JC virus genotyping using formalin-fixed, paraffin-embedded renal tissues

Recently genotyping of JC virus (JCV) DNA in renal tissue was reported to be useful to identify the geographic origin of unidentified cadavers. In the above study, autopsied tissue samples without storage or stored in a frozen state were used. This study examined JCV DNA sequence modifications caused by formalin-fixation, in an attempt to elucidate whether formalin-fixed, paraffin-embedded tissue samples can also be used to determine the genotypes of JCV DNA in the kidney. In four cases, a 610 bp typing region of the JCV genome was PCR-amplified from renal tissues stored for 1 year in three different states: frozen at -80 degrees C [Amaker, B.H., Chandler, F.W., Huey, L.O., Colwell, R.M., 1997. Molecular detection of JC virus in embalmed, formalin-fixed, paraffin-embedded brain tissue. J. Forensic Sci., 1157-1159], formalin-fixed, paraffin-embedded [Ault, G.S., Stoner, G.L., 1992. Two major types of JC virus defined in progressive multifocal leukoencephalopathy brain by early and late coding region DNA sequences. J. Gen. Virol. 73, 2669-2678], and soaked in 5% formalin [Baksh, F.K., Finkelstein, S.D., Swalskey, P.A., Stoner, G.L., Ryschkewitsch, C.F., Randhawa, P.R., 2001. Molecular genotyping of BK and JC virus in human polyomavirus-associated interstitial nephritis after renal transplantation. Am. J. Kidney Dis. 38 (2), 354-365]. The amplified fragments were cloned, and the resultant clones were sequenced. In frozen samples, single sequences ('original' sequences) were detected in all cases. In formalin-fixed, paraffin-embedded samples, not only the original sequences but also those with 1-6 base substitutions were detected. From formalin-soaked samples, the original sequences and those with 1-5 and 10-13 substitutions were detected. The genotyping of JCV DNA was not hampered by the presence of 1-6 substitutions, but a shift in JCV genotypes was observed in sequences with 10-13 substitutions. Thus, it was concluded that the genotypes of JCV DNA in the kidney can be determined only with specimens stored in a frozen state or formalin-fixed for a short time.     

Non-A, non-B hepatitis related AN6520 Ag is a normal cellular protein mainly expressed in liver. II

Detection of AN6520 Ag/Ab in human sera had indicated a close association with non-A, non-B hepatitis (NANBH). In this study, we investigated the immunochemical nature of AN6520 Ag and measured the amounts in various human and chimpanzee organs in order to clarify the association with NANBH. AN6520 Ag was found to be composed of polypeptide(s) with an apparent molecular weight of 45,000 daltons (45 kD), which are noncovalently linked together. Human antibodies in convalescent sera from NANBH patients as well as monoclonal antibodies were found to recognize only the high-order structure of the antigen, whereas rabbit antibody recognized both the high-order structure and the reduced form of 45 kD polypeptide(s). AN6520 Ag could be detected in most of the livers tested including those without any liver damage and fetal livers; their amounts varied considerably from each other. The antigen could be detected also in organs other than liver, but in contrast to liver, the amounts were small and did not vary as much between individuals. From the data of immunoblotting using rabbit antibody, our observed variation of antigen content in liver was considered to be due to the difference in expression of 45 kD polypeptide(s). Although no specific relationship was found between the amount of the antigen in liver and NANBH, the antigen was found to increase several times in livers of chimpanzees after the inoculation of NANBH virus. These data suggest that AN6520 Ag is a normal cellular protein existing mainly in liver and that its quantity may vary under some conditions such as NANBH.     

HLA-DP and susceptibility to insulin-dependent diabetes mellitus in Japanese

ABSTRACT: Human leukocyte antigen (HLA) genes are candidates for susceptibility genes in insulin-dependent diabetes mellitus (IDDM). Recently, the association of DR and DQ with IDDM has been reported, but the role of HLA-DP genes remains uncertain. To address the question, we analyzed the DPB1 gene of 20 Japanese IDDM patients and 30 control subjects using a combination of polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis (PCR-RFLP method). DPB1*0501 was the most frequent allele both in Japanese patients and control subjects. There was no appreciable association between IDDM and the DPB1 allele in Japanese. The absence of association between IDDM and DP, in spite of the known association between this disease and both DR and DQ, suggests that the HLA locus (loci) telomeric to DP encodes susceptibility to IDDM.     

