Mitochondria and autoimmunity in primary biliary cirrhosis

Primary biliary cirrhosis is an enigmatic autoimmune liver disease that predominantly affects women and is characterized by antimitochondrial antibodies and specific destruction of small bile ducts. Interestingly, patients with this disease not only have high titer antibodies to mitochondria, but also highly directed, liver-specific CD4 and CD8 cells directed at the same mitochondrial autoantigens. These mitochondrial autoantigens are all members of the 2-oxo dehydrogenase complex family and include the E2 component of pyruvate dehydrogenase as the major autoantigen. Moreover, the epitopes recognized by CD4, CD8 T cells and autoantibody, are all directed within the same region, namely the lipoyl domain of pyruvate dehydrogenase complex-E2. All cells in the body have mitochondria but there appear to be specific destruction of biliary cells. We believe that this specific destruction is secondary to a highly directed mucosal response that focuses on biliary cells because of the involvement of a polymeric immunoglobulin receptor, the presence of immunoglobulin A in mucosal secretions, and the unique apoptotic properties of biliary epithelium.     

CLIP-170 family members: a motor-driven ride to microtubule plus ends

CLIP-170 family proteins regulate microtubule plus end dynamics. Two reports published in this issue of Developmental Cell show that Bik1 and tip1p, the CLIP-170-like proteins of budding and fission yeast, are carried to microtubule plus ends by kinesin motor proteins. These findings indicate a complex interplay between microtubule-associated proteins and suggest a novel mechanism by which kinesin proteins stabilize microtubules.     

Effect of turpentine oil on C-reactive protein (CRP) production in rainbow trout (Oncorhynchus mykiss)

The effect of turpentine oil on C-reactive protein (CRP) production was studied in rainbow trout (Oncorhynchus mykiss). Serum CRP concentration was estimated by sandwich enzyme-linked immunosorbent assay using anti-rainbow trout CRP monoclonal antibody (mAb) AC4 and polyclonal antibody. Intracellular CRP was demonstrated by flow cytometry using anti-trout CRP mAb. Hepatocytes, head kidney macrophages, spleen lymphocytes and peripheral blood lymphocytes showed reaction against AC4, but RTG-2 fibroblastic line cells, derived from rainbow trout gonad did not. This is the first report on the detection of intracellular CRP in fish. CRP levels decreased significantly 1 day after intramuscular injection of turpentine oil and remained low for 14 days. Significant decreases in the expression of CRP in hepatocytes, head kidney macrophages and spleen lymphocytes after injection of turpentine oil were found. The reduction of serum CRP concentration after turpentine oil injection may be attributed to decreases in intracellular CRP synthesis.     

A series of immune responses leading to the induction ofT cell IL-12/IL-18 responsiveness in patientswith relatively large tumor burdens

The induction of interleukin-12 (IL-12) responsiveness in T cells depends on T cell receptor (TCR) triggering, and is regarded as a parameter of recently TCR-sensitized T cells. Here, we investigated whether IL-12 responsiveness could be detected in freshly prepared T cells from tumor-bearing patients, and if so whether such patients exhibited additional immunological parameters related to IL-12 responsiveness. CD4(+) and CD8(+) T cell populations from an appreciable proportion of tumor-bearing patients exhibited high levels of IL-12 responsiveness as evaluated by IL-12-stimulated interferon-gamma (IFN-gamma) production. T cell populations with high IL-12 responsiveness were observed in the group of patients with moderate to large tumor mass or tumor metastases rather than in patients with small tumors. The frequency of such a T cell population was also lower in post-surgery tumor-free patients, showing the correlation between IL-12 responsiveness and the presence of a certain extent of tumor burden. More importantly, a higher incidence of IL-12 responsiveness was observed in tumor-bearing patients exhibiting detectable plasma IL-12 levels, and correlated with IL-18 responsiveness. T cell IL-12 and IL-18 responsiveness is induced by TCR triggering and subsequent IL-12 stimulation respectively. Furthermore, TCR-triggered T cells stimulate antigen-presenting cells (APC) to produce IL-12. Therefore, the present observations suggest that an immune response loop from TCR sensitization to the induction of IL-12/IL-18 responsiveness via IL-12 production operates in tumor-bearing patients, particularly in those with relatively large tumor burdens.     

