Southern hybridization analysis of DNA polymorphism in the HLA-D region

Restriction fragment-length polymorphisms (RFLP) were systematically analyzed by Southern hybridization with restriction endonuclease-digested genomic DNA from 28 HLA-homozygous B cell lines with Dw1-Dw19 specificity using the DR beta and DQ beta chain cDNAs as probes. These probes detected polymorphic fragments unique to each HLA-DR specificity. Furthermore, the DQ beta chain probes permitted us to distinguish between different Dw specificities with an identical DR type much more efficiently than with the DR beta chain probe. Distribution analysis of restriction fragments hybridizing to DR beta in relation to the DR and DQ specificities showed several sets of them forming ten clusters, some of which correlate with DRw53, DQw1, and DR alleles. This DNA typing technique allows the direct definition of HLA types at the gene level and provides a powerful tool for isolating genes controlling HLA-associated diseases.     

Age-related changes in the levels of voltage-dependent calcium channels and other synaptic proteins in rat brain cortices

Neurotransmitter release from synapses is one of the most important interneuronal signaling in the nervous system. We previously reported that aging decreases depolarization-induced acetylcholine release in rat brain synaptosomes. To investigate the mechanisms underlying the age-related decrements of neurotransmission, we determined the levels of the alpha1 subunit proteins of voltage-dependent calcium channels (VDCCs) and three synaptic proteins that relate to exocytotic processes using synaptosomes prepared from cerebral cortices of young (6-month-old) and aged (27-month-old) rats. Immunoblotting analyses revealed that the protein levels of alpha1A (P/Q-type) and alpha1B (N-type) subunits in aged rats were 38% and 43% lower than the levels of young rats, respectively, but the levels of the alpha1C (L-type) subunit were not different between young and aged. On the contrary, the levels of synaptotagmin-1, synaptophysin and syntaxin were not significantly different between the two age groups in the synaptosomal preparations. These results suggest that synaptic density does not change much in the cerebral cortex in normal aging, and that the reduction of P/Q-type and N-type VDCCs, both of which participate in neurotransmitter release, is one of the causes for the decrease of neurotransmission at aged synapses.     

CC chemokine receptor 4 ligand production by bronchoalveolar lavage fluid cells in cigarette-smoke-associated acute eosinophilic pneumonia

We examined the production of macrophage-derived chemokine (MDC/CCL22) and thymus- and activation-regulated chemokine (TARC/CCL17) by bronchoalveolar lavage fluid (BALF) cells in cigarette-smoke-associated acute eosinophilic pneumonia (CS-AEP). The CC Chemokine Receptor 4 (CCR4) ligand levels in BALF from patients with CS-AEP were considerably higher than those in healthy volunteers and correlated well with Th2 cytokine levels. Interleukin-4 enhanced CCR4 ligand production. MDC expression was observed in CD68-positive cells from patients with CS-AEP and in healthy control smokers. In contrast, TARC expression in CD68- or CD1a-positive cells was detected only in CS-AEP. An in vivo cigarette smoke challenge test induced increases in CCR4 ligands in the BALF and in the cultured supernatant of BALF adherent cells. These results suggest that alveolar macrophages and dendritic cells contribute to the pathogenesis of CS-AEP by generating CCR4 ligands, probably in response to cigarette smoke.     

Practical application of nutritional assessment protein and inflammatory marker in the nutrition support team (NST)

Recently nutrition support team (NST) has been established for the purpose of prevention of complications which are caused by nutrition disorders and reduction of the medical expenses. Although physical examinations and blood biochemical data had been used as the indexes evaluating nutritional of patients, they were not suitable for the evaluation for the short-term in-patient. On the contrary, serum albumin (ALB) has been wildly used as a nutritional marker. However, it is impossible to evaluate nutrition state for the short-term in-patient and acute phase disease patient accurately, because the plasma half-life is 21 days and it takes long time to detect the change in nutritional state by its value. Rapid turnover proteins (RTP), whose plasma half-life is shorter, has paid attention to evaluate nutritional state for the short-term in-patients and acute phase disease patients. Although, prognostic inflammatory and nutritional index (PINI) was considered as a useful maker for evaluating inflammatory and nutritional states using the concentrations of transthyretin (TTR), a RTP, alpha1-acid glycoprotein (alpha1-AG), a chronic inflammation marker, C reactive protein (CRP), a acute inflammation marker, and ALB, However, it has several pitfalls. We newly made serum amyloid A (SAA) index using SAA instead of CRP. When we compared SAA index with PINI in many diseases, it turned out that SAA index became a more effective index which reflected the patient condition than did PINI. As for this index, it is expected to be used by NST while further alternation may be needed.     

Prevalence of asthma in adults in Menda town rural-mountain area in Japan]

Several reports have suggested that the prevalence of asthma in adults is currently increasing. However, recent prevalence of asthma has not reported in Japan, especially in rural-mountain areas. To investigate the prevalence of asthma in adults in Japan, we conducted clinical epidemiological research on 5066 inhabitants of Menda town, in a rural-mountain area of Japan. The study population comprised 98.7% of adults in the town, including senior high school students whose age were more than 15 years old. The prevalence of asthma among adults was 3.6%. The ratio of prevalence in males to prevalence in females was 1.44. Peaks prevalences were observed in the age ranges of 15-19 and > 70 years old in males, and 15-19, 40-49 and > 70 years old in females.     