Mutation analysis of two Japanese patients with Fanconi-Bickel syndrome

Fanconi-Bickel syndrome (FBS), or glycogen storage disease type XI, is a rare autosomal recessive disorder characterized by hepatorenal glycogen accumulation, Fanconi nephropathy, and impaired utilization of glucose and galactose. Recently, this disease was elucidated to link mutations in the glucose transporter 2 (GLUT2) gene. Only three mutations in three FBS families have been reported. Therefore, it is important to elucidate mutations in the GLUT2 gene in FBS by answering the question of whether the syndrome is a single gene disease. In this report, we describe two patients in two unrelated families clinically diagnosed with FBS. No mutation in the entire protein coding region of the GLUT2 gene was detected in patient 1, which suggested that no mutation existed in the GLUT 2 gene, or that some mutations had affected the expression of the GLUT 2 gene. In patient 2, a novel homozygous nonsense mutation (W420X, Trp at codon 420 to stop codon) was detected. These results support the correlation between GLTU2 gene mutation and FBS syndrome. However, many patients must be analyzed to determine whether other genes are involved in FBS. Received: July 16, 1999 / Accepted: September 3, 1999     

Human marrow cells-derived cultured bone in porous ceramics

From four patients (mean age, 60 years; range 51-76 years), 3 ml of bone marrow was collected from the ilium. The marrow was cultured to concentrate and expand the marrow mesenchymal cells on a culture dish. The cultured cells were then subcultured either on another culture dish or in porous areas of hydroxyapatite ceramics in the presence of dexamethasone and beta-glycerophosphate (osteogenic medium). The subcultured tissues on the dishes were analyzed by scanning electron microscopy (SEM), and subcultured tissues in the ceramics were implanted intraperitoneally into athymic nude mice. Vigorous growth of spindle-shaped cells and a marked formation of bone matrix beneath the cell layers was observed on the subculture dishes by SEM. The intraperitoneally implanted ceramics with cultured tissues revealed thick layer of lamellar bone together with active osteoblasts lining in many pore areas of the ceramics after 2 months. The in vitro bone formation on the culture dishes and in vivo bone formation in porous ceramics were detected in all cases. These results indicate that we can assemble an in vitro bone/ceramic construct, and due to the porous framework of the ceramic, the construct has osteogenic potential similar to that of autologous cancellous bone. A significant benefit of this method is that the construct can be made with only a small amount of aspirated marrow cells from aged patients with little host morbidity.     

Establishment and characterization of αs1-casein–specific T-cell lines from patients allergic to cow’s milk: Unexpected higher frequency of CD8 T-cell lines

To study cow’s milk allergy at the cellular level, we assessed the reactivity of peripheral blood mononuclear cells from patients allergic to cow’s milk to α_s1-casein, which is one of the major allergens in cow’s milk. Proliferation of the cells to α_s1-casein activation showed a rather weak response. Therefore to understand T-cell reactivity to α_s1-casein in more detail, we prepared α_s1-casein–specific T-cell lines from patients allergic to cow’s milk and established 26 T-cell lines. These T-cell lines could be classified into three groups by analyzing their surface marker expression: those containing predominantly CD4⁺CD8^- T cells, those containing both CD4⁺CD8^- and CD4^-CD8⁺ T cells, and those containing predominantly CD4^-CD8⁺ T cells. The CD8⁺ T cells were obtained at an unexpectedly higher frequency from the patients. These T-cell lines produced interferon-γ and IL-4. These results suggest that CD8⁺ T cells specific for α_s1-casein and CD4⁺ T cells were primed by the stimulation with α_s1-casein in patients allergic to milk and that both T cells may play a key role in the onset, progression of, or recovery from cow’s milk allergy. (J ALLERGY CLIN IMMUNOL 1996;97:1342-9.)     

