Neutralizing antibody responses to human herpesviruses 6 and 7 do not cross-react with each other, and maternal neutralizing antibodies contribute to sequential infection with these viruses in childhood

Seroprevalence of human herpesvirus 6 (HHV-6) and HHV-7 infections is very high throughout the world, and almost all people are exposed first to HHV-6 and second to HHV-7 in their childhood. However, it is not clear whether the neutralizing (NT) antibody response between each virus is cross-reactive or not. To elucidate the NT antibody response between each virus, 55 serum samples from an adult group (subjects 22 to 88 years old) and 60 serum samples from a young group (subjects 2 to 18 years old) were examined by a dot blot method for detecting viral late antigen. Thirty-nine serum samples obtained from cord bloods and a few serum samples obtained from pediatric patients with exanthem subitum were also examined to assess the maternal transferred NT antibodies against each virus. The NT antibody titers against HHV-7 in the adult group remained high throughout all the individuals, and none were negative. Those against HHV-6 were high values in the young group but low values, including negative values (three samples), in the adult group. These results suggested that the NT antibody response to either HHV-6 or HHV-7 in each individual was specific to each virus and did not cross-react with each other. In the adult group, the NT antibody response to HHV-6 decreased, while that to HHV-7 remained high throughout all the individuals. Maternal transferred NT antibody titers against HHV-7 were higher and remained longer after birth than those of HHV-6, and these findings were in accord with the clinical observation that HHV-6 infection usually occurs earlier than HHV-7 infection.     

Oxidative polymerization of 2,6-dimethylphenol with semiconducting organic polymeric metal complexes containing the bis(ethylene-1,2-dithiolato)copper (II) structure, 1

In the oxidative polymerization of 2,6-dimethylphenol ( 8 ) the catalytic activities of the following semiconducting organic polymeric metal complexes were studied: polymeric complexes containing bis(ethylene-1,2-dithiolato)Cu(II) or Fe(III) complexes and copper(II) phthalocyanine structures ( 3a – c ), catalysts consisting of bis(ethylene-1,2-dithiolato)-Cu(II) or Fe(III) complex structures and hemiporphyrazine type structures ( 5a – c ), and also polymers containing a copper complex of dimercaptomaleic acid monoamide ( 7 ). It was found that a suspension of the insoluble bis(ethylene-1,2-dithiolato)Cu(II) polymeric complexes of type 3 catalyzes the polymerization in the presence of oxygen in pyridine at room temperature producing poly[oxy-(2,6-dimethyl-1,4-phenylene)] ( 9 ) in very good yields. Polymeric complexes of type 5 were inactive by themselves. However, we showed that bis(ethylene-1,2-dithiolato)Cu(II) polymeric complexes of both the 3 and 5 type possess great activities if they are used in the presence of small amounts of copper(II)-pyridine complex in pyridine solution (the chosen concentration was so low that the Cu(II)-pyridine complex alone had only a small activity). Bis(ethylene-1,2-dithiolato)Fe(III) complexes of both the 3 and 5 type were inactive even in the presence of the Cu(II)-pyridine complex.     

Direct attachment of fibronectin to tresyl chloride-activated titanium

The aim of this study was to bind fibronectin directly to a titanium surface treated with tresyl chloride (2,2,2-trifluoroethanesulfonyl chloride) for the development of a strong connection of a dental implant to subepithelial connective tissues and/or peri-implant epithelia. Basic terminal OH groups of mirror polished titanium were allowed to react with tresyl chloride at 37 degrees C for 2 days. The tresylated titanium disk was then immersed into a fibronectin/phosphate-buffered saline solution for 24 h at 37 degrees C. The activation reaction of the basic OH of titanium with tresyl chloride was confirmed by S2p, F1s, and O1s spectra using X-ray photoelectron spectroscopy and -O-S-O2- bonds using Fourier transform infrared reflection-absorption spectroscopy. After the reaction of fibronectin with titanium, the X-ray photoelectron spectroscopy revealed the remarkable effect of the activation of terminal OH groups with the tresyl chloride treatment. The N1s peak derived from the attached fibronectin still remained after 60 s of argon-ion sputtering after tresyl chloride treatment. In contrast, the N1s peak of the specimen not treated with tresyl chloride almost disappeared after only 10 s of argon-ion etching. Fibronectin, a well-known cell-adhesive protein, could easily be attached to the titanium surface by use of the tresyl chloride activation technique.     

