Differential pulmonary toxicity and autoantibody formation in genetically distinct mouse strains following combined exposure to silica and diesel exhaust particles.

BACKGROUND: Inhalation of airborne particulate matter, such as silica and diesel exhaust particles, poses serious long-term respiratory and systemic health risks. Silica exposure can lead to silicosis and systemic autoimmune diseases, while DEP exposure is linked to asthma and cancer. Combined exposure to silica and DEP, common in mining, may have more severe effects. This study investigates the separate and combined effects of occupational-level silica and ambient-level DEP on lung injury, inflammation, and autoantibody formation in two genetically distinct mouse strains, thereby aiming at understanding the interplay between genetic susceptibility, particulate exposure, and disease outcomes. Silica and diesel exhaust particles were administered to mice via oropharyngeal aspiration. Assessments of lung injury and host response included in vivo lung micro-computed tomography, lung function tests, bronchoalveolar lavage fluid analysis including inflammatory cytokines and antinuclear antibodies, and histopathology with particle colocalization. RESULTS: The findings highlight the distinct effects of silica and diesel exhaust particles (DEP) on lung injury, inflammation, and autoantibody formation in C57BL/6J and NOD/ShiLtJ mice. Silica exposure elicited a well-established inflammatory response marked by inflammatory infiltrates, release of cytokines, and chemokines, alongside mild fibrosis, indicated by collagen deposition in the lungs of both C57BL/6J and NOD/ShilLtJ mice. Notably, these strains exhibited divergent responses in terms of respiratory function and lung volumes, as assessed through micro-computed tomography. Additionally, silica exposure induced airway hyperreactivity and elevated antinuclear antibody levels in bronchoalveolar lavage fluid, particularly prominent in NOD/ShiLtJ mice. Moreover, antinuclear antibodies correlated with extent of lung inflammation in NOD/ShiLTJ mice. Lung tissue analysis revealed DEP loaded macrophages and co-localization of silica and DEP particles. However, aside from contributing to airway hyperreactivity specifically in NOD/ShiLtJ mice, the ambient-level DEP did not significantly amplify the effects induced by silica. There was no evidence of synergistic or additive interaction between these specific doses of silica and DEP in inducing lung damage or inflammation in either of the mouse strains. CONCLUSION: Mouse strain variations exerted a substantial influence on the development of silica induced lung alterations. Furthermore, the additional impact of ambient-level DEP on these silica-induced effects was minimal.

ENT2 facilitates brain endothelial cell penetration and blood-brain barrier transport by a tumor-targeting anti-DNA autoantibody.

The blood-brain barrier (BBB) prevents antibodies from penetrating the CNS and limits conventional antibody-based approaches to brain tumors. We now show that ENT2, a transporter that regulates nucleoside flux at the BBB, may offer an unexpected path to circumventing this barrier to allow targeting of brain tumors with an anti-DNA autoantibody. Deoxymab-1 (DX1) is a DNA-damaging autoantibody that localizes to tumors and is synthetically lethal to cancer cells with defects in the DNA damage response. We found that DX1 penetrated brain endothelial cells and crossed the BBB, and mechanistic studies identify ENT2 as the key transporter. In efficacy studies, DX1 crosses the BBB to suppress orthotopic glioblastoma and breast cancer brain metastases. ENT2-linked transport of autoantibodies across the BBB has potential to be exploited in brain tumor immunotherapy, and its discovery raises hypotheses on actionable mechanisms of CNS penetration by neurotoxic autoantibodies in CNS lupus.

Low-Molecular-Weight Heparin Enhanced Therapeutic Effects of Human Adipose-Derived Stem Cell Administration in a Mouse Model of Lupus Nephritis.

