Trends in Systemic Sclerosis Mortality Over Forty-Eight Years, 1968-2015: A US Population-Based Study.

OBJECTIVE: To identify secular trends associated with systemic sclerosis (SSc) mortality over a 48-year period. METHODS: Using national mortality data compiled by the Centers for Disease Control and Prevention's Wide-Ranging Online Data for Epidemiologic Research, and population data from the US Census Bureau, we calculated an age-standardized mortality rate (ASMR) for SSc and non-SSc (all other causes), and we also calculated the ratio of the SSc ASMR to the non-SSc ASMR for each year from 1968 to 2015. We then used a joinpoint regression model to evaluate mortality trends overall and by sex and race. RESULTS: From 1968 to 2015, there were 46,798 deaths with SSc recorded as the "underlying" cause of death and 106,058,839 non-SSc deaths. There were an additional 9,063 deaths with SSc recorded as a "contributing" cause of death from 1999 to 2015. Whereas the non-SSc ASMR decreased throughout the 48-year time period, the SSc ASMR increased from 1968 to 2000, followed by decreases each year from 2001 to 2015. The SSc ASMR also decreased for deaths where SSc was a contributing cause from 1999 to 2015. Women and Black persons had higher SSc ASMRs and SSc ASMR to non-SSc ASMR ratios than men and White persons, respectively. Additionally, SSc ASMRs and SSc ASMR to non-SSc ASMR ratios increased at higher rates in women and White persons than in men and Black persons, respectively, during the initial three decades. CONCLUSION: Mortality attributable to SSc increased from 1968 to 2000, followed by a steady decline from 2001 to 2015. However, SSc mortality relative to non-SSc mortality remains high. SSc mortality has disproportionately changed by sex and race over the 48-year period assessed in the present study.

Quantitative Proteomics Using Formalin-fixed, Paraffin-embedded Biopsy Tissues in Inflammatory Disease.

BACKGROUND: Investigations in human disease pathogenesis have been hampered due to paucity of access to fresh-frozen tissues (FFT) for use in global, data-driven methodologies. As an alternative, formalin-fixed, paraffin-embedded (FFPE) tissues are readily available in pathology banks. However, the use of formalin for fixation can lead to the loss of proteins that appear during inflammation, thus introducing an inherent sample bias. To address this, we compared FF and FFPE tissue proteomics to determine whether FFPE-tissue can be used effectively in inflammatory diseases. METHODS: Adjacent kidney slices from lupus nephritic mice were processed as FFPE or FFTs. Their tissue lysates were run together using proteomics workflow involving filter-aided sample preparation, in-solution dimethyl isotope labeling, StageTip fractionation, and nano-LC MS/MS through an Orbitrap XL MS. RESULTS: We report a >97% concordance in protein identification between adjacent FFPE and FFTs in murine lupus nephritic kidneys. Specifically, proteins representing pathways, namely, 'systemic lupus erythematosus', 'interferon-α', 'TGF-ß', and 'extracellular matrix', were reproducibly quantified between FFPE and FFTs. However, 12%-29% proteins were quantified differently in FFPE compared to FFTs, but the differences were consistent across experiments. In particular, certain proteins represented in pathways, including 'inflammatory response' and 'innate immune system' were quantified less in FFPE than in FFTs. In a pilot study of human FFPE tissues, we identified proteins relevant to pathogenesis in lupus nephritic kidney biopsies compared to control kidneys. CONCLUSION: This is the first report of lupus nephritis kidney proteomics using FFPE tissue. We concluded that archived FFPE tissues can be reliably used for proteomic analyses in inflammatory diseases, with a caveat that certain proteins related to immunity and inflammation may be quantified less in FFPE than in FFTs.

Brief Report: Lupus-An Unrecognized Leading Cause of Death in Young Females: A Population-Based Study Using Nationwide Death Certificates, 2000-2015.

