, present in serum and gastric juice, can block intrinsic factor from binding to bind vitamin B12 and hinder absorption of the vitamin by cubulin receptors in the ileum. With the vanishing Schillings test, increasing reliance is placed on autoantibody to intrinsic factor for the diagnosis of pernicious anaemia. However the sensitivity and specificity of commercial enzyme-linked immunosorbent assay (ELISAs) for diagnosis of the anaemia remains unknown. Both autoantibodies can antedate development of pernicious anaemia, but their predictive value is diminished because not all patients with either one or both of these antibodies will necessarily go on to develop anaemia.

Haematopoietic stem cell gene therapy to treat autoimmune disease.

Autoimmune diseases affect approximately 6% of the population and are characterised by a pathogenic immune response that targets self-antigens. Well known diseases of this nature include type 1 diabetes, systemic lupus erythematosus, rheumatoid arthritis and multiple sclerosis. Treatment is often restricted to replacement therapy or immunosuppressive regimes and to date there are no cures. The strategy of utilising autologous or allogeneic haematopoietic stem cell transplantation to treat autoimmunity and induce immunological tolerance has been trailed with various levels of success. A major issue is disease relapse as the autoimmune response is reinitiated. Cells of the immune system originate from bone marrow and have a central role in the induction of immunological tolerance. The ability to isolate and genetically manipulate bone marrow haematopoietic stem cells therefore makes these cells a suitable vehicle for driving ectopic expression of defined autoantigens and induction of immunological tolerance.