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.     

Fine structure of hepatic sinusoids and their development in human embryos and fetuses

The fine structure of the hepatic sinusoids of 81 human embryos and fetuses and their development from 5 to 12 weeks gestation were studied. At 5 weeks gestation, sinusoid-like structures and Kupffer-like cells were observed between liver cell cords. Between 6 and 8 weeks gestation the sinusoids were completely developed. Definite Kupffer cells appear at this developmental stage, when the bone marrow has not yet formed. Floating macrophages form cell aggregates in the sinusoids which contact endothelial cells and settle as Kupffer cells. Erythroblastophagia is observed in Kupffer cells and macrophages. The endothelial linings are closed, with the attenuated cell processes and intercellular junctions between the adjoining endothelial cells. No transition was observed between Kupffer cells and endothelial cells. The findings suggest that Kupffer cells in the human embryo are extrahepatic in origin and that they reach the sinusoids via the circulatory system. Ito cells, which store fat, originate from mesenchymal cells in the septum transversum.     

Different corticostriatal projections from two parts of the cortical masticatory area in the rabbit

The cortical masticatory area (CMA) elicits rhythmic jaw movements in response to repetitive stimulation and is involved in the control of mastication. Based on jaw movement patterns, the CMA is divided into two parts. One is the part of the CMA in which a T-pattern similar to jaw movements during food transport in natural mastication is evoked by electrical stimulation. The other is more dorsomedially located, and during chewing a C-pattern similar to jaw movements can be induced. However, it is still not known which region of the putamen receives projections from the CMA and whether projections originate from both parts of the CMA. In this study, electrophysiological and histological experiments were undertaken in rabbits to investigate projections from the CMA to the putamen. Both experiments showed that the ventral region of the putamen received projections from the CMA. The density of the projections from the CMA area inducing the T-pattern seemed to be higher than that from the area inducing the C-pattern. Furthermore, the peak latency of the evoked potentials from stimulation of the CMA area inducing the T-pattern was shorter than that from stimulation of the area inducing the C-pattern. The data obtained from the present study indicate the functional role of the ventral region of the putamen in the regulation of mastication, and further suggest that the corticostriatal pathway is involved in the transition between behavioral jaw movement patterns.     

Cytopathic astrovirus isolated from porcine acute gastroenteritis in an established cell line derived from porcine embryonic kidney.

A cytopathic astrovirus was isolated from pigs with acute diarrhea in an established cell line that was derived from porcine embryonic kidneys with the aid of trypsin. The virus showed a distinct cytopathic effect characterized by an enlargement of cells and the appearance of fine granules in the cytoplasm. Porcine astrovirus was shown to have an RNA genome, as determined by the effect of 5-iodo-2'-deoxyuridine on its replication, and five polypeptides with molecular masses of 13,000, 30,000, 31,000, 36,000, and 39,000 daltons; and it was shown to be stable to lipid solvents and heating at 50 degrees C for 30 min but somewhat labile to acid (pH 3.0). The buoyant density of the isolate determined in CsCl was 1.35 g/ml. Seroconversion to the virus was evident in the paired serum specimens obtained from pigs with diarrhea that were housed at the farm where the disease occurred. The neutralization test on serum specimens collected randomly from 128 adult pigs of eight herds revealed that 50 of the serum specimens were positive for antibody to porcine astrovirus, although there was considerable variation in the prevalence among herds, ranging from 0 to 83%. Hysterectomy-produced, colostrum-deprived, 4-day-old pigs developed mild diarrhea after oral exposure to porcine astrovirus propagated in the cell culture; and the virus was isolated again from diarrheal stool specimens.     

Unique properties of fetal lymphoid progenitors identified according to RAG1 gene expression

RAG1/GFP knockin mice were exploited to isolate and characterize fetal lymphoid progenitors. CD11b and IL-7Ralpha are expressed in a developmental stage-dependent fashion, revealing how substantial numbers of early lymphoid progenitors were discarded or neglected in previous studies. The myeloerythroid potential of fetal progenitors in clonal assays declined in synchrony with activation of the RAG1 locus but was not completely extinguished. Lymphoid differentiation corresponded to patterns of gene expression previously found for adult marrow, but no fraction of fetal liver was enriched with respect to B + T progenitors. Also, unlike adults, fetal lymphoid progenitors transiently expressed endothelial cell markers. These findings help to reconcile discrepancies in previous reports and suggest that the fetal immune system arises via unique mechanisms.     