Background: Lupus nephritis is a life-threatening complication in systemic lupus erythematosus (SLE), but the efficiency of current therapies involving corticosteroids, immunosuppressants, and biological agents is limited. Adipose-derived mesenchymal stem cells (ASCs) are gaining attention as a novel treatment for inflammation in SLE. Low-molecular-weight heparin (LMWH) exhibits multiple functions including anti-inflammatory, anti-fibrotic, and cell function-promoting effects. LMWH stimulation is expected to increase the therapeutic effect of ASCs by promoting cellular functions. In this study, we investigated the effects of LMWH on ASC functions and the therapeutic effect of LMWH-activated human-ASCs (hep-hASCs) in an SLE mouse model. Methods: The cellular functions of human-derived ASCs stimulated with different LMWH concentrations were observed, and the optimum LMWH dose was selected. The mice were assigned to control, human-ASC, and hep-hASC groups; treatments were performed on week 20. Twenty-six week-old mice were sacrificed, and urine protein score, serum blood urea nitrogen, creatinine (Cr), anti-ds DNA IgG antibody, and serum IL-6 levels were analyzed in each group. Mice kidneys were evaluated via histological examination, immunohistochemical staining, and gene expression levels. Results: LMWH significantly promoted ASC migration and proliferation and hepatocyte growth factor production and upregulated immunomodulatory factors in vitro. Hep-hASC administration resulted in significant disease activity improvement including proteinuria, serum Cr and IL-6 levels, anti-ds DNA IgG antibody, glomerulonephritis, and immune complex in mice. Inflammation and fibrosis in kidneys was significantly suppressed in the hep-hASC group; the gene expression levels of TNF-alpha, TIMP-2, and MMP-2 was significantly downregulated in the hep-hASC group compared with the control group. Conclusions: Hep-hASC exhibited higher anti-inflammatory and anti-fibrotic effects than hASCs and may be a candidate tool for SLE treatment in future.

Infusion of anti-DFS70 antibodies prolonged survival of lupus-prone mice.

BACKGROUND: Systemic-lupus-nephritis is a chronic autoimmune disease characterized by immune complex deposition and a flare of autoantibodies and leading to renal injury. OBJECTIVES: To expose anti-Dense-Fine-Speckled-70 (DFS70)-antibodies to genetically-prone-lupus-mice. METHODS: NZBXW/F1 female mice were monitored for the onset of glomerulonephritis by proteinuria upon infusion of anti-DFS70 (40 µg/mouse), commercial-human-IgG (cIgG) or phosphate-buffered-saline (PBS) as controls. The survival time was detected by mice death. Circulating anti-dsDNA were tested by ELISA. Proteinuria, was defined by a standard semi-quantitative-Bayer-Multistix-dipstick. Kidney histology was analyzed by periodic-acid-Schiff-PAS staining. RESULTS: A significantly higher percentage of anti-DFS70-infused mice exhibited prolonged survival time as compared with cIgG and PBS-subjected mice (p < 0.022). One mouse out of 10 mice injected with anti-DFS70-antibodies died at week 36, whereas, 6 out of 10 mice subjected with PBS found dead at this time. Eighty percent of anti-DFS70 injected mice did not show severe glomerulonephritis by histology. CONCLUSIONS: anti-DFS70 attenuated the progression of glomerulonephritis and prolonged the survival time. Circulating anti-DFS70-autoantibodies may confer a protective role against renal injury in murine-lupus-nephritis. Our data may propose a novel therapy approach for lupus patients.

Anti-double Stranded DNA Antibodies: Origin, Pathogenicity, and Targeted Therapies.

