ROUTINE LOW AND HIGH RESOLUTION TYPING OF THE HLA-DRB GENE USING THE PCR-MPH (MICROTITRE PLATE HYBRIDIZATION) METHOD

We describe HLA-DRB1 typing using polymerase chain reaction-based microtitre plate hybridization (PCR-MPH), which can process large numbers of samples. MPH typing is similar to an enzyme-linked immunosorbent assay (ELISA), in which a tandemly ligated sequence-specific oligonucleotide is immobilized on microtitre wells. The typing procedure consisted of two steps. In the first, PCR-MPH with 16 probes was performed to determine the specificities of the serological levels (DR1, DR2, DR3, DR4, DR11, DR12, DR13, DR14, DR7, DR8, DR9 and DR10) after generic amplification (‘low resolution typing’). In the second step, DR1, DR2, DR4, DR 12/8 and DR3/11/13/14 were group-specifically amplified based on the results of the first PCR-MPH, and microtitre plate hybridization proceeded in a similar manner to the first step (‘high resolution typing’). Low resolution typing was completed within 2 h after generic amplification, and the results of high resolution typing were obtained in another 3.5 h after amplification. The allelic types classified using PCR-MPH were completely concordant with those obtained by PCR- single-strand conformation polymorphism or PCR-restriction fragment length polymorphism.     

Distribution of 5'-nucleotidase activity in greater omentum of rats

The aim of the present study is to demonstrate the cellular basis of 5'-nucleotidase (5'-Nase) activity in the greater omentum of rats. Enzyme histochemistry for 5'-Nase showed that lymphatic vessels in the omentum as well as lymphocytes in the milky spots were positively stained. Electron microscopic observation revealed-5'-Nase activity at the luminal surface of the lymphatic endothelial cells, pinocytotic vesicles in the endothelial cells and the surface of fibroblasts located at the intercellular space of adipose cells. Fibroblasts extended long cytoplasmic processes toward adipose cells and inflammatory cells. These findings suggest that lymphatic endothelial cells as well as fibroblasts in the omentum may play an important role in regulation of metabolism and immune mechanisms in the greater omentum by supplying adenosin.     

Molecular cloning and expression analysis of a macrophage-colony stimulating factor receptor-like gene from rainbow trout, Oncorhynchus mykiss

The M-CSF and its receptor (M-CSFR, CSF-1R or c-fms proto-oncogene) system were initially implicated as essential in mammals for normal monocyte development as well as for pregnancy. To allow a comparison with the M-CSF and M-CSFR system of an oviparous animal, we cloned a M-CSFR-like gene from rainbow trout (Oncorhynchus mykiss). The gene was cloned from a cDNA library of head kidney. It contained an open reading frame encoding 967 amino acids with a predicted size of 109 kDa. The putative amino acid sequence of rainbow trout M-CSFR showed 54% amino acid identity to fugu (Takifugu rubripes) M-CSFR, 52% to zebrafish (Danio rerio) M-CSFR and 40% to mouse (Mus musculus) and human (Homo sapiens) M-CSFR. The M-CSFR-like gene was constitutively expressed in head kidney, kidney, intestine, spleen and blood. The gene was detected especially in the ovary of immature female rainbow trout. These results suggest that a M-CSFR-like receptor may be involved in female reproductive tracts even in an oviparous animal like fish.     

Immunohistochemical localization of glomerular basement membrane antigens in various renal diseases

Summary The immunofluorescent localization of glomerular basement membrane (GBM) antigens was examined in 52 specimens from normal kidneys and in various renal diseases using antisera to human GBM HGBM), IV type collagen (IV Col) and P3 antigen, a rat nephritogen. Anti-HGBM serum normally stained the GBM and the mesangium in a restrictive pattern, anti-IV Col serum stained the GBM and the mesangium in a wider pattern and anti-P3 serum stained only the GBM. In mesangial proliferative glomerulonephritis, including IgA nephropathy pathy and Henoch-Schönlein nephritis, the widened mesangial areas were stained with anti-HGBM and anti-IV Col sera. In membranous nephropathy, the punched-out lesions of thickened GBM were demonstrated with the three antisera in moderate cases and a double linear distribution with fine granulation with anti-HGBM and anti-IV Col sera were revealed in one severe case. In membranoproliferative glomerulonephritis, the expanded mesangium and thickened capillary walls were stained with anti-HGBM and anti-IV Col sera, while the outer line of glomerular capillary walls was only positive with anti-P3 serum. In crescentic glomerulonephritis, the collapsed glomerular tufts were stained normally with anti-HGBM and anti-P3 sera and weakly with anti-IV Col serum. In diabetic nephropathy, anti-HGBM serum stained the GBM in a double linear distribution without reacting with the expanded mesangium; anti-IV Col serum stained the mesangium and the GBM in a less clear double linear fashion while anti-P3 serum stained the GBM as single line. Thin membrane disease and Alport's syndrome had normal reactivity with all antisera. However, in one case of Alport's syndrome anti-HGBM and anti-P3 sera stained the GBM in a focal and segmental pattern, while normal staining with anti-IV Col serum was found. In lesions with adhesions and crescents the staining was positive for HGBM and IV Col and negative for P3; obsolescent glomeruli were stained with anti-HGBM and anti-P3 sera, and had diminished staining with anti-IV Col serum.The identification of the various structural glomerular antigens is useful in the classification of certain types of glomerular diseases. Further insight into the mechanisms underlying these conditions may be obtained in this way.     

Genetic control of resistance to Mycobacterium intracellulare infection in mice.

