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Background To reduce the risk of human parvovirus B19 (B19V) transmission through contaminated blood for transfusion and plasma-derived products, the Japanese Red Cross (JRC) Blood Centers introduced B19V antigen screening by chemiluminescent enzyme immunoassay (CLEIA-B19V) in 2008. Study Design and Methods Donor samples that were positive by CLEIA-B19V screening were tested for B19V DNA. The sensitivity of CLEIA-B19V was tested using samples of all three genotypes and B19V DNA–positive donations. B19V DNA–positive donations and pooled plasma were quantitatively assayed for B19V DNA. B19V DNA–positive donations were phylogenetically analyzed by polymerase chain reaction direct sequencing. Results The sensitivity of CLEIA-B19V was inferred to be approximately 6.3 log IU/mL with the genotype samples and 6.4 log IU/mL with B19V DNA–positive donor samples. Of 417 CLEIA-B19V–positive samples from 1,035,560 donations in Hokkaido, Japan, 101 were positive for B19V DNA. The 198 strains of B19V DNA–positive donations in Hokkaido over the past 15 years clustered exclusively with Genotype 1. After introduction of CLEIA-B19V, the viral load for B19V DNA in all 772 pooled plasma for fractionation from donors in nationwide Japan did not exceed 4 log IU/mL. Conclusion CLEIA-B19V can detect all three genotypes of B19V (viral load >6.3 log IU/mL) and limit the viral load (

SCA1 is a dominant spinocerebellar ataxia (SCA) and a multi-systemic syndrome caused by abnormal expansion of unstable CAG repeat in a novel gene located on chromosome 6p22-p23. We clinically studied 35 Japanese SCA1 patients who were assumed to have come from a common origin. The age at onset ranged from 15-63 years, and significantly correlated with CAG repeat units of mutant alleles. Ataxia was the initial symptom, and the majority of patients had a similar history of signs and symptoms. Nystagmus was at first minimal, later attenuated, and a slow saccade followed. Limb tendon reflexes were mostly hyperactive and depressed with the development of diffuse amyotrophy. The cardinal feature was ataxia-hyperreflexia-late slow saccade syndrome with terminal amyotrophy. Although the phenotype of SCA1 overlaps with those of other dominant SCAs, some facets of the neurological events differ from either SCA2 with ataxia-hyporeflexia-slow saccade syndrome, or early-onset Machado-Joseph disease with dystonia-bradykinesia-spasticity syndrome.

In an attempt to study the gene products of the HLA complex, a monoclonal antibody, named HU-30, was produced by immunizing BALB/c mice with a cultured human B lymphoblastoid cell line, Shi-C3 (Aw24, Aw31, Bw51, Bw52, DR2, DR blank, MT1, MT2, MB3). HU-30 belonged to the IgG2 subclass and was active in complement dependent cytotoxicity. When the serological specificity was evaluated with a panel of 15 cultured human lymphoblastoid cell lines, it was found that HU-30 detected a polymorphic determinant, common to HLA-DR1 and 2, with much stronger cytotoxic activity against HLA-DR2 positive B cell lines. When HU-30 was tested against a panel of B cells from 84 healthy donors at a dilution of 2(-13), it gave positive reactions only with cells typed as HLA-DR2. Furthermore, sequential coprecipitation studies indicated that the HU-30 determinant was borne on the molecules carrying the HLA-DR determinants. Thus, HU-30 appears to be of great value as a tissue typing reagent monospecific for HLA-DR2.

Two Leu2(-), Leu3(+), Leu4(+) human cytotoxic T lymphocyte (CTL) clones, BE-11 and AF-3, were generated against Epstein-Barr virus (EBV)-transformed cell line GI (Dw8/DRw8/DQWa homozygous). Blocking experiments with various monoclonal antibodies (MoAbs) revealed that the former recognized the DR molecule and the latter recognized the DQ molecule, respectively. Panel studies showed that CTL clone BE-11 lysed not only DRw8-positive cells but also DR1-positive ones. CTL clone AF-3 exhibited cytotoxicity against only Dw8/DRw8/DQWa typed cells. Until now, such specificities have not been defined serologically or biochemically. These results demonstrated that the previously unknown DR and DQ specificities could be defined by CTL clones, suggesting that CTL clones might be especially valuable tools for investigating the structural polymorphism of HLA antigens.

The HLA—B8 antigen is one of the characteristic antigens associated with organ specific autoimmune diseases among Caucasians. Among the Japanese, HLA—B8 positives have been assumed to be extremely rare. By using some HLA-B8 typing sera, a new HLA-B antigen, HLA-Bw4 positive, was found in the Japanese. This new HLA-B antigen, tentatively called HOK-1, showed a phenotype frequency of 3.7% and a gene frequency of 1.9 ± 0.6%. This antigen has a strong linkage disequilibrium with HLA-Cw1 among the Japanese.

A new HLA-D specificity was found in the Japanese population in two different laboratories. Japanese cell YT, found at Stanford, California, was A9,BW22J,CW1 and cell Wa, found at Sapporo, Japan, was A9,BW22J homozygous. They were shown to be HLA-D identical to the homozygous Japanese cell AH which submitted to the VIth International Histocompatibility Testing Workshop (Workshop number 2-001). This specificity was common in the Japanese (gf = 0.089) but completely absent from 62 Caucasians tested. Strong association of this specificity with HLA-BW22J was demonstrated.

