Dwain L. Thiele

Amyloid-β peptide levels in brain are inversely correlated with insulysin activity levels in vivo

Factors that elevate amyloid-β (Aβ) peptide levels are associated with an increased risk for Alzheimers disease. Insulysin has been identified as one of several proteases potentially involved in Aβ degradation based on its hydrolysis of Aβ peptides in vitro. In this study, in vivo levels of brain Aβ40 and Aβ42 peptides were found to be increased significantly (1.6- and 1.4-fold, respectively) in an insulysin-deficient gene-trap mouse model. A 6-fold increase in the level of the γ-secretase-generated C-terminal fragment of the Aβ precursor protein in the insulysin-deficient mouse also was found. In mice heterozygous for the insulysin gene trap, in which insulysin activity levels were decreased ≈50%, brain Aβ peptides were increased to levels intermediate between those in wild-type mice and homozygous insulysin gene-trap mice that had no detectable insulysin activity. These findings indicate that there is an inverse correlation between in vivo insulysin activity levels and brain Aβ peptide levels and suggest that modulation of insulysin activity may alter the risk for Alzheimers disease.

Ethnic Differences in the Prevalence of Cryptogenic Cirrhosis

OBJECTIVES:Nonalcoholic steatohepatitis (NASH) associated with obesity and type 2 diabetes mellitus (DM) is postulated to be the cause of most cases of cryptogenic cirrhosis (CC). While ethnic differences in the prevalence of obesity and DM in the United States are well documented, there is little information regarding prevalence of CC or NASH among different U.S. ethnic groups. This study was performed to assess the demographic characteristics of patients with CC at a U.S. county hospital with a racially and ethnically diverse patient population.METHODS:Medical records and pathology databases were reviewed to identify patients at Parkland Memorial Hospital, Dallas County, Texas from 1990 to 2001 with CC or cirrhosis attributed to NASH.RESULTS:Forty-one patients (12 men, 29 women) were found to meet these criteria. Of these, 68% were obese (BMI ≥ 30) and/or had type 2 DM and 74% of liver biopsies revealed one or more features of NASH. Of patients with CC 68% were Hispanic while only 7% were African American, despite the fact that Hispanics comprised <26% and African Americans >40% of adult medicine patients. Prevalence of CC among Hispanic and African American patients was 3.1-fold higher and 3.9-fold lower, respectively, than among European American patients despite similar prevalence of DM among Hispanics and African Americans.CONCLUSIONS:These findings support the hypothesis that NASH associated with obesity and DM is responsible for the majority of cases of CC among Hispanics and European Americans. However, the current findings also indicate that this form of cirrhosis is unexpectedly rare among African Americans.

Prevalence studies of GB virus-C infection using reverse transcriptase-polymerase chain reaction

Among the three recently described GB viruses (GBV‐A, GBV‐B, and GBV‐C), only GBV‐C has been linked to cryptogenic hepatitis in man. Because of the limited utility of currently available research tests to determine antibody response to GBV‐C proteins, the prevalence of GBV‐C RNA in human sera was studied using reverse transcription‐polymerase chain reaction (RT‐PCR). The prevalence of GBV‐C is higher among volunteer blood donors with elevated serum alanine aminotransferase (ALT) levels (3.9%) than among volunteer blood donors with normal ALT levels (0.8%). Higher rates were also noted among commercial blood donors (12.9%) and intravenous drug users (16.0%). GBV‐C was frequently detected in residents of West Africa, where the prevalence was >10% in most age groups. Approximately 20% of patients diagnosed with either acute or chronic hepatitis C virus (HCV) were found to be positive for GBV‐C RNA. In addition, GBV‐C RNA sequences were detected in individuals diagnosed with non‐A‐E hepatitis, with clinical courses ranging from mild disease to fulminant hepatitis. Fourteen of sixteen subjects with or without clinically apparent hepatitis were positive for GBV‐C RNA more than 1 year after the initial positive result.

Purification and characterization of dipeptidyl peptidase I from human spleen

The lysosomal hydrolase, dipeptidyl peptidase I (DPPI), was purified from human spleen and its enzymatic activity characterized. The enzyme was purified to apparent homogeneity by a combination of differential pH solubility, heat-treatment, affinity chromatography on concanavalin A-agarose and p-hydroxymercuribenzoate-agarose, and gel filtration chromatography on Sephacryl S-300. This procedure resulted in a 1100-fold purification of DPPI protein with a yield of approximately 2% of the total DPPI activity. The enzyme was characterized as a glycoprotein with a pI of 5.4, a molecular mass of 200,000 Da as determined by gel filtration under nondenaturing conditions, and a subunit size of 24,000 Da. Amino acid sequence analysis of peptides isolated from cyanogen bromide and trypsin digests of the 24,000-Da subunit revealed extensive sequence similarity between human and rat DPPI. Purified DPPI exhibited both hydrolytic and transpeptidase (polymerase) activity. DPPI exhibited activity against a variety of dipeptide substrates including peptides with either non-polar or polar residues in the P1 position. In contrast to the reported substrate specificity of bovine and murine DPPI, the human enzyme exhibited a modest preference for peptides with nonpolar residues in the P1 position. DPPI content was found to be highest among cytotoxic lymphocytes and myeloid cells. The high level of DPPI expression in these cell populations correlates with their sensitivity to the toxic effects of leucyl-leucine methyl ester, a substrate for DPPI.