Neutralizing activity of the antibodies against two kinds of envelope glycoproteins of Sendai virus

Summary Murine monoclonal antibodies against the fusion (F) and hemagglutininneuraminidase (HN) proteins of Sendai virus (SV) were prepared and studied on their antiviral activities, particularly on the neutralization of infectivity. On the analysis with solid phase competitive ELISA, 26 anti-HN antibodies were divided into at least four groups (HN-I, -II, -III and -IV). Antigenic sites recognized by the HN-I, -II, and -III group antibodies topographically separate from each other. Sites recognized by the HN-IV group antibodies overlaps partially with ones recognized by the HN-I, HN-II and -III group antibodies. The antibodies belonging to the HN-III group highly neutralize the infectivity of SV and weakly or not at all inhibit the hemagglutination (HA). In contrast, the HN-IV group antibodies strongly inhibit HA, but weakly neutralize the infectivity. Adsorption of SV to chicken red blood cells or L cells is inhibited by the HN-IV antibodies, but scarcely by the HN-III antibodies. On the other hand, incubation with HN-III antibodies of HeLa cells that have been preadsorbed with SV at 4° C, followed by culture at 37° C, causes inhibition of infection, but the HN-IV antibodies do not effectively interfere with such infection.The competitive ELISA showed that 17 anti-F antibodies were divided into two groups (F-I and -II). Two antigenic sites recognized by the antibodies, however, seem to be near to each other because a certain competition is observed between the antibodies of both groups. Two of the seven antibodies belonging to the F-II group inhibit the hemolysis activity and also neutralize the infectivity of SV, but the other five F-II antibodies do not. One of the anti-F antibodies has a low HI activity, and, in competition tests, competes with one of the anti-HN antibodies (HN-IV).With 2 Figures     

Identification of the defects in the hemagglutinin gene of two temperature-sensitive mutants of A/WSN/33 influenza virus

Two temperature-sensitive mutants of WSN influenza virus, ts-61S and ts-134, possess defects in the hemagglutinin (HA) gene. These defects are characterized as a defective intracellular transport of the HA at the nonpermissive temperature and a marked thermolability. The nucleic acid sequences of the HA gene of these two viruses, as well as a series of revertant viruses, were determined. The deduced amino acid sequences demonstrate that the HA of ts-61S varied from the wild type protein by three amino acids while that of ts-134 differed by two residues. For both mutants, analysis of revertant viruses indicated that the phenotype of transport inhibition at the nonpermissive temperature and heat lability were associated with a single amino acid change in the globular portion of the molecule. In the case of ts-61S, the critical change in the HA was the replacement of a serine residue at position 110 with that of a proline. The mutational defect in the HA of ts-134 was due to the substitution of a tyrosine residue at position 159 with that of a histidine residue. Four of five revertants of ts-134 were suppressor revertants, of which some of the compensatory changes did not restore thermostability to the HA.     

Detection of human papillomavirus DNA in invasive cervical cancers by the polymerase chain reaction and its clinical significance.

In order to detect human papillomavirus (HPV) DNA in invasive cervical cancers, three different polymerase chain reactions to amplify different subgenomic fragments of HPV DNA were carried out on DNA extracted from 93 formalin-fixed and paraffin-embedded tumor tissues. This study detected HPV DNA in 54 cases (58.1%), which broke down to HPV 16 in 39 (41.9%) cases, HPV 18 in six (6.4%), HPV 52 in three, HPV 33 in one and unclassified HPV type in the remainder. Histopathologically, squamous cell carcinomas frequently contained HPV 16, whereas, HPV 18 was present in adenocarcinoma, adenosquamous cell carcinoma and small cell carcinoma of the cervix. Clinicopathological study revealed that HPV 16 and 18 DNA found were more frequently than other HPV subtypes in premenopausal patients. Moreover, HPV 18 DNA-positive cancers had a relatively high recurrence rate. These results indicate that cervical cancers might be clinically influenced by the difference in subtypes of the infecting HPV.