A novel adenovirus expressing human 4-1BB ligand enhances antitumor immunity

4-1BB ligand (4-1BBL), a member of the tumor necrosis factor (TNF) superfamily, interacts with 4-1BB (CDw137) expressed on activated T cells and delivers a costimulatory signal for T cell activation and growth. Various studies have demonstrated a role for murine 4-1BB in immune function, but relatively few investigations of human 4-1BB have been conducted. Here we report on the construction of a recombinant E1/E3-deleted adenovirus encoding human 4-1BBL (Ad4-1BBL) and its stimulation of antitumor immunity. Ad4-1BBL was able to efficiently infect several human adenocarcinoma cell lines and induce 4-1BBL expression on the cell surface within 24 h, this enhancing the antitumor activity not only of lymphokine-activated killer cells with a T cell phenotype (T-LAK) but also naive peripheral blood mononuclear cells (PBMC). This antitumor activity with T-LAK cells was further enhanced by addition of bispecific antibody (BsAb; anti-MUC1xanti-CD3). Cocultivation of Ad4-1BBL-infected tumor cells with either T-LAK cells or PBMC resulted in significant elevation of interferon-gamma (IFN-gamma), interleukin-2 (IL-2), and granulocyte-macrophage colony-stimulating factor (GM-CSF) production. Furthermore, remarkable tumor growth inhibition was observed in cholangiocarcinoma-grafted severe combined immunodeficient (SCID) mice to which Ad4-1BBL and T-LAK cells were administered when tumor size exceeded 5 mm in diameter. These results provide strong evidence in support of the efficacy of adenovirally delivered 4-1BBL for genetic immunotherapy of cancer.     

Recombinant bovine herpesvirus-1 expressing p23 protein of Cryptosporidium parvum induces neutralizing antibodies in rabbits

In order to develop a vaccine against cryptosporidiosis in cattle, we constructed a recombinant bovine herpesvirus-1 (BHV-1) expressing an immunodominant surface protein, p23, of Cryptosporidium parvum sporozoites. In the recombinant virus, the p23 gene under the control of a CAG promoter and a gene coding for an enhanced green fluorescent protein were integrated into the gG gene of BHV-1. Despite a low frequency of homologous recombination, cloning of the recombinants was easy because of the specific fluorescence of the plaques formed by recombinants. These plaques were among the plaques of the nonfluorescent parental virus. All clones selected for fluorescence also contained the p23 gene. In MDBK cells infected with the recombinant BHV-1, the antibody against the p23 protein recognized the p23 protein as an approximately 23-kDa specific band in Western blotting analysis. Rabbits immunized with the recombinant produced IgG against the p23 protein. It was also demonstrated that the sera of immunized rabbits reduced infection of C. parvum sporozoites in HCT-8 cells. The serum of an immunized rabbit reduced infection compared with the normal rabbit serum control. These results indicate that the recombinant BHV-1 induces neutralizing antibodies in rabbits.     

Macrophage-tropic simian/human immunodeficiency virus chimeras use CXCR4, not CCR5, for infections of rhesus macaque peripheral blood mononuclear cells and alveolar macrophages

After the nearly complete and irreversible depletion of CD4(+) T lymphocytes induced by highly pathogenic simian/human immunodeficiency virus chimeric viruses (SHIVs) during infections of rhesus monkeys, tissue macrophages are able to sustain high levels (>10(6) viral RNA copies/ml) of plasma viremia for several months. We recently reported that the virus present in the plasma during the late macrophage phase of infection had acquired changes that specifically targeted the V2 region of gp120 (H. Imamichi et al., Proc. Natl. Acad. Sci. USA 99:13813-13818, 2002); some of these SHIV variants were macrophage-tropic (M-tropic). Those findings have been extended by examining the tropic properties, coreceptor usage, and gp120 structure of five independent SHIVs recovered directly from lymph nodes of late-stage animals. All of these tissue-derived SHIV isolates were able to infect alveolar macrophages. These M-tropic SHIVs used CXCR4, not CCR5, for infections of rhesus monkey PBMC and primary alveolar macrophages. Because the starting highly pathogenic T-tropic SHIV inoculum also utilized CXCR4, these results indicate that the acquisition of M-tropism in the SHIV-macaque system is not accompanied by a change in coreceptor usage. Compared to the initial T-tropic SHIV inoculum, tissue-derived M-tropic SHIVs from individual infected animals carry gp120s containing similar changes (specific amino acid deletions, substitutions, and loss of N-linked glycosylation sites), primarily within the V1 and/or V2 regions of gp120.     

