A missense mutation in damage-specific DNA binding protein 2 is a genetic risk factor for ocular squamous cell carcinoma in Belgian horses.

BACKGROUND: Belgian horses are commonly affected with ocular squamous cell carcinoma (SCC), the most common cancer of the equine eye. A missense mutation in damage-specific DNA binding protein 2 (DDB2 c.1013C>T, p.Thr338Met) has been established as a recessive genetic risk factor for ocular SCC in the Haflinger breed. A sample of Belgian horses with unknown SCC phenotype was shown to possess this variant at a similar frequency to the Haflinger breed. Retrospective studies indicate that chestnut coat colour may predispose to the development of SCC. OBJECTIVES: To determine if DDB2 c.1013C>T is a risk factor for ocular SCC in a strictly phenotyped sample of Belgian horses. To investigate associations between coat colour loci genotypes and ocular SCC. STUDY DESIGN: Retrospective and prospective case identification, genetic investigation. METHODS: Genomic DNA was isolated from blood, hair or formalin-fixed paraffin-embedded tissue from 25 Belgian horses with histologically confirmed ocular SCC and 18 unaffected Belgian horses. Association testing of 34 single nucleotide variants from 11 genomic loci and genotyping for DDB2 c.1013C>T and coat colour alleles were performed. Exons of DDB2 were sequenced in four cases and two controls. Associations were analysed by Chi-square or Fisher's exact tests and relative risk was calculated. RESULTS: Homozygosity for DDB2 c.1013C>T was significantly associated with ocular SCC (P = 7.4 × 10-7 ). Seventy-six per cent of affected horses were homozygous for the variant. Relative risk for homozygous horses developing SCC was 4.0 (P = 1.0 × 10-4 ). Sequencing DDB2 did not identify a variant more concordant with disease phenotype. An association between disease and coat colour loci was not identified. MAIN LIMITATIONS: Phenotyping was determined at a single timepoint. Each included horse genotyped as chestnut, so association with this MC1R variant could not be investigated. CONCLUSIONS: A missense variant, DDB2 c.1013C>T, p.Thr338Met, is a risk factor for ocular SCC in Belgian horses. A genetic risk test is commercially available.

Selective HDAC6 inhibition decreases early stage of lupus nephritis by down-regulating both innate and adaptive immune responses.

We have demonstrated previously that histone deacetylase (HDAC6) expression is increased in animal models of systemic lupus erythematosus (SLE) and that inhibition of HDAC6 decreased disease. In our current studies, we tested if an orally active selective HDAC6 inhibitor would decrease disease pathogenesis in a lupus mouse model with established early disease. Additionally, we sought to delineate the cellular and molecular mechanism(s) of action of a selective HDAC6 inhibitor in SLE. We treated 20-week-old (early-disease) New Zealand Black (NZB)/White F1 female mice with two different doses of the selective HDAC6 inhibitor (ACY-738) for 5 weeks. As the mice aged, we determined autoantibody production and cytokine levels by enzyme-linked immunosorbent assay (ELISA) and renal function by measuring proteinuria. At the termination of the study, we performed a comprehensive analysis on B cells, T cells and innate immune cells using flow cytometry and examined renal tissue for immune-mediated pathogenesis using immunohistochemistry and immunofluorescence. Our results showed a reduced germinal centre B cell response, decreased T follicular helper cells and diminished interferon (IFN)-γ production from T helper cells in splenic tissue. Additionally, we found the IFN-α-producing ability of plasmacytoid dendritic cells was decreased along with immunoglobulin isotype switching and the generation of pathogenic autoantibodies. Renal tissue showed decreased immunoglobulin deposition and reduced inflammation as judged by glomerular and interstitial inflammation. Taken together, these studies show selective HDAC6 inhibition decreased several parameters of disease pathogenesis in lupus-prone mice. The decrease was due in part to inhibition of B cell development and response.

Renal-infiltrating CD11c+ cells are pathogenic in murine lupus nephritis through promoting CD4+ T cell responses.