OBJECTIVE: Mortality statistics from the Centers for Disease Control and Prevention (CDC) are used for planning health care policy and allocating resources. The CDC uses these data to compile its annual ranking of leading causes of death based on a selected list of 113 causes. Systemic lupus erythematosus (SLE) is not included on this list. Since the ranking is a useful tool for assessing the relative burden of cause-specific mortality, this study was undertaken to rank SLE deaths among the CDC's leading causes of death to see whether SLE is a significant cause of death among females. METHODS: Death counts for the female population of the US were obtained from the CDC's Wide-ranging Online Data for Epidemiologic Research database and then grouped by age and race/ethnicity. Data on the leading causes of death were obtained from the Web-based Injury Statistics Query and Reporting System database. RESULTS: During 2000-2015, there were 28,411 deaths of females with SLE recorded as an underlying or contributing cause of death. SLE ranked among the top 20 leading causes of death in females between 5 and 64 years of age. SLE ranked tenth among those ages 15-24 years, fourteenth among those ages 25-34 years and 35-44 years, and fifteenth among those ages 10-14 years. For African American and Hispanic females, SLE ranked fifth among those ages 15-24 years, sixth among those ages 25-34 years, and eighth or ninth among those ages 35-44 years, after the 3 common external injury causes of death were excluded from the analysis. CONCLUSION: SLE is among the leading causes of death in young females, underscoring its impact as an important public health issue.

46-Year Trends in Systemic Lupus Erythematosus Mortality in the United States, 1968 to 2013: A Nationwide Population-Based Study.

BACKGROUND: No large population-based studies have been done on systemic lupus erythematosus (SLE) mortality trends in the United States. OBJECTIVE: To identify secular trends and population characteristics associated with SLE mortality. DESIGN: Population-based study using a national mortality database and census data. SETTING: United States. PARTICIPANTS: All U.S. residents, 1968 through 2013. MEASUREMENTS: Joinpoint trend analysis of annual age-standardized mortality rates (ASMRs) for SLE and non-SLE causes by sex, race/ethnicity, and geographic region; multiple logistic regression analysis to determine independent associations of demographic variables and period with SLE mortality. RESULTS: There were 50 249 SLE deaths and 100 851 288 non-SLE deaths from 1968 through 2013. Over this period, the SLE ASMR decreased less than the non-SLE ASMR, with a 34.6% cumulative increase in the ratio of the former to the latter. The non-SLE ASMR decreased every year starting in 1968, whereas the SLE ASMR decreased between 1968 and 1975, increased between 1975 and 1999, and decreased thereafter. Similar patterns were seen in both sexes, among black persons, and in the South. However, statistically significant increases in the SLE ASMR did not occur among white persons over the 46-year period. Females, black persons, and residents of the South had higher SLE ASMRs and larger cumulative increases in the ratio of the SLE to the non-SLE ASMR (31.4%, 62.5%, and 58.6%, respectively) than males, other racial/ethnic groups, and residents of other regions, respectively. Multiple logistic regression showed independent associations of sex, race, and region with SLE mortality risk and revealed significant racial/ethnic differences in associations of SLE mortality with sex and region. LIMITATIONS: Underreporting of SLE on death certificates may have resulted in underestimates of SLE ASMRs. Accuracy of coding on death certificates is difficult to ascertain. CONCLUSION: Rates of SLE mortality have decreased since 1968 but remain high relative to non-SLE mortality, and significant sex, racial, and regional disparities persist. PRIMARY FUNDING SOURCE: None.

Production of ß-galactosidase from streptococcus thermophilus for galactooligosaccharides synthesis.

Efficiency of different methods for disruption of Streptococcus thermophilus cells, isolated from different dairy products, to release ß-galactosidase and synthesis of GOS by extracted enzyme using whey supplemented with different concentrations of lactose as a substrate was studied. Unlike most other studies on GOS synthesis which used only one method of cell disruption and only few microbial strains, we compared five different cell disruption methods and used 30 strains of S. thermophilus in order to find out the most effective method and efficient strain for production of ß-galactosidase. Appreciable amount of GOS (53.45 gL(-1)) was synthesized at a lactose concentration of 30 %, using enzyme (10 U mL(-1) of reaction medium), extracted from S. thermophilus within a very short incubation time of 5 h at a temperature of 40 °C and pH 6.8. S. thermophilus is heavily employed in the preparation of fermented dairy products but this study extends the use of this organism for the production of GOS, a potential prebiotic.

Hypoglycaemic effect of galactooligosaccharides in alloxan-induced diabetic rats.

