Role of B70/B7-2 in CD4+ T-cell immune responses induced by dendritic cells.

Dendritic cells (DC) are potent antigen-presenting cells (APC). However, the molecular basis underlying this activity remains incompletely understood. To address this question, we generated murine monoclonal antibodies (mAb) against human peripheral blood-derived DC. One such antibody, designated IT209, stained differentiated DC and adherent monocytes, but failed to stain freshly isolated peripheral blood mononuclear cells (PBMC). The antigen recognized by IT209 was identified as B70 (B7-2; also recently identified as CD86). Using this mAb we studied the role of B70 in CD4+ T-cell activation by DC in vitro. IT209 partly inhibited the proliferative response of CD4+ T cells to allogeneic DC and to recall antigens, such as tetanus toxoid (TT) and purified protein derivative (PPD) of tuberculin, presented by autologous DC. More importantly, the mAb had a potent inhibitory effect on the primary response of CD4+ T cells to autologous DC pulsed with human immunodeficiency virus (HIV) gp160 or keyhole limpet haemocyanin (KLH). Adherent monocytes, despite their expression of B70, failed to induce T-cell responses to these antigens. IT209-mediated inhibition of CD4+ T-cell responses was equivalent to that produced by anti-CD25 mAb, whereas an anti-CD80 mAb was only marginally inhibitory and did not augment the effect of IT209. These findings indicate that the B70 antigen plays an important role in DC-dependent CD4+ T-cell activation, particularly in the induction of primary CD4+ T-cell responses to soluble antigens. However, since activated monocytes, despite their expression of B70, failed to prime naive T cells to these antigens, our results suggest that additional molecules contribute to the functions of DC in CD4+ T-cell activation.     

Effects of ruthenium red on membrane ionic currents in urinary bladder smooth muscle cells of the guinea-pig

 Three major ionic currents, Ca²⁺-dependent K⁺ current (I _K-Ca), delayed rectifier type K⁺ current (I _kd) and Ca²⁺ current (I _Ca), were activated by depolarization under whole-cell clamp in single smooth muscle cells isolated from guinea-pig urinary bladder. Externally applied ruthenium red (RuR) reduced the amplitude of I _K-Ca and I _Ca at 0 mV (IC₅₀ values were 4.2 and 5.6 μM, respectively), but did not affect I _Kd. Spontaneous transient outward currents (STOCs) and caffeine-induced outward currents (I _caf) at –30 mV were reduced by external 10 μM RuR. When 10 μM RuR was added to the pipette solution, I _K-Ca during depolarization, STOCs and I _caf significantly decreased with time. RuR did not change the unitary current amplitude of the large-conductance Ca²⁺-dependent K⁺ (BK) channels, but reduced the open probability of the channel under excised patch-clamp recording mode. RuR reduced the channel activity more effectively from the cytosolic face than from the other. This inhibition decreased when the cytosolic Ca²⁺ concentration was increased. These results indicate that RuR blocks BK and Ca²⁺ channels in urinary bladder smooth muscle cells. The decrease in I _K-Ca, STOCs and I _caf by RuR is attributable to the direct inhibition of BK channel activity, probably in addition to the inhibition of Ca²⁺ release from storage sites. The direct inhibition of BK channel activity by RuR may be related to the interaction of RuR with the Ca²⁺-binding sites of the channel protein. Received: 15 October 1997 / Received after revision and accepted: 25 November 1997     

Lamivudine and IFN-beta sequential therapy in HBe antigen-positive patients with chronic hepatitis B virus genotype C infection.