Lupus nephritis (LN) is a major manifestation of systemic lupus erythematosus (SLE), causing morbidity and mortality in 40-60% of SLE patients. The pathogenic mechanisms of LN are not completely understood. Recent studies have demonstrated the presence of various immune cell populations in lupus nephritic kidneys of both SLE patients and lupus-prone mice. These cells may play important pathogenic or regulatory roles in situ to promote or sustain LN. Here, using lupus-prone mouse models, we showed the pathogenic role of a kidney-infiltrating CD11c+ myeloid cell population in LN. These CD11c+ cells accumulated in the kidneys of lupus-prone mice as LN progressed. Surface markers of this population suggest their dendritic cell identity and differentiation from lymphocyte antigen 6 complex (Ly6C)low mature monocytes. The cytokine/chemokine profile of these renal-infiltrating CD11c+ cells suggests their roles in promoting LN, which was confirmed further in a loss-of-function in-vivo study by using an antibody-drug conjugate (ADC) strategy targeting CX3 CR1, a chemokine receptor expressed highly on these CD11c+ cells. However, CX3 CR1 was dispensable for the homing of CD11c+ cells into lupus nephritic kidneys. Finally, we found that these CD11c+ cells co-localized with infiltrating T cells in the kidney. Using an ex- vivo co-culture system, we showed that renal-infiltrating CD11c+ cells promoted the survival, proliferation and interferon-γ production of renal-infiltrating CD4+ T cells, suggesting a T cell-dependent mechanism by which these CD11c+ cells promote LN. Together, our results identify a pathogenic kidney-infiltrating CD11c+ cell population promoting LN progression, which could be a new therapeutic target for the treatment of LN.

Pneumonia Caused by Klebsiella spp. in 46 Horses.

BACKGROUND: Klebsiella spp. are implicated as a common cause of bacterial pneumonia in horses, but few reports describe clinical presentation and disease progression. HYPOTHESIS/OBJECTIVES: To describe the signalment, clinicopathologic data, radiographic and ultrasonographic findings, antimicrobial susceptibility, outcome, and pathologic lesions associated with Klebsiella spp. pneumonia in horses. ANIMALS: Forty-six horses from which Klebsiella spp. was isolated from the lower respiratory tract. METHODS: Retrospective study. Medical records from 1993 to 2013 at the William R. Pritchard Veterinary Medical Teaching Hospital, University of California, Davis were reviewed. Exact logistic regression was performed to determine if any variables were associated with survival to hospital discharge. RESULTS: Survival in horses <1 year old was 73%. Overall survival in adults was 63%. For adults in which Klebsiella pneumoniae was the primary isolate, survival was 52%. Mechanical ventilation preceded development of pneumonia in 11 horses. Complications occurred in 25/46 horses, with thrombophlebitis and laminitis occurring most frequently. Multi-drug resistance was found in 47% of bacterial isolates. Variables that significantly impacted survival included hemorrhagic nasal discharge, laminitis, and thoracic radiographs with a sharp demarcation between marked caudal pulmonary alveolar infiltration and more normal-appearing caudodorsal lung. CONCLUSIONS AND CLINICAL IMPORTANCE: Klebsiella spp. should be considered as a differential diagnosis for horses presenting with hemorrhagic pneumonia and for horses developing pneumonia after mechanical ventilation. Multi-drug resistance is common. Prognosis for survival generally is fair, but is guarded for adult horses in which K. pneumoniae is isolated as the primary organism.

Laser Capture Microdissection of Feline Streptomyces spp Pyogranulomatous Dermatitis and Cellulitis.

Suspected Streptomyces spp infections were identified in 4 cats at UC Davis Veterinary Medical Teaching Hospital between 1982 and 2011. Three had ulcerated, dark red mycetomas involving the dermis, subcutis, and fascia with fistulous tracts and/or regional lymphadenopathy. One cat had pyogranulomatous mesenteric lymphadenitis. Granulomatous inflammation in all cats contained colonies of Gram-positive, non-acid-fast organisms. All 4 cats failed to respond to aggressive medical and surgical treatment and were euthanized. Laser capture microdissection (LCM) was used to selectively harvest DNA from the affected formalin-fixed, paraffin-embedded (FFPE) tissues. Cloned amplicons from LCM-derived tissue confirmed the presence of Streptomyces spp in the dermatitis cases. Amplicons from the remaining cat with peritoneal involvement aligned with the 16S ribosomal RNA gene for Actinomycetales. Usually considered a contaminant, Streptomyces spp can be associated with refractory pyogranulomatous dermatitis and cellulitis in cats with outdoor access. LCM is useful in the diagnosis of bacterial diseases where contamination may be an issue.