Diversity of Hepatitis G Virus within a Single Infected Individual

The extent of population diversity among GB virus C (GBV-C)/hepatitis G virus (HGV) within a persistently infected individual (Iw) was investigated by sequence analysis of multiple clones generated from polymerase chain reaction (PCR)-amplified products of cDNA analogous to fragments of 5 non-coding region (5NC), envelope region 1/2 (E1/E2) and non-structural region 3 (NS3) of viral genome. Although nucleotide substitutions were more common in coding regions than in the 5NC region, there was no region corresponding to the hypervariable region of hepatitis C virus in the E1/E2 region. Transition substitution exceeded transversion by 7 to 12-fold, and 79.4% of substitutions were synonymous. This bias against substitutions producing amino acid replacements and the use of Pfu DNA polymerase with an error rate 10 times lower than the observed frequency of substitution, suggests that most substitutions were not artefactual. This data suggests that individual genomes of HGV within an infected individual may differ from each other at 0.23–0.84% nucleotide position and at 0.42–0.61% amino acid position.     

Total hematological analysis system as a screening test for hematological malignancy

Measurement of complete blood cell count and white blood cell differentiation is an essential laboratory test and the most important screening test for hematological malignancy. Recently, several automated blood cell analyzers have been developed to improve accuracy and precision. When flag messages generated in the presence of morphological abnormalities of the samples are displayed, manual revision is necessary. In our laboratory, the manual revision rate has been 35-40%. Therefore blood cell analyzers are useful in screening for abnormalities as well as greatly reducing expensive and time-consuming manual differential procedures. In addition, automated blood cell analyzers can provide several types of useful information including the leukocyte distribution scattergram. However, most such information is not utilized in the clinical field. In the future, a total hematological analysis system will be constructed so that all information provided by automated blood cell analyzer and by manual methods are available.     

Properties of the omega-hydroxylation system of docosahexaenoic or arachidonic acid in brain or liver homogenate of rat

The homogenate of a brain or liver obtained from a 1–55-day-old rat was incubated with NADPH and docosahexaenoic or arachidonic acid as the substrate. ω-Hydroxydocosahexaenoic or ω-hydroxyeicosatetraenoic acid from an incubation mixture of the homogenate was detected on a selected-ion monitoring chromatogram of reversed phase-HPLC-thermospray-mass spectrometry. ω-Hydroxylation activity in the brain homogenate considerably increased with growth up to 55 days. Activity in the liver homogenate decreased much with growth up to 55 days. ω-Hydroxylation activity in homogenates of rat brain gray matter, white matter, medula oblongata and cerebellum was much the same. ω-Hydroxylation activity of docosahexaenoic acid in rat brain homogenate was maximal at pH 7.5–8.0 in 50 mM Tris-HCL buffer and was inhibited by CO gas, metyrapone, ADP-Fe³⁺, heat treatment at 100°C for 5 min and without NADPH. Based on these results, it is suggested that ω-hydroxylation activity is associated with cytochrome P-450 without NADPH-ADP-Fe³⁺-dependent lipid peroxidation, and the ω-hydroxylation system may be a metabolic pathway of the fatty acids in adult rat brain or neonatal rat liver. Since ω-hydroxyeicosatetraenoic acid produces relaxation of artery, it is suggested that blood flow changes in rat brain or liver with growth are caused by ω-hydroxylation activity changes in these organs with growth.     

Two color flow cytometry of lymphocytes from patients with adult T-cell leukemia

Lymphocytes from eight patients with adult T-cell leukemia were analyzed by two color flow cytometry. Monoclonal antibodies (Leu 3 a, Leu 8, Leu 2 a and Leu 15) labelled with fluorescein isothiocyanate or phycoerythrin were used. The purpose was to identify the subsets of the lymphocytes as helper, suppressor/inducer, suppressor or cytotoxic by the surface marker of the cells. All eight patients had antibodies for ATLA. Proviral DNA in the lymphocytes was found in six patients. Summarising the results, OKT4-positive ATL cells were all of the helper T-cell subset, not the inducer subset. OKT8-positive ATL cells were also positive for OKT4 and were all of the cytotoxic T cell subset, not the suppressor subset. In two patients, some ATL cells had both OKT4 and OKT8 on the same cells, especially in the lymph nodes. In our study, ATL cells from eight cases of ATL had all of the helper T subset. These results suggest that the target cells of the human T cell leukemia/lymphoma virus type will be helper T cells.