Chymase participates in chronic dermatitis by inducing eosinophil infiltration

An epicutaneous application of 2,4-dinitrofluorobenzene (DNFB) to a mouse ear caused a transient skin swelling, and the repetition of the challenge enlarged the contact dermatitis. The repeated challenge with DNFB also induced eosinophil infiltration on the application site. Administration of a chymase inhibitor significantly inhibited the ear swelling as well as eosinophil accumulation. An intradermal injection of human chymase to the mouse ear also elicited transient skin swelling and eosinophil infiltration, both of which were augmented in proportion to the number of injections. Human serum albumin and heat-inactivated chymase failed to induce such skin reactions, suggesting the participation of proteolytic activity of the enzyme. In addition, chymase stimulated eosinophil migration in vitro in a concentration-dependent manner. Taken together, these observations suggest that mast cell chymase may contribute to development of the DNFB-induced dermatitis, probably by promoting eosinophil infiltration. It is therefore possible that chymase plays a role in pathogenesis of chronic dermatitis such as atopic dermatitis.     

Primary carcinosarcoma of the vagina

Primary carcinosarcoma of the vagina is a very rare tumor, with only eight cases diagnosed as carcinosarcoma in the literature that we are aware of. We recently encountered a case of primary carcinosarcoma of the vagina in a 75-year-old woman. The patient had a history of hysterectomy and bilateral ovariectomy for uterine corpus cancer at 55 years of age. Recurrence of the cancer was suspected 17 years after the operation and irradiation therapy was performed, but the patient died 3 years after the recurrence. Autopsy revealed a mass lesion in the pelvic cavity that originated in the vagina. Histological examination showed that the tumor contained anaplastic carcinoma, rhabdomyosarcoma, leiomyosarcoma and chondrosarcoma components, and it was diagnosed as carcinosarcoma. The histological diagnosis of the uterine corpus cancer was well-differentiated adenocarcinoma, and there was no sarcomatous component. The carcinosarcoma occurred 17 years after the hysterectomy, and it was concluded to be a primary carcinosarcoma of the vagina. This is the first case of primary vaginal carcinosarcoma in which the epithelial and sarcomatous components were clearly identified histologically and immunohistochemically.     

Identification of a large novel imprinted gene cluster on mouse proximal chromosome 6

Mice with maternal duplication of proximal chromosome 6 die in utero at an early embryonic stage. Recently, two imprinted genes, paternally expressed Sgce and maternally expressed Asb4, were identified in this region. This report analyzes the imprinting status of genes within a 1-Mb region containing these two genes. Peg10, which is next to Sgce, shows complete paternal expression, like Sgce. Conversely, Neurabin, Pon2, and Pon3 show preferential maternal expression at embryonic stages, although they all show biallelic expression in neonatal tissues. These results demonstrate that there is a large novel imprinted gene cluster in this region. 5'-RACE (Rapid Amplification of cDNA Ends) analysis of Peg10 revealed the existence of a novel first exon separate from the second exon, which encoded two putative ORFs similar to the viral Gag and Pol proteins. A differentially methylated region established in sperm and eggs is located just within the region containing the two first exons of Peg10 and Sgce, and may play an important role in regulating the two paternally expressed genes: Peg10 and Sgce.     

Localization of two cholinergic markers, choline acetyltransferase and vesicular acetylcholine transporter in the central nervous system of the rat: in situ hybridization histochemistry and immunohistochemistry

Choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) are proteins that are required for cholinergic neurotransmission. Present knowledge concerning the organization of cholinergic structures has been derived primarily from immunohistochemistry for ChAT. In the present study, we investigated the distribution of mRNAs and the corresponding proteins for ChAT and VAChT by in situ hybridization histochemistry and immunohistochemistry. The patterns of distribution of perikarya containing ChAT mRNA, ChAT protein, VAChT mRNA and VAChT protein were similar in most regions, and co-localization in the same neuron of mRNAs for ChAT and VAChT, that of ChAT mRNA and ChAT protein, and that of VAChT mRNA and VAChT protein were demonstrated. However, in the cerebral cortex and hypothalamus, ChAT-immunoreactive perikarya were present, but they did not contain mRNAs for ChAT and VAChT, and VAChT protein. On the other hand, in the cerebellum, Purkinje cell bodies contained VAChT mRNA and VAChT protein, but they did not contain either ChAT mRNA or ChAT protein. Axon bundles were clearly revealed by immunohistochemistry for ChAT, but they were not detected by that for VAChT. Both ChAT and VAChT antibodies revealed preterminal axons and terminal-like structures. In the forebrain, they were present in the olfactory bulb, nucleus of the lateral olfactory tract, olfactory tubercle, lateral septal nucleus, amygdala, hippocampus, neocortex, caudate-putamen, thalamus and median eminence of the hypothalamus. In the brainstem, they were localized in the superior colliculus, interpeduncular nucleus and some cranial nerve motor nuclei, and further in the ventral horn of the spinal cord. These results indicate strongly that ChAT and VAChT are expressed in most of the cholinergic neurons, and that immunohistochemistry for VAChT is as useful to detect cholinergic terminal fields as that for ChAT.     

Role of tight junctions of polarized epithelial MDCK cells in the replication of herpes simplex virus type 1

Before completion of polarization, Madin-Darby canine kidney (MDCK) cells showed high infectivity and progeny production of herpes simplex virus type 1 infection. After polarization or formation of tight junctions, the infectivity and virus replication in MDCK cells was restricted significantly. The disruption of tight junctions by depletion of Ca²⁺ resulted in increasing virus infectivity and productivity. Mechanical disruption of tight junctions by scratching the cell monolayers with injection needle allowed markedly the replication of HSV-I in the cells aligned along the injured area. In polarized MDCK cells the progeny were released preferentially from the apical surface of the cells. These data suggest that because polarized MDCK cells mimic the epithelial cell layers, this cell line is helpful for determining the factors which regulate viral transmission in the human body. © Wiley-Liss, Inc.     

Ethnic differences in allele frequency of autoimmune-disease-associated SNPs

Several multiple, large-scale, genetic studies on autoimmune-disease-associated SNPs have been reported recently: peptidylarginine deiminase type 4 (PADI4) in rheumatoid arthritis (RA); solute carrier family 22 members 4 and 5 (SLC22A4 and 5) in RA and Crohns disease (CD); programmed cell death 1 (PDCD1) in systemic lupus erythematosus (SLE), type 1 diabetes mellitus (T1D), and RA; and protein tyrosine phosphatase nonreceptor type 22 (PTPN22) in T1D, RA, and SLE. Because these reports on association were not always evaluated in multiple ethnic groups and because ethnic difference in allele frequency of the variants has been also reported, we investigated allele frequencies of nine SNPs in four autoimmune-disease-associated loci in Caucasian, African-descent, and Japanese populations. Although SNPs in PADI4 had similar allele frequency among three groups [maximal difference 11%; (P >0.05)], the other three loci revealed statistically significant allele frequency differences (maximal difference 39% (P <0.00001), 13% (P <0.00001), and 8% (P <0.00001) in SLC22A4, PDCD1, and PTPN22, respectively). Of note, three SNPs in the three loci that had allele frequency more than 8% in the Caucasian population were either not polymorphic at all or extremely rare in the Japanese population. Our data suggest that ethnic variations of polymorphisms should be evaluated in detail, and differences should be incorporated into investigations of susceptibility variants for common diseases.     

