Using the mouse interleukin 3 (IL-3) receptor cDNA as a probe, we obtained a homologous cDNA (KH97) from a cDNA library of a human hemopoietic cell line, TF-1. The protein encoded by the KH97 cDNA has 56% amino acid sequence identity with the mouse IL-3 receptor and retains features common to the family of cytokine receptors. Fibroblasts transfected with the KH97 cDNA expressed a protein of 120 kDa but did not bind any human cytokines, including IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF). Interestingly, cotransfection of cDNAs for KH97 and the low-affinity human GM-CSF receptor in fibroblasts resulted in formation of a high-afflity receptor for GM-CSF. The dissociation rate of GM-CSF from the reconstituted high-affinity receptor was slower than that from the low-affinity site, whereas the association rate was unchanged. Cross-linking of '12I-labeled GM-CSF to fibroblasts cotransfected with both cDNAs revealed the same cross-linking patterns as in TF-1 cells-i.e., two major proteins of 80 and 120 kDa which correspond to the low-affinity GM-CSF receptor and the KH97 protein, respectively. These results indicate that the highaffinity GM-CSF receptor is composed of at least two components in a manner analogous to the IL-2 receptor. We therefore propose to designate the low-affinity GM-CSF receptor and the KH97 protein as the a and ( subunits of the GM-CSF receptor, respectively.

SummaryWe established six T cell clones specific for pyruvate dehydrogenase complex (PDC)-E2 peptides from four different patients with primary biliary cirrhosis using 33 different peptides of [17][18][19][20] amino acid residues corresponding to human PDC-E2 as stimulating antigens. The minimal T cell epitopes of these six T cell clones were all mapped to the same region of the PDC-E2 peptide 163-176 (GDLLAEIETDKATI), which corresponds to the inner lipoyl domain of PDC-E2. The HLA restriction molecules for this epitope were all identified as HLA DRB4 0101. The common essential amino acids of this epitope for these T cell clones were E, D, and K at positions 170, 172, and 173, respectively; other crucial amino acids for this epitope differed in each T cell clone. In addition, the alanine-substituted peptides at positions 170 and 173, but not 172, inhibited the proliferation of all T cell clones induced by the original peptide of human PDC~E2 163-176, indicating that amino acid D at position 172 is a critical MHC~binding site for all T cell clones tested. Interestingly, all T cell clones reacted to PDC-E2 peptide 36-49 (GDLIAEVETDKATV), which corresponds to the outer lipoyl domain of human PDC-E2. Furthermore, one T cell clone cross-reacted with exogenous antigens such as Escherichia coli PDC-E2 peptide 31-44/134-147/235-248 (EQSLITVEGDKASM), which has an EXDK sequence. This is a definite demonstration of the presence of molecular mimicry at the T cell clonal level in human autoimmune diseases. It is also considered possible to design peptide-specific immunotherapy based on the findings of T cell autoepitopes in primary biliary cirrhosis.