Background and Objectives Virus inactivation and removal are important prerequisites to ensure the safety of plasma derivatives. For virus inactivation and removal in our coagulation factor VIII (FVIII) product, CROSS EIGHT M®, the production process consists of solvent–detergent (S/D) treatment, two chromatography steps and virus filtration with a 35-nm pore-size filter. However, the clearance of non-enveloped viruses was not as good as that of enveloped viruses because non-enveloped viruses are resistant to S/D treatment and are too small to be removed by the filter. In this study, in order to improve the viral safety of the FVIII products, we attempted to replace the 35-nm pore-size virus filter with a 20-nm filter. Materials and Methods The virus-filtration process was validated for the removal of enveloped and non-enveloped model viruses. Several factors that might affect the FVIII yield on filtration were investigated to obtain a higher recovery. The biochemical properties of the FVIII products produced with the 20-nm pore-size filter were compared with those produced by the 35-nm filter. Results Virus filters of 20-nm pore size effectively removed the small non-enveloped viruses when compared with the 35-nm pore-size virus filter. The permeability of FVIII through the 20-nm pore-size filter was inversely proportional to the concentration of FVIII at filtration, and directly proportional to the amount of postfiltration solution. No differences were observed in the biochemical properties of both FVIII products, such as the structure and stability of the FVIII, the contents and multimeric structure of von Willebrand factor (vWF), and FVIII activation by thrombin. Conclusions The virus-clearance efficiency of the FVIII product, CROSS EIGHT M®, was markedly increased, in particular against small non-enveloped viruses, by changing the virus filter pore size from 35 nm to 20 nm. It was possible to implement the 20-nm pore-size filter without variation of the biochemical properties or a serious loss of FVIII.

The frequency of HLA class I and II phenotypes was determined among 23 patients with positive gelatin IgE, eight of whom developed anaphylaxis, 18 patients who did not have gelatin IgE but who experienced non-immediate reactions after exposure to gelatin. HLA-DR9, which is unique to Orientals, was present in 56.5% of the gelatin IgE positive patients, as compared to control population frequency of 24% (P < 0.002). In the non-immediate reaction group, who did not generate IgE, phenotype distribution resembled controls. HLA-DR9 positive individuals have a relative risk of 4.1 for developing gelatin allergy with positive IgE.

To determine whether human endogenous retroviruses are implicated in the pathogenesis of inflammatory vascular diseases of unknown etiology, we examined mRNA expression of a human endogenous retrovirus, HERV-R, which has a long open reading frame in the env region, in cultured human vascular endothelial and smooth muscle cells stimulated in the presence of various cytokines. mRNA of HERV-R was always evident in these cells but not in fibroblastic cells. Levels of expression in vascular endothelial cells were significantly regulated by treatment with tumor necrosis factor-alpha, interleukin (IL)-1alpha, and IL-1beta as up-regulators and interferon-gamma as a down-regulator. These observations are interpreted to mean that HERV-R expression may be up- or down-regulated at sites of inflammation in vessels in vivo and hence may play a pathogenetic role in inflammatory vascular diseases in humans, perhaps similar to endogenous retroviruses in mouse models of polyarteritis nodosa in humans.

Since NAT is a highly sensitive technique for detecting low amounts of viral genomes, it is applied to the screening of source plasma to reduce the risk of viral infection through plasma-derived blood products.For this purpose we have used an NAT test for HBV, HCV and HIV by PCR in pooled plasma from of 500 individuals since November, 1997.Because nucleic acid extraction is the most time-consuming and laborious job in this NAT test, an automated method was sought to reduce the operation time and human error. This would allow processing of more samples than the currently used extraction.Recently, Roche Diagnostic K. K. has developed an automated extraction system named GT-12 and an extraction reagent for HCV RNA. In this study we compared GT-12 with the manual technique. The results show GT-12 extraction caused no contamination and had good reproducibility without any inter-day variation. Although GT-12 is less sensitive than the manual extraction method, it is sensitive enough to detect 100IU/ml as required by the CPMP guideline (CPMP/BWP/390/97). Operation time of extraction by GT-12 was about 2/3 of that by manual extraction. Introduction of GT-12 may contribute to the increased efficiency of the NAT test.

We investigated virus inactivation by two albumin products (Sekijyuji Albumin® and Sekijyuji Albumin 25® derived from human plasma with heat treatment at 60°C in liquid-phase (pasteurization) using several enveloped and non-enveloped viruses as marker viruses. Enveloped viruses were sindbis virus (SIN), vesicular stomatitis virus (VSV), herpes simplex virus type-1 (HSV-1) and human immunodeficiency virus type-1 (HIV-1). Non-enveloped viruses were polio virus (Polio V) and hepatitis A virus (HAV). The infectivity of all viruses except HAV was lost after 1hr heating, with logarithmic reduction value (LRV) for SIN, VSV, HSV-1, HIV-1 and Polio V of >5.1, >5.7, >4.3, >3.8 and >5.9, respectively. No significant difference in LRVs were observed between the two products. The infectivity of HAV, however, remained at 104.2-5.0 TCID50 even after 10hr heating and LRV stayed in the range of 1.9 to 2.7.

