Genetic association of Fc receptor-like 3 polymorphisms with susceptibility to primary biliary cirrhosis: ethnic comparative study in Japanese and Italian patients.

A functional variant in the Fc receptor-like 3 (FCRL3) gene is associated with the susceptibility to several autoimmune diseases. In this study, we examined whether the FCRL3 is associated with susceptibility to primary biliary cirrhosis (PBC) by comparing the two different ethnic groups, Japanese and Italians. We enrolled 232 patients with PBC and 230 controls in Japanese, and 216 PBC and 180 controls in Italians. Minor allele frequency of fcrl3_3 (-169 T>C) in the patients with PBC and controls was 0.20 and 0.09 in Japanese and 0.24 and 0.21 in Italians, respectively. We found a significant association of fcrl3_3 with PBC only in Japanese (P = 9.64 × 10(-7) ). These findings support the presence of common FCRL3-related pathological pathways in several autoimmune diseases, especially in Asians.

Viral level is an indicator of long-term outcome of hepatitis B virus e antigen-negative carriers with persistently normal serum alanine aminotransferase levels.

The association between viral level and the long-term outcomes of hepatitis B virus (HBV) carriers who test negative for hepatitis B virus e antigen (HBeAg) but have persistently normal serum alanine aminotransferase levels (PNALT) remains unclear. We examined hepatocarcinogenesis, hepatitis reactivation, predictive factors and the time course of HBV DNA levels during follow-up in 104 HBeAg-negative Japanese carriers with PNALT. During a mean follow-up period of 6.4 ± 3.4 years, 5 patients (4.8%) had hepatocarcinogenesis and 14 (13.5%) had hepatitis reactivation. At 5 and 10 years, the cumulative rates of hepatocarcinogenesis were 2.4% and 9.9%, while those of hepatitis activation were 13.7% and 15.5%, respectively. An HBV DNA level of ≥5 log10 copies/mL was the sole predictor of hepatocarcinogenesis with a univariate analysis. An HBV DNA level of ≥5 log10 copies/mL and an alanine aminotransferase (ALT) level of >20 to ≤40 IU/L were independent predictors of hepatitis reactivation in a Cox model. Because there was no association between hepatocarcinogenesis and ALT activity, the HBV DNA level was considered an essential predictor. In addition, the baseline HBV DNA level was related to the future level and was not subject to wide fluctuations. Our results showed that an HBV DNA level of ≥5 log10 copies/mL predicts subsequent hepatocarcinogenesis and hepatitis reactivation in HBeAg-negative carriers with PNALT. As the baseline HBV DNA level reflects the future level, appropriate clinical management according to the viral level is expected to decrease future risk.

Cloning of human neuroblastoma cells in methylcellulose culture.

An in vitro methylcellulose technique was used in an attempt to culture neuroblastoma cells from 25 bone marrows from eight children with neuroblastoma. Colonies appeared within 5 days in histologically positive bone marrows. Light microscopy, linearity study, and marker study provided evidence for the neuroblastoma origin of the colonies. These colonies could be distinguished from other colonies under the inverted microscope because of its distinct feature. In one case, the characteristic morphology of neuroblastoma was shown in 3 days of culture, while histological evidence is absent. The diagnosis of neuroblastoma was confirmed by subsequent catecholamine determination. All histologically negative specimens formed no colonies, while all positive specimens formed more than three colonies. Potential application of this culturing technique for monitoring of bone marrow involvement and differential diagnosis in children with neuroblastoma is presented.

Primary structure of human hepatocellular carcinoma-associated aldehyde dehydrogenase.

Tumor-associated aldehyde dehydrogenase (T-ALDH) is strongly expressed in hepatocellular carcinoma (HCC) but undetectable in normal liver. In the present study, this enzyme from human HCC, HCC T-ALDH, was purified and the partial amino acid sequences (384 residues) determined by direct protein sequencing matched the amino acid sequence (453 residues) deduced from cloned HCC T-ALDH cDNAs with an open reading frame. The coding sequences of HCC T-ALDH cDNA, human stomach ALDH3A1 cDNA [Hsu et al., J. Biol. Chem. 267 (1992) 3030-3037] and human squamous cell carcinoma (SCC) T-ALDH cDNA (Schuuring et al., GenBank I.D. M74542) matched one another except for discrepancies at four positions, with consequent P12R, I27F and S134A substitutions. R and A were found in HCC and SCC T-ALDHs, whereas P and S were present in stomach ALDH3A1. To confirm that these discrepancies would have general occurrence, coding sequences of HCC T-ALDH cDNAs from six patients and stomach ALDH3A1 cDNAs from two individuals were examined and all were found to encode ALDH3A1 having R, I and A at protein positions 12, 27 and 134, respectively, indicating HCC T-ALDH to be variant ALDH3A1 which is common in human stomach tissues.

