Differentiation of subtypes B and E of human immunodeficiency virus type 1 by polymerase chain reaction using novel env gene primers

Novel sets of env gene PCR primers for distinguishing human immunodeficiency virus type 1 (HIV-1) subtypes B and E were designed. These primers anneal to different regions of the env gene and amplify DNA fragments of distinct sizes in a subtype-specific manner. Blood samples from 11 HIV-1 carriers in Thailand and 46 carriers in Japan were examined by PCR. The new env primers detected HIV-1 proviral DNA in 100% (11/11) and 88% (37/42) of the subtype B and E infection cases, respectively. The env primers also detected proviral DNA in saliva and breast milk samples in seven of 11 cases and two of three cases, respectively. The PCR subtyping results matched completely with those obtained by nucleotide sequencing of the env V3 region. The results suggest that the PCR using the env primers designed in this study may be an accurate and cost-effective method for differentiating subtypes B and E of HIV-1 in a large number of clinical samples. However, subtype E specific primer cross-react with subtype A, C, G, the new primer in this study is useful for regions in South East Asia where subtype E is predominant.     

Association of genetic variation of the RIL gene,encoding a PDZ-LIM domain protein and localized in 5q31.1, with low bone mineral density in adult Japanese women

Twin and family studies had shown that genetic factors are important determinants of bone mass. Multiple genes might be involved. One candidate gene, the reversion-induced LIM gene (RIL), is a PDZ and LIM-domain-containing protein and has been localized within the cytokine cluster of chromosome 5 (5q31.1). In a genetic study of 370 adult Japanese women, we investigated the correlation between radial bone mineral density (BMD) and a genetic variation (−3333T→C) of the 5'-flanking region of RIL gene. A significant association was identified between the RIL variation −3333T→C and radial BMD (r=0.15, P=0.003). The variation of the RIL locus may be an important determinant of osteoporosis.     

HTL V-I from Iranian Mashhadi Jews in Israel is phylogenetically related to that of Japan,India, and South America rather than to that of Africa and Melanesia

A new endemic focus of human T-lymphotropic virus type I (HTL V-I) was recently reported among Mashhadi Jews, a group of immigrants from northeastern Iran to Israel. We extracted DNAs from fresh peripheral blood mononuclear cells (PBMCs) and/or gargle mouthwash from 10 HTL V-I carriers, who consisted of members of one family, and HTL V-I-associated myelopathy (HAM) and adult T-cell leukemia (ATL) patients. Long terminal repeat (LTR) regions of proviral DNAs were sequenced and analyzed phylogenetically. In a phylogenetic tree, all the Mashhadi HTL V-I isolates belonged to subtype A, one of the three subtypes of the cosmopolitan type of HTL V-I, and made a tight cluster distinct from the other isolates of subtype A from Japan, India, the Caribbean Basin, and South America. Although a few nucleotide substitutions were observed among the clones sequenced, no characteristic sequence variation was found in different disease manifestations, even in one family or different sources of DNA preparation.     

Induction of blood-brain barrier properties in immortalized bovine brain endothelial cells by astrocytic factors

The blood-brain barrier (B-BB) protects the free passage of substances into the brain and maintains the homeostasis of the central nervous system. It is commonly accepted that astrocytes surrounding brain endothelial cells influence the B-BB formation and the exhibition of B-BB function of capillaries. To begin the in vitro study on the B-BB, it is essential to obtain a homogenous and sufficient supply of brain endothelial cells as well as astrocytes. We thus immortalized the bovine brain endothelial cell (BBEC) by transfection of the SV40 large T antigen and obtained a single clone, t-BBEC-117, which retained the brain endothelial cell phenotype. Astrocyte in co-culture was found to tighten the intercellular contacts of the immortal cells resulting in a reduced L-glucose permeability, and its conditioned medium (CM) augmented a B-BB phenotype, alkaline phosphatase (ALP) activity. Among known astrocytic factors, only fibroblast growth factor-basic (bFGF) could mimic the actions of astrocytes as measured by L-glucose permeability and ALP activity. Moreover, anti-bFGF antibody canceled 90% of ALP activation by astrocyte CM. Basic FGF, however, failed to induce other B-BB phenotypes such as the expressions of multidrug resistance (mdr) and glucose transporter (GLUT-1) genes. These data suggest that bFGF is one of the most plausible astrocytic factors to induce the B-BB properties of immortal brain endothelial cells together with some unknown factors in the astrocyte CM.     

