Novel single nucleotide polymorphisms of the human nuclear factor kappa-B 2 gene identified by sequencing the entire gene

The nuclear factor kappa-B 2 (NFKB2) gene is a member of the NFKB/Rel gene family, which is known to be a pivotal regulator of the acute phase of the inflammatory response and of immune responses. We identified three novel single nucleotide polymorphisms (SNPs) and determined their allelic frequencies, as determined by the sequencing of 48 alleles of the entire gene in a Japanese population sample. Two of the three polymorphisms were identified at nucleotide (nt) position 1837 (T/C) and nt position, 1867 (GG/G) in the upstream region of the gene. The other polymorphism was identified at nt position 2584 (G/T) within intron 1. These polymorphisms will be useful in genetic studies of the processes involved in inflammatory responses and in bone differentiation. Received: October 17, 2000 / Accepted: October 23, 2000     

Reference ranges for haematology, biochemical profile and electrophoresis in a single herd of Ragusana donkeys from Sicily (Italy)

Donkeys, an endangered species, have recently gained a new application with the use of their milk to feed humans with allergic processes. The Ragusana donkey breed from Sicily is used to produce milk for humans with allergic diseases. In order to evaluate the hygienic, nutritional and management measures on a farm of Ragusana donkeys, complete blood counts, extended biochemical profiles and serum protein electrophoresis, as part of metabolic profile test (MPT), were performed in Ragusana donkeys. Fifty-four donkeys were studied and grouped according to their age, (1) 29 females and a single stallion (n=30), (2) young females, 1 – 3 years old (n=10) and (3) young of both sexes under 1 year old (n=14). The RBC count, RDW value, Lymp, and Mono counts, and PDW values were statistically greater in donkeys under one year old than in adult donkeys, while the Seg Neu count was lower. The CPK, ALP, iPhos, and HCO₃, values were statistically higher in the group of donkeys under 1 year of age than adult donkeys while Cl and LDH values were statistically lower in donkeys under 1 year than adult donkeys. Additionally, statistically significant increased values for CPK, ALP, Alb, Chol, iPhos, HCO₃, and UIBC in young donkeys under 1 year when compared with young donkeys, 1 – 3 years were observed. A statistically significant decreased value for Urea and an increased value for Crea in young donkeys, 1 – 3 years old were found as compared to adults. The serum protein fractions recognised by electrophoresis were: albumin, alpha globulin (subdivided into alpha-1 and alpha-2-globulins), beta globulin, and gamma globulin. In the alpha-1-globulin region three small peaks were constantly noticed, and alpha-2-globulins were statistically different between the three groups being greater in young donkeys under 1 year of age. The results obtained were used both to establish reference ranges and a data bank for the farm of Ragusana donkeys for future needs in assessing the metabolic status and health of the animals.     

Human and Murine Immune Responses to a Novel Leishmania major Recombinant Protein Encoded by Members of a Multicopy Gene Family

Vaccination of BALB/c mice with Leishmania major promastigote culture filtrate proteins plus Corynebacterium parvum confers resistance to infection with L. major. To define immunogenic components of this protein mixture, we used sera from vaccinated mice to screen an L. major amastigote cDNA expression library. One of the immunoreactive clones thus obtained encoded a novel protein of L. major with a molecular mass of 22.1 kDa. The predicted amino acid sequence of this clone exhibited significant homology to eukaryotic thiol-specific-antioxidant (TSA) proteins. Therefore, we have designated this protein L. major TSA protein. Southern blot hybridization analyses indicate that there are multiple copies of the TSA gene in all species of Leishmania analyzed. Northern blot analyses demonstrated that the TSA gene is constitutively expressed in L. major promastigotes and amastigotes. Recombinant TSA protein containing an amino-terminal six-histidine tag was expressed in Escherichia coli with the pET17b system and was purified to homogeneity by affinity chromatography. Immunization of BALB/c mice with recombinant TSA protein resulted in the development of strong cellular immune responses and conferred protective immune responses against infection with L. major when the protein was combined with interleukin 12. In addition, recombinant TSA protein elicited in vitro proliferative responses from peripheral blood mononuclear cells of human leishmaniasis patients and significant TSA protein-specific antibody titers were detected in sera of both cutaneous-leishmaniasis and visceral-leishmaniasis patients. Together, these data suggest that the TSA protein may be useful as a component of a subunit vaccine against leishmaniasis.     