Systemic lupus erythematosus (SLE) is characterized by high-titer serological autoantibodies, including antibodies that bind to double-stranded DNA (dsDNA). The origin, specificity, and pathogenicity of anti-dsDNA antibodies have been studied from a wider perspective. These autoantibodies have been suggested to contribute to multiple end-organ injuries, especially to lupus nephritis, in patients with SLE. Moreover, serum levels of anti-DNA antibodies fluctuate with disease activity in patients with SLE. By directly binding to self-antigens or indirectly forming immune complexes, anti-dsDNA antibodies can accumulate in the glomerular and tubular basement membrane. These autoantibodies can also trigger the complement cascade, penetrate into living cells, modulate gene expression, and even induce profibrotic phenotypes of renal cells. In addition, the expression of suppressor of cytokine signaling 1 is reduced by anti-DNA antibodies simultaneously with upregulation of profibrotic genes. Anti-dsDNA antibodies may even participate in the pathogenesis of SLE by catalyzing hydrolysis of certain DNA molecules or peptides in cells. Recently, anti-dsDNA antibodies have been explored in greater depth as a therapeutic target in the management of SLE. A substantial amount of data indicates that blockade of pathogenic anti-dsDNA antibodies can prevent or even reverse organ damage in murine models of SLE. This review focuses on the recent research advances regarding the origin, specificity, classification, and pathogenicity of anti-dsDNA antibodies and highlights the emerging therapies associated with them.

Fast RBC loading by fluorescent antibodies and nuclei staining dye and their potential bioanalytical applications.

This study was designed to load different antibodies (Abs) and a fluorescent dye onto the red blood cell (RBC) surface. We have used fluorescein isothiocyanate (FITC)-conjugate anti-human Ab, CD22-PE (B-cell marker-phycoerythrin Ab), and 4',6-diamidino-2-phenylindole (DAPI) for insertion over the RBC surface. In a first step, conjugation experiments were performed: in dimethyl sulfoxide (DMSO), RBCs were conserved and modified by succinic anhydride to create an additional -COOH group, and then activated with 3-(3-dimethylaminopropyl)carbodiimide-N-hydroxysuccinimide (EDC-NHS) in 2-(N-morpholino) ethanesulfonic acid hydrate buffer for insertion of labeled Abs or DAPI. In a second step, fluorescence signals were evaluated by microscopy and the mean fluorescence intensities of cell lysates were measured by spectrofluorometry. The results showed clear evidence for adsorption of FITC- and PE-labeled Abs to activated conserved RBCs. DAPI was adsorbed well also to DMSO-conserved RBCs without the need for an activation step. The DMSO conservation step was enough to create reactive RBCs for insertion of specific Abs and fluorescent dyes. The additional modification by succinic anhydride and activation with EDC-NHS resulted in two- to seven-fold increase in fluorescence signals, indicating a much higher RBC loading capacity. These Ab- and fluorescent dye-functionalized RBCs have potentially high application in developing new biomedical diagnostic and in vitro assay techniques.

Local MicroRNA Modulation Using a Novel Anti-miR-21-Eluting Stent Effectively Prevents Experimental In-Stent Restenosis.

OBJECTIVE: Despite advances in stent technology for vascular interventions, in-stent restenosis (ISR) because of myointimal hyperplasia remains a major complication. APPROACH AND RESULTS: We investigated the regulatory role of microRNAs in myointimal hyperplasia/ISR, using a humanized animal model in which balloon-injured human internal mammary arteries with or without stenting were transplanted into Rowett nude rats, followed by microRNA profiling. miR-21 was the only significantly upregulated candidate. In addition, miR-21 expression was increased in human tissue samples from patients with ISR compared with coronary artery disease specimen. We systemically repressed miR-21 via intravenous fluorescein-tagged-locked nucleic acid-anti-miR-21 (anti-21) in our humanized myointimal hyperplasia model. As expected, suppression of vascular miR-21 correlated dose dependently with reduced luminal obliteration. Furthermore, anti-21 did not impede reendothelialization. However, systemic anti-miR-21 had substantial off-target effects, lowering miR-21 expression in liver, heart, lung, and kidney with concomitant increase in serum creatinine levels. We therefore assessed the feasibility of local miR-21 suppression using anti-21-coated stents. Compared with bare-metal stents, anti-21-coated stents effectively reduced ISR, whereas no significant off-target effects could be observed. CONCLUSION: This study demonstrates the efficacy of an anti-miR-coated stent for the reduction of ISR.