Chromosome pattern in juvenile chronic myelogenous leukemia, myelodysplastic syndrome, and acute leukemia associated with neurofibromatosis.

We studied chromosomes of BM cells from four neurofibromatosis (NF) patients with leukemia. One patient had a normal diploid karyotype in the chronic phase of juvenile chronic myelogenous leukemia (JCML). When the the leukemia evolved into the accelerated phase, she had cells with 46,XX,-7,+der(7)t(3;7)(q21;p22); the abnormalities resulted in a partial 7p deletion. In another patient with JCML, BM cells in the accelerated phase had 45,XY,-7. The abnormal cells with monosomy 7 disappeared from the BM after chemotherapy but reappeared later in the course. Another patient developed refractory anemia with excess of blasts in transformation (RAEB-T) and had cells with 46,XX,-6,+r(6)(p23?q21?); the abnormalities resulted in partial 6p and 6q deletions. The other patient with ANLL had cells with 45,XX,-7. Our findings and review of data on nine other patients suggest that BM cells of NF patients with JCML in chronic phase have no microscopically detectable chromosome changes and that cells with chromosomal deletion emerge when JCML evolve into the accelerated or blast phase. Thus, deletion of the whole or part of certain chromosomes, such as chromosomes 6, 7, etc., may be an important step towards the evolution of JCML cells or the development of de novo acute leukemias in NF patients.

An autopsy case of troglitazone-induced fulminant hepatitis.

OBJECTIVE: To study an autopsy case of troglitazone-induced fulminant hepatitis. CASE REPORT: A 58-year-old man contracted severe hepatitis 2 months after administration of troglitazone for the treatment of type 2 diabetes. Liver damage progressed gradually, even after withdrawal of the agent. Despite intensive therapy, the hepatitis became fulminant and the patient died 8 weeks after the onset of liver damage. Autopsy revealed massive hepatic necrosis and cholestasis with inflammatory cell infiltration. The pathological findings and the positive result of the drug-induced lymphocyte stimulation test for troglitazone indicated that the liver damage was mediated by hypersensitivity to troglitazone. CONCLUSIONS: One report has attributed troglitazone-induced liver damage in less severe cases to idiosyncratic reactions. However, the present case indicates that troglitazone can induce hypersensitivity resulting in fulminant hepatitis. Careful monitoring of serum liver enzymes during troglitazone therapy is therefore essential.

Characterization of natural killer cells cultured from human bone marrow cells.

Human bone marrow (BM) cells, depleted of nylon wool-adherent cells, T cells, and natural killer (NK) cells, were cultured in medium containing recombinant interleukin 2 (rIL2). After 21 or 24 days in culture, numerous lymphoid cells with multiple azurophilic granules and a morphology similar to large granular lymphocytes (LGL) were found. Two-color analysis of surface phenotype showed many of these cells to be NKH1-positive and a limited number of cells had other NK markers such as CD16, CD2, or CD8. The CD3 antigen was not coexpressed with NKH1. The cultured BM cells were cytotoxic for K562, Daudi, and Raji cell lines. The NKH1+, CD2-, CD3-, CD16- cells were sorted and, in addition to having the LGL morphology, were found to be cytotoxic for K562 cells (NK [K562]). The generation of NK(K562) activity was significantly suppressed by 5-bromodeoxyuridine plus ultraviolet light treatment, indicating that DNA synthesis is required. These experiments suggest that the described culture conditions allow differentiation of progenitor cells, into immature, but functionally active, NK cells.

Dominant expansion of human T cells in non-obese diabetic/severe combined immunodeficiency mice implanted with human bone fragments.

