Differential expression of human beta-defensin 2 in keratinized and non-keratinized oral epithelial lesions; immunohistochemistry and in situ hybridization

Human beta-defensin(hBD)-2, an antimicrobial peptide, is produced by various epithelial cells. Because hBD-2 expression in the oral epithelium has not been assessed, we investigated its localization in normal oral epithelium and epithelial lesions. hBD-2 expression was studied using immunohistochemistry and in situ hybridization on formalin-fixed, paraffin-embedded tissue sections from 30 cases of squamous cell carcinoma and 6 cases of leukoplakia. Immunostaining for hBD-2 was more intense in hyperkeratinized than in ortho- or non-keratinized epithelium. In contrast, signals for hBD-2 mRNA were frequently stronger in non-keratinized epithelium than in hyper- or ortho-keratinized epithelium. The results suggest that keratinization in oral epithelium plays an important role in the biological function of hBD-2 both at the mRNA level and in the retention of the peptide in the epithelium.     

Immunocytochemical Evidence of Amyloid-enhancing Factor (AEF) in Polymorphonuclear Leukocytes

Amyloid enhancing factor (AEF) was extracted from spleens of mice that had received amyloidogenic stimulation. Sephacryl S 300 gel filtration of the crude AEF yielded five fractions, among which strong AEF activity was present in the first peak (Fl), and confirmed by an amyloid induction experiment. An anti AEF antiserum was obtained from a rabbit by immunization with Fl. This antibody reacted strongly with splenic polymorphonuclear leukocytes (PML) from mice given amyloidogenic stimulation, and weakly with those from normal untreated mice. Isoelectric focusing (IEF) analysis of both Fl and sera from mice given amyloidogenic stimulation was performed. A single band was observed on IEF analysis of Fl, whereas many bands were seen on IEF analysis of the sera. After the substances in the gel had been transferred to nitrocellulose membranes by capillary blotting, the membranes were made to react with the anti-AEF antiserum. The results suggested that AEF is a high molecular-weight substance derived from PML and increases in the serum at the time of, or shortly prior to, amyloid deposition in the spleen. Acta Pathol Jpn 39: 349∼355, 1989.     

Heat interaction parameter of polystyrene solutions determined by inverse gas-liquid chromatography

The interaction between polymer and solvent in highly concentrated polymer solutions was studied by inverse gas-liquid chromatography as a function of the molecular weight of polymer. The heat interaction parameter was estimated for the systems polystyrene(PS)-benzene, -toluene, -pyridine, -ethylbenzene, and -anisole. It was found that the heat interaction parameter for the concentrated polystyrene systems PS-toluene, PS-pyridine, and PS-benzene exhibits a similar behavior as in the dilute polystyrene solutions determined by calorimetry at 298,15 K. Further, the heat interaction parameter in both the concentrated and dilute polymer solutions is considerably dependent on the molecular weight of polymer.     

A promoter variant of the ATP-binding cassette transporter A1 gene alters the HDL cholesterol level in the general Japanese population

To investigate the effects of polymorphisms in the ATP-binding cassette transporter A1 (ABCA1) gene on the high-density lipoprotein cholesterol (HDL-C) level and the incidence of myocardial infarction (MI), we performed association studies. Sequence analysis identified 14 polymorphisms in the promoter region of ABCA1. After considering linkage disequilibrium, three polymorphisms in the promoter region and 11 polymorphisms from the JSNP database were determined in 1,880 subjects recruited from the Suita Study, representing the general population in Japan. We evaluated the association between the ABCA1 genotype and HDL-C level adjusted not only for standard factors, but also for genetic factors including ApoA1 and ApoE genotypes. Of the 14 polymorphisms tested, the G(–273)C (P=0.0074), C(–297)T (P=0.0195), and IMS-JST071749 (P=0.0093) polymorphisms were significantly associated with the HDL-C level in the Suita population. We could reconfirm that the G(–273)C genotype was influential in another set of subjects (P=0.0310, n=743). However, the distribution of the ABCA1 G(–273)C genotype in subjects with MI (n=598) was not different from that in the control population (n=801). These results indicate that ABCA1 G(–273)C has a significant effect on the HDL-C level in the general Japanese population, but not on the incidence of MI.     