Aggressive local therapy combined with systemic chemotherapy provides long-term control in grade II stage 2 canine mast cell tumour: 21 cases (1999-2012).

This retrospective case series evaluates the outcome of 21 dogs with grade II stage 2 mast cell tumour (MCT) treated with adequate local therapy and adjuvant systemic chemotherapy (prednisone, vinblastine and CCNU). The median survival for all dogs was 1359 days (range, 188-2340). Median disease-free interval was 2120 days (149-2325 days). Dogs treated with surgery and chemotherapy had shorter survival (median, 1103 days; 188-2010 days) than those that underwent surgery, radiation therapy and chemotherapy as part of their treatment (median, 2056 days; 300-2340 days). Two patients had local recurrence in the radiation field and four patients had de novo MCT. Distant metastasis was not observed in any dogs. The results of this study suggest that, in the presence of loco-regional lymph node metastasis in grade II MCT, the use of prednisone, vinblastine and CCNU after adequate local-regional therapy can provide a median survival in excess of 40 months.

Immunohistochemical characteristics of normal canine eyes.

Immunohistochemistry is widely utilized in diagnostic laboratories to study neoplastic and nonneoplastic diseases. Knowledge of the immunohistochemical characteristics of normal tissue is essential for interpretation of immunoreactivity in pathologic conditions. In this study, immunohistochemistry was performed with a broad panel of diagnostically relevant antibodies on 4 normal canine globes--namely, vimentin, pan-cytokeratin (AE1/AE3), cytokeratin 7, cytokeratin 8/18, cytokeratin 20, α-smooth muscle actin, muscle specific actin, desmin, Melan-A, microphthalmia transcription factor, S-100, glial fibrillary acidic protein, triple neurofilaments, neuron-specific enolase, chromogranin A, synaptophysin, laminin and CD31. Results include cytokeratin immunoreactivity limited to the conjunctival epithelium, corneal epithelium, and retinal pigment epithelium; distinct patterns of immunopositivity of muscle markers; and widespread immunoreactivity for vimentin and most neural/neuroendocrine markers. These findings in normal eyes provide the basis for interpretation of ocular immunohistochemistry in dogs. Published immunophenotypes of primary ocular neoplasms are also reviewed.

Equine degenerative myeloencephalopathy in Lusitano horses.

BACKGROUND: Equine degenerative myeloencephalopathy (EDM) is a neurodegenerative disorder that has been previously associated with low vitamin E concentrations. OBJECTIVE: To describe the clinical, electrophysiologic, and pathologic features of EDM in a group of related Lusitano horses. ANIMALS: Fifteen Lusitano horses. PROCEDURES: Neurologic examinations were conducted, and serum vitamin E concentrations were measured. Three neurologically abnormal horses were further evaluated by ophthalmologic examination, electroretinography, electroencephalography, muscle and nerve biopsies, and post-mortem examination. RESULTS: Six horses appeared neurologically normal, 6 were neurologically abnormal, and 3 had equivocal gait abnormalities. Abnormal horses demonstrated ataxia and paresis. An inconsistent menace response was noted in 4 neurologically abnormal horses and in 1 horse with equivocal findings. All horses had low serum vitamin E concentrations (<1.5 ppm). Ophthalmologic examinations, electroretinograms, electroencephalograms, and muscle and peripheral nerve biopsies were unremarkable in 3 neurologically abnormal horses. At necropsy, major neuropathological findings in these horses were bilaterally symmetric, severe, neuro axonal degeneration in the gracilis, cuneatus medialis, cuneatus lateralis, and thoracicus nuclei and bilaterally symmetric axonal loss and demyelination mainly in the dorsolateral and ventromedial tracts of the spinal cord. A diagnosis of EDM was made based on these findings. Pedigree analysis identified 2 sires among the affected horses. CONCLUSIONS AND CLINICAL RELEVANCE: Equine degenerative myeloencephalopathy is a neurodegenerative disorder that causes ataxia and, in severe cases, paresis, in young Lusitano horses. The disease appears to have a genetic basis, and although vitamin E deficiency is a common finding, low serum vitamin E concentrations also may occur in apparently unaffected related individuals.