OBJECTIVE: To establish an in vivo animal model in which human T cells develop and function normally, a step toward developing new vaccines or chemical compounds that modulate immune functions and toward understanding T-cell immunity in humans. MATERIALS AND METHODS: Human bone fragments were implanted into non-obese diabetes/severe combined immunodeficiency (NOD/SCID) mice. The presence of human blood cells in the peripheral blood of these mice was monitored periodically by immunostaining and fluorescence-activated cell sorting. RESULTS: After implantation of bone fragments, dominant expansion of human T lymphocytes, rather than myeloid and B cells, was observed over a 3-month period. In some cases, the proportion of human T cells rose to 40% of the peripheral blood mononuclear cells. These T cells showed CD4/CD8 ratios similar to those observed in human peripheral blood lymphocytes and had a broad repertoire of rearranged T-cell receptor genes. Graft-versus-host reaction was not noted in any organ analyzed. To assess the suitability of NOD/SCID mice implanted with human bone fragments (hu-bone-NOD/SCID mice) as an in vivo model for HIV infection, the mice were infected with a T-lymphotropic strain of HIV-1 (NL4-3) at 7 weeks posttransplant. Serum p24 gag was detected at 2 weeks after inoculation, after which total CD4-positive cell numbers declined, as seen clinically in patients infected with HIV. CONCLUSION: Although the precise mechanism is yet to be determined by which predominant expansion of human T cells occurs in hu-bone-NOD/SCID mice, such mice appear likely to serve as a useful and versatile model for studies involving human T-cell immunity.

Applications of retrovirus-mediated expression cloning.

We have recently established a novel expression cloning system using retroviral vectors. The system is based on a high-efficiency packaging cell line, BOSC23, and a simplified retroviral vector, pBabeX, carrying no selection marker. cDNA libraries, constructed in the pBabeX vector, are transiently transfected into BOSC23 cells. The supernatant contains more than 3X10(6)/mL, which would cover large complexities of cDNA libraries. The retrovirus stock gave 100% infection efficiency in NIH3T3 cells and 5-40% infection efficiency in various hematopoietic cell lines. In contrast to the conventional expression cloning system, in which it is necessary to transfect cDNA libraries transiently into particular cell types such as COS cells, retrovirus-mediated expression cloning allows us to transduce cDNAs into a wide variety of cell types. This method therefore makes it possible to select cells expressing a cDNA of interest by various functional assays. When combined with polymerase chain reaction (PCR)-driven random mutagenesis, this system is also useful in searching for mutations of various molecules that will result in alterations of their functions.

Oxymetholone therapy in patients with familial antithrombin III deficiency.

Three patients with familial antithrombin III (ATIII) deficiency, who also have histories of thromboembolism, were treated with oxymetholone in combination with warfarin. Thrombolysis was observed in one patient with acute thrombosis of inferior vena cava during the oxymetholone and warfarin therapy. No further thromboembolic episodes occurred in these patients after initiation of warfarin with or without oxymetholone. The levels of plasma ATIII, alpha 1-antitrypsin, plasminogen and Cl-inactivator were significantly increased in all patients after the introduction of oxymetholone therapy. This suggests that oxymetholone augments anticoagulant and fibinolytic activity. Hence we consider that oxymetholone in combination with warfarin may be possible thrombolytic therapy in patients with familial ATIII deficiency.

Variable expression on lung cancer cell lines of HLA-A2-binding MAGE-3 peptide recognized by cytotoxic T lymphocytes.

Cytotoxic T lymphocytes (CTL) specific for HLA-A2-binding MAGE-3 peptide (FLWGPRALV) were generated by repetitive stimulation of PBMC with the peptide in the presence of EBV-transformed B blasts and IL-2. Using these CTL, we investigated the expression of the HLA-A2-binding MAGE-3 peptide on lung cancer cell lines. Of 14 cell lines investigated, 1-87, PC-9, OU-LC-KI, 11-18 and LK87 were derived from HLA-A2 positive patients. But cytofluorometry analysis showed that 1-87, PC-9 and OU-LC-KI, but not 11-18 or LK87 expressed the HLA-A2 antigen. All five cell lines expressed MAGE-3 gene mRNA. Twelve of thirteen CTL lines from two HLA-A2 positive donors showed no cytotoxicity against any of the 14 lung cancer cell lines. CTL line TI-1 showed cytotoxicity against 1-87 but not against any of the other cell lines. Treatment of 1-87 with IFN-gamma greatly augmented the cytotoxicity of TI-1 and induced it in the other 12 CTL lines, confirming the expression of the peptide on 1-87. No cytotoxicity was induced by IFN-gamma treatment of PC-9 or OU-LC-KI. However, PC-9 and OU-LC-KI pulsed with the peptide were killed efficiently by all of the CTL lines, suggesting no expression of the peptide on those cells. A low level of cytotoxicity was induced on 11-18 but not LK87 by IFN-gamma treatment, although expression of the HLA-A2 antigen was not observed by cytofluorometry. These findings showed that expression of the HLA-A2-binding MAGE-3 peptide recognized by CTL was variable on lung cancer cell lines.