This study was conducted to assess the effect of prebiotic galactooligosaccharides (GOS) on alloxan-induced diabetes in male Sprague-Dawley (SD) rats. Diabetes was induced by administration of alloxan (100 mg/kg) and rats were divided in 4 groups: normal control group (NCG), prebiotic control group (PCG), diabetic control group (DCG) and diabetic prebiotic group (DPG). While PCG and DPG were fed with GOS supplemented (10% w/w) diet, NCG and DCG were administered with basal diet. Rats were sacrificed after 42 d for collection of blood and liver. Faecal samples were collected at the interval of 7 d throughout the study for measurement of lactobacilli and coliform count. Feeding of GOS decreased or delayed the severity of diabetes by amelioration of diabetes associated markers including fasting blood glucose, haemoglobin, glycosylated haemoglobin triglycerides, total cholesterol, low density lipoproteins, creatinine and urea. GOS was also found to improve the levels of antioxidative enzymes (superoxide dismutase, catalase and glutathione peroxidase) in liver and blood. Improvement in lactobacilli count along with a concomitant decrease in coliform count was observed in GOS fed groups.

HRES-1/Rab4-mediated depletion of Drp1 impairs mitochondrial homeostasis and represents a target for treatment in SLE.

OBJECTIVE: Accumulation of mitochondria underlies T-cell dysfunction in systemic lupus erythematosus (SLE). Mitochondrial turnover involves endosomal traffic regulated by HRES-1/Rab4, a small GTPase that is overexpressed in lupus T cells. Therefore, we investigated whether (1) HRES-1/Rab4 impacts mitochondrial homeostasis and (2) Rab geranylgeranyl transferase inhibitor 3-PEHPC blocks mitochondrial accumulation in T cells, autoimmunity and disease development in lupus-prone mice. METHODS: Mitochondria were evaluated in peripheral blood lymphocytes (PBL) of 38 SLE patients and 21 healthy controls and mouse models by flow cytometry, microscopy and western blot. MRL/lpr mice were treated with 125 µg/kg 3-PEHPC or 1 mg/kg rapamycin for 10 weeks, from 4 weeks of age. Disease was monitored by antinuclear antibody (ANA) production, proteinuria, and renal histology. RESULTS: Overexpression of HRES-1/Rab4 increased the mitochondrial mass of PBL (1.4-fold; p=0.019) and Jurkat cells (2-fold; p=0.000016) and depleted the mitophagy initiator protein Drp1 both in human (-49%; p=0.01) and mouse lymphocytes (-41%; p=0.03). Drp1 protein levels were profoundly diminished in PBL of SLE patients (-86±3%; p=0.012). T cells of 4-week-old MRL/lpr mice exhibited 4.7-fold over-expression of Rab4A (p=0.0002), the murine homologue of HRES-1/Rab4, and depletion of Drp1 that preceded the accumulation of mitochondria, ANA production and nephritis. 3-PEHPC increased Drp1 (p=0.03) and reduced mitochondrial mass in T cells (p=0.02) and diminished ANA production (p=0.021), proteinuria (p=0.00004), and nephritis scores of lupus-prone mice (p<0.001). CONCLUSIONS: These data reveal a pathogenic role for HRES-1/Rab4-mediated Drp1 depletion and identify endocytic control of mitophagy as a treatment target in SLE.

Prevalence of subclinical atherosclerosis is increased in systemic sclerosis and is associated with serum proteins: a cross-sectional, controlled study of carotid ultrasound.

OBJECTIVES: SSc is associated with an increased prevalence of atherosclerosis (ATS). This study assessed the prevalence of subclinical ATS as measured by carotid US and explored serum proteins to identify potential biomarkers of SSc-ATS. METHODS: Forty-six SSc female patients and 46 age- and ethnicity-matched controls underwent carotid US to assess the presence of plaque and carotid intima media thickness (CIMT). Abstracted data included demographics, ATS risk factors and serum measurements [cholesterol, proinflammatory high-density lipoprotein (piHDL), CRP, lipoproteins]. Serum cytokines/proteins analyses included circulating type I IFN activity by quantifying IFN-inducible genes, soluble junctional adhesion molecule A (sJAM-A) and 100 serum proteins by using a microplate-based multiplex platform. Proteins significant at P < 0.05 on bivariate analyses for the presence of plaque were used to develop a composite measure. RESULTS: Patients with SSc had more plaque (45.6% vs 19.5%, P = 0.01) but similar CIMT compared with controls. Multiplex analysis detected significant associations between serum proteins of inflammation, vasculopathy and fibrosis with ATS in SSc, including IL-2, IL-6, CRP, keratinocyte growth factor, intercellular adhesion molecule 1, endoglin, plasminogen activator inhibitor 1 and insulin-like growth factor binding protein 3 associated with carotid plaque. Myeloid progenitor inhibitory factor 1, serum amyloid A, thrombomodulin, N-terminal pro-brain natriuretic peptide (BNP), and Clara cell secretory protein 16 kD correlated with CIMT. The median composite score for the plaque group was 6 and for the no plaque group it was 2 (P < 0.0001). CONCLUSION: Patients with SSc have a higher prevalence of carotid plaque than matched controls, and patients with SSc-plaque vs patients without plaque have elevated serum proteins implicated in both vasculopathy and fibrosis. Further studies are needed to evaluate the role of these proteins in SSc compared with healthy controls.