The susceptibilities of various strains of mice to a highly pathogenic strain of Mycobacterium intracellulare, the Mino strain, were determined by intravenous injection of 5 X 10(6) bacteria. CFU were counted on days 1 and 21 of infection. Among 10 strains of mice, C57BL/6, C57BL/10, BALB/c, B10.BR, B10.A, and B10.D2 were susceptible, whereas DBA/2, A/J, CBA, and C3H/He were resistant. In the susceptible mouse strains, the number of bacteria increased during 21 days of infection, whereas no bacterial growth was observed in the resistant strains. Susceptible mice showed weak but positive delayed-type hypersensitivity to M. intracellulare purified protein derivative 20 days after injection of bacteria. Resistant mice developed no delayed-type hypersensitivity. Histological examination showed severe granulomatous lesions in livers or spleens of the susceptible mice after M. intracellulare injection. Analysis of F1 hybrids of susceptible and resistant strains and of F2 and backcross mice showed that the resistance to M. intracellulare seems to be controlled genetically by a single dominant gene. The pattern of distribution of resistance to M. intracellulare among the mouse strains was consistent with that of natural resistance to Mycobacterium bovis to BCG. Thus, resistance to M. intracellulare infection may be regulated by a gene linked to the Bcg gene on chromosome 1.     

Autoreactive CD4- CD8- alpha beta T cells to vaccinate adjuvant arthritis.

Studies suggested that experimental autoimmune diseases can effectively be prevented and treated by application of normal autoreactive T cells or autoreactive T cells in an attenuated form. In this study, several autoreactive CD4- CD8- T-cell clones (A2, A6, and A13 cells) were isolated for the first time from the draining lymph nodes of Lewis rats with adjuvant arthritis (AA). Surprisingly, intraperitoneal inoculation with A13 cells, but not A2 or A6 cells protected rats from AA both clinically and histologically. It was demonstrated that A13 cells were CD4- CD8- alpha beta T cells, and showed proliferative responses to irradiated syngeneic spleen cells (antigen-presenting cells; APC). Interestingly, A13 cells proliferated against concanavalin A (Con A) and staphylococcal enterotoxin B (SEB), but did not show any proliferation to Mycobacterium tuberculosis (Mt), or its 65 000 MW heat-shock protein (HSP). Rats protected from AA by inoculation with A13 cells showed a specific anti-idiotypic delayed-type hypersensitivity reaction compared with other autoreactive T cells (A2 or A6 cells). These findings demonstrate that AA can be suppressed by autoreactive CD4- CD8- alpha beta T cells, and these cells may be used as therapeutic agents in experimental autoimmunity.     

IMMUNOELECTRON MICROSCOPIC LOCALIZATION OF THYROGLOBULIN IN HUMAN FOLLICULAR ADENOMA

An electron microscopic immunohistochemical localization of thyroglobulin (TG) using PAP methods has been made in 15 cases of cold follicular adenoma. All cases of follicular adenoma showed organ specific functions such as synthesis, storage, reabsorption, and hydrolysis of thyroglobulin except for an area composed of follicular cells with trabecular arrangement. Immuno-reaction product for TG was precisely demonstrated in follicular lumina, subapical vesicles and reabsorbed colloid droplets. The reaction product observed in the follicular lumen was clearly demarcated from the cytoplasm of the follicular cells by the apical plasma membrane. The subapical vesicles ranging approximately from 50 mμ to 300 mμ in diameter were rarely observed in follicular adenoma and some of them fused with the reabsorbed colloid droplets. The reabsorbed colloid droplets usually had the intense reaction product and hydrolyzed colloid droplets had a vacuole containing floccular low electron dense materials. There is no reaction product in rough endoplasmic reticulum and Golgi complexes.     

Functionality of chimeric Rev proteins of HIV/SIV

Studies on functional compatibility of various Rev proteins derived from all known human and simian immunodeficiency virus subgroups have shown that this essential gene product is not always exchangeable among the viruses. In an attempt to map the region of Rev proteins responsible for the observed nonreciprocal complementation, hybrid genomic Rev expression vectors were constructed by exchanging the first and second exons ofrev genes, and were examined for their abilities to activate reporter clones by transfection. With one exception, the second coding exon ofrev gene determined the functional specificity of Rev proteins.     

Compared colonizing and immunizing efficiency of toxinogenic (A+ B+) Vibrio cholerae and an A- B+ mutant (Texas Star-SR) studied in adult rabbits

Four strains of Vibrio cholerae O1 were compared for their ability to colonize and immunize adult rabbit intestine. Three were virulent, toxinogenic (A+ B+) isolates, and one, an A- B+ mutant (Texas Star-SR), was derived by mutagenesis with nitrosoguanidine. When given orally to nonimmune rabbits, virulent strains colonized the small bowel with similar efficiency, whereas Texas Star-SR colonized poorly. Rabbits fed less than 50 CFU of an A+ B+ strain developed marked serotype-specific resistance to recolonization. In contrast, Texas Star-SR evoked resistance to reinfection less efficiently, with a minimum immunizing dose of 10(5) CFU when given once or 10(3) CFU when given twice. Oral inoculation with an A+ B+ strain also evoked vigorous, dose-dependent mucosal antitoxin responses; comparable inocula of Texas Star-SR were much less effective, causing antitoxin responses that were 90 to 95% smaller. Finally, rabbits inoculated once with 10(4) CFU of an A+ B+ strain were markedly protected against experimental cholera or fecal shedding of V. cholerae when challenged with 10,000 times the 50% effective dose of a virulent strain by the RITARD technique. In contrast, an inoculum of 10(4) CFU of Texas Star-SR was nonprotective, and 10(10) CFU was only partially protective. These studies reveal the remarkable efficiency with which virulent V. cholerae evokes intestinal immunity to recolonization or experimental cholera and show that the A- B+ mutant, Texas Star-SR, is substantially less effective.