Relationship between HLA-D and in vitro and in vivo responsiveness to Candida allergen.

In vitro reaction to Candida allergen was studied in 100 normal healthy Japanese and related to HLA. A significant association was found between the low responder group (< 7,000 c.p.m.) and HLA-B15 and the high responder group (> 7,000 c.p.m.) and HLA-B7, as well as Dwl. Four HLA-D homozygote cells of types HLA-Dwl and DHO, were tested; these fitted in the high responder group. HLA-DYT and DEn fitted into the low responder group. Responsiveness to Candida allergen corresponded to skin test (r = 0.884, P < 0.01). From these results, in vitro lymphocyte reaction to Candida allergen and skin tests showed close correlation and an association with HLA antigens, HLA-D in particular.     

A nonsynonymous polymorphism in the human fatty acid amide hydrolase gene did not associate with either methamphetamine dependence or schizophrenia

Genetic contributions to the etiology of substance abuse and dependence are topics of major interest. Acute and chronic cannabis use can produce drug-induced psychosis resembling schizophrenia and worsen positive symptoms of schizophrenia. The endocannabinoid system is one of the most important neural signaling pathways implicated in substance abuse and dependence. The fatty acid amide hydrolase (FAAH) is a primary catabolic enzyme of endocannabinoids. To clarify a possible involvement of FAAH in the etiology of methamphetamine dependence/psychosis or schizophrenia, we examined the genetic association of a nonsynonymous polymorphism of the FAAH gene (Pro129Thr) by a case-control study. We found no significant association in allele and genotype frequencies of the polymorphism with either disorder. Because the Pro129Thr polymorphism reduces enzyme instability, it is unlikely that dysfunction of FAAH and enhanced endocannabinoid system induce susceptibility to either methamphetamine dependence/psychosis or schizophrenia.     

Modified PCR-RFLP method for HLA-DPB1 and -DQA1 genotyping.

We previously developed a new technique for HLA class II genotyping by digestion of polymerase chain reaction-amplified genes with restriction endonucleases (PCR-RFLP method). This PCR-RFLP method is an efficient and convenient typing technique for class II alleles. However, small fragments or bands located close to each other on polyacrylamide gels sometimes prevent precise analysis of the RFLP bands. Furthermore, the restriction enzymes we have reported in the previous papers are not sufficient to identify the genotypes of all heterozygous individuals. Here, we report an improved PCR-RFLP method using some informative restriction enzymes which have either a single cleavage site or, alternatively, no cleavage site in the amplified DNA region, depending on the HLA alleles, making reading of RFLP band patterns much easier. Each second exon of the HLA-DQA1 or -DPB1 gene was selectively amplified from genomic DNAs of 70 HLA-homozygous B-cell lines and 100 healthy Japanese by PCR. Amplified DNAs were digested with restriction endonucleases and then subjected to electrophoresis assaying simply for cutting, or no cutting, of the DNA. ApaLI, HphI, BsaJI, FokI, MboII and Mn1I can discriminate eight alleles of the DQA1 gene. Similarly 19 alleles of the DPB1 gene can be discriminated with Bsp1286I, FokI, DdeI, BsaJI, BssHII, Cfr13I, RsaI, EcoNI, and AvaII enzymes. This modified PCR-RFLP method can be successfully applied to heterozygotes. Thus, the method is technically simpler and more practical for routine HLA typing work than our previous PCR-RFLP method.     

Histological evaluation of medial patellofemoral ligament reconstructed using the Leeds-Keio ligament prosthesis

In a total of 15 knees from 15 patients undergoing medial patellofemoral ligament reconstruction using an artificial substitute and a medial retinaculum slip coverage for recurrent patellar dislocation, the reconstructed ligaments were histologically evaluated using hematoxilin-eosin and elastic van Gieson stains. The artificial substitute was a mesh-type Leeds-Keio ligament. The mean age of the patients at the time of surgery was 20 years (range; 13-38 years). The mean interval from initial reconstruction to gathering was 53 months (range; 11-109 months). In the tissue over the artificial ligament, longitudinally aligned collagen fiber bundles with spindle-shaped nuclei were formed in all specimens except one specimen of 11 months postsurgery, but it seemed to be mature ligament only in specimens more than 60 months postsurgery. The tissue inside the artificial ligament was immature as a whole in all specimens, although 13 out of the 15 specimens had regularly aligned collagen fiber bundles slightly or in some portions.     