Activation of AMPK inhibits inflammation in MRL/lpr mouse mesangial cells.

Recent reports show that 5-amino-4-imidazole carboxamide riboside (AICAR), a pharmacological activator of AMP-activated protein kinase (AMPK), inhibits the lipopolysaccharide (LPS)-induced production of proinflammatory cytokines. MRL/MPJ-Fas(lpr) (MRL/lpr) mice show an intrinsic decreased threshold for the production of inflammatory mediators when stimulated. In our current studies, we sought to determine if AMPK activation would inhibit inflammatory mediator production in stimulated kidney mesangial cells. Cultured mesangial cells from MRL/lpr mice were treated with AICAR and stimulated with LPS/interferon (IFN)-gamma. AICAR decreased dose-dependently inducible nitric oxide synthase (iNOS), cyclooxygenase-2 and interleukin-6 production in LPS/IFN-gamma-stimulated mesangial cells. Mechanistically, AICAR inhibited the LPS/IFN-gamma-stimulated PI3K/Akt signalling inflammatory cascade but did not affect LPS/IFN-gamma-mediated inhibitory kappa B phosphorylation or nuclear factor (NF)-kappaB (p65) nuclear translocation. Treatment with the adenosine kinase inhibitor 5'-iodotubercidin blocked the ability of AICAR to activate AMPK and prevented AICAR from inhibiting the LPS/IFN-gamma-stimulated PI3K/Akt pathway and attenuating iNOS expression. Taken together, these observations suggest that AICAR inhibits LPS/IFN-gamma-induced Akt phosphorylation through AMPK activation and may serve as a potential therapeutic target in inflammatory diseases.

Feline pulmonary Langerhans cell histiocytosis with multiorgan involvement.

Histiocytic proliferative diseases are uncommon in cats, although recently a progressive histiocytosis of the skin with terminal involvement of internal organs has been described in cats. Here we describe 3 cats (2 males and 1 female) with pulmonary Langerhans cell histiocytosis (PLCH). The cats were euthanized due to progressive respiratory clinical symptoms and deterioration. Macroscopically, extensive, multifocal to confluent, pulmonary masses were evident. Infiltration of pancreas (2 cats), kidneys (1 cat), liver (1 cat), as well as tracheobronchial, hepatosplenic, or mesenteric lymph nodes (2 cats) was observed by gross or microscopic examination. The infiltrating cells had histiocytic morphology with cytologic atypia characterized by anisokaryosis and hyperchromasia regionally within infiltrated tissues. Lesional histiocytes expressed vimentin, CD18, and E-cadherin. Expression of E-cadherin was usually markedly reduced in extra-pulmonary lesions, which is consistent with possible down-regulation of E-cadherin associated with distant migration from the lung. Transmission electron microscopy demonstrated intracytoplasmic organelles consistent with Birbeck's granules of Langerhans cells in the lesional histiocytes in all cats, except in the pancreas of one cat. These findings were compatible PLCH with limited organ involvement of humans. It remains unproven whether feline PLCH represents a reactive or neoplastic cell proliferation.

An enhanced postnatal autoimmune profile in 24 week-old C57BL/6 mice developmentally exposed to TCDD.

Developmental exposure of mice to the environmental contaminant and AhR agonist, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), causes persistent postnatal suppression of T cell-mediated immune responses. The extent to which prenatal TCDD may induce or exacerbate postnatal autoimmune disease remains unknown. In the present study, time-pregnant high affinity AhR C57BL/6 mice received a single oral administration of 0, 2.5, or 5 microg/kg TCDD on gestation day (gd) 12. Offspring of these mice (n=5/gender/treatment) were evaluated at 24 weeks-of-age and showed considerable immune dysregulation that was often gender-specific. Decreased thymic weight and percentages of CD4(+)CD8(+) thymocytes, and increased CD4(+)CD8(-) thymocytes, were present in the female but not male offspring. Males but not females showed decreased CD4(-)CD8(+) T cells, and increased Vbeta3(+) and Vbeta17a(+) T cells, in the spleen. Males but not females also showed increased percentages of bone marrow CD24(-)B220(+) B cell progenitors. Antibody titers to dsDNA, ssDNA and cardiolipin displayed increasing trends in both male and female mice, reaching significance for anti-dsDNA in both genders and for ssDNA in males at 5 microg/kg TCDD. Immunofluorescent staining of IgG and C3 deposition in kidney glomeruli increased in both genders of prenatal TCDD-exposed mice, suggestive of early stages of autoimmune glomerulonephritis. Collectively, these results show that exposure to TCDD during immune system development causes persistent humoral immune dysregulation as well as altered cell-mediated responses, and induces an adult profile of changes suggestive of increased risk for autoimmune disease.