The high-affinity receptor for human granulocyte/macrophage colony-stimulating factor (hGM-CSF) is composed of two subunits, a and (3. The a subunit binds GM-CSF with low affinity, whereas the ( subunit does not bind GM-CSF by itself. The a and (3 subunits together form the high-affinity GM-CSF receptor. The (3 subunit has extensive sequence homology with the mouse interleukin 3 (IL-3) receptor (AIC2A) and its homologue (AIC2B) that does not bind IL-3 or other cytokines including GM-CSF. To examine the function of these receptor components, we expressed the a subunit of the hGM-CSF receptor with the human (3 subunit or the mouse AIC2A or AIC2B in a mouse IL-3-dependent pro-B-cell line, Ba/F3, and in a mouse IL-2-dependent T-cell line, CTLL2.Coexpression of the a and (3 subunits in Ba/F3 and CTLL2 cells resulted in high-affinity hGM-CSF binding and growth response to low concentrations of hGM-CSF. Whereas Ba/F3 cells expressing the a subunit alone proliferated in response to high concentrations of hGM-CSF, CTLL2 cells expressing the a subunit alone did not respond to hGM-CSF at all. Since Ba/F3 cells express endogenous AIC2A and AIC2B whereas CTLL2 expresses neither of them, we examined the possibility that either AIC2A or AIC2B is involved in the formation of a functional GM-CSF receptor. The expression of the human a subunit with AIC2B, but not with AIC2A, in CTLL2 cells conferred a growth response to hGM-CSF. These results indicate that the (3 subunit of the GM-CSF receptor is required for generation of growth signals and that AIC2B is likely the (3 subunit of the mouse GM-CSF receptor.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) plays a critical role in growth and differentiation of myeloid cells. We previously reconstituted high-affinity human GM-CSF receptor (hGM-CSFR) in a pro-B cell line, BA/F3, by cotransfecting a-and 1U-chain cDNA clones and showed that the reconstituted receptor could transduce growth-promoting signals. The high-affinity hGM-CSFR was also reconstituted in mouse NIH 3T3 cells, but its ability to transduce signals in fibroblasts remained undetermined. In the present study, we further characterized signal transduction by the reconstituted hGM-CSFR in both NIH 3T3 cells and BA/F3 cells. We found that the reconstituted hGM-CSFR transduces signals in NIH 3T3 fibroblasts and BA/F3 cells in response to hGM-CSF to activate transcription of the c-fos, c-jun, and c-myc proto-oncogenes. hGM-CSF also induces protein tyrosine phosphorylation and DNA synthesis in both cell types. These results indicated that hGM-CSFR is functional in fibroblasts, that signal transduction via hGM-CSFR in fibroblasts involves tyrosine kinase(s), and that association of hGM-CSFR with a factor(s) specific to hematopoietic cell lineage is not essential to transduce growth-promoting signals.

Abstract. The aims of this retrospective survey were to determine the epidemiologic distribution of hepatitis B virus (HBV) genotypes and analyze the genotype-related clinical differences among Japanese patients with chronic HBV infection. The 158 surveyed patients with chronic HBV infection lived in Fukuoka and Okinawa were serially tested for serum alanine aminotransferase (ALT) and hepatitis B e antigen (HBeAg). Follow-up was for a period of 10.8 ± 6.4 years (mean ± SD). The HBV genotypes were determined in sera by an enzyme-linked immunosorbent assay and detection of HBV DNA in serum was done by the transcription-mediated amplification−hybridization protection assay. Genotypes B and C were found in 58 (36.7%) and 100 (63.3%) of the patients, respectively. Genotype B was predominant in Okinawa (B ‫ס‬ 86.9%, C ‫ס‬ 13.1%), whereas genotype C was predominant in Fukuoka (B ‫ס‬ 5.2%, C ‫ס‬ 94.8%). The HBeAg positivity and ALT abnormality rates at the start of the observation period were significantly higher in patients with genotype C (66.0% and 84.0%) than in patients with genotype B (34.5% and 22.4%) (P < 0.05, respectively). The annual rate of spontaneous HBeAg disappearance in patients with genotype B was much higher than in patients with genotype C (8.38% versus 2.34%, respectively). Patients with genotype C who were continuously HBeAg negative from entry had a significantly higher ALT abnormality (58.8%) than those with genotype B (19.2%) (P < 0.05). Interestingly, patients with genotype C who became HBeAg negative after treatment with interferon had a high ALT abnormality (58.8%). All patients with an ALT abnormality were positive for HBV DNA in their serum. These findings indicate that patients with HBV genotype C have more severe liver deterioration because of the delay of HBeAg disappearance and continued HBV replication after HBeAg disappearance.

The hepatitis C virus (HCV) virion is associated with lipoproteins and immunoglobulins in the sera of patients with chronic hepatitis C; however, an accurate binding rate of HCV to lipoproteins or immunoglobulins has not yet been elucidated. Therefore, the accurate binding rate of HCV to low-density lipoproteins (LDL), high-density lipoproteins (HDL), and immunoglobulins was measured quantitatively by a real-time PCR assay. The immunoglobulin binding rate of HCV was found to be greater than 97.5% in most patients, as compared with an LDL binding rate of greater than 80% in most patients. In contrast, the HDL binding rate was greater than 98% in the genotype 2a/2b patients, while it varied in the genotype 1b patients. The genotype 2a/2b HCV not only had a higher LDL binding rate but also had a strikingly higher HDL binding rate than that of the genotype 1b HCV. These lipoprotein binding rates correlated neither to any patient's variables, including the serum apolipoprotein levels, nor to the viral load or the hypervariable region 1 (HVR 1) amino acid sequences. Most of the HCV virions in the sera of such patients have been shown to be associated simultaneously with immunoglobulins and LDL and/or HDL, but not exclusively.

