Michael H. Walter

1,25-Dihydroxyvitamin D3 suppresses TLR8 expression and TLR8-mediated inflammatory responses in monocytes in vitro and experimental autoimmune encephalomyelitis in vivo.

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) suppresses autoimmunity and inflammation; however, the mechanism of its action has not been fully understood. We sought in this study to determine whether the anti-immune/anti-inflammatory action of 1,25(OH)2D3 is in part mediated through an interplay between 1,25(OH)2D3 and toll-like receptor (TLR)7/8 signaling. 1,25(OH)2D3 treatment prior to and/or following experimental autoimmune encephalomyelitis (EAE) induction effectively reduced inflammatory cytokine expression in the spinal cord and ameliorated EAE. These effects were accompanied with a reduction in expression of several TLRs with the most profound effect observed for TLR8. The expression of TLR8 adaptor protein MyD88 was also significantly reduced by 1,25(OH)2D3. To determine the molecular mechanism by which 1,25(OH)2D3 suppresses EAE induction of TLR8 and inflammatory cytokine expression, we evaluated whether 1,25(OH)2D3 can directly inhibit TLR8 signaling and the resulting inflammatory responses in human THP-1 monocytes. 1,25(OH)2D3 treatment not only significantly reduced TLR8 expression but also the expression or activity of MyD88, IRF-4, IRF-7 and NF-kB in monocytes challenged with TLR8 ligands. TLR8 promoter-luciferase reporter assays indicated that 1,25(OH)2D3 decreases TLR8 mRNA level in part via inhibiting TLR8 gene transcription activity. As a result of inhibition on TLR8 signaling cascade at various stages, 1,25(OH)2D3 significantly diminished the TLR8 target gene expression (TNF-α and IL-1β). In summary, our novel findings suggest that TLR8 is a new target of 1,25(OH)2D3 and may mediate the anti-inflammatory action of 1,25(OH)2D3. Our findings also point to a destructive role of TLR8 in EAE and shed lights on pathogenesis of multiple sclerosis.

Utilization of Baclofen in Maintenance of Alcohol Abstinence in Patients with Alcohol Dependence and Alcoholic Hepatitis with or without Cirrhosis

AIM To report the efficacy and safety of baclofen in improving clinical state in patients with alcoholic hepatitis. METHOD Single center, open, retrospective study analyzing the effects of baclofen utilized over 12 months in patients with alcoholic hepatitis with or without cirrhosis and alcohol dependence on these liver parameters: aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin (Tbili), prothrombin time (PT), international normalized ratio (INR), albumin and Model for End-Stage Liver Disease (MELD) score. RESULTS Out of 40 patients, 35 were treated with baclofen. On average, baclofen was used for 5.8 months. A significant decrease in the mean AST, ALT, Tbili, INR, PT and MELD score was seen when comparing pre-baclofen use compared with post-baclofen use. Of the 35 patients who were started on baclofen, 34 (97%) remained abstinent. There were no serious adverse events. CONCLUSIONS Baclofens safety and efficacy in improving the clinical condition patients with alcoholic liver disease has been supported. Randomized prospective studies with longer duration of baclofen in this population may further optimize its use and corroborate efficacy.

Targeted 25-hydroxyvitamin D3 1α-hydroxylase Adoptive Gene Therapy Ameliorates DSS-induced Colitis Without Causing Hypercalcemia in Mice

Systemic 1,25(OH)2D3 treatment ameliorating murine inflammatory bowel diseases (IBD) could not be applied to patients because of hypercalcemia. We tested the hypothesis that increasing 1,25(OH)2D3 synthesis locally by targeting delivery of the 1α-hydroxylase gene (CYP27B1) to the inflamed bowel would ameliorate IBD without causing hypercalcemia. Our targeting strategy is the use of CD11b(+)/Gr1(+) monocytes as the cell vehicle and a macrophage-specific promoter (Mac1) to control CYP27B1 expression. The CD11b(+)/Gr1(+) monocytes migrated initially to inflamed colon and some healthy tissues in dextran sulfate sodium (DSS) colitis mice; however, only the migration of monocytes to the inflamed colon was sustained. Adoptive transfer of Gr1(+) monocytes did not cause hepatic injury. Infusion of Mac1-CYP27B1-modified monocytes increased body weight gain, survival, and colon length, and expedited mucosal regeneration. Expression of pathogenic Th17 and Th1 cytokines (interleukin (IL)-17a and interferon (IFN)-α) was decreased, while expression of protective Th2 cytokines (IL-5 and IL-13) was increased, by the treatment. This therapy also enhanced tight junction gene expression in the colon. No hypercalcemia occurred following this therapy. In conclusion, we have for the first time obtained proof-of-principle evidence for a novel monocyte-based adoptive CYP27B1 gene therapy using a mouse IBD model. This strategy could be developed into a novel therapy for IBD and other autoimmune diseases.Systemic 1,25(OH)2D3 treatment ameliorating murine inflammatory bowel diseases (IBD) could not be applied to patients because of hypercalcemia. We tested the hypothesis that increasing 1,25(OH)2D3 synthesis locally by targeting delivery of the 1α-hydroxylase gene (CYP27B1) to the inflamed bowel would ameliorate IBD without causing hypercalcemia. Our targeting strategy is the use of CD11b+/Gr1+ monocytes as the cell vehicle and a macrophage-specific promoter (Mac1) to control CYP27B1 expression. The CD11b+/Gr1+ monocytes migrated initially to inflamed colon and some healthy tissues in dextran sulfate sodium (DSS) colitis mice; however, only the migration of monocytes to the inflamed colon was sustained. Adoptive transfer of Gr1+ monocytes did not cause hepatic injury. Infusion of Mac1-CYP27B1-modified monocytes increased body weight gain, survival, and colon length, and expedited mucosal regeneration. Expression of pathogenic Th17 and Th1 cytokines (interleukin (IL)-17a and interferon (IFN)-α) was decreased, while expression of protective Th2 cytokines (IL-5 and IL-13) was increased, by the treatment. This therapy also enhanced tight junction gene expression in the colon. No hypercalcemia occurred following this therapy. In conclusion, we have for the first time obtained proof-of-principle evidence for a novel monocyte-based adoptive CYP27B1 gene therapy using a mouse IBD model. This strategy could be developed into a novel therapy for IBD and other autoimmune diseases.