Anti-dsDNA antibodies induce inflammation via endoplasmic reticulum stress in human mesangial cells.

BACKGROUND: Anti-dsDNA antibodies play an important role in the pathogenesis of lupus nephritis (LN). Endoplasmic reticulum (ER) stress is a physical reaction under stressful condition and can cause inflammation when stimulation is sustained. This study investigated the roles of ER stress in anti-dsDNA antibody-induced inflammation response in human mesangial cells (HMCs). METHOD: Anti-dsDNA antibodies isolated from LN patients were used to stimulate HMCs. The expression of GRP78, PERK, p-PERK, p-eIF2α, ATF4, p-IRE1α, ATF6 and CHOP in HMCs was measured by western blot. NF-κB activation was detected by examining nuclear translocation of NF-κB p65. The expression and production of IL-1ß, TNF-α and MCP-1 were examined by qPCR and ELISA. RESULTS: Flow cytometry and cellular ELISA showed that anti-dsDNA antibodies can bind to HMCs. The binding was not inhibited by blockage of Fc receptor. Anti-dsDNA antibody stimulation significantly enhanced the expression of GRP78, p-PERK, p-eIF2α and ATF4 in HMCs. However, no significant increase in the expression of p-IRE1α and ATF6 was found. In addition, anti-dsDNA antibodies also significantly increased the activation of NF-κB and upregulated the expression of IL-1ß, TNF-α and MCP-1, which were suppressed by pretreatment of HMCs with chemical ER stress inhibitor 4-PBA. Transfection of specific ATF4 siRNA also significantly reduced the activation of NF-κB and expression of proinflammatory cytokines. CONCLUSION: Anti-dsDNA antibodies induce NF-κB activation and inflammation in HMCs via PERK-eIF2α-ATF4 ER stress pathway.

A nucleolytic lupus autoantibody is toxic to BRCA2-deficient cancer cells.

Cancer cells with defects in DNA repair are highly susceptible to DNA-damaging agents, but delivery of therapeutic agents into cell nuclei can be challenging. A subset of lupus autoantibodies is associated with nucleolytic activity, and some of these antibodies are capable of nuclear penetration. We hypothesized that such antibodies might have potential as therapeutic agents targeted towards DNA repair-deficient malignancies. We identified the lupus autoantibody 5C6 as a cell-penetrating nucleolytic antibody and found that 5C6 has a differential effect on a matched pair of BRCA2-proficient and deficient DLD1 colon cancer cells. 5C6 selectively induced γH2AX in, and suppressed the growth of, the BRCA2-deficient cells. These findings demonstrate the potential utility of 5C6 in targeted therapy for DNA repair-deficient malignancies and strengthen the rationale for studies of additional lupus autoantibodies in order to identify the best candidates for development as therapeutic agents. In addition, the toxic effect of 5C6 on BRCA2-deficient cells provides further support for the hypothesis that some lupus autoantibodies contribute to the lower risk of specific cancers associated with systemic lupus erythematosus.

Association of human papilloma virus with atypical and malignant oral papillary lesions.

OBJECTIVE: This study aimed to examine atypical and malignant papillary oral lesions for low- and high-risk human papillomavirus (HPV) infection and to correlate HPV infection with clinical and pathologic features. STUDY DESIGN: Sections of 28 atypical papillary lesions (APLs) and 14 malignant papillary lesions (MPLs) were examined for HPV by in situ hybridization and for p16 and MIB-1 by immunohistochemistry; 24 conventional papillomas were studied for comparison. RESULTS: Low-risk HPV was found in 10 of 66 cases, including 9 APLs and 1 papilloma. All low-risk HPV-positive cases showed suprabasilar MIB-1 staining, and the agreement was statistically significant (P < .0001). Diffuse p16 staining combined with high-risk HPV was not seen in any of the cases. A subset of HPV(-) APLs progressed to carcinoma. CONCLUSIONS: Oral papillary lesions are a heterogeneous group. Low-risk HPV infection is associated with a subset of APLs with a benign clinical course. Potentially malignant APLs and MPLs are not associated with low- or high-risk HPV.

