Howard Dang

Autoimmunity associated with TGF-beta1-deficiency in mice is dependent on MHC class II antigen expression.

The progressive inflammatory process found in transforming growth factor beta1 (TGF-beta1)-deficient mice is associated with several manifestations of autoimmunity, including circulating antibodies to nuclear antigens, immune complex deposition, and increased expression of both class I and class II major histocompatibility complex (MHC) antigens. The contribution of MHC class II antigens to the genesis of this phenotype has been determined by crossing the TGF-beta1-null [TGF-beta1(-/-)] genotype into the MHC class II-deficient [MHC-II(-/-)] background. Mice homozygous for both the TGF-beta1 null allele and the class II null allele [TGF-beta1(-/-);MHC-II(-/-)] are without evidence of inflammatory infiltrates, circulating autoantibodies, or glomerular immune complex deposits. Instead, these animals exhibit extensive extramedullary hematopoiesis with progressive splenomegaly and adenopathy, surviving only slightly longer than TGF-beta1(-/-);MHC-II(+/+) mice. The role of CD4+ T cells, which are also absent in MHC class II-deficient mice, is directly demonstrated through the administration of anti-CD4 monoclonal antibodies in class II-positive, TGF-beta1(-/-) mice. The observed reduction in inflammation and improved survival emphasize the significance of CD4+ cells in the pathogenesis of the autoimmune process and suggest that the additional absence of class II antigens in TGF-beta1(-/-);MHC-II(-/-) mice may contribute to their extreme myeloid metaplasia. Thus, MHC class II antigens are essential for the expression of autoimmunity in TGF-beta1-deficient mice, and normally may cooperate with TGF-beta1 to regulate hematopoiesis.

A conserved idiotype and antibodies to retroviral proteins in systemic lupus erythematosus.

22 of 61 systemic lupus erythematosus (SLE) patients produced antibodies to the p24 gag protein of HIV-1 demonstrated by Western blotting. 20 of these 22 patients (91%) also express the 4B4 idiotype (Id 4B4) previously identified on a human anti-Sm monoclonal antibody called 4B4. This represents an enrichment for this Id (seen in only 52% of SLE patients generally). Eight of these 22 SLE patients also have anti-Sm antibody activity. Sm partially inhibits the antibody binding of p24 gag suggesting immunologic cross-reactivity between the retroviral antigen p24 gag and the autoantigen Sm. Anti-Id 4B4 also inhibits p24 gag antibody binding by as much as 40%. Finally the monoclonal antibody 4B4 showed cross-reactivity to Sm and p24 gag. The following points emerge from our studies: (a) SLE patients make antibodies to p24 gag of HIV-1, (b) there is a relationship between immunity to p24 gag and a conserved idiotype, and (c) anti-Sm antibodies can cross-react with p24 gag.

The role of apoptosis in the initiation of the autoimmune response in Sjogren's syndrome

The aetiopathogenesis of autoimmune disorders has remained elusive despite much effort over many years devoted to possible genetic, viral and hormonal mechanisms [1,2]. Although genes, viruses, and sex hormones are no doubt involved, a key factor analogous to the role of oncogenes in cancer has escaped detection in autoimmunity. Apoptosis, or programmed cell death (PCD), a critical mechanism preserved throughout evolution to assure both morphologic modelling in fetal life as well as elimination of damaged cells throughout life, may be that key factor in development of autoimmunity [3].

Are endogenous retroviruses involved in human autoimmune disease

A role for viruses in the etiopathogenesis of human autoimmune diseases has long been suspected but has not yet been proven. In Sjögrens syndrome (SS), there is continuing experimental support for the possible involvement of Epstein-Barr virus. Since the advent of AIDS, there is also great interest in retroviruses and autoimmune disease. We previously reported that 30% of SS patients and 36% of systemic lupus erythematosus (SLE) patients have serum antibodies to the p24 gag protein of HIV-1. We now report that two mechanisms classic for retroviruses (molecular mimicry and immunosuppression) may be operative in SS and SLE. The p24 gag protein shares a proline-rich epitope with the Sm nucleoprotein to which many SLE patients have antibodies. The impaired lymphocyte activation seen in peripheral blood T cells in SS patients is also seen in a human T cell line infected with an A-type retroviral particle linked to SS. Many studies suggest that endogenous retroviral sequences are important in immunoregulation. We now suggest that endogenous retroviral sequences may also be important in the etiology and pathogenesis of SS and SLE.