Spinocerebellar ataxia type 6 (SCA6) is caused by small CAG repeat expansion in the gene encoding the alpha1A-voltage-dependent-calcium channel subunit (CACNLIA4) on chromosome 19p13, and is a subgroup of the late-onset pure cerebellar ataxia (ADCA III). To investigate the prevalence of SCA6 in the Japanese, we analyzed this mutation in 23 families and 12 probands with ADCA III. The specificity and stability of the CAG repeat were examined in additional individuals and families with other miscellaneous dominant SCAs. The CAG expansion of SCA6 gene was exclusively observed in 12 of 23 families (52%) and 12 proband cases with ADCA III, but not in others. The CAG repeat was 21-33 in the disease-associated alleles (n=56), and 4-18 in normal alleles (n=1148). Expanded alleles were stable during transmission, and a significant inverse correlation for CAG repeat number with age at onset was noted. Our results indicate that SCA6 shares approximately half of the ADCA III in the Japanese, and that gene mutations causing the remaining, have yet to be identified.

Autosomal dominant spastic paraplegia (ADSP) is a genetically heterogenous disorder. To date, 3 loci of ADSP have been identified on chromosome 2p, 14q, and 15q, but specific gene mutations remain unknown. To determine the genetic background of ADSP in the Japanese, we studied a large 3-generation pedigree, clinically and genetically. Of the 36 individuals clinically examined, 15 were affected. The main feature in the affected individuals was a slowly progressive spastic paraplegia, associated with upper limb hyperreflexia (58%), reduction of vibration sense (27%) and bladder disturbance (13%). Age at onset ranged from 13 to 50 years with a mean of 30.3 +/- 14.2 (SD). There were 6 parent-child pairs with anticipation and at least 3 others with 'anti-anticipation'. Linkage with 14q and 15q ADSP loci was excluded, and a highly significant lod score was obtained only in the case of the 2p locus (Zmax = 3.53 for D2S400/D2S352, at theta = 0.00). Our study is the first to confirm the existence of 2p-linked ADSP in the Japanese. There is a significant variety in age at onset and disease severity in these 2p-linked families, but the implication for underlying ADSP mutation is not clear.

To investigate the pathogenesis of human T lymphocyte virus type I (HTLV-I)- related diseases, the env-pX gene of HTLV-I was introduced into the germline of inbred Wistar-King-Aptekman-Hokudai rats. A wide spectrum of collagen vascular diseases was evident in the transgenic rats, including chronic destructive arthritis similar to rheumatoid arthritis, necrotizing arteritis mimicking polyarteritis nodosa, polymyositis, myocarditis, dermatitis, and chronic sialoadenitis and dacryoadenitis resembling Sjögren's syndrome in humans. Thymic atrophy with the depletion of CD4 and CD8 double-positive thymocytes was also observed. In these animals, a number of autoantibodies, including high titers of rheumatoid factor, were present in the serum. We propose that the HTLV-I env-pX gene region may play a pathogenetic role in the development of collagen vascular and autoimmune diseases associated with autoimmune phenomenon.

Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominant, neurodegenerative disorder that affects the cerebellum and other areas of the central nervous system. We have devised a novel strategy, the direct identification of repeat expansion and cloning technique (DIRECT), which allows selective detection of expanded CAG repeats and cloning of the genes involved. By applying DIRECT, we identified an expanded CAG repeat of the gene for SCA2. CAG repeats of normal alleles range in size from 15 to 24 repeat units, while those of SCA2 chromosomes are expanded to 35 to 59 repeat units. The SCA2 cDNA is predicted to code for 1,313 amino acids-with the CAG repeats coding for a polyglutamine tract. DIRECT is a robust strategy for identification of pathologically expanded trinucleotide repeats and will dramatically accelerate the search for causative genes of neuropsychiatric diseases caused by trinucleotide repeat expansions.

To identify the markers tightly linked to Machado-Joseph disease (MJD) and to investigate whether a limited number of ancestral chromosomes are shared by Japanese MJD pedigrees, a detailed linkage analysis employing D14S55, D14S48, D14S67, D14S291, D14S280, AFM343vf1, D14S81, D14S265, D14S62, and D14S65 was performed. The results of multipoint linkage analysis as well as detection of critical recombination events indicate that the gene for MJD is localized in a 4-cM region between D14S280-D14S81. We found strong linkage disequilibria at AFM343vf1 and D14S81, and association of a few common haplotypes with MJD. These results indicate that there is an obvious founder effect in Japanese MJD and suggest the possibility of the existence of predisposing haplotypes which are prone to expansions of CAG repeats. 47 refs., 3 figs., 2 tabs.