Tumor necrosis factor inhibitor ameliorates murine intestinal graft-versus-host disease☆☆☆

BACKGROUND & AIMS Transfer of T helper cells from DBA/2 mice to irradiated allogeneic B6D2F1 mice leads to development of colonic graft-versus-host disease with pathological features of inflammatory bowel disease. To examine the role of tumor necrosis factor (TNF) in graft-versus-host disease enteropathy, an adenoviral vector encoding a TNF inhibitor protein was administered. METHODS Irradiated B6D2F1 mice were infused with DBA/2 bone marrow and spleen cells. Mice then received either a control beta-galactosidase-encoding adenovirus or an adenovirus encoding a TNF inhibitor, composed of the extracellular domain of the human 55-kilodalton TNF receptor linked to the murine immunoglobulin G1 heavy chain. Mucosal permeability to sucralose and colonic histology were assessed 14 and 25 days after transplantation. RESULTS Less diarrhea was observed in DBA/2 --> B6D2F1 mice expressing the TNF inhibitor, and colonic sections from these mice had significantly less inflammation and epithelial cell abnormalities. In TNF inhibitor recipients, mucosal permeability to sucralose was similar to that in nonirradiated control mice and significantly less than in recipients of the control adenovirus. CONCLUSIONS TNF inhibition decreases the severity of enteropathy in the DBA/2 --> B6D2F1 murine model of colonic graft-versus-host disease.

Seroprevalence of GB virus C and persistence of RNA and antibody

Exposure to GB virus C (GBV‐C) was determined in several U.S. populations by both reverse‐transcription‐polymerase chain reaction (RT‐PCR) and by an enzyme linked immunosorbent assay (ELISA) for antibodies to mammalian cell‐expressed GBV‐C envelope protein, E2 (GBV‐C E2). Most individuals exposed to GBV‐C were either RNA positive/ELISA negative or ELISA positive/RNA negative. Exposure, therefore, was measured as the sum of GBV‐C RNA positive and GBV‐C E2 antibody positive specimens, and was higher in commercial plasmapheresis donors (40.5%) than in volunteer blood donors (5.5%). In intravenous drug users (IVDUs), GBV‐C exposure was 89.2%. Serial bleed specimens tested for GBV‐C RNA indicate that some patients remain viremic for at least 3 years and fail to produce detectable antibodies to GBV‐C E2. In other exposed individuals who tested negative for GBV‐C RNA, antibodies to E2 appear to be similarly long‐lived (greater than 3 years) with a fairly constant titer (ranging in reciprocal endpoint dilution from 336 to 21,504). Since the detection of GBV‐C RNA and GBV‐C E2 antibody are mutually exclusive in most exposed individuals, studies pertaining to incidence and prevalence of GBV‐C infection require both antibody and nucleic acid detection. J. Med. Virol. 53:167–173, 1997.

Virally Infected Hepatocytes Are Resistant to Perforin-Dependent CTL Effector Mechanisms

Cell-mediated cytotoxicity plays an important role in the clearance of noncytopathic viruses from infected tissues. Perforin-dependent cytotoxic mechanisms have been noted to play an important role in the clearance of infections from multiple extrahepatic organs. In contrast, mice with defects in the Fas/Fas ligand (FasL)-mediated cytotoxicity pathway exhibit delayed clearance of adenovirus from the liver without apparent delay in the clearance of viral infections from extrahepatic organs. The present studies examined the role of cytotoxic effector mechanisms in intrahepatic immune responses to a replication-defective, recombinant β-galactosidase-encoding adenovirus (AdCMV-lacZ). Delayed clearance of AdCMV-lacZ from the livers of FasL-defective B6.gld mice, but not perforin-deficient B6.pfp−/− mice, was noted despite no significant differences in initial hepatic CD8+ T cell IFN-γ or TNF responses or in activation of intrahepatic cytotoxic lymphocytes cells capable of killing AdCMV-lacZ-infected fibroblast targets. In contrast, AdCMV-lacZ-infected hepatocyte targets were far more sensitive to killing by intrahepatic cytotoxic lymphocytes from B6.pfp−/− than from B6.gld mice, and residual levels of virus-specific killing of hepatocyte targets by FasL-defective B6.gld CTL were blocked by TNF inhibition. These results suggest that inherent resistance of hepatocytes to cytotoxicity mediated by perforin-dependent mechanisms leaves Fas/FasL-dependent, cell-mediated cytotoxicity as the major pathway for CTL-mediated killing of virally infected hepatocytes and accounts for the more prominent role of perforin-independent anti-viral mechanisms in immune responses in the liver.