Hemolytic crisis after excessive ingestion of fava beans in a male infant with G6PD Canton.

After ingesting fava beans, a 26-month-old Chinese-Japanese male infant showed a sickly complexion and yellowish-brownish skin and was hospitalized. Severe hemolytic anemia was observed on admission, and transfusion of 200 ml of packed red cells was required. Red cell enzyme assay revealed that the patient and the mother were deficient in glucose-6-phosphate dehydrogenase (G6PD). Subsequent molecular analysis showed that the patient had a missense mutation 1376 G to T (G6PD Canton) and his mother was a homozygote for the mutation. The patient was a son of a Chinese (Taiwanese) mother and a Japanese father. Although G6PD deficiency is rare in the original Japanese population, the number of "imported" cases could be rising rapidly. This is the first reported Japanese case of G6PD deficiency with G6PD Canton.

Treatment of infant acute lymphoblastic leukemia in Japan. Childhood Leukemia Study Group of the Ministry of Health and Welfare (Kouseisho).

Although current chemotherapeutic regimens cure as many as 70% of children with acute lymphoblastic leukemia (ALL), infants continue to show a poor outcome. In this paper, we describe the outcome in 37 ALL infants treated between 1989 and 1995 in Japan. Patients had characteristic findings of infant ALL, including hyperleukocytosis > 100 x 10(9)/l (15/37, 41%), blast cells with a CD10-negative phenotype (30/37, 81%), and 11q23/MLL involvement (21/37, 57%). Seven were treated according to Aggressive Treatment Research Group protocol, 15 according to the Ministry of Health and Welfare protocol, and 15 according to protocols of other institutions. The 3-year overall event-free survival (EFS) was 33%. The EFS was 13% for infants aged < 26 weeks at diagnosis and 43% for infants aged > 26 weeks. Infants who had blast cells with CD10 negative phenotype with 11q23/MLL involvement were also associated with poor prognosis. However, infants with CD10 positive blasts without 11q23/MLL involvement had a better outcome (EFS 75%). These results suggest that intensive chemotherapy is effective for patients with good prognostic factors, but for infants with poor prognostic factors a more aggressive approach such as stem cell transplantation might be necessary.

Dopamine release in the medial preoptic area during male copulatory behavior in rats.

In vivo microdialysis was employed to measure the extracellular concentrations of dopamine (DA) in the medial preoptic area (MPOA) of rats during male sexual activity to look for a correlation with sexual activity. During copulation, the concentration of DA in the MPOA was significantly increased. A significant difference was detected in changes of DA between copulators and non-copulators. These findings were consistent with the assertion that DA neuron activity in the MPOA facilities male copulatory behavior.

Hepatitis G virus infection from needle-stick injuries in hospital employees.

A new RNA virus, hepatitis G virus (HGV) is known to be transmissible by blood transfusion. The aim of this study was to assess whether HGV is an occupational risk to hospital employees as a result of exposure to needle-stick injuries. Among 220 cases of needle-stick injuries, 21 employees were contaminated with HGV. Initially none of the 21 recipients were HGV positive. Fourteen of the 21 recipients were followed up and further tested for HGV RNA and serum anti-envelope (E2) specific antibody. None of the 21 recipients exposed to HGV developed liver function abnormalities, but one of the 14 recipients became positive for HGV RNA after the injury. Anti-E2 was negative in all recipients tested. These findings suggests a low clinical risk of occupational exposure to HGV in hospital employees. Nevertheless HGV is transmissible by needle-stick injury.

Transfusion transmitted virus infection in patients on maintenance haemodialysis and in hospital workers.