Identification of the binding sites to monoclonal antibodies on A/USSR/90/77 (H1N1) hemagglutinin and their involvement in antigenic drift in H1N1 influenza viruses

We have determined nucleotide sequences of the HA1 portion of the hemagglutinin (HA) gene of the parental A/USSR/90/70 (H1N1) virus and its eight variants selected in vitro with six monoclonal antibodies to study antigenic determinants. The HA1 gene of one of the variants (B-1-23) was cloned in bacteria and its nucleotide sequence was determined by the Maxam-Gilbert method. The nucleotide sequence of the variant was confirmed by the dideoxy chain termination method. The gene sequences of the other viruses were determined by the latter method. Three variants with reduced reactivity in HI test only with the selecting antibodies possessed one amino acid substitution. On the other hand, most other variants which had the changed reactivity to multiple antibodies in HI test possessed more than one substitution. Comparison of the amino acid sequences of the HA1 molecule, deduced from the nucleotide sequences, suggested that the monoclonal antibodies W18 and 264 reacted with epitopes located on the area involving amino acid residues 125C and 189-190, respectively, whereas, the antibodies 22 and 70 reacted with epitopes involving amino acid residues 129, 132, and 157. The epitope recognized by antibody 110 overlapped with that of W18, and the epitope recognized by antibody 385 was located on the area involving at least amino acid residues 129, 159, and 189, which overlapped with some of the above epitopes. The sequence analysis with B-1-23 variant selected with antibody 264 clearly showed that in A/USSR/77 viruses, a single substitution at amino acid residue 190 effectively changes the epitope and caused a significant antigenic variation detectable by postinfection ferret sera.     

A girl with 1p36 deletion syndrome and congenital fiber type disproportion myopathy

Chromosome 1p36 deletion syndrome is characterized by hypotonia, moderate to severe developmental and growth retardation, and characteristic craniofacial dysmorphism. Muscle hypotonia and delayed motor development are almost constant features of the syndrome. We report a 4-year-old Japanese girl with 1p36 deletion syndrome whose muscle pathology showed congenital fiber type disproportion (CFTD) myopathy. This is the first case report of 1p36 deletion associated with CFTD. This association may indicate that one of the CFTD loci is located at 1p36. Ski proto-oncogene −/− mice have phenotypes that resemble some of the features observed in patients with 1p36 deletion syndrome. Because fluorescent in situ hybridization analysis revealed that the human SKI gene is deleted in our patient, some genes in 1p36, including SKI proto-oncogene, may be involved in muscle hypotonia and delayed motor development in this syndrome. Received: March 4, 2002 / Accepted: July 7, 2002     

Retroviral gene therapy of collagen-induced arthritis by local delivery of IL-4

Rheumatoid arthritis (RA) is an autoimmune arthritis, for which treatment options remain limited. This study investigated the potential role of adoptive cellular gene therapy as a novel means for treating the RA animal model collagen-induced arthritis (CIA). Adoptive transfer of antigen-specific T-cell hybridomas retrovirally transduced to express IL-4 1 day before booster immunization significantly reduced the number of inflamed joints. Cell transfer after clinical onset of disease had no therapeutic effect. Bioluminescence imaging showed that the hybridomas migrated to the inflamed joints, thus delivering the regulatory protein locally at the site of inflammation. The homing was, at least in part, due to chemotaxis in response to proinflammatory chemokines that are expressed in inflamed joints. There were no significant changes in the cytokine milieu of the draining lymph nodes, nor in the systemic levels of anti-collagen antibodies in treated mice. We conclude that the beneficial clinical effects observed in our model were most likely based on the local action(s) of IL-4 in the inflamed joints and that the local delivery (and effects) of regulatory cytokines, like IL-4, constitutes a novel and effective method of preventing organ-specific autoimmune disease and of minimizing systemic adverse effects.     

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.