Prolonged ectopic calcification induced by BMP-2-derived synthetic peptide

Bone morphogenetic protein-2 (BMP-2) promotes the formation and regeneration of bone and cartilage, and therefore constitutes the most promising candidate for a bone repair material. However, it also has a wide range of functions, such as in organogenesis and apoptosis. Therefore, we investigated a novel synthetic peptide corresponding to residues 73-92 of BMP-2. This peptide bound to a BMP-2-specific receptor and elevated both alkaline phosphatase activity and osteocalcin mRNA in the murine cell line, C3H10T1/2. The 73-92 peptide also induced ectopic calcification when conjugated to a covalently crosslinked alginate gel. Here we report that the 73-92 peptide-conjugated alginate gel showed prolonged ectopic calcification for up to 7 weeks in rat calf muscle. In contrast, rhBMP-2-impregnated collagen gel showed maximum ectopic calcification at 3 weeks, and the calcified products that had formed disappeared after 5 weeks. Histological examination showed that the 73-92 peptide-conjugated alginate gel induced many osteoblast-like cells and few osteoclasts. In contrast, rhBMP-2-impregnated collagen gel induced many osteoclasts. These results suggest that the 73-92 peptide on alginate gel remains active at the implanted site, continuously induces differentiation of osteoblast precursor cells into osteoblasts, and activates osteoblasts to promote ectopic calcification.     

Development of a rapid PCR method using the insertion sequence IS1203 for genotyping Shiga toxin-producing Escherichia coli O157

We developed a rapid PCR method utilizing the diversity of the insertion site IS1203 for genotyping Shiga toxin-producing Escherichia coli (STEC) O157 (IS1203 PCR typing). DNA fragments digested by PvuII, which cut IS1203 at one site, were ligated with themselves and detected by PCR with outward-facing primer pairs for IS1203. To minimize nonspecific bands, nested PCR was also performed. Two fingerprinting patterns produced from the upstream or downstream regions of IS1203 were obtained within 1 or 2 days. By combining the two patterns, 79 STEC O157 isolates were classified into 39 types, which were then classified into 36 subtypes by pulsed-field gel electrophoresis (PFGE). The discriminatory power of IS1203 PCR typing (D = 0.974) is similar to that of PFGE (D = 0.981). This method can be used for rapid and simplified genotyping.     

Gorlin syndrome with ulcerative colitis in a Japanese girl

We present the case of a 14-year-old Japanese girl who had both Gorlin syndrome and ulcerative colitis. She had complained of blood stools for 6 months and severe scoliosis from her infancy. Physical examination revealed multiple nevi, palmar and plantar pits, jaw cysts, and calcification of the falx cerebri, leading to the diagnosis of Gorlin syndrome. Total colonoscopy revealed an edematous and spotty bleeding mucosa extending from the anus to the transverse colon. Histological examination was also compatible with ulcerative colitis. Thus, we diagnosed her as having Gorlin syndrome with ulcerative colitis. Gene analysis revealed a mutation, 1247InsT, in the human patched gene (PTCH), resulting in the truncation of PTCH protein. Since Gorlin syndrome and ulcerative colitis are rare disorders in childhood, this association is interesting, suggesting a correlation between the hedgehog signaling and intestinal disorders.     

Simplified Quantitative Assay System for Measuring Activities of Drugs against Intracellular Legionella pneumophila

We developed a new simple assay for the quantitation of the activities of drugs against intracellular Legionella pneumophila. The cells of a murine macrophage-like cell line (J774.1 cells) allowed the intracellular growth and replication of the bacteria, which ultimately resulted in cell death. The infected J774.1 cell monolayers in 96-well microplates were first treated with antibiotics and were further cultured for 72 h. The number of viable J774.1 cells in each well was quantified by a colorimetric assay with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and an enzyme-linked immunosorbent assay reader. The number of growing bacteria in each well was also determined by counting the numbers of CFU on buffered charcoal yeast extract-α agar plates. Viable J774.1 cell counts, determined by the colorimetric assay, were inversely proportional to the number of intracellular replicating bacteria. The minimum extracellular concentrations (MIECs) of 24 antibiotics causing inhibition of intracellular growth of L. pneumophila were determined by the colorimetric assay system. The MIECs of beta-lactams and aminoglycosides were markedly higher than the MICs in buffered yeast extract-α broth. The MIECs of macrolides, fluoroquinolones, rifampin, and minocycline were similar to the respective MICs. According to their intracellular activities, clarithromycin and sparfloxacin were the most potent among the macrolides or fluoroquinolones tested in this study. Our results indicated that the MTT assay system allows comparative and quantitative evaluations of the intracellular activities of antibiotics and efficient processing of a large number of samples.     