Mitotic index is predictive for survival for canine cutaneous mast cell tumors.

Mitotic index (MI) is an indirect measure of cell proliferation that has been demonstrated to be a strong predictor of outcome for several human and canine cancers. The purpose of this study was to evaluate the utility of MI as a predictor of biologic behavior and survival in dogs with cutaneous mast cell tumors (MCTs). Medical records from 148 dogs with histologically confirmed MCTs were reviewed. Information regarding tumor grade, local recurrence, metastatic disease, date of death/last follow-up, and outcome was obtained. The region of the tumor with the highest overall mitotic activity was chosen for evaluation, and the MI value was defined as the number of mitotic figures/10 high-power fields (400x, 2.7 mm(2)). A Cox proportional hazards regression model was used to compare MI with survival data. A Mann-Whitney test was used to compare MI on the basis of the development of local recurrence and metastatic disease. The MI correlated directly with tumor grade (P < .0001). The median survival time for dogs with an MI < or =5 was significantly longer (70 months) than for those with an MI >5 (2 months), regardless of grade (P < .001). For grade II tumors with an MI < or =5, the median survival time (MST) was 70 months, compared with 5 months for those with an MI >5 (P < .001). For grade III tumors with an MI < or =5, the MST was not reached, compared with <2 months for those with an MI >5 (P < .001). In conclusion, MI is a strong predictor of overall survival for dogs with cutaneous MCTs and should be included as a prognostic indicator when determining therapeutic options.

Impact of different cell isolation techniques on lymphocyte viability and function.

The outcome of immunological assays is markedly influenced by the method of isolation of lymphocytes. It is, therefore, important to comparatively assess various techniques of isolation of lymphocytes, an aspect thus far not thoroughly addressed. In particular, the potential of isolation techniques to influence cell recovery, viability, and function has not yet been evaluated. These studies were designed to determine the effect of different mechanical tissue dissociation methods on the viability and function of lymphocytes. Following spleen and thymus removal, the lymphoid organs were dissociated by one of four different tissue dissociation techniques: metallic screen, sheer force slide, commercial stomacher, or plunger-screen. Cells were then enumerated and a trypan blue exclusion technique and 7-amino-actinomycin D (7-AAD) were both employed to assess viability. Mitogen-induced lymphocyte proliferation was measured using the Alamar Blue assay. Cell viability and lymphocyte surface antigen expression were assessed using flow cytometry. No significant differences in lymphocyte viability, morphology, or surface antigen expression were observed among the different techniques. Likewise, cellular apoptosis and necrosis were comparable across all the techniques. However, mitogen induced splenic T-cell proliferation was higher in cells collected using the metallic screen and plunger-screen isolation methods as compared to the sheer force slide or commercial stomacher procedures. These data suggest that cell recovery, morphology, and viability are not affected by isolation techniques. However, lymphocyte function, as assessed by mitogen induced proliferation, was negatively affected by the sheer force slide or commercial stomacher isolation techniques.

Complement component C3 is not required for full expression of immune complex glomerulonephritis in MRL/lpr mice.