Sequential treatment with lamivudine and interferon (IFN) has induced sustained biochemical and virologic responses in the majority of patients with chronic hepatitis B in France. However, the efficacy of sequential treatment in patients with chronic hepatitis B virus (HBV) genotype C infection has not been evaluated. Twenty-four HBe antigen-positive patients were treated with 100 mg lamivudine alone for 16-32 weeks, then with both 6 MU IFN-beta and lamivudine for 4 weeks, and lastly with IFN-beta alone for 20 weeks. Sustained response was achieved in 7 (29%) patients 24 weeks after the end of therapy. No lamivudine-resistant variants emerged in any patient. Hepatitis flare occurred in 3 patients after the withdrawal of lamivudine, but none had decompensation. The patients with sustained response were significantly younger at baseline (p = 0.033) and had a significantly lower HBV DNA level at the start of IFN (p = 0.020) than those without sustained response. In conclusion, the rate of response to sequential therapy with lamivudine and IFN in HBe antigen-positive patients with HBV genotype C infection was lower than the rate reported previously. Patients who were young or who had a favorable virologic response to lamivudine were more likely to have a sustained response.     

Hyperplastic epithelial foci in honeycomb lesions in idiopathic pulmonary fibrosis

Seventy-two cases of idiopathic pulmonary fibrosis (IPF) were examined from 2856 consecutive autopsy cases at the Japanese Red Cross Medical Center in Tokyo from 1973-1996. Primary lung cancer had arisen in 31 of 72 cases of IPF (43%), significantly higher than the incidence in cases without IPF (8.1%) and in the cases with non-IPF chronic lung diseases (11.9%). Hyperplastic epithelial foci in the honeycomb lesions of IPF cases were significantly more prominent in the lower than in the upper lobe, in cases with or without lung cancer, and they were more prominent in the lower lobe of IPF with than in those without cancer. The length of hyperplastic epithelial foci in the lower lobe of IPF cases was longer than that in interstitial pneumonia-associated with collagen vascular diseases. There was a higher PCNA labeling index of hyperplastic epithelial foci in IPF cases than in cases of interstitial pneumonia-associated with collagen vascular diseases. The PCNA labeling index was almost the same between smokers and nonsmokers with IPF. Overexpression of p53 was observed in hyperplastic epithelial foci in honeycomb lesion of IPF. DNA ploidy analysis of hyperplastic epithelial foci in the paraffin sections of 12 IPF cases revealed aneuploidy patterns in eight cases. These results strongly suggest that accelerated cell proliferation occurs in the honeycomb lesion of IPF, and that regenerative epithelia becomes susceptible to carcinogenic agents in addition to the smoking effect.     

CD69-null mice protected from arthritis induced with anti-type II collagen antibodies

CD69, known as an early activation marker antigen on T and B cells, is also expressed on platelets and activated neutrophils, suggesting certain roles in inflammatory diseases. In order to address the role of CD69 in the pathogenesis of arthritis, we established CD69-null mice. CD69-null mice displayed a markedly attenuated arthritic inflammatory response when injected with anti-type II collagen antibodies. Cell transfer experiments with neutrophils, but not T cells or spleen cells, from wild-type mice into CD69-null mice restored the induction of arthritis. These results indicate a critical role for CD69 in neutrophil function in arthritis induction during the effector phase. Thus, CD69 would be a possible therapeutic target for arthritis in human patients.     

Inferring alternative splicing patterns in mouse from a full-length cDNA library and microarray data

Although many studies on alternative splicing of specific genes have been reported in the literature, the general mechanism that regulates alternative splicing has not been clearly understood. In this study, we systematically aligned each pair of the 21,076 cDNA sequences of Mus musculus, searched for putative alternative splicing patterns, and constructed a list of potential alternative splicing sites. Two cDNAs are suspected to be alternatively spliced and originating from a common gene if they share most of their region with a high degree of sequence homology, but parts of the sequences are very distinctive or deleted in either cDNA. The list contains the following information: (1) tissue, (2) developmental stage, (3) sequences around splice sites, (4) the length of each gapped region, and (5) other comments. The list is available at http://www.bioinfo.sfc.keio.ac.jp/intron. Our results have predicted a number of unreported alternatively spliced genes, some of which are expressed only in a specific tissue or at a specific developmental stage.     