Machado-Joseph disease (MJD) is an autosomal dominant neurodegenerative disorder caused by unstable expansion of a CAG repeat in the MJD1 gene at 14q32.1. To identify elements affecting the intergenerational instability of the CAG repeat, we investigated whether the CGG/GGG polymorphism at the 3' end of the CAG repeat affects intergenerational instability of the CAG repeat. The [expanded (CAG)n-CGG]/[normal (CAG)n-GGG] haplotypes were found to result in significantly greater instability of the CAG repeat compared to the [expanded (CAG)n-CGG]/[normal (CAG)n-CGG] or [expanded (CAG)nGGG]/[normal (CAG)n-GGG] haplotypes. Multiple stepwise logistic regression analysis revealed that the relative risk for a large intergenerational change in the number of CAG repeat units (-2 or2) is 7.7-fold (95% CI: 2.5-23.9) higher in the case of paternal transmission than in that of maternal transmission and 7.4-fold (95% CI: 2.4-23.3) higher in the case of transmission from a parent with the [expanded (CAG)n-CGG]/[normal (CAG)n-GGG] haplotypes than in that of transmission from a parent with the [expanded (CAG)n-CGG]/[normal (CAG)n-CGG] or [expanded (CAG)n-GGG]/[normal (CAG)n-GGG] haplotypes. The combination of paternal transmission and the [expanded (CAG)n-CGG]/[normal (CAG)n-GGG] haplotypes resulted in a 75.2-fold (95% CI: 9.0-625.0) increase in the relative risk compared with that of maternal transmission and the [expanded (CAG)n-CGG]/[normal (CAG)n-CGG] or [expanded (CAG)n-GGG]/[normal (CAG)n-GGG] haplotypes. The results suggest that an inter-allelic interaction is involved in the intergenerational instability of the expanded CAG repeat.

SCA1 is caused by expansion of an unstable CAG triplet repeat in a novel gene located on the short arm of chromosome 6. In 126 Japanese individuals from 12 pedigrees with SCA1, studies were done to determine if they carried this mutant gene. All the affected and pre-symptomatic individuals, determined by haplotype segregation analyses, carried an abnormally expanded allele with the range of 39-63 repeat units. This repeat size inversely correlated with the age at onset. However, contrary to reported results, size of the repeat did not correlate with gender of the transmitting parent. Therefore, the CAG triplet repeat instability on paternal transmission is not likely to be fundamental to SCA1.

To reduce the risk of human parvovirus B19 (B19V) transmission through contaminated blood for transfusion and plasma-derived products, the Japanese Red Cross (JRC) Blood Centers introduced B19V antigen screening by chemiluminescent enzyme immunoassay (CLEIA-B19V) in 2008.Donor samples that were positive by CLEIA-B19V screening were tested for B19V DNA. The sensitivity of CLEIA-B19V was tested using samples of all three genotypes and B19V DNA-positive donations. B19V DNA-positive donations and pooled plasma were quantitatively assayed for B19V DNA. B19V DNA-positive donations were phylogenetically analyzed by polymerase chain reaction direct sequencing.The sensitivity of CLEIA-B19V was inferred to be approximately 6.3 log IU/mL with the genotype samples and 6.4 log IU/mL with B19V DNA-positive donor samples. Of 417 CLEIA-B19V-positive samples from 1,035,560 donations in Hokkaido, Japan, 101 were positive for B19V DNA. The 198 strains of B19V DNA-positive donations in Hokkaido over the past 15 years clustered exclusively with Genotype 1. After introduction of CLEIA-B19V, the viral load for B19V DNA in all 772 pooled plasma for fractionation from donors in nationwide Japan did not exceed 4 log IU/mL.CLEIA-B19V can detect all three genotypes of B19V (viral load6.3 log IU/mL) and limit the viral load (4 log IU/mL) in pooled plasma, and thus such screening has further reduced the risk of transfusion-transmitted B19V infection. These results show that CLEIA-B19V screening at the JRC Blood Centers can be an alternative approach to comply with recommendations regarding B19V in the United States and Europe.

We did a linkage study of 2 multigenerational pedigrees with dominant olivopontocerebellar atrophy (OPCA) other than SCA1, with chromosome 12q microsatellites. Multipoint linkage analysis led to the conclusion that the disease locus locates within the 6.2 cM interval between IGF1 and D12S84/D12S105. This result coincides with that of Cuban ataxia pedigrees designated as SCA2. Our study provides genetic evidence that dominant OPCA in the Japanese consists of at least two genetically different disorders; SCA1 and SCA2.

Human T lymphocyte virus type I (HTLV-I) can be transmitted into several inbred strains of newborn and adult rats by inoculating newly established HTLV-I-immortalized rat T cell lines or the human T cell line MT-2. The transmission efficiency exceeds 80%, regardless of strain differences or the age at transmission. The production of anti-HTLV-I antibodies significantly differs among the strains and depends on the age at the time of transmission. Rats neonatally inoculated with HTLV-I-positive rat or human cells generally become seronegative HTLV-I carriers throughout their lives, whereas adult rats inoculated with HTLV-I-positive cells at 16 wk of age become seropositive HTLV-I carriers. The HTLV-I provirus genome is present in almost all organs, regardless of whether the carriers are seronegative or seropositive. According to antibody titers to HTLV-I, there are three groups of inbred rat strains: ACI, F344, and SDJ (high responders); WKA, BUF, and LEJ (intermediate responders); and LEW (low responder). Three of three 16-mo-old seronegative HTLV-I carrier rats of the WKA strain developed spastic paraparesis of the hind legs. Neuropathological examinations revealed that the lesions were confined primarily to the lateral and anterior funiculi of the spinal cord. Both myelin and axons were extensively damaged in a symmetrical fashion, and infiltration with massive foamy macrophages was evident. The most severe lesions were at levels of the thoracic cord and continued from the cervical to the lumbar area. These histopathological features as well as clinical symptoms largely parallel findings in humans with HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). These HTLV-I carrier rats, in particular the WKA rats described above, can serve as a useful animal model for investigating virus-host interactions in the etiopathogenesis of HTLV-I-related immunological diseases, particularly HAM/TSP.