Complement activation and tissue deposition of complement fragments occur during disease progression in lupus nephritis. Genetic deficiency of some complement components (e.g., Factor B) and infusion of complement inhibitors (e.g., Crry, anti-C5 Ab) protect against inflammatory renal disease. Paradoxically, genetic deficiencies of early components of the classical complement pathway (e.g., C1q, C4, and C2) are associated with an increased incidence of lupus in humans and lupus-like disease in murine knockout strains. Complement protein C3 is the converging point for activation of all three complement pathways and thus plays a critical role in biologic processes mediated by complement activation. To define the role of C3 in lupus nephritis, mice rendered C3 deficient by targeted deletion were backcrossed for eight generations to MRL/lpr mice, a mouse strain that spontaneously develops lupus-like disease. We derived homozygous knockout (C3(-/-)), heterozygous (C3(+/-)), and C3 wild-type (C3(+/+)) MRL/lpr mice. Serum levels of autoantibodies and circulating immune complexes were similar among the three groups. However, there was earlier and significantly greater albuminuria in the C3(-/-) mice compared with the other two groups. Glomerular IgG deposition was also significantly greater in the C3(-/-) mice than in the other two groups, although overall pathologic renal scores were similar. These results indicate that C3 and/or activation of C3 is not required for full expression of immune complex renal disease in MRL/lpr mice and may in fact play a beneficial role via clearance of immune complexes.

Prostaglandin J(2) inhibition of mesangial cell iNOS expression.

Mesangial cells from MRL/lpr mice, a model of lupus, overproduce nitric oxide (NO) compared to controls. J series prostaglandins (PG) and thiazolidinediones block LPS stimulation of NO production via the activation of peroxisome proliferator-activator receptor-gamma (PPAR-gamma) in macrophages but utilize an alternative mechanism in microglial cells. We investigated the mechanism by which PGJ(2) inhibits NO production in LPS/IFN-gamma-stimulated MRL/lpr mesangial cells. Our results demonstrated that LPS/IFN-gamma addition to MRL/lpr mesangial cells stimulated iNOS activation, expression of p-38 kinase and p44/42 MAPK, and NF-kappaB translocation to the nucleus. Both pioglitazone, a specific PPAR-gamma agonist, and PGJ(2) blocked NO production, iNOS protein expression, and iNOS mRNA transcription. PGJ(2) failed to inhibit nuclear NF-kappaB translocation or p44/42 MAPK or p-38 kinase induction in stimulated mesangial cells. These data suggest that PGJ(2) blocks iNOS expression and subsequent NO production in mesangial cells via a PPAR-gamma-mediated mechanism either by interfering with NF-kappaB transcriptional activity or by an NF-kappaB-independent mechanism.

Cystic fibrosis transmembrane conductance regulator currents in guinea pig pancreatic duct cells: inhibition by bicarbonate ions.

BACKGROUND & AIMS: Cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels play an important role in HCO(3)(-) secretion by pancreatic duct cells (PDCs). Our aims were to characterize the CFTR conductance of guinea pig PDCs and to establish whether CFTR is regulated by HCO(3)(-). METHODS: PDCs were isolated from small intralobular and interlobular ducts, and their Cl(- )conductance was studied using the whole-cell patch clamp technique. RESULTS: Activation of a typical CFTR conductance by adenosine 3',5'-cyclic monophosphate (cAMP) was observed in 114 of 204 cells (56%). A larger (10-fold), time- and voltage-dependent Cl(-) conductance was activated in 39 of 204 cells (19%). Secretin had a similar effect. Coexpression of both conductances in the same cell was observed, and both conductances had similar anion selectivity and pharmacology. Extracellular HCO(3)(-) caused a dose-dependent inhibition of both currents (K(i), approximately 7 mmol/L), which was independent of intracellular and extracellular pH, and the PCO(2) and CO(3)(2-) content of the bathing solutions. CONCLUSIONS: Two kinetically distinct Cl(-) conductances are activated by cAMP in guinea pig PDCs. Because these conductances are coexpressed and exhibit similar characteristics (anion selectivity, pharmacology, and HCO(3)(-) inhibition), we conclude that CFTR underlies them both. The inhibition of CFTR by HCO(3)(-) has implications for the current model of pancreatic ductal HCO(3)(-) secretion.

Inhibition of mesangial cell nitric oxide in MRL/lpr mice by prostaglandin J2 and proliferator activation receptor-gamma agonists.