Primary biliary cirrhosis (PBC) is a chronic cholestatic T-cell-mediated autoimmune mechanisms are considered liver disease characterized by the feature of chronic nonsupto be involved in the pathogenesis of primary biliary cirrhosis purative destructive cholangitis and the presence of antimito-(PBC). In the previous study, we identified the immunodomichondrial antibodies (AMA). 1 Lymphocytes, mainly T cells, nant T-cell epitope on the E2 component of pyruvate dehydropredominantly infiltrate around the interlobular bile ducts genase complex (PDC-E2) in patients with PBC who have in the portal tracts. 2 HLA class II antigens are aberrantly HLA-DRB4*0101. In this report, we revealed that the freexpressed on the surface of the interlobular bile duct epithequency of the T cells reactive to the human PDC-E2 163-176 lial cells 3 and the expression of adhesion molecules are also peptide is significantly increased in the peripheral blood of increased. 4 Furthermore, pyruvate dehydrogenase complex patients with PBC as compared with healthy subjects. We (PDC) or PDC-like molecules are present on the luminar also confirmed that these T cells were all restricted with HLAsurface of the interlobular bile duct epithelial cells. 5,6 These DRB4*01 (DR53) by using HLA-DR-transfected L cells.findings indicate that T-cell-mediated mechanisms are inThese results together with the evidence that the immunovolved in the development of PBC. dominant B-cell epitope overlaps with the human T-cell epi-In fact, T cells in the peripheral blood and/or in the liver tope of the PDC-E2 antigen indicate that the T cells reactive tissue of the patients with PBC have been shown to proliferate to this epitope are closely associated with the pathogenesis in response to PDC or E2 component of PDC (PDC-E2), of PBC at least in patients who have HLA-DR53. Therefore, which is a major self-antigen recognized by AMA. 5,[7][8][9][10][11][12][13][14] In the we analyzed the T-cell receptor (TCR) Vb sequence of the previous study, we revealed that the human PDC-E2 163-five different T-cell clones and the three T-cell clones derived 176 (GDLLAEIETDKATI; one-letter amino acid) is the imfrom three patients with PBC and healthy subjects, respecmunodominant T-cell epitope restricted with HLA-DR53 tively, which are reactive to the human PDC-E2 163-176 pep-(DRB4*01). 15 Moreover, this T-cell epitope is overlapped tide in the context of HLA-DR53. The Vb-and the Jb-gene with the immunodominant B-cell epitope, 9 indicating that usages were diverse among the T-cell clones (Vb11-Jb1.4, the human PDC-E2 163-176 is a critical epitope that induces Vb8-Jb1.2, Vb12-Jb2.1, Vb10-Jb1.5, and Vb20-Jb2.1) in paautoimmune response in PBC. tients with PBC. By contrast, in the third complementarityThe analysis of the T-cell receptor (TCR) of autoimmune determining region (CDR3), G was frequently found and GXG T cells is important to clarify the molecular mechanism of or GXS motif was identified in all T-cell clones. Moreover, TCR recognition of antigenic peptide-major histocompatibil-R...

A 21-year-old woman was diagnosed as having Graves' disease in April, 1995. Thiamazole was administered; about a month later the patient had a skin rash and propylthiouracil (PTU) was given instead. Two months after commencing PTU, she rapidly developed jaundice, accompanied by severe liver damage. The drug-induced lymphocyte stimulating test was positive for PTU and she was diagnosed as having severe hepatitis induced by PTU. After pulse therapy with 500 mg of methylprednisolone was given for 3 days, liver function test results were gradually improved, and became normalized 1 1/2 months after admission. The pathology findings of the liver biopsy sample taken before administration of corticosteroid showed necrosis of hepatocytes predominantly around the central veins (i.e., zone 3 necrosis), and moderate to severe infiltration of lymphocytes and neutrophils in portal areas and lobules. Severe hepatic damage due to PTU is rare; 25 cases have been reported so far in the English-language literature. When we use PTU for patients with hyperthyroidism, we should keep in mind that severe liver damage induced by PTU can be fatal, and we should therefore diagnose it earlier by liver biopsy and lymphocyte stimulating test.