Interaction of age and specific saliva component output on caries

Background and aims: The purpose of this study was to explore the relationship between individual salivary components, dental caries and age, utilizing the data from the Oral Health: San Antonio Longitudinal Study of Aging (OH:SALSA). Methods: The study population comprised a well-defined stratified sample of 811 dentate men and women. Subjects were divided into four age groups from 35 to 75+ years old. Unstimulated and stimulated submandibular/sublingual saliva flow rates, unstimulated and stimulated parotid saliva flow rates, total protein, 6 individual proteins and 4 inorganic constituents were measured. Specific salivary components were lactoferrin, secretory IgA, albumin, lysozyme, mucin, cystatin, K+, Ca+2, Na+ and Cl−. Caries measurements were the DMFT Index for crowns and for roots, Tooth Health Index for crowns and roots, Tooth caries, Root caries and Tooth restoration. The data on saliva components were square root transformed for linearity prior to analysis. Analysis was carried out in two stages. Partial correlation was performed, in order to identify significant relationships between specific salivary components and caries measurements, controlling for age group. In the second stage, using caries measurement as the dependant variable, the effects of age, flow rate and specific salivary component output (product of flow rate and concentration) were examined. Results: Significant associations were found between caries, age and specific individual submandibular/sublingual salivary proteins (lactoferrin, albumin, lysozyme, mucin and cystatin) and specific inorganic constituents (K+, Ca+2, Na+ and Cl− ). Conclusions: Changes in submandibular/sublingual salivary component output during aging are correlated with high caries prevalence. These changes in saliva components over age may represent caries risk indicators.

FAS (CD95)-TRANSDUCED SIGNAL PREFERENTIALLY STIMULATES LUPUS PERIPHERAL T LYMPHOCYTES

Fas (CD95) is a cell surface receptor whose biological function in circulating peripheral T cells is not well understood. To address the question of abnormal T cell sensitivity to Fas stimulation in systemic lupus erythematosus (SLE), we studied Fas‐transduced stimulation and apoptosis in peripheral blood T cells from patients with SLE and normal control. Immobilized anti‐Fas monoclonal antibodies (mAb) (imCH‐11; IgM type) significantly stimulated SLE T cell proliferation compared to T cells from normal donors and patients with rheumatoid arthritis ( p < 0.003 and p < 0.005, respectively). The soluble form of CH‐11 and other immobilized anti‐Fas mAb (UB‐2, ZB‐4; IgG type) failed to stimulate lupus T cells while immobilized human Fas ligand did. Furthermore, imCH‐11 induced IL‐2 and IL‐6 mRNA expression. However, imCH‐11 activation failed to induce expression of the T cell activation surface molecules CD25 and CD69. Addition of exogenous ceramide, a second messenger for Fas‐mediated apoptosis signaling, also induced T cell proliferation in SLE and normal controls. Moreover, fumonisin B1, a specific ceramide synthase inhibitor, and caspase inhibitors markedly suppressed imCH‐11 induced T cell proliferation, suggesting that the ceramide pathway may be involved in Fas‐transduced stimulation signals in SLE T cells. These results show that SLE T cells have an alteration in the Fas signal transduction pathway leading to cell proliferation. This defect may be important in Fas‐mediated peripheral immune homeostasis.

Role for notch signaling in salivary acinar cell growth and differentiation

The Notch pathway is crucial for stem/progenitor cell maintenance, growth and differentiation in a variety of tissues. The Notch signaling is essential for Drosophila salivary gland development but its role in mammalian salivary gland remains unclear. The human salivary epithelial cell line, HSG, was studied to determine the role of Notch signaling in salivary epithelial cell differentiation. HSG expressed Notch 1 to 4, and the Notch ligands Jagged 1 and 2 and Delta 1. Treatment of HSG cells with inhibitors of γ‐secretase, which is required for Notch cleavage and activation, blocked vimentin and cystatin S expression, an indicator of HSG differentiation. HSG differentiation was also associated with Notch downstream signal Hes‐1 expression, and Hes‐1 expression was inhibited by γ‐secretase inhibitors. siRNA corresponding to Notch 1 to 4 was used to show that silencing of all four Notch receptors was required to inhibit HSG differentiation. Normal human submandibular gland expressed Notch 1 to 4, Jagged 1 and 2, and Delta 1, with nuclear localization indicating Notch signaling in vivo. Hes‐1 was also expressed in the human tissue, with staining predominantly in the ductal cells. In salivary tissue from rats undergoing and recovering from ductal obstruction, we found that Notch receptors and ligands were expressed in the nucleus of the regenerating epithelial cells. Taken together, these data suggest that Notch signaling is critical for normal salivary gland cell growth and differentiation. Developmental Dynamics 238:724–731, 2009.

Distinct pathways of ERK activation by the muscarinic agonists pilocarpine and carbachol in a human salivary cell line.