Targeting Non-classical Myelin Epitopes to Treat Experimental Autoimmune Encephalomyelitis

Qa-1 epitopes, the peptides that bind to non-classical major histocompatibility complex Ib Qa-1 molecules and are recognized by Qa-1-restricted CD8+ regulatory T (Treg) cells, have been identified in pathogenic autoimmune cells that attack myelin sheath in experimental autoimmune encephalomyelitis (EAE, an animal model for multiple sclerosis [MS]). Additionally, immunization with such epitopes ameliorates the EAE. However, identification of such epitopes requires knowledge of the pathogenic autoimmune cells which are largely unknown in MS patients. Hence, we asked whether the CD8+ Treg cells could directly target the myelin sheath to ameliorate EAE. To address this question, we analyzed Qa-1 epitopes in myelin oligodendrocyte glycoprotein (MOG that is a protein in myelin sheath). Here, we report identification of a MOG-specific Qa-1 epitope. Immunization with this epitope suppressed ongoing EAE, which was abrogated by CD8+ T cell depletion. Additionally, the epitope immunization activated the epitope-specific CD8+ T cells which specifically accumulated in the CNS-draining cervical lymph nodes. Finally, CD8+ T cells primed by the epitope immunization transferred EAE suppression. Hence, this study reveals a novel regulatory mechanism mediated by the CD8+ Treg cells. We propose that immunization with myelin-specific HLA-E epitopes (human homologues of Qa-1 epitopes) is a promising therapy for MS.

7042 Role of diagnostic and therapeutic ercp in infants, children and adolscents.

ERCP in adults is highly successful and effective with low complication rate. However its role in the pediatric pts is still evolving. The purpose of this study was to evaluate the indications, safety and efficacy of diagnostic (Dx) and therapeutic (Rx) ERCP in infants, children and adolescents. Methods: All ERCPs performed between 1980-1999, on infants, children, and adolescents, up to the age of 20 were retrospectively reviewed. Indications, success in selective cannulation, findings on cholangiogram (CG) and pancreatogram (PG), Rx pancreaticobiliary procedures performed and complications were reviewed. Results: 110 ERCPs (out of 150), performed on 85 pts (32 Male, 53 Female), mean age 16±5yrs (range 8 month to 20 yrs), are presented here. Referring physicians included pediatric-GI (25%), adult-GI (16%), surgeon (11%), and primary physician (48%). All ERCPs were performed by an adult gastroenterologist. Adult side-view Dx Olympus duodenoscope, was used in 80% of the cases. Procedure was well tolerated by all pts. Number of pts receiving conscious sedation vs general anesthesia, were 87% and 13% respectively. Indications included, CBD stone (suspected or +IOC) 31%, recurrent pancreatitis 31%, abnormal LFTs 13%, stent placement 11%, malabsorption 9%, and other 5%. Selective bile duct (BD), and pancreatic duct (PD) cannulation was successful in 90% and 93% respectively. CG findings included, normal 41%, stone 32%, stricture 11%, bile leak 9%, dilated BD 4%, and choledochal cyst 3%. PG findings included, normal 67%, chronic pancreatitis 22%, stricture 15%, dilated PD 6%, panc divisum 5%, and stone 2%. A total of 93 Rx procedures ( Biliary 84, Pancreatic 9) were performed on 57 ERCPs (52%). Biliary Rx procedures included, 30 sphincterotomies, 11 stents, 25 stone extractions, 6 sphincter dilations , 6 stent removals , 5 stricture dilations and 1 brushing. Pancreatic Rx procedures included, 8 stents, and 1 stone removal. There were 8 (7%) ERCP related complications (6 pancreatitis, 2 cholangitis), 5 after Dx and 3 after Rx ERCP (p=NS). There was no procedure related mortality. Conclusion: ERCP in infants, children and adolescents is safe and well tolerated. Similar to adults, success in cannulation and performance of therapeutic procedure remains high. Further prospective studies on long-term outcome of Rx ERCP in pediatric population are warranted.