TNF-TNFR2 Interactions Are Critical for the Development of Intestinal Graft-Versus-Host Disease in MHC Class II-Disparate (C57BL/6J→C57BL/6J × bm12)F1 Mice

TNF-TNFR2 interactions promote MHC class II-stimulated alloresponses while TNF-TNFR1 interactions promote MHC class I-stimulated alloresponses. The present studies were designed to evaluate whether TNF-TNFR2 interactions were involved in the in vivo generation of CD4+ T cell-mediated intestinal graft-versus-host disease (GVHD) in the (C57BL/6J (hereafter called B6)→B6 × B6.C-H-2bm12 (bm12))F1 GVHD model. Briefly, 5 × 106 splenic CD4+ T lymphocytes from B6.TNFR2−/− or control B6 mice were transferred with 1–2 × 106 T cell-depleted B6 bone marrow cells (BMC) to irradiated MHC class II-disparate (bm12 × B6)F1 mice. Weight loss, intestinal inflammation, and the surface expression of CD45RB (memory marker) on intestinal and splenic lymphocytes were assessed. IL-2 and IFN-α mRNA levels in intestinal lymphocytes were assessed by nuclease protection assays. A significant reduction in weight loss and intestinal inflammation was observed in recipients of the TNFR2−/−CD4+ SpC. Similarly, a significant decrease was noted in T cell numbers and in CD45RBlow (activated/memory) expression on intestinal but not CD4+ T cells in recipients of TNFR2−/−CD4+ spleen cells. IL-2 and IFN-α mRNA levels were reduced in the intestine in the recipients of TNFR2−/− splenic CD4+ T cells. These results indicate that TNF-TNFR2 interactions are important for the development of intestinal inflammation and activation/differentiation of Th1 cytokine responses by intestinal lymphocytes in MHC class II-disparate GVHD while playing an insignificant role in donor T cell activation in the spleen.

The role of cell surface recognition structures in the initiation of MHC-unrestricted ‘promiscuous’ killing by T cells

CD3+ T cells mediate relatively promiscuous patterns of major histocompatibility complex (MHC)-unrestricted target cell lysis following activation. Cell-cell contact between target and effector cells is essential in this form of cytotoxicity. Although the T-cell receptor (TCR)-CD3 molecular complex can transmit signals that initiate MHC unrestricted T-cell killing, recognition of targets by the TCR is not essential for this form of cytotoxicity. In this review by Dwain Thiele and Peter Lipsky, a model of the triggering of T cells to effect MHC-unrestricted cytotoxicity is proposed.

The role of TNF-TNFR2 interactions in generation of CTL responses and clearance of hepatic adenovirus infection

Deficiency or inhibition of tumor necrosis factor (TNF) significantly prolongs hepatic expression of recombinant adenoviral vectors. To explore mechanisms responsible for this observation, the present studies examined the effects of TNF versus TNF receptor 1 (TNFR1) or TNFR2 deficiency on the course of antiviral‐immune responses to a replication‐deficient, β‐galactosidase‐encoding recombinant adenovirus (AdCMV‐lacZ). Clearance of AdCMV‐lacZ was significantly delayed in TNF‐deficient mice. Less pronounced but significant delays in AdCMV‐lacZ clearance were observed in TNFR2‐deficient but not TNFR1‐deficient mice. Numbers of interferon‐γ expressing intrahepatic lymphocytes (IHL) were similar in AdCMV‐lacZ‐infected, TNF‐deficient, TNFR1‐deficient, TNFR2‐deficient, and control mice. However, IHL isolated from AdCMV‐lacZ‐infected, TNF‐deficient or AdCMV‐lacZ‐infected, TNFR2‐deficient mice exhibited decreased levels of FasL expression and adenovirus‐specific cytolytic T lymphocyte (CTL) activity. Similar defects in allo‐specific killing of Fas‐sensitive hepatocyte targets by TNF‐deficient or TNFR2‐deficient but not TNFR1‐deficient CTL were also noted. No defects in generation of allo‐specific cytotoxicity directed against perforin‐sensitive target cells were noted in TNF‐, TNFR1‐, or TNFR2‐deficient lymphocytes. These findings indicate that TNF/TNFR2 interactions facilitate generation of FasL‐dependent CTL effector pathways that play an important role in in vivo antiviral‐immune responses in the liver.