To investigate the pathogenetic role of human T-lymphocyte virus type I (HTLV-I) in central nervous system disease, a rat model for HTLV-I-associated myelopathy/tropical spastic paraparesis, designated as HAM rat disease, was examined with regard to chronological neuropathology, from early asymptomatic phase to late disease. In the thoracic spinal cord of rats with HTLV-I infection, the first event was the appearance of apoptosis of oligodendrocytes beginning at 7 months after induced infection, thereafter followed by the appearance of white matter degeneration, increase of macrophages/activated microglia and of gemistocytic astrocytes at 12, 15 and 20 months, respectively. In the spinal cord, HTLV-I provirus DNA was evident as early as 4 months after the infection, and HTLV-I pX and the tumor necrosis factor (TNF)-alpha messages began to be expressed at age 7 months, just before or at the same time as the appearance of apoptotic cells. Collective evidence suggests that the apoptotic death of oligodendrocytes, which may be induced either directly by the local expression of HTLV-I or indirectly by TNF-alpha, through the transactive function of p40Tax, is the major cause of chronic progressive myeloneuropathy in Wistar-King-Aptekman-Hokudai rats with HTLV-I infection.

To investigate the pathogenetic role of human T lymphocyte virus type I (HTLV-I) in central nervous system disease, a rat model for HTLV-I-associated myelopathy/tropical spastic paraparesis, designated as HAM rat disease, has been established. Wistar-King-Aptekman-Hokudai strain rats with induced HTLV-I infection develop a chronic progressive myeloneuropathy with paraparesis of hind limbs after an incubation period of 15 months. In the affected spinal cord in these rats, white matter degeneration, demyelination and vacuolar change with microglia/macrophage infiltration are present as are the provirus DNA and the virus mRNA. To identify infected cells in the affected lesions, we carried out in situ hybridization of amplified fragments of the provirus DNA by polymerase chain reaction on thin sections, plus immunohistochemistry on the same sections. The provirus DNA was localized in some microglia/macrophages in the spinal cord lesion. In addition, the HTLV-I provirus was clearly evident not only in ED-1-negative lymphoid cells but also in ED-1-positive macrophages from lymph nodes. These observations suggest that cells of microglia/macrophage lineage may be one of dominant viral reservoirs in the spinal cords and lymph nodes in HAM rat disease. These infected microglia/macrophages may relate to cause the myeloneuropathy through neurotoxic cytokine synthesis.

Spinocerebellar ataxia-2 (SCA2) is an autosomal dominant ataxia caused by an abnormal CAG repeat expansion in a novel gene on chromosome 12q24.1. The size of the mutant allele is unstable during transmission, and correlates inversely with age at onset. We studied eight Japanese SCA2 families, including 28 patients, to assess the effect of repeat length on the phenotype features of SCA2. Frequencies of slow eye movements (SEM), reflex activity, dementia, choreiform movements, and axial tremor correlated significantly with CAG repeat size. Parkinsonism was seen in a man homozygote for SCA2 mutation. The clinical variety of SCA2 is apparently influenced by the size of the mutant allele, as is the case in other CAG repeat disorders.

In an attempt to clarify the biological nature of a human endogenous retrovirus (HERV), HERV-R, which is a single-copy type of HERVs and is conserved as a full-length viral sequence, the expression of HERV-R mRNA in normal autopsied systemic organs was examined by Northern blot analysis. The expression showed different levels among individuals, with the adrenal glands expressing the highest level of HERV-R among all organs tested, except for the placenta. In various adrenal tumors, HERV-R was expressed at high levels in all cortical adenomas but less so in pheochromocytomas. In situ hybridization revealed the expression of HERV-R to be localized in all layers of the adrenal cortex, but not in the medulla. This high-level expression of HERV-R in the adrenal cortex may possibly relate to differentiation and/or steroid production by adrenocortical cells.

We studied the relationship between the number of CAG repeat units in the MJD1 gene and clinical features of Machado-Joseph disease (MJD) in eight patients from two generations of a Japanese MJD family. Because of lack of characteristic clinical signs of MJD such as dystonia, bulging eyes or facial myokymia, clinical diagnosis of MJD in this family was difficult to make prior to molecular testing for the CAG repeat expansion in the MJD1 gene. All the patients exhibited maternal transmission of MJD, and the intergenerational change in the number of CAG repeat units in the MJD1 gene was very small (+0.5±0.3, mean±S.E.M., n=4) in spite of marked genetic anticipation (−17.0 years/generation). In the present family, the degree of anticipation per repeat unit in maternal transmissions was much larger than that in maternal transmissions in the other six MJD families. This indicates that some maternal factors other than the increase of the number of CAG repeat units, which is known to be the basis of anticipation, may play a role in genetic anticipation in this MJD family.