Immunopathological similarities between IgA nephropathy and Henoch-Schoenlein purpura (HSP) nephritis

A study on the immunopathological similarities between IgA nephropathy and Henoch-Schoenlein purpura (HSP) nephritis is described. Various examinations were performed as follows. (1) Pathological studies: light microscopic findings and immunofluorescent staining; (2) Measurement of the levels of IgA in pharyngeal washings and sera, and those of IgA quantitated by radial immunodiffusion; (3) Elution studies: renal biopsy specimens obtained from patients with IgA nephropathy and HSP nephritis were treated with citrate buffer (pH 3.2) and the "eluate" was neutralized by sodium hydroxide. The "eluate" was then applied to the acid-treated sections obtained from the same and other patients with IgA nephropathy as well as sections from patients with HSP nephritis and other glomerular diseases. The sections were stained with FITC-conjugated heavy chain specific antihuman IgA antisera and then examined with a fluorescent microscope. There were no differences in pathological findings of IgA nephropathy and HSP nephritis in the light microscopic and immunofluorescent examinations. The levels of IgA in pharyngeal washings and sera were significantly increased in patients with both diseases. IgA antibodies deposited in kidneys from patients with HSP nephritis crossreacted with kidneys from some patients with IgA nephropathy, and vice versa. However, antibodies from patients with IgA nephropathy and HSP nephritis did not react with normal glomeruli or other nephritic glomeruli. It is concluded that there are some immunopathological similarities between IgA nephropathy and HSP nephritis.     

Possible role of autoantibodies against nephrin in an experimental model of chronic graft-versus-host disease

Nephrin, a product of the NPHS1 gene, is a component of the slit diaphragms that are found between glomerular foot processes and is a crucial element for glomerular filtration barrier. Recently, nephrin has been focused in a number of studies of proteinuria development including various types of acquired glomerular diseases including minimal change nephrotic syndrome and membranous nephropathy. However, the precise role of nephrin in such acquired glomerular diseases is still unknown. To analyse the role of nephrin further, two kinds of anti-nephrin antibodies were raised in the rabbits and applied to an experimental mouse model of chronic graft-versus-host disease, in which (C57BL/10 x DBA/2) F1 mice developed clinically apparent severe proteinuria with significant glomerular lesions 7 weeks after parental DBA/2 cell transfer. Antibody-sandwich ELISA detected anti-nephrin antibodies during week 2 to week 6, with the peak at week 2 or week 4. Colocalization of nephrin and IgG on week 4, week 6, and week 8 was revealed by confocal microscopic analysis, suggesting that in situ immune complex formation with nephrin in glomerular lesion. Taken together, it seems to be suggested nephrin and its autoantibody have a certain role in the development of glomerular lesion in our model mice.     

Infection of macaques with chimeric simian and human immunodeficiency viruses containing Env from subtype F

Chimeric simian and human immunodeficiency viruses (SHIVs) are useful for investigating the pathogenicity of human immunodeficiency virus (HIV-1) and to develop an anti-HIV-1 vaccine. We attempted to construct SHIVs containing Env from various subtypes, because almost all SHIVs which have been reported so far have Env from HIV-1 that belongs to subtype B. Two infectious SHIVs containing Env from two strains of HIV-1, CMR304 and CMR306, which belong to subtype F and A, respectively, were newly obtained. These SHIVs essentially showed a coreceptor usage and a neutralization pattern that were similar to those of the parental HIV-1s. In macaque PBMC, SHIVcmr304 replicated with kinetics similar to that of prototypic SHIV-NM-3rN with HIV-1 NL432 Env, but SHIVcmr306 replicated poorly. Inoculation of four rhesus macaques with SHIVcmr304 resulted in an increase of plasma viral load in all the macaques, though viral RNA copies were 100-fold lower than that in the infection with NM-3rN. This SHIV containing Env from HIV-1 subtype F will be a valuable source for the analysis of HIV-1 subtype F and the evaluation of vaccine candidates as a genetically divergent challenge virus.     