[Antibodies to DNA stimulates death of mononuclear cells of healthy persons in vitro].

We have shown that polyclonal antibody IgG to native DNA leads to increased levels of apoptosis of moonuclear cells of healthy persons in vitro. Possible biological role of antibodies to DNA is discussed.

[Influence of antibodies to DNA on MDCK cells and their intracellular localization].

Objects of the study were highly purified by liquid chromatography antibodies to DNA (IgG class) from the blood serums of healthy donors, patients with systemic lupus erythematosus (SLE) or rheumatoid arthritis (RA), and clinically healthy relatives of RA patients. It has been shown that, depending on the pathology, these antibodies differ in the DNA-hydrolyzing activity and differently affect the number of viable cells, morphology and chromatin of MDCK cells after co-culture. The antibodies to DNA were located in the area of nuclei. Biological role of the antibodies to DNA is discussed.

The lupus-derived anti-double-stranded DNA IgG contributes to myofibroblast-like phenotype in mesangial cells.

Anti-double-stranded DNA (dsDNA) antibodies have been indicated to play a major role in the pathogenesis of lupus nephritis (LN), which is characterized by mesangial alterations, including phenotypic changes. To explore the effects of anti-dsDNA antibodies on the phenotype of mesangial cells (MCs), the anti-dsDNA IgG in sera and histological features of glomeruli were analyzed in the mice models of immune-complex glomerulonephritis. The MCs were cultured in vitro with the addition of anti-dsDNA or non-anti-dsDNA IgG. Compared to the anti-dsDNA-negative controls, the serum positive mice had increased extracellular matrix accumulation and higher alpha-smooth muscle actin expression in the mesangial region. The anti-dsDNA IgG enhanced the synthesis of transforming growth factor beta, alpha-smooth muscle actin, and fibronectin, and even induced the myofibroblast-like morphological features in cultured MCs. Our results indicated that anti-dsDNA antibodies contribute to the phenotypic changes in MCs, which suggests another mechanism of renal injuries in LN induced by anti-dsDNA antibodies.

A novel monoclonal antibody against human CD80 and its immune protection in a mouse lupus-like disease.

Blockade of the interactions between CD28/CTLA-4 and their ligands, CD80 (B7, B7.1)/CD86 (B70, B7.2), is an attractive means to induce antigen-specific peripheral tolerance in autoimmune disease and organ transplantation. In this study, we generated and characterized a monoclonal antibody (Clone 4E5) against human CD80. 4E5 could recognize both human and mouse CD80 and suppress mixed lymphocyte reaction in vitro. To investigate their potency for clinical use, we further administrated 4E5 to a mouse lupus-like disease model (C57BL/J6) induced by Pristane. 4E5 could inhibit the immune response and attenuate the severity of lupus-like disease. The data showed 4E5 function and suggested that blockade of CD80/CD28 co-stimulatory signal pathway with 4E5 is a promising strategy to decelerate the progression of lupus-like disease and other autoimmune diseases.

An RNA-hydrolyzing recombinant antibody exhibits an antiviral activity against classical swine fever virus.

Some proteins with ribonuclease (RNase) activity have been shown to suppress viral replication. A well-characterized recombinant antibody, 3D8 single-chain variable fragment (3D8 scFv), has RNA-hydrolyzing and cell-penetrating activities. Here, we investigated antiviral activity of 3D8 scFv against classical swine fever virus (CSFV). In a cell line expressing 3D8 scFv (C26), intracellular RNA-hydrolysis activity was higher compared to control PK-15 cells and viral replication was strongly suppressed at the viral RNA level, with the evidence of independency of IFN-alpha/beta induction. Exogenous treatment of 3D8 scFv, prior to or post-CSFV infection, was also shown to suppress CSFV replication at the viral RNA level. These observations suggest that antiviral activity of 3D8 scFv may be due to the intrinsic RNase activity of 3D8 scFv, which is capable of targeting viral RNA genomes or transcripts.