Development of sutureless vascular connecting system for easy implantation of small-caliber artificial grafts

A novel sutureless vascular connecting system, an assembly with a delivery rod, an introducing sheath, and a connecting device, was developed for easy implantation of small-caliber vascular grafts less than 2 mm in internal diameter. A microporous stainless tube (length 2 mm, external diameter 1.6 mm, wall thickness 65 µm, pore diameter 400 µm, pore-to-pore distance 500 µm) was designed to serve as a connecting device. The feasibility of the system was tested using two types of preliminary animal experiments. One animal model consisted of graft implantation into the rat abdominal aorta (1.5 mm in diameter). The connecting device was inserted into the proximal and distal ends of the aorta through the introducing sheath by pushing the delivery rod with the connecting device placed over it. Subsequently, the aortic segments were inserted into both ends of model grafts made of segmented polyurethane (1.8 mm in internal diameter) and were fixed with banding silk threads from the exterior. The procedure was completed within 20 min without requiring specialized microsurgery techniques. Blood leakage and obstruction did not occur. The second model consisted of an end-to-end anastomosis between rabbit common carotid arteries (2 mm in diameter), which was performed within several minutes of blood flow interruption. Scanning electron microscopy demonstrated that the luminal surface of the device was fully covered with endothelial cells (ECs) after 1 week as a result of transluminal ingrowth of native ECs through the micropores in the device. This endothelialization may prevent early thrombus-induced occlusion. This simple and “easy-to-learn” technique will promote the development of small-caliber arterial grafts, and furthermore, it may have potential for clinical application.     

Expression cloning of gamma interferon-inducing antigens of Mycobacterium avium subsp. paratuberculosis

Three recombinant proteins, Map10, Map39, and Map41, produced based on nucleotide sequences obtained from the screening of Mycobacterium avium subsp. paratuberculosis genomic library expressed in Escherichia coli significantly elicited gamma interferon production in peripheral blood mononuclear cells from infected cattle. Two of these proteins were members of the PPE protein family.     

Antigen-induced tolerance by intrathymic modulation of self-recognizing inhibitory receptors

CD1d-restricted invariant natural killer T cell (iNKT cells) have a limited T cell receptor (TCR) repertoire and share characteristics common to T cells and natural killer cells. While intrathymic selection facilitates the production of T cells carrying self major histocompatibility complex-restricted TCRs, natural killer cells carry an appropriate repertoire of self major histocompatibility complex-recognizing receptors to avoid self-reactivity. Here we show that chronic exposure to specific glycolipid antigen resulted in iNKT cell disappearance and thymus-dependent repopulation of iNKT cells with increased expression of inhibitory Ly-49 molecules that resulted in impaired responsiveness. Thymic selection of peripheral Ly-49-expressing iNKT cell repertoire inhibited cytokine production and other functions in vivo. These observations emphasize the acquisition of self-recognizing inhibitory receptors on NKT cells as a previously unknown mechanism of thymic tolerance after chronic antigen exposure.     

Simple,rapid, and accurate determination of deletion mutations by automated dna sequencing of heteroduplex fragments of the adenomatous polyposis coli (APC) gene generated by PCR amplification

Germline mutations in patients with familial adenomatous polyposis were analyzed by polymerase chain reaction (PCR) amplification of the adenomatous polyposis coli gene. PCR products from heterozygous patients for deletions of this gene formed four distinct bands on polyacrylamide gel electrophoresis. The four fragments were subsequently purified and both strands of each fragment were directly sequenced, using an automated DNA sequencer and the same primers as those for PCR amplification. It was found that the two slower migrating fragments were “bulge” heteroduplexes, while the other two were homoduplexes made up of two wild-type strands and two deletion-mutant strands, respectively. The sites of deletions in the adenomatous polyposis coli gene could be exactly determined in four of the five patients. In an attempt to identify deletion-carriers of familial adenomatous polyposis at the presymptomatic stage, a family study was also carried out, and two children were found to have the same mutations as those of their affected parents. The direct sequencing of heteroduplex fragments generated during PCR amplification is a potentially useful method for detecting mutations of not only the adenomatous polyposis coli gene but also many other genes of genetic diseases. © 1993 Wiley-Liss, Inc.