Ras homolog gene family H (RhoH) deficiency induces psoriasis-like chronic dermatitis by promoting TH17 cell polarization

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Direct in vivo protein transduction into a specific restricted brain area in rats

Attempts at protein transduction into specific restricted brain areas have remained unsuccessful. We attempted targeted, direct in vivo protein transduction by microinjecting beta-galactosidase (beta-gal) with hemagglutinating virus of Japan envelope (HVJ-E) vector into the rat nucleus tractus solitarius (NTS). The medulla oblongata including the NTS was removed 6h post-injection and cryostat sections were histochemically stained to detect beta-gal enzymatic activity. beta-gal-positive cells were present in these sections as was beta-gal activity determined by colorimetric analysis. beta-gal-positive cells were not present in the rats microinjected only beta-gal protein without HVJ-E vector. Our findings suggest that direct in vivo protein transduction into specific restricted brain areas is possible. The type of targeted delivery system we present may have wide applications in the administration of therapeutic proteins to the central nervous system.     

Flow cytometric and functional analysis of mononuclear cells infiltrating the liver in experimental autoimmune hepatitis.

Experimental autoimmune hepatitis was produced by immunizing Wistar rats with syngeneic liver proteins. Mononuclear cells infiltrating the liver tissue were identified by immunohistochemical techniques using monoclonal antibodies specific for subpopulations of rat lymphocytes. The strong infiltration of CD8+ cytotoxic T lymphocytes (CTL) were found in the portal areas. Subpopulations of mononuclear cells infiltrating the liver, spleen cells and peripheral blood lymphocytes were identified by flow cytometry. Flow cytometric analysis revealed the presence of CD5- and CD8+ lymphocytes in the liver tissues. Mononuclear cells infiltrating the liver were isolated from Wistar rats having autoimmune hepatitis to determine whether those exhibit cytotoxicity against syngeneic hepatocytes; they exhibited cytotoxicity against isolated syngeneic hepatocytes, but failed to lyse K562 cells, syngeneic concanavalin A-activated splenocytes and allogeneic hepatocytes. Depletion of CD8+ T cells significantly reduced the cytotoxic ability of mononuclear cells infiltrating into the liver against syngeneic hepatocytes. These findings support the idea that liver cell injury in experimental autoimmune hepatitis may at least in part be mediated by CTL.     

Supplementation Effect of Early Pregnancy Factor-Positive Serum into Bovine In Vitro Fertilization Culture Medium

PROBLEM: Early pregnancy factor (EPF) has been detected in pregnant bovine serum, and its activity appeared from 24 to 48 hr after insemination. However, in bovine in vitro fertilization (IVF), an EPF-like substance(s) had been detected in the culture medium of fertilized ovum. Therefore, we think that EPF and EPF-like substance(s) are very important materials for the development of the embryo. In this study, we examined the development of the embryo when fertilized bovine ova were cultured with IVF culture medium supplemented with EPF-positive or -negative serum. METHOD OF STUDY: EPF activity of each serum (fetal calf serum [FCS], calf serum [CS], estrus bovine serum, and pregnant bovine serum) was assessed by the bovine-rosette inhibition test. The sera were supplemented in TCM-199 culture medium, and IVF bovine ova were cultured with the media for 6 or 7 days, respectively. The culture media of each group were evaluated for EPF activity by the bovine-rosette inhibition test 48 hr after IVF. The cleavage rate of each group was calculated at 48 hr, and 6 or 7 days after IVF. The culture medium of cumulus cells was also tested for EPF activity. RESULTS: Only the pregnant bovine sera were EPF positive. All the culture media 48 hr after IVF became EPF positive. Additionally, the culture medium of cumulus cells did not have EPF activity. There was no significant difference in the cleavage rate of the EPF-positive and - negative sera 48 hr after IVF. However, the cleavage rate of EPF-positive sera tended to be higher than the negative sera. In contrast, the blastocyst development rates of EPF-positive sera were significantly higher than those of the negative sera 6 to 7 days after IVF (P < 0.05). CONCLUSIONS: The data suggest that an EPF-like substance(s) may be secreted from the in vitro fertilized bovine ovum but not from the cumulus cell, and that the EPF in the pregnant serum may accelerate the development of the bovine embryo in IVF.