The gene for SCA2 has been mapped to chromosome 12q23-q24.1, but the mutant gene remained to be identified. When studying a Japanese family with SCA2, we noted that clinical features and disability varied among patients, with the central feature being progressive ataxia-slow eye movement-hyporeflexia syndrome. Additional symptoms were parkinsonism with minor cerebellar deficits, and severe ataxia with choreoathetosis. Our experience plus related literature documentation indicates that choreoathetosis is not so rare at the advanced stage of the disease, with onset at an early age, and that the variety of SCA2 phenotype depends on age at onset and duration of the disorder.

The pathogenetic roles of human T lymphocyte virus type I (HTLV-I) and cytokines were investigated in HTLV-I-induced myeloneuropathy in Wistar-King-Aptekman-Hokudai rats. In the nervous system, pX messenger RNAs of HTLV-I were selectively expressed in the diseased spinal cord and peripheral nerves but not in the unaffected cerebrum and cerebellum, even though proviral DNAs were consistently identified in these tissues. Among several cytokines examined, mRNA expression and production of tumor necrosis factor (TNF)-alpha in the spinal cord and cerebrospinal fluid correlated positively with the development of spinal cord lesions. The collective evidence strongly suggests that selective activation of HTLV-I, in particular Tax expression and production of TNF-alpha induced by HTLV-I infection in target spinal cord and peripheral nerves, is causally related to apoptotic death of oligodendrocytes and Schwann cells, a major pathogenetic pathway of the HTLV-I-induced myeloneuropathy.

Machado-Joseph disease (MJD) is caused by abnormal expansion of an unstable CAG repeat in a novel gene locating on chromosome 14q32.1. We analysed this CAG repeat polymorphism with 66 Japanese MJD patients. All the patients were selectively associated with abnormal expansion of the CAG repeat. Repeat length of the mutant allele did not overlap that of normal allele and closely correlated with not only age at onset but also with clinical phenotypes. CAG repeat size is apparently related to a wide variety of phenotypic presentations in MJD.

Spinocerebellar ataxia 1 (SCA1) is caused by expansion of an unstable CAG triplet repeat located on the short arm of chromosome 6. Precise mapping has shown a positional relationship to closely linked markers in the order of D6S109-D6S274-D6S288-SCA1-AM10GA-D6S89+ ++-EDN1 from centromere to telomere. The haplotype which cosegregated with the disease was determined in 12 Japanese pedigrees with SCA1. Although the alleles of the SCA1 haplotype varied from pedigree to pedigree, depending on the distance from the SCA1 locus, the affected and presymptomatic subjects carried the same alleles at D6S288 and D6S274. All the families with SCA1 had migrated from either Miyagi or Yamagata Prefectures, neighbouring areas in the Tohoku District, the northern part of Honshu which is the main island of Japan. It seems highly likely that SCA1 in the Japanese, at least those residing in Hokkaido, derives from a single common ancestry.

SUMMARY We addressed the question of whether or not expression of human endogenous retroviruses (ERV). ERV3 and λ4–1, is related to the pathogenesis of rheumatoid arthritis (RA). In genomic Southern hybridization, there were no significant differences between RA patients and healthy volunteers with regard to frequencies of restriction fragment length polymorphism (RFLP) patterns, for either ERV3 or λ4–1. By Northern blot analysis using fresh synovial tissues, cultured synovial cells, and peripheral blood mononuclear cells (PBMC) from patients with RA, we noted two molecular species of ERV3 mRNAs of 3·5 kb and 9·0 kb sizes, and one single molecular species of λ4–1 mRNAs of 4·2 kb size. The expression was detected not only in RA patients but also in synovial cells from osteoarthritis (OA) as a non-RA control and PBMC from healthy volunteers, and was not related to RA activities or treatments. Although ERV3 and λ4–1 expression may not be directly associated with the pathogenic pathway of RA, the possibility exists that human ERV may have a causative role in autoimmune diseases, including RA. We also examined the effect of cytokines on the transcriptional regulation of ERV3. Although the level of ERV3 expression in cultured synovial cells did not change with IL-1β treatment, the level for cultured proximal tubular epithelial cells (hKEC) was up-regulated.