Steroid hormone receptors in breast cancer

The significance of hormone receptor (estrogen(ER)- and progesterone receptor (PgR) assay was described with special reference to the treatment of advanced and early breast cancer. Fifty to sixty percent of ER-positive breast cancer responds to endocrine treatment, while only about 10% of ER-negative cancer do. Advanced breast cancer patients with ER-positive tumors survive longer than those with ER-negative tumors, mainly because of better response to therapy. The clinical benefit of assaying the hormone receptors in primary breast cancer was discussed, particularly concerning with the relapse-free and overall survivals of the patients after primary operation. The findings suggest the possibility of selecting operable breast cancer patients for the most appropriate adjuvant using ER and PgR.     

Development of a PCR method for rapid identification of new Streptococcus mutans serotype k strains

In a previous study, we isolated and characterized a new serotype k of Streptococcus mutans from human blood and oral cavities. Analysis of the genes involved in biosynthesis of the serotype-specific polysaccharide of serotype k strains revealed that the serotype k-specific nucleotide alignment was commonly present in the 5' region of the rgpF gene (350 bp from the initial sequence) compared to the reference strains, and then a method for rapid identification of serotype k strains was developed by use of PCR with primers designed on the basis of the sequence of the variable region. PCR assays with primers specific for amplification of serotype k strains showed a negative reaction with serotype c, e, and f strains and a positive reaction with serotype k strains, with the sensitivity for identification of the serotype k strains shown to range from 5 to 50 cells. Next, the frequency of positive reactions for serotype k-specific primers was surveyed with DNA taken from saliva samples from 200 subjects (2 to 18 years of age), and 10 of those showed a positive reaction, which was higher than the frequency in our previous survey with a serological method. In addition, all saliva samples from subjects with serotype k strains in our previous study were shown to be positive with the serotype k-specific primers. These results indicate that this new PCR method is effective for identification of subjects with S. mutans serotype k.     

Inhibition of experimental metastasis and cell adhesion of murine melanoma cells by chondroitin sulfate-derivatized lipid, a neoproteoglycan with anti-cell adhesion activity

Chondroitin sulfate dipalmitoylphosphatidylethanolamine (CS-PE), when immobilized onto substratum, inhibited the adhesion of B16F10 mouse melanoma cells to fibronectin-coated dishes (anti-adhesion activity). CS-PE showed the most potent anti-adhesion activity for the melanoma cells among various GAG-PEs. CS-PE also inhibited the adhesion of B16F10 cells to Matrigel and the invasion of the cells into Matrigel. In the in vivo system of experimental metastasis, administration of B16F10 cells with CS-PE into C57BL/6 mice significantly inhibited lung metastasis. The inhibition degree of CS or hyaluronic acid-PE was lower than CS-PE. CS-PE administered intravenously into mice before the injection of B16F10 cells also inhibited metastasis. Pretreatment of B16F10 cells with CS-PE caused some but a lower degree of inhibition. When CS-PE was injected intravenously into mice, more binding in the lung was found than when CS was injected. CS-PE but not CS inhibited the retention in the lung of fluorochrome-labeled B16F10 cells when injected intravenously into mice. Since there was no significant effect of CS-PE on the viability and growth of B16F10 cells, the results suggest that CS-PE immobilized onto the subendothelial matrix may prevent melanoma cells from adhering to the subendothelial substrata of lung capillaries and inhibit subsequent invasion processes of metastasis.     