Murine stromal cell line HESS-5 maintains reconstituting ability of Ex vivo-generated hematopoietic stem cells from human bone marrow and cytokine-mobilized peripheral blood

Human bone marrow (BM) or mobilized peripheral blood (mPB) CD34(+) cells have been shown to loose their stem cell quality during culture period more easily than those from cord blood (CB). We previously reported that human umbilical CB stem cells could effectively be expanded in the presence of human recombinant cytokines and a newly established murine bone marrow stromal cell line HESS-5. In this study we assessed the efficacy of this xenogeneic coculture system using human BM and mPB CD34(+) cells as materials. We measured the generation of CD34(+)CD38(-) cells and colony-forming units, and assessed severe-combined immunodeficient mouse-repopulating cell (SRC) activity using cells five days after serum-free cytokine-containing culture in the presence or the absence of a direct contact with HESS-5 cells. As compared with the stroma-free culture, the xenogeneic coculture was significantly superior on expansion of CD34(+)CD38(-) cells and colony-forming cells and on maintenance of SRC activity. The PKH26 study demonstrated that cell division was promoted faster in cells cocultured with HESS-5 cells than in cells cultured without HESS-5 cells. These results indicate that HESS-5 supports rapid generation of primitive progenitor cells (PPC) and maintains reconstituting ability of newly generated stem cells during ex vivo culture irrespective of the source of samples. This xenogeneic coculture system will be useful for ex vivo manipulation such as gene transduction to promote cell division and the generation of PPC and to prevent loss of stem cell quality.     

Cloning and sequencing of cDNAs corresponding to mite major allergen Der f II

A cDNA library corresponding to mite protein was screened employing anti-Der f II antibody. Two possible clones were obtained, which contained plasmids, pFL1 and pFL11, respectively. Both plasmids had insertions of about 500 base pairs. The DNA sequences of the two insertions were determined, from which the amino acid sequences were deduced. The amino acid sequence of the purified native Der f II protein could be determined to 45 residues from the N-terminus. As a result of comparison, we concluded that the cDNAs prepared from live mite Dermatophagoides farinae corresponded to the mite allergen, Der f II.     

Diagnostic value of measurement of RNA in platelets by fluorescence flow cytometry]

We analyzed the RNA in platelets by fluorescence flow cytometry after staining with thiazole orange(TO) in whole blood samples from hematologically normal subjects and patients with thrombocytopenia. The percentage of TO-positive platelets and their mean fluorescence channel number in 32 control subjects were 6.2 +/- 2.5% (mean +/- SD) and 6.9 +/- 0.7, respectively. In 11 patients with idiopathic thrombocytopenic purpura, the percentage of fluorescently labeled platelets was significantly elevated (p less than 0.05) to 21.5 +/- 14.3%. By contrast, the proportion of positively stained platelets in 14 patients with thrombocytopenia due to impaired platelet production did not significantly differ from that of the controls, whereas the absolute counts of TO-positive platelets were significantly lowered (p less than 0.05). In both patient groups, the mean fluorescence channel numbers of TO-positive platelets were significantly elevated to 16.1 +/- 16.8 and 6.9 +/- 0.7, respectively (p less than 0.05, 0.005). We conclude that flow cytometric analysis of platelets after staining with TO provides information on the thrombopoietic activity in thrombocytopenic disorders. The main advantages of this method for clinical use are its simplicity and the rapidity.     

Gastric motility in patients with recurrent gastric ulcers.

The existence of abnormal gastric motility in gastric ulcer disease remains controversial. The aim of this study was to characterize gastric motility in patients with recurrent gastric ulcers. Studies were performed in 10 control subjects and in 24 patients with recurrent active gastric ulcer disease as diagnosed by gastrointestinal endoscopy. Gastric motility was evaluated by cutaneous electrogastrography (EGG) and by gastric semi-liquid meal emptying. The EGG was recorded before and after ingestion of a test meal containing 20 mg/kg of acetaminophen. Patients with a dominant EGG frequency of greater than 0.06 Hz were defined as tachygastria, while those with a frequency of less than 0.04 Hz were defined as bradygastria. A transient frequency decrease, called postprandial dip (PD), was identified visually. The degree of gastric emptying was determined from the serum acetaminophen concentration 45 minutes after the meal. Control subjects showed no irregularity in their dominant EGG frequency in tither fasting or postprandial states. PD was observed in 8 control subjects. In patients presenting with active gastric ulcers, abnormal patterns in the dominant EGG frequency (either as tachygastria or bradygastria) were observed in 14 of the 24 patients when fasting and in 15 of them in the postprandial state. After successful treatment, the number of patients with abnormal patterns in their dominant EGG frequency remained unchanged, while PD was observed in 11 patients. No significant difference was observed in the EGG power ratio as a result of successful treatment. Gastric emptying was significantly delayed compared with controls in both the active and healed stages. These findings suggest that abnormal gastric motility, including gastric electrical abnormalities and delayed gastric emptying, plays an important role in the pathophysiology of recurrent gastric ulcers.