Induction of TNF-alpha-converting enzyme-ectodomain shedding by pathogenic autoantibodies.

The release of the soluble form of tumor necrosis factor (TNF)-alpha from the plasma membrane occurs through the activation of the secretase tumor necrosis factor-alpha-converting enzyme (TACE). The current study was designed to examine whether the anti-Ro/SSA autoantibodies (Abs) are capable to regulate TACE expression in non-neoplastic human salivary gland epithelial cells (SGEC) cultures. We investigated the effect of anti-Ro/SSA Abs on the localization and abundance of cell-surface TACE and on TACE pro-domain-shedding and activation. In addition, the potential physiological consequences of TNF-alpha blockage by the biological agent Adalimumab on post-translational regulation of TACE are discussed. Anti-Ro/SSA Abs were purified from IgG fractions of patients with primary Sjögren's syndrome, using Sepharose 4B-Ro/SSA affinity columns. Flow cytometry, reverse transcription-PCR, western blot and immunohistochemistry were used to study TACE expression on SGEC and TACE regulation by Abs. Our study demonstrated a dose-dependent increase of TACE messenger RNA (mRNA) expression in anti-Ro/SSA Abs-treated SGEC, followed by internalization, pro-domain shedding and activation of TACE protein, suggesting that increased TACE activity is necessary for the release of TNF-alpha observed in anti-Ro/SSA Abs-stimulated SGEC. Adalimumab treatment brought TACE mRNA and surface TACE expression to levels than those observed in untreated SGEC. These data suggest that the effect of anti-Ro/SSA Abs on TACE expression and intracellular distribution is exerted by TNF-alpha production.

Tumor necrosis factor inhibitors block apoptosis of human epithelial cells of the salivary glands.

Inhibition of tumor necrosis factor-alpha (TNF-alpha) in organ-specific autoimmune disease is proving efficacious for a large number of patients. A wide array of biological agents has been designed to inhibit TNF-alpha, such as adalimumab (fully humanized) and etanercept (soluble TNF-alpha receptor fusion constructs p75 subunit). Recently, we suggested that anti-Ro and anti-La autoantibodies (Abs) isolated from patients with Sjögren's syndrome, an autoimmune rheumatic disease, are able to trigger cell death through extrinsic apoptotic mechanisms in human salivary gland epithelial cells (SGEC). We analyzed if primary human SGEC cultures, established from biopsy of labial minor salivary glands, are able to produce TNF-alpha, an inductor of the extrinsic apoptotic pathway, when treated with anti-Ro autoantibodies. A comparative study was performed to test the efficacy of adalimumab and etanercept to block TNF-alpha-mediated apoptosis. ELISA assay and RT-PCR were employed to visualize TNF-alpha production, and apoptosis was evaluated by DNA ladder and flow cytometry. We found that cell treatment with anti-Ro autoantibodies determines TNF-alpha production that reaches a maximum at 16 h and is decreased (P < 0.05) at 24 and 48 h. Adalimumab seems to be more efficacious than etanercept in blocking TNF-alpha-mediated apoptosis. The YOPRO-1 (+) and propidium iodide (-) method revealed 60% of apoptotic cells after 24 h of incubation with anti-Ro compared with 15% of apoptotic cells treated with anti-Ro plus adalimumab and 25% of apoptotic cells treated with anti-Ro plus etanercept. The antiapoptotic effect of adalimumab and etanercept was supported by inhibition of DNA laddering induced by anti-Ro Abs. These data validate the therapeutic efficacy of the anti-TNF reagents in the treatment of autoimmune disorders.