Cholinergic-muscarinic receptor agonists are used to alleviate mouth dryness, although the cellular signals mediating the actions of these agents on salivary glands have not been identified. We examined the activation of ERK1/2 by two muscarinic agonists, pilocarpine and carbachol, in a human salivary cell line (HSY). Immunoblot analysis revealed that both agonists induced transient activation of ERK1/2. Whereas pilocarpine induced phosphorylation of the epidermal growth factor (EGF) receptor, carbachol did not. Moreover, ERK activation by pilocarpine, but not carbachol, was abolished by the EGF receptor inhibitor AG-1478. Downregulation of PKC by prolonged treatment of cells with the phorbol ester PMA diminished carbachol-induced ERK phosphorylation but had no effect on pilocarpine responsiveness. Depletion of intracellular Ca2+ ([Ca2+]i by EGTA did not affect ERK activation by either agent. In contrast to carbachol, pilocarpine did not elicit [Ca2+]i mobilization in HSY cells. Treatment of cells with the muscarinic receptor subtype 3 (M3) antagonist N-(3-chloropropyl)-4-piperidnyl diphenylacetate decreased ERK responsiveness to both agents, whereas the subtype 1 (M1) antagonist pirenzepine reduced only the carbachol response. Stimulation of ERKs by pilocarpine was also decreased by M3, but not M1, receptor small interfering RNA. The Src inhibitor PP2 blocked pilocarpine-induced ERK activation and EGF receptor phosphorylation, without affecting ERK activation by carbachol. Our results demonstrate that the actions of pilocarpine and carbachol in salivary cells are mediated through two distinct signaling mechanisms-pilocarpine acting via M3 receptors and Src-dependent transactivation of EGF receptors, and carbachol via M1/M3 receptors and PKC-converging on the ERK pathway.

Risk factors influencing antibody responses to kaposi's sarcoma-associated herpesvirus latent and lytic antigens in patients under antiretroviral therapy

Background:Kaposis sarcoma-associated herpesvirus (KSHV) seropositivity and lytic antibody titer are predictors for Kaposis sarcoma. Methods:We examined demographic, viral, and immunologic factors that influence KSHV latent and lytic antibodies in HIV-infected patients. Results:Detection rate of KSHV latent but not lytic antibodies was lower in patients with CD4 cells/mm3 less than 200 than greater than 200 (odds ratio [OR], 0.26; 95% confidence interval [CI], 0.11-0.61) and CD8 cells/mm3 less than 400 than greater than 400 (OR, 0.26; 95% CI, 0.07-0.67). Overall seropositivity rate was higher in patients with CD4 cells/mm3 less than 200 than greater than 200 (OR, 2.34; 95% CI, 1.37-4.02) and HIV copies/mL greater than 400 than less than 400 (OR, 1.70; 95% CI, 1.09-2.65). Lytic antibody level was inversely correlated with CD4 count (P < 0.001). Lytic seropositivity (OR, 2.47; 95% CI, 1.35-4.50) and antibody level (adjusted difference mean optical density, 0.324; 95% CI, 0.16-0.46) were higher in patients with HIV infection greater than 15 than less than 15 years. Hispanics had higher lytic seropositivity rate (OR, 1.71; 95% CI, 1.07-2.73) and antibody level (adjusted difference mean optical density, 0.111; 95% CI, 0.03-0.18) than non-Hispanics. Conclusions:Lower CD4 and CD8 counts impair antibody response to KSHV latent antigens. Immune deterioration, long-term HIV infection, and Hispanic status are risk factors for Kaposis sarcoma predictors.

INDUCTION OF TRANSCRIPTION FACTORS IN HUMAN T LYMPHOCYTES BY ASPIRIN-LIKE DRUGS

Aspirin-like drugs (ALD) induce calcium mobilization, an essential component of T cell activation, but do not induce the biosynthesis of IL-2. To understand the extent to which ALD may mimic mitogenic stimulation, we studied cytoplasmic and nuclear signaling steps in ALD-treated T cells. We found that ALD induce a transient activation of protein kinase (PKC) but have no effect (in comparison to anti-CD3 antibodies) on protein tyrosine phosphorylation nor on PCL gamma 1 tyrosine phosphorylation. ALD-induced calcium mobilization and PKC activation are independent of tyrosine protein kinase activity as shown by the lack of effect of herbimycin, a tyrosine-protein kinase-specific inhibitor. Although we detected no IL-2 mRNA in ALD-treated cells, the nuclei of these cells contain proteins capable of binding to three regulatory sequences in the IL-2 promoter region: NFAT, NF kappa B, and AP-1. These binding activities are expressed only in activated T cells. The expression of AP-1 depended on calcium mobilization and PKC activation. These data suggest that ALD cause transient but significant changes in T cell transmembrane signaling, although some events induced by stimulation with anti-CD3 antibodies are not induced by ALD. The signal is transmitted to the nucleus and induces DNA-binding activity by several transcription factors. However, the ALD stimulus is not capable of causing complete T cell activation.