Microglia produce IFN-gamma independently from T cells during acute toxoplasmosis in the brain.

We previously reported a requirement of interferon-gamma (IFN-gamma) production by both T cells and cells other than T or natural killer (NK) cells in the brain for prevention of toxoplasmic encephalitis. In the present study, we examined whether microglia, the resident macrophage system in the brain, produce IFN-gamma in response to infection with Toxoplasma gondii in SCID and wild-type BALB/c mice. IFN-gamma was detected in the culture supernatants of microglia purified from the brains of SCID mice that had developed toxoplasmic encephalitis due to reactivation of infection. A significant increase in numbers of IFN-gamma-expressing microglia was also observed by immunostaining for this cytokine in the brains of SCID and BALB/c mice during the acute stage of acquired infection, and those numbers decreased in the later stage of infection in the BALB/c animals. These results indicate that microglia produce IFN-gamma in the presence and absence of T cells in response to reactivated or acute acquired infection in the brain. Because IFN-gamma is the essential effector molecule to control tachyzoites and because this cytokine is a potent inducer of expression of chemokines and MHC antigens important for recruitment and activation of T cells, IFN-gamma production by microglia might play a critical role in the early stage of tachyzoite proliferation in the brain by limiting parasite growth and initiating subsequent T cell immunity.     

Neurite extension of DRG neurons by gicerin expression is enhanced by nerve growth factor

Gicerin, a cell adhesion molecule, is expressed in dorsal root ganglion (DRG) and sciatic nerves during chick development. This molecule re-appears in these tissues after an injury to the sciatic nerve. In the present study, we investigated the expression of nerve growth factor (NGF) in the regenerating sciatic nerve of chicks and the effects of NGF on the expression and neurite activities of gicerin in DRG. In the sciatic nerve after a crush injury, the expression of NGF and gicerin increased in the Schwann cells and in the nerve fibers, respectively. NGF promoted the neurite projections from in vitro DRG on the gicerin ligands, which were inhibited by anti-NGF antibody. The gicerin mRNA expression increased in the DRG with NGF, which was inhibited by the co-incubation with anti-NGF antibody. These results indicate that NGF might therefore enhance the expression of gicerin in DRG, thereby promoting the gicerin-dependent neurite extension during sciatic nerve regeneration.     

Embryonic development of the pituitary gland in the chick

Pituitary glands of chicken, from stages 20 (70 approximately 72 h of incubation) to 46 (20 days) of Hamburger and Hamilton (1951), were studied by immunocytochemical and histological stainings and India ink injection into blood vessels. Using the distribution pattern of 6 types of immunoreactive adenohypophyseal cells and the location of pituitary stalk as guideposts, we found how specific areas in the epithelium of Rathke's pouch differentiate into specific regions of the adenohypophysis at 20 days. In the sagittal plane, the walls of Rathke's pouch were tentatively divided into the upper part (A(1) + A(2)) and lower part (A(3)) of the anterior wall, and the posterior wall (P(1) + P(2) + P(3)). The cephalic lobe was mainly assembled by the proliferation of parenchymal cells in the areas A(2) + A(3) + P(2) of Rathke's pouch epithelia at 3 days of incubation. The caudal lobe was derived from A(1) + P(1) + P(3). The pars tuberalis was derived from A(1) + A(2). Thus, the avian adenohypophysis is established at 13 days, though the blood supply to the pars distalis is established at 20 days. Therefore, the cephalic lobe and caudal lobe of the pars distalis and the pars tuberalis of the chicken adenohypophysis are derived from specific areas of the cell cords of Rathke's pouch at 3 days of incubation.     

Functional evaluation of lung by Xe-133 lung ventilation scintigraphy before and after lung volume reduction surgery (LVRS) in patients with pulmonary emphysema

We evaluated the respiratory functions of patients with pulmonary emphysema who underwent lung volume reduction surgery (LVRS) by the mean transit time (MTT) with Xe-133 lung ventilation scintigraphy, forced expiration volume in 1 sec (FEV1.0), residual volume (RV), distance walked in 6 min (6-min walk), and the Hugh-Jones classification (H-J classification) before and after LVRS. In 69 patients with pulmonary emphysema (62 men, 7 women; age range, 47-75 years; mean age, 65.4 years +/- 6.1, preoperative H-J classification, III (two were II)-V) who underwent LVRS, all preoperative and postoperative parameters (MTT 3 weeks after LVRS and the others 3 months after LVRS) were judged statistically by the Wilcoxon signed-ranks test and Odds ratio. Every postoperative parameter was improved with a significant difference (P < 0.05) compared to preoperative parameters. MTT at 3 weeks after LVRS was not associated with %FEV1.0 and the H-J classification at 3 months after LVRS, but was associated with RV and a 6-min walk at 3 months after LVRS. MTT was useful for the clinical evalution of aerobic capability after LVRS.     