MRL/Mp-lpr/lpr (MRL/lpr) mice develop immune complex glomerulonephritis similar to human lupus. Glomerular mesangial cells are key modulators of the inflammatory response in lupus nephritis. When activated, these cells secrete inflammatory mediators including NO and products of cyclooxygenase perpetuating the local inflammatory response. PGJ2, a product of cyclooxygenase, is a potent in vitro inhibitor of macrophage inflammatory functions and is postulated to function as an in vivo inhibitor of macrophage-mediated inflammatory responses. We hypothesized that in lupus, a defect in PGJ2 production allows the inflammatory response to continue unchecked. To test this hypothesis, mesangial cells were isolated from MRL/lpr and BALB/c mice and stimulated with IL-1beta or LPS plus IFN-gamma. In contrast to the 2- to 3-fold increase in PGJ2 production by stimulated BALB/c mesangial cells, supernatant PGJ2 did not increase in MRL/lpr mesangial cell cultures. NO production in stimulated MRL/lpr and BALB/c mesangial cells, was blocked by PGJ2 and pioglitazone. These studies suggest that abnormalities in PGJ2 production are present in MRL/lpr mice and may be linked to the heightened activation state of mesangial cells in these mice.

Prospective measure of serum 3-nitrotyrosine levels in systemic lupus erythematosus: correlation with disease activity.

Systemic lupus erythematosus (SLE) is a prototypical autoimmune disease. Overproduction of nitric oxide (NO) has been implicated in its pathogenesis. Several retrospective studies have indicated a correlation between serum nitrate and nitrite (NOx) and disease activity. This measure of NO production can be falsely elevated by exogenous dietary and medication sources of NOx and variably reduced by serum thiols. These variables can make NOx a less reliable tool for studying the role of NO in SLE. Peroxynitrite, a by-product of NO and superoxide, nitrates tyrosine moieties. The resulting 3-nitrotyrosine (3NT) serves as a long-term indicator of NO-mediated protein modifications that is not affected by exogenous sources of NOx or serum thiols. We hypothesized that for these reasons serum 3NT levels would correlate with lupus disease activity more significantly than serum NOx. To address this hypothesis, we prospectively evaluated lupus disease activity, serum protein 3NT levels, and NOx levels in a cohort of lupus patients at 3-month intervals. Serum 3NT correlated with disease activity among African-Americans, while NOx correlated with disease activity among Caucasians. Subjects with active lupus nephritis had higher levels of serum 3NT than those without renal disease. Immunohistochemical analysis of renal biopsies from subjects with active proliferative lupus nephritis revealed renal expression of inducible NO synthase. The results of this study suggest that overproduction of NO may play a pathogenic role in SLE and lupus nephritis. Serum 3NT may be a useful, new tool for studying the contributions of NO to the pathogenesis of SLE.

Purinoceptor activation of chloride transport in cystic fibrosis and CFTR-transfected pancreatic cell lines.

The regulation of chloride efflux from cystic fibrosis pancreatic adenocarcinoma cells (CFPAC-1) and wild-type CFTR-transfected CFPAC-1 cells (TPAC) was compared. Forskolin (10 microM) stimulated chloride efflux from the corrected TPAC cells but not from CFPAC-1 cells. Chloride efflux from both cell types was activated by thapsigargin (0.5 microM). The nucleotides ATP and UTP and the non-hydrolyzable ATP analogue, adenosine 5'-O-(3-thio) triphosphate (ATPgammaS), stimulated chloride efflux from both cell types. None of the other P2 purinoceptor agonists investigated elicited a response. The order of potency was ATP > or = UTP > or = ATPgammaS. Adenosine (10-100 microM) activated choride efflux from the TPAC but not the CFPAC cell line with no increase in intracellular cyclic AMP. Small but statistically significant inhibitions of the adenosine-(50 microM)-stimulated increase in chloride efflux were elicited by the A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (CPX, 100 nM) and the A2 receptor antagonist 3,7-dimethyl-1-propylargylxanthine (DMPX, 10 microM). The A2A receptor antagonist 8-(3-chlorostyryl)caffeine (CSC, 100 nM) had no significant effect. These results provide evidence for the regulation of chloride efflux by P2Y2 purinoceptors in genetically-corrected and CF pancreatic cell lines. Studies with adenosine receptor antagonists indicate some possible involvement of A1 and A2 (but not A2A) receptors in the adenosine stimulation of chloride efflux, but the relatively small effects of the inhibitors coupled with lack of increase in cyclic AMP and a response only in the CFTR-transfected cells also suggests a possible direct effect of adenosine on CFTR.