Micromechanical properties of demineralized dentin collagen with and without adhesive infiltration

In a previous study, we reported the upper limit of Young's modulus of the unprotected protein at the dentin/adhesive interface to be 2 GPa. In this study, to obtain a more exact value of the moduli of the components at the d/a interface, we used demineralized dentin collagen with and without adhesive infiltration. The prepared samples were analyzed using micro-Raman spectroscopy (micro RS) and scanning acoustic microscopy (SAM). Using an Olympus UH3 SAM (Olympus Co., Tokyo), measurements were recorded with a 400 MHz burst mode lens (120 degrees aperture angle; nominal lateral resolution, 2.5 microm). A series of calibration curves were prepared using the relationship between the ultrasonically measured elastic moduli of a set of known materials and their SAM response. Finally, both the bulk and bar wave elastic moduli were computed for a set of 13 materials, including polymers, ceramics, and metals. These provided the rationale for using extensional wave measurements of the elastic moduli as the basis for extrapolation of the 400 MHz SAM data to obtain Young's moduli for the samples: E = 1.76 +/- 0.00 GPa for the collagen alone; E = 1.84 +/- 0.65 GPa for the collagen infiltrated with adhesive; E = 3.4 +/- 1.00 GPa for the adhesive infiltrate.     

Role of complement in acute tubulointerstitial injury of rats with aminonucleoside nephrosis.

The present work was designed to elucidate the in vivo role of complement in the proteinuria-associated tubulointerstitial injury. Rats were intravenously injected with puromycin aminonucleoside, and massive proteinuria was observed within 5 days. Prominent tubulointerstitial injury characterized by proximal tubular degeneration, tubular dilatation, and leukocyte infiltration were observed 7 days after injection. C3 and C5b-9 were observed in the luminal side of proximal tubular cells. Renal function, assessed by inulin and para-aminohippurate clearance, was significantly decreased. To-assess the role of complement in this model, rats were injected with either cobra venom factor or soluble recombinant human complement receptor type 1 starting at day 3. These manipulations significantly improved tubulointerstitial pathology and para-aminohippurate clearance without affecting the degree of proteinuria. Deposition of C3 and C5b-9 was not detected in the kidney of rats depleted of complement by cobra venom factor. In rats treated with soluble complement receptor, C3 was still detected in the tubules, but deposition of C5b-9 was not observed. Soluble complement receptor was detected at the site of C3 deposition and in the urine. These data strongly suggest that complement plays a pivotal role in proteinuria-associated tubulointerstitial injury and that systemic complement depletion or inhibition of complement in the tubular lumen may diminish the tubulointerstitial damage.     

Effect of skin temperature on the ion reabsorption capacity of sweat glands during exercise in humans

The effect of skin temperature on the ion reabsorption capacity of sweat glands during exercise in humans is unknown. In this study, eight healthy subjects performed a 60-min cycling exercise at a constant intensity (60% VO_2max) under moderate (25°C) and cool (15°C) ambient temperatures at a constant relative humidity of 40%. The sweating rate (SR), index of sweat ion concentration (ISIC) by using sweat conductivity, esophageal temperature (Tes), mean skin temperature, and heart rate (HR) were measured continuously under both ambient temperatures. The SR and ISIC were significantly lower at the cool ambient temperature versus the moderate temperature. There were no significant differences in the changes in HR and esophageal temperature between these ambient temperature conditions, while the mean skin temperature was significantly lower at the cool ambient temperature by almost 3°C (P<0.05). The slopes of the relationships between Tes and the SR and ISIC were significantly lower and the thresholds of these relationships were significantly higher at the cool ambient temperature (P<0.05). The ion reabsorption capacity of the sweat glands was significantly lower (P<0.05) in a cool environment (0.21±0.04 vs. 0.52±0.06 mg/cm²/min at 15 and 25°C, respectively) as evaluated using the relationships for SR and ISIC. The results suggest that the ion reabsorption capacity of the sweat glands is influenced by skin temperature during exercise in humans.