IntroductionIn Sjögren's syndrome, keratoconjunctivitis sicca (dry eye) is associated with infiltration of lacrimal glands by leukocytes and consequent losses of tear-fluid production and the integrity of the ocular surface. We investigated the effect of blockade of the lymphotoxin-beta receptor (LTBR) pathway on lacrimal-gland pathology in the NOD mouse model of Sjögren's syndrome.MethodsMale NOD mice were treated for up to ten weeks with an antagonist, LTBR-Ig, or control mouse antibody MOPC-21. Extra-orbital lacrimal glands were analyzed by immunohistochemistry for high endothelial venules (HEV), by Affymetrix gene-array analysis and real-time PCR for differential gene expression, and by ELISA for CXCL13 protein. Leukocytes from lacrimal glands were analyzed by flow-cytometry. Tear-fluid secretion-rates were measured and the integrity of the ocular surface was scored using slit-lamp microscopy and fluorescein isothiocyanate (FITC) staining. The chemokine CXCL13 was measured by ELISA in sera from Sjögren's syndrome patients (n = 27) and healthy controls (n = 30). Statistical analysis was by the two-tailed, unpaired T-test, or the Mann-Whitney-test for ocular integrity scores.ResultsLTBR blockade for eight weeks reduced B-cell accumulation (approximately 5-fold), eliminated HEV in lacrimal glands, and reduced the entry rate of lymphocytes into lacrimal glands. Affymetrix-chip analysis revealed numerous changes in mRNA expression due to LTBR blockade, including reduction of homeostatic chemokine expression. The reduction of CXCL13, CCL21, CCL19 mRNA and the HEV-associated gene GLYCAM-1 was confirmed by PCR analysis. CXCL13 protein increased with disease progression in lacrimal-gland homogenates, but after LTBR blockade for 8 weeks, CXCL13 was reduced approximately 6-fold to 8.4 pg/mg (+/- 2.7) from 51 pg/mg (+/-5.3) in lacrimal glands of 16 week old control mice. Mice given LTBR blockade exhibited an approximately two-fold greater tear-fluid secretion than control mice (P = 0.001), and had a significantly improved ocular surface integrity score (P = 0.005). The mean CXCL13 concentration in sera from Sjögren's patients (n = 27) was 170 pg/ml, compared to 92.0 pg/ml for sera from (n = 30) healthy controls (P = 0.01).ConclusionsBlockade of LTBR pathways may have therapeutic potential for treatment of Sjögren's syndrome.

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Here we present a preclinical model to assess drug-drug interactions due to inhibition of glucuronidation. Treatment with the antiepileptics phenobarbital (PB) or phenytoin (PH) has been associated with increased incidence of acetaminophen (APAP) hepatotoxicity in patients. In human hepatocytes, we found that the toxicity of APAP (5 mM) was increased by simultaneous treatment with phenobarbital (2 mM) or phenytoin (0.2 mM). In contrast, pretreatment with PB for 48 h prior to APAP treatment did not increase APAP toxicity unless both drugs were present simultaneously. Cells treated with APAP in combination with PB or PH experienced decreases in protein synthesis as early as 1 h, ultrastructural changes by 24 h, and release of liver enzymes by 48 h. Toxicity correlated with inhibition of APAP glucuronidation. PB or PH also inhibited APAP glucuronidation in rat and human liver microsomes and expressed human UGT1A6, 1A9, and 2B15. As with intact hepatocytes, PB and PH were neither hydroxylated nor glucuronidated, suggesting the direct inhibition of UGTs. Our findings suggest that, in multiple drug therapy, an inhibitory complex between UGT and one of the drugs can lead to decreased glucuronidation and increased systemic exposure and toxicity of a coadministered drug.

The objective of this study was to determine whether Toll-like receptor 4 (TLR4) has a role in alcohol-mediated acetaminophen (APAP) hepatotoxicity. TLR4 is involved in the inflammatory response to endotoxin. Others have found that ethanol-mediated liver disease is decreased in C3H/HeJ mice, which have a mutated TLR4 resulting in a decreased response to endotoxin compared with endotoxin-responsive mice. In the present study, short-term (1 wk) pretreatment with ethanol plus isopentanol, the predominant alcohols in alcoholic beverages, caused no histologically observed liver damage in either C3H/HeJ mice or endotoxin-responsive C3H/HeN mice, despite an increase in nitrotyrosine levels in the livers of C3H/HeN mice. In C3H/HeN mice pretreated with the alcohols, subsequent exposure to APAP caused a transient decrease in liver nitrotyrosine formation, possibly due to competitive interaction of peroxynitrite with APAP producing 3-nitroacetaminophen. Treatment with APAP alone resulted in steatosis in addition to congestion and necrosis in both C3H/HeN and C3H/HeJ mice, but the effects were more severe in endotoxin-responsive C3H/HeN mice. In alcohol-pretreated endotoxin-responsive C3H/HeN mice, subsequent exposure to APAP resulted in further increases in liver damage, including severe steatosis, associated with elevated plasma levels of TNF-alpha. In contrast, alcohol pretreatment of C3H/HeJ mice caused little to no increase in APAP hepatotoxicity and no increase in plasma TNF-alpha. Portal blood endotoxin levels were very low and were not detectably elevated by any of the treatments. In conclusion, this study implicates a role of TLR4 in APAP-mediated hepatotoxicity.

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