In seropositive HTLV-I carrier rats of the WKAH strain inoculated with 2 x 10(7) MT-2 cells at 3-6 months of age, chronic progressive myeloneuropathy, tentatively designated as HTLV-I-associated myelopathy (HAM) rat disease, occurred when the rats were 19-23 months old. Clinical and pathological findings were basically identical to those of seronegative HAM rats of the same strain neonatally inoculated with MT-2 cells. It appears that a high dose of MT-2 cells (10(8) cells) is more effective for the induction and acceleration of HAM rat disease. Seronegative and seropositive carriers of other strains (F344, ACI, and LEW), WKAH rats inoculated with HUT-78 (a human T cell line without HTLV-I infection), and untreated WKAH rats at comparable ages did not develop HAM rat disease, thereby indicating that development of this disease is caused by HTLV-I infection and is under strict genetic restriction of the host strain. Chronological examination of HAM rat disease induced by 10(7) MT-2 inoculation into newborn rats showed that the spinal cord lesion began to develop by 12 months of age. T cells were absent in the affected spinal cord throughout the disease process. There was morphological evidence of apoptotic death of oligodendrocytes in the affected spinal cord. Apoptosis was also confirmed by the specific nick end labeling of the nuclear fragmentation in situ, and the apoptotic oligodendrocytes confined to the demyelinating foci, and the number of apoptotic cells positively correlated with severity of the spinal cord lesion.(ABSTRACT TRUNCATED AT 250 WORDS)

The genetic factors involved in the development of insulin-dependent diabetes mellitus (IDDM) have been found to be associated with genes in the major histocompatibility complex (MHC) located on chromosome 6. DQ antigens appear to be the most relevant for the development of IDDM: HLA-DQw2 and DQw3 in Caucasians1,2 and HLA-DQw4 and DQw9 in Japanese.3 Moreover, position 57 of the DQβ chain and position 52 of the DQα chain were found to be associated with IDDM susceptibility in Caucasians. Homozygosity for non-aspartic acid (non-Asp-57) was observed in 90% of the IDDM patients and none were homozygous for aspartic acid (Asp-57).2,5

Attempts to isolate the Huntington disease (HD) gene based on its position have been frustrated by apparently contradictory recombination events in HD pedigrees that have predicted two non-overlapping candidate regions: 100 kb at the telomere of the short arm of chromosome 4, and a 2.2 Mb region located internally at 4p16.3. The proximal location is also supported by the detection of a linkage disequilibrium between HD and some restriction fragment length polymorphisms (RFLPs) at the D4S95, D4S98, and D4S127 loci. In the present study, a proximal marker D4S95 showed tight linkage to the disease locus in Japanese pedigrees (Zmax = 3.31, theta max = 0.00), while distal markers D4S115 and D4S111 did not. Particularly, a two point linkage analysis between D4S111 and HD yielded a lod score -2.01 for theta = 0.015. This result leads to the exclusion, as a possible region of localization of the HD gene, of more than 3 cM of the genome around D4S111 locus. At the same time our results favor aforementioned proximal location as a candidate location for the HD gene.

A genomic HLA-G clone named 7.0E was isolated from a Japanese placenta. The deduced amino acid sequence of the 7.0E was identical to two HLA-G genomic clones and two cDNA clones previously described. The DNA sequences of alpha 1 and alpha 2 domains of the HLA-G gene from 5 cell lines also encoded the same amino acids. However, a 14 bp insertion, ATTTGTTCATGCCT, was present in the 3' untranslated region of 7.0E compared with the originally described HLA-G clone (HLA 6.0). Polymerase chain reaction (PCR)/single strand conformational polymorphism (SSCP) analysis of exon 8 allowed the HLA-G gene to be classified into two alternative types, G6.0 and 7.0 E, those correlated to the absence or the presence of the 14 bp stretch. Each group had minor sequence variant(s), and the alleles of the 7.0E-type were more heterogeneous than those of the G6.0-type. The 14 bp deletion is present only in the G6.0-type of HLA-G alleles among HLA class I genes. Thus it was suggested that G6.0 alleles were generated after diversification of the HLA-G.

Fresh and cultured leukemia cells from an adult T-cell leukemia (ATL) patient which possessed gag and env gene defective human T-cell leukemia virus type I (HTLV-I) provirus genome were molecularly analyzed. Cells from both fresh and the established cell line, named KB-1 showed identical surface markers of helper T cells, expressed the interleukin 2 (IL-2) receptor and had an identical defective (HTLV-I) provirus genome with deletions of the gag and env genes involving pX gene exon 2. The KB-1 cells grew vigorously in vitro , even in the absence of IL-2 and the culture supernatant of KB-1 contained a large amount of IL-2. Neither pX mRNA nor p40(TAX) protein was detected in the KB-1 cells. The collective evidence suggests that the pX gene was not functioning in this particular ATL case. The biological function of the HTLV-I genes, especially the pX gene is discussed in relation to the early and late leukemogenesis of ATL.

A flow-cytometric technique was developed to measure the frequency of variant lymphocytes lacking expression of HLA-A2 or A24 allele products among donors heterozygous for HLA-A2 or A24. It was found that the variant frequency of lymphocytes in peripheral blood was of the order of 10−4 and increased with donor age. Molecular analyses of mutant clones revealed that about one-third were derived from somatic recombinations and that the remaining two-thirds did not show any alterations after Southernblotting analysis. In contrast, mutants obtained after in vitro X-ray mutagenesis study were found to be mostly derived from large chromosomal deletions. A small-scale study on atomic bomb survivors did not show a significant dose effect.