Prevention of autoimmune symptoms in autoimmune-prone mice by elimination of B-1 cells

Our recent studies on an autoantibody-transgenic mouse linedemonstrated that peritoneal B-1 cells are responsible for autoimmunesymptoms. However, whether B-1 cells in the peritoneum are generallyinvolved in the pathogenesis of autoimmune disease remains controversial.To test the possible involvement of peritoneal B-1 cells inautoimmune symptoms of autoimmune-prone NZB mice, we eliminatedthe peritoneal cells by hypotonic shock with repeated I.p. injectionof distilled water every 7 days into neonatal or 8-week-oldNZB mice. By this treatment, B-1 cells, which self- renew withinthe peritoneal cavity, are expected to be preferentially eliminated,while other peritoneal cells can be easily supplied from bonemarrows after this treatment indeed, in distilled water-treatedold NZB mice, the number of B-1 cells decreased in spleen aswell as in lamina propria of the gut but the numbers of conventionalB cells and T cells did not change. Moreover, the productionof autoantibodies against erythrocytes significantly decreasedand the occurrence of autoimmune hemolytic anemia was reducedin 12-month-old treated NZB mice. Similarly, the eliminationof peritoneal cells of NZB/NZW (NZB/W) F₁; mice by water injectiondecreased anti-DNA IgG antibodies in the sera and reduced thepathological changes of the kidney. These results suggest thatperitoneal B-1 cells may be a source of autoantibody-producingcells in autoimmune diseases of NZB and NZB/W F₁; mice.     

Expression of insulin-like growth factor 1 receptor in primary breast cancer: immunohistochemical analysis

Insulin-like growth factor-1 receptor (IGF-1R) has been implicated in regulation in tumor growth. The results of previous studies performed by radioimmunoassay are conflicting, and the prognostic significance of IGF-1R expression in primary breast cancer is still controversial. IGF-1R expression was evaluated in formalin-fixed, paraffin-embedded tissue of 210 primary breast cancer patients by using anti-IGF-1R antibody. The clinicopathologic variables and 5-year disease-free survival were studied, and their correlations between IGF-1R expressions were investigated. IGF-1R overexpression was observed in 43.8% of tumors. IGF-1R overexpression had no correlation with prognosis or with other clinicopathologic parameters, such as age, tumor size, nodal status, histologic grade, hormone receptor status, and human epidermal growth factor 2 status. Though its prognostic value in breast cancer is limited, immunohistochemical evaluation of IGF-1R by using this monoclonal antibody may be useful in translational research using archived material.     

ATP regulation of ciliary beat frequency in rat tracheal and distal airway epithelium

Ciliary beat frequency (CBF) was measured by video-optical microscopy in rat tracheal and distal airway ciliary cells using a slice preparation. In tracheal ciliary cells (tracheal slice), ATP or 2-methylthio ATP (MeSATP) increased CBF, which was inhibited by suramin (100 microm, an inhibitor of purinergic receptor). Ionomycin (5 microm) or thapsigargin (2 microm) increased CBF similarly. Ca2+-free solution or addition of Ni2+ (1 mm) decreased CBF gradually by approximately 25% and subsequent stimulation with ATP (10 microm) increased CBF transiently. The purinergic agonist experiments demonstrated that ATP increases CBF in tracheal ciliary cells via both P2X and P2Y receptors. ATP increased the intracellular calcium concentration ([Ca2+]i) in tracheal ciliary cells. However, in distal airway ciliary cells (lung slice), ATP did not increase CBF and [Ca2+]i, although a Ca2+-free solution decreased CBF, and ionomycin (5 microm) or thapsigargin (2 microm) increased it. Moreover, acetylcholine (100 microm) did not increase CBF in distal airway ciliary cells, although it increased CBF in tracheal ciliary cells. Terbutaline (10 microm), a selective beta2-adrenergic agonist, increased CBF in both tracheal and distal airway ciliary cells. These observations suggest that the Ca2+-mobilization mechanisms via purinergic or muscarinic receptors of the distal airway ciliary cell may be different from those of the tracheal ciliary cell. In conclusion, the CBF increase is differently regulated in the tracheal and distal airway epithelia of the rat.