A newly discovered DNA virus, transfusion transmitted virus (TTV), was isolated from a post-transfusional hepatitis patient in Japan. A high prevalence (32-46%) of TTV infections in patients receiving maintenance haemodialysis (HD) has been reported but the occupational risk of TTV on HD units has not yet been determined. We determined the prevalence of TTV in workers in the same HD unit and the risk factors for TTV infection in HD patients, using logistic regression analysis. The prevalence of TTV DNA was 59.6% in 198 HD patients, significantly higher than that in the HD unit (13 of 39, 33.3%;P= 0.002) and non-HD healthcare workers (20 of 75, 26.7%; P= 0.001). A logistic regression analysis showed that male gender and negative test results for hepatitis G virus RNA were risk factors for TTV infection, but prior blood transfusion and duration of HD were not. Stepwise selection of multiple regression analysis showed that the presence of hepatitis C virus RNA was the only significant predictor for high serum ALT activity, and that the presence of TTV DNA was not. These results indicate that TTV is one of the prevalent human viruses transmissible either parenterally or nonparenterally in HD patients, but the occupational risk of TTV infection in HD unit workers is as low as in other healthcare workers. The pathogenic effects of TTV on the liver appear to be limited.

Effects of long-term psychological stress on sexual behavior and brain catecholamine levels.

The effects of long-term psychological stress on sexual behavior and brain catecholamines were investigated in rats. Stress was applied using the communication box developed by Ogawa and Kuwabara (1996), and a psychological stress group (n = 12), a physical stress group (n = 5), and a control group (n = 5) were established. Stress was applied for 1 hour every day for 10 consecutive weeks. Sexual behavior was observed before the start of exposure to stress and 2, 4, 6, 8, and 10 weeks thereafter. The results showed that long-term psychological stress impaired the sexual behavior of male rats. Long-term psychological stress decreased the concentrations of catecholamine and its metabolites in the brain, especially in the medial preoptic area (MPOA). Thus, we hypothesized that low catecholamine neurotransmission in the brain results in impairment of male rat sexual behavior. We then tried to restore the impaired sexual behavior by administration of a cerebral-activating drug, indeloxazine hydrochloride. The administration of indeloxazine hydrochloride for a 3-week period restored the sexual behavior that had been impaired by long-term psychological stress. These present results suggest that impairment of neurotransmission in the central nervous system could be a cause of sexual dysfunction, and activation of neurotransmission may result in restoration of impaired male sexual behavior.

Thrombotic thrombocytopenic purpura in autoimmune hepatitis.

A 19-year-old woman presented with clinical manifestations of sudden, fulminant thrombotic thrombocytopenic purpura associated with autoimmune hepatitis and autoimmune thrombocytopenic purpura. Although thrombotic thrombocytopenic purpura may, rarely, be associated with systemic lupus erythematosus and other autoimmune diseases, to our knowledge, the syndrome has never been described in association with autoimmune hepatitis. In this patient, too, the etiology of thrombotic thrombocytopenic purpura associated with autoimmune disease remains elusive. The patient was treated with corticosteroid, which brought about no improvement in her condition, and she died of multiorgan failure. Diagnosis is challenging, but prompt diagnosis is necessary because thrombotic thrombocytopenic purpura is a life-threatening syndrome whose prognosis has been improved significantly by early plasmapheresis treatment.

CRST syndrome (calcinosis cutis, Raynaud's phenomenon, sclerodactyly, and telangiectasia) associated with autoimmune hepatitis.

A case of Raynaud's phenomenon, was complicated with autoimmune hepatitis (AIH) during the clinical course, and subsequently with gangrene of the fingertips caused by CRST syndrome (calcinosis cutis, Raynaud's phenomenon, sclerodactyly and telangiectasia). The presence of anticentromere antibodies is rare in AIH; to date, there has been only one report of a combination of AIH and CRST syndrome. This combination of the two diseases has been identified only in Japan.

Immunoglobulin-complexed aspartate aminotransferase.

We report a case of increased aspartate aminotransferase (AST, EC 2.6.1.1; GOT) in a 17-year-old girl which persisted for 3 years. The patient was healthy, but a high level of serum AST was detected during a school health check. Further examination revealed that AST was increased to as high as 259 IU/l while alanine aminotransferase (ALT) was normal. Immunoelectrosyneresis and immunoprecipitation methods revealed that this atypical AST combined with IgG--kappa, lambda globulin and formed macromolecular complexes. Including the present case, 26 cases of IgG-complexed AST have been reported. It is important to be aware of this syndrome, and thereby avoid unnecessary examinations and therapies.