Occurrence of embryonal kidney tumors in patients with primitive neuroectodermal tumors, so-called central nervous system-renal neoplasia has been reported. An infant who presented with masses in the right lateral ventricle and the cerebellar vermis is reported. Histological examination showed primitive neuroectodermal tumors. Further investigation revealed tumors in the bilateral kidneys, which were removed subtotally and pathologically shown to be Wilms' tumors. The patient was then treated with anticancer drugs and irradiation. However, he developed lung metastases from the renal tumors and expired. At autopsy, a small tumor was found in the inferior horn of the left lateral ventricle. Histological finding showed a primitive neuroectodermal tumor. Also, bilateral large masses of recurrent Wilms' tumors, multiple metastases to the lungs and peritoneal dissemination were found. There is no evidence that this association is based on the selective neoplastic transformation of embryonal cells of similar histogenetic or cytogenetic origin. Several reports demonstrate the presence of embryonal cells in the nervous tissue which could imply a neuroepithelial origin for Wilms' tumors.

We studied allotypes of the fourth component of complement (C4) and factor B in 76 patients with Sjogren's syndrome (SS) and in 63 normal subjects. C4A-null (C4AQ0) was found in 10 of 28 patients who had primary SS, compared with 1 of 63 control subjects (P less than 0.005). In contrast, no significant difference in the frequency of any C4 allotype was observed between patients with secondary SS and control subjects. An association of HLA-DRw53 with primary SS in Japanese patients has been reported. Since there is no linkage disequilibrium between DRw53 and C4AQ0, it is possible that at least 2 genes in the major histocompatibility complex may determine susceptibility to the development of primary SS in the Japanese population.

To study the gene products of the HLA complex, we produced two monoclonal antibodies, termed HU-18 and HU-23. They were active in complement-dependent cytotoxicity and detected B-cell alloantigens encoded by a locus (or loci) linked to HLA. When three types of HLA-DR4 homozygous B-cell lines with different HLA-D specificities were tested for reactivity with HU-18 and HU-23, they displayed distinct reaction patterns depending on the HLA-D specificities they possessed: EBV-Wa (HLA-DYT homozygous), negative for both HU-18 and HU-23; KT2 and KOB (HLA-DKT2 homozygous), positive only for HU-18; and ER (HLA-Dw4 homozygous), positive for both. These differential reaction patterns were further confirmed by testing against a panel of 17 HLA-DR4-positive peripheral blood lymphocytes with known HLA-D specificities. Thus, these monoclonal antibodies allow us to identify HLA-DYT, HLA-DKT2, and HLA-Dw4 solely by serologic methods. This is the first clearcut serologic identification of these three HLA-DR4-associated HLA-D specificities, which have been indistinguishable by conventional serology and identified only by cellular techniques. It is hoped that immunochemical investigations using HU-18 and HU-23 will advance our understanding of the HLA-D region on a molecular level.

Islet Cell-Cytoplasmic Antibodies (ICA) in serum samples obtained from 616 Japanese diabetics were examined. The patients included 296 subjects with insulin-dependent diabetes (IDDM) and 320 with noninsulin dependent diabetes (NIDDM). The number of ICA-positive cases found in 96 subjects in whom the duration of IDDM was under one year was found to be 50% (48/96), though in subjects with a duration over one year, it was only 14.5% (29/200). The prevalence of ICA-positive cases in the NIDDM group was 2.2% (7/320), and none of the nondiabetics had ICA in their serum. Moreover, none of 45 first-degree relatives of 19 patients with IDDM of whom 5 were positive and others were negative for ICA had ICA. Concerning the relationship between HLA-type and ICA in IDDM, there was a tendency for the prevalence of BW54, DR4 and MT3 of HLA to be higher in the ICA-negative group than in the ICA-positive group or the non-diabetic group. In 28 patients with IDDM, both ICA and ICSA were checked. Of 21 patients positive for ICA, only 3 had ICSA, although 3 out of 7 patients with negative ICA were positive for ICSA. Therefore no correlation was found between ICA and ICSA.

Polymorphism in the genes encoding the alpha (alpha), beta (beta) and gamma (gamma) chains of the human T-cell receptors was analyzed both in population and family studies. Against twelve unrelated Japanese, several out of the 15 restriction endonucleases tested, revealed restriction fragment length polymorphism. The segregation of the polymorphic fragments were confirmed among 15 members of three families. In most of the cases paternal and/or maternal haplotypes could be assigned. By testing the polymorphic enzymes among the random healthy Japanese, the frequency of each polymorphic fragment was then determined. Although the polymorphism found in this study was similar to that reported in Caucasians, some differences were observed. Such differences are discussed. The restriction fragment length polymorphism in both population and family studies, derived from alpha, beta and gamma chains of the T-cell receptor found in this report, might be useful markers for genetic analysis of the T-cell function in relation to immunological disorders.

Possible association between HLA antigens and antibody response to rubella vaccine was examined in 71 seronegative adult females, aged from 18 to 23, immunized with QEF vaccine. High responders (HAI antibody titer greater than or equal to 1:32) with HLA-B15 were significantly higher in frequency than those without HLA-B15 (p < 0.05). On the other hand, subjects with HLA-A9 had a tendency toward low immune responsiveness, including all the three non-responders. These results suggest that the antibody response to rubella vaccine may be influenced by the host-genetic factors relating to HLA antigens.