Characterization of hydrogen peroxide removal reaction by hemoglobin in the presence of reduced pyridine nucleotides

Hydrogen peroxide removal rates by hemoglobin were enhanced in the presence of reduced pyridine nucleotides. The species which had the activity to oxidize pyridine nucleotides was purified from human blood and identified as hemoglobin A. Hydrogen peroxide removal rates by hemoglobin A without reduced pyridine nucleotides at 0.2 mM hydrogen peroxide were 0.87+/-0.11 micromol/s/g hemoglobin, and the removal rates using 0.2 mM NADH and NADPH were 2.02+/-0.20 and 1.96+/-0.31 micromol/s/g hemoglobin, respectively. We deduced that the removal reaction by hemoglobin included formations of methemoglobin and the ferryl radical and reduction of the latter with pyridine nucleotides. The hydrogen peroxide removal ability by hemoglobin was less than that by catalase but was larger than that by glutathione peroxidase-glutathione reductase system at 0.2 mM hydrogen peroxide. Under acatalasemic conditions, it was suggested that NAD(P)H were important factors to prevent the oxidative degradation of hemoglobin.     

A novel DNA polymerase inhibitor and a potent apoptosis inducer: 2-mono-O-acyl-3-O-(alpha-D-sulfoquinovosyl)-glyceride with stearic acid

Sulfo-glycolipids in the class of sulfoquinovosyl diacylglycerol (SQDG) including the stereoisomers are potent inhibitors of DNA polymerase alpha and beta. However, since the alpha-configuration of SQDG with two stearic acids (alpha-SQDG-C(18)) can hardly penetrate cells, it has no cytotoxic effect. We tried and succeeded in making a permeable form, sulfoquinovosyl monoacylglycerol with a stearic acid (alpha-SQMG-C(18)) from alpha-SQDG-C(18) by hydrolysis with a pancreatic lipase. alpha-SQMG-C(18) inhibited DNA polymerase activity and was found to be a potent inhibitor of the growth of NUGC-3 cancer cells. alpha-SQMG-C(18) arrested the cell cycle at the G1 phase, and subsequently induced severe apoptosis. The arrest was correlated with an increased expression of p53 and cyclin E, indicating that alpha-SQMG-C(18) induced cell death through a p53-dependent apoptotic pathway.     

Upregulation of cAMP is a new functional signal pathway of Klotho in endothelial cells

We measured angiotensin I-converting enzyme (ACE) activity in a human endothelial cell to characterize the intracellular signal pathways of Klotho. COS-1 cells transfected with naked mouse membrane-form klotho plasmid DNA (pCAGGS-klotho) translated proper Klotho protein. This translated Klotho protein was secreted into the culture medium. Furthermore, ACE activity in human umbilical vein endothelial cells (HUVEC) was upregulated when HUVEC were co-cultured with COS-1 cells that were pre-transfected with pCAGGS-klotho. The conditioned medium from COS-1 cells pre-transfected with pCAGGS-klotho also dose-dependently upregulated ACE in HUVEC. In addition, the conditioned medium induced time- and dose-dependent enhancement of cAMP production in HUVEC. Rp-cAMP, an inhibitor of cAMP-dependent protein kinase A (PKA), inhibited the upregulation of ACE by Klotho protein. Our results suggest that mouse membrane-form Klotho protein acts as a humoral factor to increase ACE activity in HUVEC via a cAMP-PKA-dependent pathway. These findings may provide a new insight into the mechanism of Klotho protein.