Analysis of photopolymerization behavior of UDMA/TEGDMA resin mixture and its composite by differential scanning calorimetry

The aim of this study was to investigate the extent of polymerization (Ep) in terms of polymerization rate of UDMA/TEGDMA resin mixtures and its composite resin, by using a differential scanning calorimeter (DSC) technique employing a photopolymerization apparatus. The resin mixtures used in this study consisted of urethane dimethacrylate (UDMA) as a base monomer and triethyleneglycol dimethacrylate (TEGDMA) as a low viscosity monomer. The concentration of TEGDMA in the mixed monomer was varied to 0, 20, 40, 60, 80, and 100 mol %. Additionally, using a base monomer consisting of 60 mol % UDMA and 40 mol % TEGDMA, four kinds of composites with silica filler of 0, 20, 40, 60, and 70 wt %, were prepared in this study. The general reaction profile and Ep values were obtained from the DSC curves. Increasing the concentration of TEGDMA resulted in a decrease in the viscosity of the UDMA/TEGDMA mixture, a delay in the time to maximum polymerization rate, and an increase in the Ep values of the resin mixtures. Furthermore, Ep values decreased with increasing filler content between 0 and 60 wt % but did not decrease further between 60 and 70 wt %.     

Construction of Canine Herpesvirus Vector Expressing Foreign Genes Using a lacZ-TK Gene Cassette as a Double Selectional Marker

An improved method for constructing canine herpesvirus (CHV) recombinants expressing foreign genes by using the lacZ-TK gene cassette as a double selectional marker was developed. A recombinant CHV carrying the lacZ-TK gene at a targeted gene locus was constructed and used as a parental virus for generating new recombinants. The parental virus formed blue plaques and was sensitive to TK-specific drugs, while newly generated recombinants, in which the lacZ-TK gene was replaced with the desired foreign gene, become both resistant to the TK-specific drugs and formed white plaques. Recombinants were isolated by using the combination of drug selection and color selection. This improved method allows construction of recombinant CHV with great ease, because the drug selection can enrich the frequency of recombinant CHV from 0.01–0.1% to 10–80%. This method was employed to construct a recombinant CHV that expressed rabies virus (RV) glycoprotein (G protein). This revised version was published online in July 2006 with corrections to the Cover Date.     

Immunocytochemical characteristics of human osteosarcoma cell line HS-Os-1: possible implication in apoptotic resistance against irradiation

In our previous study, we examined reactive oxygen species (ROS) formation in T lymphocytes following 5 Gy irradiation. We found that ROS formation occurred immediately after irradiation, continued for several hours, and resulted in oxidative DNA damage. Therefore, the origin of the hyper-radiosensitivity of T lymphocytes seemed to be the high production of ROS in the mitochondrial DNA following irradiation. In the succeeding study, we examined radiation-induced ROS formation, oxidative DNA damage, early apoptotic changes, and mitochondrial membrane dysfunction in the human osteosarcoma cell line HS-Os-1. We found that ROS formation and oxidative DNA damage were actually scarcely seen after irradiation of up to 30 Gy in these cells, that mitochondrial membrane potential was preserved, and that apoptotic changes were not demonstrated despite the relatively high-dose irradiation of 30 Gy. In the present study, we examined the immunocytochemical characteristics of the apoptotic-resistance of the HS-Os-1 cell line against irradiation in order to clarify its possible implications regarding radiosensitivity. The results showed that these cells lack P53 and Bax protein expression, and strong peroxidase activity was confirmed in the nuclei of the cells. Moreover, SODII (manganese superoxide dismutase II) protein expression was gradually increased in spite of irradiation of up to 30 Gy. Therefore, it is concluded that HS-Os-1 cells are originally apoptotic-resistant and that the cells possess a strong ability to scavenge for free radicals. To convert these cells to a state of apoptotic-susceptibility, a powerful oxidant such as hydrogen peroxide might exert such an effect in terms of the production of hydroxyl radicals in lysosomes in the cells as shown in our previous studies. The origin of the radioresistance of the human osteosarcoma cell line HS-Os-1 is considered to to be low degree of ROS formation following irradiation, reflecting the strong scavenging ability of these cells for free radicals including hydroxyl radicals.     