Identification and characterization of the human FMN1 gene in silico

Mouse Formin (Fmn1) protein plays a key role in limb morphogenesis. Fmn1 is one of the actin regulators with scaffold function, interacting with Profilin, SRC, EMS1, FNBP1, FNBP2, FNBP3, FNBP4, WBP4 and alpha-catenin. Fmn1, Fmn2, FHOD1, FHOD3, GRID2IP and FHDC1 are non-FDD-type Formin homology proteins, while FMNL1, FMNL2, FMNL3, DIAPH1, DIAPH2, DIAPH3, DAAM1 and DAAM2 are FDD-type Formin homology proteins. Here, we identified the human FMN1 gene by using bioinformatics. The complete coding sequence of human FMN1 cDNA was determined by assembling AC055874.8 genome sequence (nucleotide position 178207-180073), AI040235 EST (complementary sequence for nucleotide position 331-156) and FLJ45135 cDNA (nucleotide position 319-3310). FMN1 isoform 1 (exons 1-18) and FMN isoform 2 (exons 1b and 3-18) were transcribed due to alternative splicing of the alternative promoter type. The FMN1 gene at human chromosome 15q13.3 was located between CKTSF1B1 (Gremlin) and RYR3 genes. The Xenopus fmn1 gene was identified within the Xenopus genome sequence CH216-24N20 (AC147835.1). The FMH1 domain (codon 1-120 of FMN1) and FMH2 domain (codon 683-835 of FMN1) were identified as novel regions conserved among human FMN1, mouse Fmn1, and Xenopus fmn1. The FMH2 domain was almost identical to the alpha-catenin binding domain of mouse Fmn1. Human FMN1 (1419 aa), showing 77.1% total amino-acid identity with mouse Fmn1, was found consisting of FMH1, FMH2, FH1 and FH2 domains. This is the first report on the identification and characterization of the human FMN1 gene as well as the FMH1 and FMH2 domains.     

Abnormal accumulation in lipopolysaccharide in biliary epithelial cells of rats with self-filling blind loop

Primary sclerosing cholangitis (PSC) is known to be frequently associated with inflammatory bowel diseases. In a rat with self-filling blind loop (SFBL), a proposed animal model for PSC, hepatobiliary inflammation has previously been demonstrated. In this study, we assessed the involvement of lipopolysaccharide (LPS), a bacterial endotoxin, in the pathogenesis of hepatobiliary inflammation of the SFBL model. The hepatic localization of LPS was examined by immunohistochemistry using an anti-lipid A antibody. The portal blood concentration of LPS was measured by an endotoxin-specific chromogenic Limulus test (Endospecy test). LPS was localized in the biliary epithelial cells (BECs) of rats with SFBL, and the portal blood concentration of LPS was significantly higher than that of sham-operated rats. Development of hepatobiliary inflammation, peribiliary fibrosis, and injury to the intestinal mucosa were histologically confirmed. Constriction in the biliary trees was radiologically demonstrated. These findings suggested that abnormal accumulation of LPS, which may be derived from portal blood, in BECs was involved in the pathogenesis of hepatobiliary inflammation with intestinal injury.     

Diversity of antisperm antibodies bound to sperm surface in male immunological infertility

PROBLEM: The presence of antisperm antibodies (ASA) in males can reduce fecundity, however, relationship between the two is disputed. This study was performed to investigate if there is diversity of ASA bound to sperm surface using immunobead test (IBT) combined with complement dependent sperm immobilization test (SIT). METHODS: The ASA bound to sperm surface were detected using the direct IBT (D-IBT) in 275 semen samples. In some cases with ASA detected by D-IBT, sperm immobilizing antibodies bound to sperm surface were also evaluated using direct SIT (D-SIT). RESULTS: The incidence of the immunoglobulin G (IgG), IgA, and IgM classes of ASA detected by D-IBT were 2.5, 1.8, and 0.4%, respectively. Totally, nine (3.3%) infertile men had ASA on the sperm surface. D-SIT was tested positive in four (66.7%) of six cases with ASA assessed by D-IBT. CONCLUSIONS: Some of the sperm-bound antibodies are associated with complement dependent sperm immobilizing antibodies, indicating that there exists a heterogeneity of sperm-bound antibodies. This result might be one of the reasons for the controversy about the relationship between ASA and immunological infertility in men.