A one-year, phase I/IIa, open-label pilot trial of imatinib mesylate in the treatment of systemic sclerosis-associated active interstitial lung disease.

OBJECTIVE: Transforming growth factor ß (TGFß) and platelet-derived growth factor (PDGF) may play a critical role in systemic sclerosis (SSc)-related interstitial lung disease (ILD), and imatinib is a potent inhibitor of TGFß and PDGF production. In this 1-year, phase I/IIa open-label pilot study of imatinib in patients with SSc-related active ILD, our primary aim was to assess the safety of imatinib; we also explored its efficacy. METHODS: We recruited 20 SSc patients with a forced vital capacity (FVC) of <85% predicted, dyspnea on exertion, and presence of a ground-glass appearance on high-resolution computed tomography. Patients received oral therapy with imatinib (up to 600 mg/day) for a period of 1 year. Adverse events were recorded, pulmonary function was tested, and the modified Rodnan skin thickness score (MRSS) was assessed every 3 months. The course of changes in lung function, the Health Assessment Questionnaire (HAQ) disability index (DI), and the MRSS were modeled over the period of study to explore treatment efficacy. RESULTS: The majority of patients were female (65%), Caucasian (75%), and had diffuse cutaneous SSc (70%). At baseline, the mean ± SD FVC % predicted was 65.2 ± 14.0 and the mean ± SD MRSS was 18.7 ± 10.1. The mean ± SD dosage of imatinib was 445 ± 125 mg/day. Of the 20 SSc patients, 12 completed the study, 7 discontinued because of adverse events (AEs), and 1 patient was lost to followup. Common AEs (≥20%) included fatigue, facial/lower extremity edema, nausea and vomiting, diarrhea, generalized rash, and new-onset proteinuria. Treatment with imatinib showed a trend toward improvement in the FVC % predicted (1.74%; P not significant) and the MRSS (3.9 units; P < 0.001). CONCLUSION: Use of high-dose daily therapy with imatinib (600 mg/day) in SSc patients with ILD was associated with a large number of AEs. Our experience with AEs suggests that dosages of imatinib lower than 600 mg/day may be appropriate and that further dose ranging analysis is needed in order to understand the therapeutic index of imatinib in SSc.

Generation of mice with a conditional allele for transforming growth factor beta 1 gene.

Transforming growth factor beta1 (TGFbeta1) is a multifunctional growth factor involved in wound healing, tissue fibrosis, and in the pathogenesis of many syndromic diseases (e.g., Marfan syndrome, Camurati-Engelmann disease) and muscular, neurological, ophthalmic, cardiovascular and immunological disorders, and cancer. Since the generation of Tgfb1 knockout mice, there has been extraordinary progress in understanding its physiological and pathophysiological function. Here, we report the generation of a conditional knockout allele for Tgfb1 in which its exon 6 is flanked with LoxP sites. As proof of principle, we crossed these mice to LckCre transgenic mice and specifically disrupted Tgfb1 in T cells. The results indicate that T-cell-produced TGFbeta1 is required for normal in vivo regulation of peripheral T-cell activation, maintenance of T-cell homeostasis, and suppression of autoimmunity.

TGFbeta1 deficiency does not affect the generation and maintenance of CD4+CD25+FOXP3+ putative Treg cells, but causes their numerical inadequacy and loss of regulatory function.