Sera from anti-Jo-1-positive patients with polymyositis and interstitial lung disease induce expression of intercellular adhesion molecule 1 in human lung endothelial cells.

OBJECTIVE: To investigate whether sera or purified IgG from patients with polymyositis (PM) and patients with dermatomyositis (DM), with or without interstitial lung disease (ILD), can activate endothelial cells (ECs). METHODS: Patients' sera were selected based on the presence or absence of anti-Jo-1, anti-SSA, or anti-U1 small nuclear RNP autoantibodies. The presence of autoantibodies was determined by line blot assays. Cultured human microvascular ECs derived from lung tissue (HMVEC-L) were incubated with sera or purified IgG from 22 patients with PM, 7 patients with DM, and 10 healthy individuals as controls. Assessment of intercellular adhesion molecule 1 (ICAM-1) expression was conducted by immunofluorescence (n=22) and by cell-based enzyme-linked immunosorbent assay (ELISA) (n=20). Serum levels of soluble ICAM-1 (sICAM-1) were determined by ELISA. RESULTS: Sera from PM patients with ILD who were positive for anti-Jo-1 autoantibodies had a significantly stronger effect on the expression of ICAM-1 by HMVEC-L in comparison with sera from healthy controls and patients with other autoantibodies. Purified IgG did not induce ICAM-1 expression. Higher serum levels of sICAM-1 were found in patients with myositis compared with healthy controls. CONCLUSION: EC activation with ICAM-1 expression could contribute to the multiorgan involvement, including the development of myositis and ILD, in patients carrying anti-Jo-1 autoantibodies. The EC-activating factors are not the autoantibodies themselves, but might be systemic factors associated with these autoantibodies.

Anti-DNA antibody induction of protein kinase C phosphorylation and fibronectin synthesis in human and murine lupus and the effect of mycophenolic acid.

OBJECTIVE: To examine fibronectin (FN) expression in human lupus nephritis and the effect of anti-DNA antibodies on transforming growth factor beta1 (TGFbeta1) and FN synthesis in cultured human mesangial cells. The effects of mycophenolic acid (MPA) on this pathway, and the effects of mycophenolate mofetil (MMF) treatment in (NZB x NZW)F(1)/J mice were also studied. METHODS: Immunohistochemical analyses of renal biopsy samples from patients with active diffuse proliferative lupus nephritis were performed. Cultured human mesangial cells were incubated with human polyclonal anti-DNA antibodies, with or without MPA. (NZB x NZW)F(1)/J mice with active nephritis were randomized to receive either MMF (100 mg/kg/day) or vehicle treatment for 12 weeks. RESULTS: Glomerular FN expression was increased in patients with lupus nephritis, and it colocalized with IgG deposition. Anti-DNA antibodies induced protein kinase Calpha (PKCalpha), PKCbetaI, and PKCbetaII activation, increased levels of bioactive TGFbeta1, and increased FN synthesis in human mesangial cells (P < 0.001 for each comparison versus control conditions). Pretreatment of anti-DNA antibodies with exogenous DNA reduced their cellular binding and abrogated their induction of TGFbeta1 and FN synthesis. Inhibition of PKC activation in human mesangial cells prior to anti-DNA antibody stimulation had no effect on cell proliferation, but resulted in significantly reduced antibody-mediated TGFbeta1 secretion and FN synthesis. MPA treatment down-regulated PKCalpha, PKCbetaI, and PKCbetaII phosphorylation, reduced levels of TGFbeta1 bioactivation, and decreased FN synthesis and deposition into the extracellular matrix. MMF treatment in (NZB x NZW)F(1)/J mice resulted in a reduction in glomerular IgG deposition, PKC activation, and FN expression, as well as an amelioration of proteinuria. CONCLUSION: Human polyclonal anti-DNA antibodies induce TGFbeta1 and FN synthesis in human mesangial cells through PKC activation, which is inhibited by MPA.