Novel mutations in TNFRSF1A in patients with typical tumor necrosis factor receptor-associated periodic syndrome and with systemic lupus erythematosus in Japanese

Molecular defects of TNFRSF1A was investigated in members of a family presenting with typical phenotypes of tumor necrosis factor receptor-associated periodic syndrome (TRAPS) and in patients with the autoimmune disorders, systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Genomic DNA from the members of a family with typical TRAPS, as well as from 100 patients with SLE, 100 patients with RA and 100 healthy individuals, was studied for mutations in exons 2, 3 and 4 of the TNFRSF1A gene. All individuals were Japanese. Three novel missense mutations were identified in the TNFRSF1A. The C70G mutation was identified in family members with typical TRAPS, which was the second case in eastern Asian population. In addition, the T61I and R104Q mutations were each identified in 2 of the 100 SLE patients. The T61I mutation was identified in one of the 100 healthy individuals. No mutations were identified in the 100 RA patients. Functional analysis revealed that PMA-induced shedding of TNFRSF1A from PBMCs was impaired in a patient carrying T61I. A larger scale of study will clarify whether these two mutations, T61I and R104Q, are associated with chronic inflammatory disorders, such as SLE, or not.     

Molecular analysis of structural protein genes of the Yamagata-1 strain of defective subacute sclerosing panencephalitis virus. III. Nucleotide sequence of the hemagglutinin gene

The full-length cDNA corresponding to the mRNA for the hemagglutinin (H) protein of the Yamagata-1 strain of the subacute sclerosing panencephalitis (SSPE) virus was cloned and the nucleotide sequence was determined. The mRNA corresponding to the H protein was composed of 1952 nucleotides and contained a single large open reading frame, which encoded 620 amino acids with a predicted molecular weight of 69,723. This cDNA clone expressed the H protein in Cos 7 cells, and the transfected cells showed hemadsorption. The nucleotide and amino-acid sequence homology with the Edmonston strain of MV were 98.0% and 96.6%, respectively. The deduced amino acid sequence had a single hydrophobic domain near the N-terminus that was long enough to serve as an anchor in the membrane. Five potential glycosylation sites were found on the H protein at identical positions as in the H protein of MV. Cysteine and proline were located at almost identical positions as those of the H protein of MV. In addition, monoclonal antibody study revealed that three epitopes, including the domains that were involved in the biological activities of the H protein of MV, were conserved on the Yamagata-1 strain. These results suggested that the H protein of the Yamagata-1 strain of defective SSPE virus is structurally and functionally similar to that of the Edmonston strain of MV.     

Humoral immunity to immunodominant epitopes of Hepatitis C virus in individuals infected with genotypes 1a or 1b

Cellular immunity against multiple Hepatitis C virus (HCV) proteins is observed in patients acutely infected with HCV most of whom later resolve infection. We wished to assess humoral immunity in patients infected with HCV 1a or 1b genotypes in relation to viral load using plasma samples from HCV-infected individuals and a panel of peptides representing immunodominant epitopes of HCV structural and nonstructural proteins. Plasma from HCV 1a- and 1b-infected patients, respectively, were divided into two groups: patients with low viral load (<==100,000 RNA copies/ml) and patients with high viral load (>/=10,000,000 RNA copies/ml). The antigens were peptides representing epitopes from immunodominant regions of HCV core, E2, NS3, and NS4 proteins, as well as the hypervariable (HVR) epitopes in E2 from genotypes 1a and 1b. Individuals infected with HCV 1a evoked a stronger immune response to many immunodominant epitopes of HCV relative to individuals infected with HCV 1b. Moreover, among individuals infected with HCV 1a, those with low viral loads mounted significantly greater responses against these epitopes than did individuals with high viral loads. Our observations demonstrate that quantitatively different antibody responses are elicited against HCV depending on the genotype of infecting virus, and suggest that humoral immunity directed against multiple immunodominant epitopes in HCV 1a-infected individuals may help lower viral load in vivo.