TGFbeta1 is considered to be required for peripheral maintenance of CD4(+)CD25(+)FOXP3(+) T(reg) cells. However, we demonstrate no reduction in the percentage of such T cells in the spleens and thymi of Tgfb1(-/-) mice. Although putative T(reg) cells, characterized as CD4(+)CD25(+)FOXP3(+)CD62L(+) T cells, are increased in Tgfb1(-/-) mice, they may be inadequate to control activated T cells since the ratio of activated T cells:putative T(reg) cells is several-fold higher in Tgfb1(-/-) mice than in control mice. We further show that whereas Tgfb1(-/-) mice that express a chicken OVA-specific TCR transgene (DO11.10) have an increase in putative T(reg) cells, there are no detectable CD4(+)CD25(+) T cells in the spleens of DO11.10 Rag1(-/-) mice suggesting that T(reg)-cell generation is self-antigen dependent regardless of whether they express Tgfb1. Finally, we demonstrate that Tgfb1(-/-) T cells remain responsive to the suppressive effect of TGFbeta1 in vitro. These data suggest that TGFbeta1 is required for the regulatory function of T(reg) cells to prevent activation of T cells and autoimmunity.

Self-antigen recognition by TGF beta1-deficient T cells causes their activation and systemic inflammation.

To investigate whether the multifocal inflammatory disease in TGFbeta1-deficient mice is caused by self-antigen (self-Ag)-specific autoreactive T cells, or whether it is caused by antigen independent, spontaneous hyperactivation of T cells, we have generated Tgfb1(-/-) and Tgfb1(-/-) Rag1(-/-) mice expressing the chicken OVA-specific TCR transgene (DO11.10). On a Rag1-sufficient background, Tgfb1(-/-) DO11.10 mice develop a milder inflammation than do Tgfb1(-/-) mice, and their T cells display a less activated phenotype. The lower level of activation correlates with the expression of hybrid TCR (transgenic TCRbeta and endogenous TCRalpha), which could recognize self-Ag and undergo activation. In the complete absence of self-Ag recognition (Tgfb1(-/-) DO11.10 Rag1(-/-) mice) inflammation and T-cell activation are eliminated, demonstrating that self-Ag recognition is required for the hyper-responsiveness of TGFbeta1-deficient T cells. Thus, TGFbeta1 is required for the prevention of autoimmune disease through its ability to control the activation of autoreactive T cells to self-Ag.

The role of invariant natural killer T cells in lupus and atherogenesis.

Systemic lupus erythematosus (SLE) is increasingly recognized as a risk factor for the development of premature atherosclerosis. The inflammatory process in both of these diseases is controlled by a variety of cell types of the innate and adaptive immune systems. Recent studies from several groups, including ours, have revealed a critical role of a unique subset of lymphocytes, termed invariant natural killer T (iNKT) cells, in the development of lupus-like autoimmunity and atherosclerosis in animal models. iNKT cells appear to play complex and divergent roles in the development of SLE and atherosclerosis. Our findings suggest that alterations in iNKT cell functions during the development of SLE may be related to the increased risk of SLE patients to develop atherosclerosis and coronary heart disease. We found that iNKT cell activation with the sponge-derived glycolipid alpha- galactosylceramide generally protects against the development of lupus-like autoimmunity in mice, whereas it exacerbates atherosclerosis. Therefore, while our studies have identified iNKT cells as potential therapeutic targets for SLE, further studies are necessary to design drugs that will avoid the underlying harmful effects of iNKT cell activation on the development of atherosclerosis.

Cellular and molecular mechanisms of regulation of autoantibody production in lupus.

The hyperactive interaction between helper T cells and autoimmune B cells in individuals predisposed to systemic lupus erythematosus (SLE) can be interrupted by induction of regulatory and suppressor T cells. Using two strategies-high dose tolerance to an immunoglobulin-derived peptide, and minigene vaccination with DNA encoding T cell epitopes presented by MHC class I molecules-our group has induced at least three types of regulatory/suppressive T cells. They include CD8+ T cells that suppress helper T cells by cytokine secretion, CD8+ T suppressors that kill B cells making anti-DNA antibodies, and peptide-binding CD4+CD25+ regulatory T cells that suppress B cells by direct cell contact. Each of these lymphocyte subsets suppresses anti-DNA antibody production and delays the onset of nephritis in BWF1 lupus-prone mice. Patients with SLE have amino acid sequences similar to those from murine anti-DNA antibodies used in these studies, and at similar locations in the VH regions of anti-DNA immunoglobulins. Therefore, strategies described here might ultimately be useful in therapy of the human disease.

The natural killer T cell ligand alpha-galactosylceramide prevents or promotes pristane-induced lupus in mice.