The effect of prednisolone on substance P-induced vascular permeability in mice

The effect of prednisolone on the substance P (SP)-induced vascular permeability increase in male ddY, WBB6 F₁–+/+ (control) and WBB6 F₁-W/W^v (no mast cell in skin or internal organs) mice was investigated. 1) SP (1–10 000 pg/site) increased vascular permeability in ddY, WBB6 F₁–+/+ and WBB6 F₁-W/W^v mice ears. 2) SP (100 pg/site)-induced vascular permeability was inhibited by prednisolone (10 mg/kg) administered intraperitoneally 3 to 12 hours prior to the elicitation of the reaction in ddY mice. When dexamethasone at a dose of 1 mg/kg was administered intraperitoneally 2 to 24 hours prior to the elicitation of the reaction, significant inhibition was observed. When prednisolone was administered intraperitoneally 8 hours prior to the elicitation of the reaction, the SP-induced capillary permeability increase in both ddY and WBB6 F₁-W/W^v mice was clearly inhibited by the drug at doses of 5 and 10 mg/kg. 3) Diphenhydramine (1 and 10 mg/kg) inhibited SP-induced vascular reaction in ddY mice but not in WBB6 F₁-W/W^v mice. 4) Atropine (10 mg/kg) inhibited SP-induced vascular reaction in both ddY and WBB6 F₁-W/W^v mice. But acetylcholine did not cause an increase of vascular permeability in ddY and WBB6 F₁-W/W^v mice ears. 5) Prednisolone (5 mg/kg) inhibited histamine- and serotonin-induced vascular permeability in ddY and WBB6 F₁-W/W^v mice ears. 6) Prednisolone (5 and 10 mg/kg) inhibited the SP-induced histamine release from ddY mice peritoneal mast cells. These results suggest that the vascular effect of SP is mediated by both mast cell dependent (release of histamine from mast cells) and mast cell independent mechanisms. Prednisolone inhibits the SP-induced vascular permeability mediated by both mechanisms in mice.     

The N-terminal flanking region of the invariant chain peptide augments the immunogenicity of a cryptic "self" epitope from a tumor-associated antigen

The N-terminal flanking region of the invariant chain peptide termed CLIP appears to have superagonistic properties interacting with the T cell receptor and the MHC class II molecule at or near the binding site for the bacterial superantigen Staphylococcal enterotoxin B (SEB). The present studies explored the hypothesis that the N-terminal segment of CLIP can augment the immunogenicity of cryptic "self" tumor-associated antigens. A chimeric construct of an MHC class II binding peptide from the c-erb oncogene (Her-2/neu) containing the N-terminal flanking region of CLIP elicited potent antitumor activity against a Her-2/neu-positive tumor in a rat model system. Comparatively, the unmodified parent peptide was ineffective. The induction of effective antitumor immunity, however, required presentation of the chimeric peptide construct on irradiated tumor cells or the peptide construct in concert with a Her-2/neu MHC class I-restricted peptide from Her-2/neu. As revealed by adoptive transfer studies, effective protective antitumor immunity in this setting required the CD4 T helper subset. Additionally, in vitro analysis revealed that immunization with the parent peptide resulted in a weak immune response to the unmodified peptide consisting of both type 1 (IL-2, IFN-gamma) and type 2 (IL-4, IL-10) cytokine-producing cells analyzed by RT-PCR (qualitative and quantitative) and by limiting dilution assay. Comparatively, immunization with the chimeric construct elicited a potent immune response to the parent peptide with predominantly type 1 cytokine-producing cells. Taken together, the results suggest that immunization with the chimeric Her-2/neu peptide induced protective antitumor immunity. Associated with this immunization strategy was the enhancement of a type 1 cytokine response.