Systemic lupus erythematosus is a systemic autoimmune disease characterized by inflammation in organs such as kidneys and presence of autoantibodies against nuclear antigens. We have previously shown that CD1d deficiency in BALB/c mice exacerbates lupus nephritis and autoantibody production induced by the hydrocarbon oil pristane. Here, we have tested the impact of activating CD1d-restricted natural killer T (NKT) cells on pristane-induced lupus-like autoimmunity in BALB/c and SJL mice. Repeated in vivo treatment of pristane-injected BALB/c mice with the NKT cell ligand alpha-galactosylceramide (alpha-GalCer) prior to the onset of florid disease suppressed proteinuria, in a manner that was dependent on CD1d and IL-4 expression. In sharp contrast, however, similar treatment of pristane-injected SJL mice with alpha-GalCer resulted in increased proteinuria. Consistent with these dichotomous effects of NKT cell activation on the development of lupus-like autoimmunity, NKT cells in BALB/c and SJL/J mice exhibited a mixed Th1/Th2 and a Th1-biased cytokine production profile, respectively. These findings demonstrate that NKT cell activation with alpha-GalCer suppresses or promotes pristane-induced lupus-like autoimmunity in mice, in a strain-dependent manner.

TGF-beta 1 regulates lymphocyte homeostasis by preventing activation and subsequent apoptosis of peripheral lymphocytes.

TGF-beta1 plays an important role in the maintenance of immune homeostasis and self-tolerance. To determine the mechanism by which TGF-beta1 prevents autoimmunity we have analyzed T cell activation in splenic lymphocytes from TGF-beta1-deficient mice. Here we demonstrate that unlike wild-type splenic lymphocytes, those from Tgfb1(-/-) mice are hyporesponsive to receptor-mediated mitogenic stimulation, as evidenced by diminished proliferation and reduced IL-2 production. However, they have elevated levels of IFN-gamma and eventually undergo apoptosis. Receptor-independent stimulation of Tgfb1(-/-) T cells by PMA plus ionomycin induces IL-2 production and mitogenic response, and it rescues them from anergy. Tgfb1(-/-) T cells display decreased CD3 expression; increased expression of the activation markers LFA-1, CD69, and CD122; and increased cell size, all of which indicate prior activation. Consistently, mutant CD4(+) T cells have elevated intracellular Ca(2+) levels. However, upon subsequent stimulation in vitro, increases in Ca(2+) levels are less than those in wild-type cells. This is also consistent with the anergic phenotype. Together, these results demonstrate that the ex vivo proliferative hyporesponsiveness of Tgfb1(-/-) splenic lymphocytes is due to prior in vivo activation of T cells resulting from deregulated intracellular Ca(2+) levels.

Effect of an exogenous trigger on the pathogenesis of lupus in (NZB x NZW)F1 mice.

OBJECTIVE: This study examined the interactions between exogenous and endogenous factors shaping the phenotype of lupus in autoimmune (NZB x NZW)F(1) mice exposed to pristane, a model environmental trigger. METHODS: Frequencies of various autoantibodies in untreated NZB/NZW mice were determined by various means (immunoprecipitation, enzyme-linked immunosorbent assay [ELISA], Crithidia luciliae kinetoplast staining). Pristane or saline was administered intraperitoneally to 9-12-week-old NZB/NZW mice, followed by serial studies of autoantibodies, total Ig levels (ELISA), and proteinuria (dipstick). RESULTS: Besides antichromatin/DNA responses, NZB/NZW mice spontaneously produced novel autoantibodies against the double-stranded RNA binding protein RNA helicase A (RHA). In contrast, NZB/NZW mice (n = 70) did not produce autoantibodies against the nuclear RNP (nRNP), Sm, Ro, or La antigens. Pristane exposure synergistically activated the production of antichromatin/DNA antibodies and dramatically accelerated renal disease. Production of anti-nRNP/Sm and Su autoantibodies also was induced, indicating that the unresponsiveness of NZB/NZW mice to these antigens can be overcome. Curiously, pristane treatment did not enhance the production of anti-RHA, suggesting that these autoantibodies are regulated differently than anti-DNA/chromatin and Sm. In contrast to previous reports that suggest a critical role of deficient interleukin-12 (IL-12) production in the pathogenesis of lupus, there was overproduction of IL-12 in the peritoneal cavity of pristane-treated NZB/NZW mice, and their spleen cells also produced large amounts of IL-12. CONCLUSION: These data lead us to propose that environmental influences exacerbate autoimmune manifestations in genetically lupus-susceptible mice through their stimulatory effects on proinflammatory cytokines, such as IL-12.