Oxidative stress-induced posttranslational modification of TRPV1 expressed in esophageal epithelial cells

Human esophageal epithelium is continuously exposed to physical stimuli or to gastric acid that sometimes causes inflammation of the mucosa. Transient receptor potential vanilloid 1 (TRPV1) is a nociceptive, Ca(2+)-selective ion channel activated by capsaicin, heat, and protons. It has been reported that activation of TRPV1 expressed in esophageal mucosa is involved in gastroesophageal reflux disease (GERD) or in nonerosive GERD symptoms. In this study, we examined the expression and function of TRPV1 in the human esophageal epithelial cell line Het1A, focusing in particular on the role of oxidative stress. Interleukin-8 (IL-8) secreted by Het1A cells upon stimulation by capsaicin or acid with/without 4-hydroxy-2-nonenal (HNE) was measured by ELISA. Following capsaicin stimulation, the intracellular production of reactive oxygen species (ROS) was determined using a redox-sensitive fluorogenic probe, and ROS- and HNE-modified proteins were determined by Western blotting using biotinylated cysteine and anti-HNE antibody, respectively. HNE modification of TRPV1 proteins was further investigated by immunoprecipitation after treatment with synthetic HNE. Capsaicin and acid induced IL-8 production in Het1A cells, and this production was diminished by antagonists of TRPV1. Capsaicin also significantly increased the production of intracellular ROS and ROS- or HNE-modified proteins in Het1A cells. Moreover, IL-8 production in capsaicin-stimulated Het1A cells was enhanced by synthetic HNE treatment. Immunoprecipitation studies revealed that TRPV1 was modified by HNE in synthetic HNE-stimulated Het1A cells. We concluded that TRPV1 functions in chemokine production in esophageal epithelial cells, and this function may be regulated by ROS via posttranslational modification of TRPV1.     

Neonatal tolerance induction in the thymus to MHC-class II-associated antigens. II. Significance of MHC antigens in anti-Mls tolerance

Specificity of anti-Mlsa tolerance induced in BALB/c (H-2d, Mlsb) neonates was investigated by a popliteal lymph node (PLN)-swelling assay for the local graft-versus-host (GVH) reaction by injecting tolerant thymus cells into the footpads of several types of F1 hybrid mice. When thymus cells were obtained from 1-week-old normal BALB/c, they evoked enlargement of PLNs of (BALB/c X DBA/2)F1 (H-2d, Mlsb/a) [CDF1] recipients and of other hybrid recipients, heterozygous in Mlsa,c,d alleles, irrespective of the major histocompatibility complex (MHC) haplotypes. The same thymus cells did not cause the response in MHC-heterozygous F1 hybrids when the hybrids were homozygous in Mlsb, identical with BALB/c mice. Therefore, the PLN response to Mls antigens, known to be closely associated with MHC-class II antigens, was not directed to the class II antigens themselves. This enabled us to examine the effects of MHC on tolerance induction to the Mls antigens. When BALB/c neonates were injected with CDF1 bone marrow cells, complete tolerance to Mlsa-H-2d antigens of CDF1 cells was induced in the thymus, while responsiveness to Mlsa antigens in the context of H-2k and H-2b antigens, was not affected. This indicates MHC-restriction of neonatal tolerance to Mls antigens. Furthermore, when Mls and H-2-heterozygous (BALB/c X AKR)F1 (H-2d/k, Mlsb/a) bone marrow cells served as the tolerogen, thymus cells of BALB/c neonates were also tolerized to Mlsa-H-2k antigens as well as to Mlsa-H-2d antigens, which suggests the involvement of MHC, probably class II antigens of tolerance-inducing cells.     

NMR analyses of polysaccharide derivatives containing amine groups

Amylose, amylopectin, hydroxyethylcellulose, methylcellulose, and cellulose were reacted with diethylaminoethyl chloride HCl salt and 3-chloro-2-hydroxy-propyltrimethylammonium chloride under aqueous alkaline conditions in order to introduce tertiary amine and quaternary ammonium groups into polysaccharides. Degrees of substitution were obtained from ¹H- or ¹³C-NMR spectra of hydrolyzates, and distributions of diethylaminoethyl groups in polysaccharides were measured by ¹³C-NMR. Since amylose, amylopectin, and hydroxyethylcellulose were soluble in the reaction media, these three polysaccharides had higher reactivity for etherifications than cellulose. Methyl-cellulose, which has hydrophobic methyl groups, had as much reactivity as cellulose. Primary hydroxyl groups, C-6, of polysaccharides had the highest reactivity for diethylaminoethylation.     

Requirement of Stat3 Signaling in the Postnatal Development of Thymic Medullary Epithelial Cells

Thymic medullary regions are formed in neonatal mice as islet-like structures, which increase in size over time and eventually fuse a few weeks after birth into a continuous structure. The development of medullary thymic epithelial cells (TEC) is dependent on NF-κB associated signaling though other signaling pathways may contribute. Here, we demonstrate that Stat3-mediated signals determine medullary TEC cellularity, architectural organization and hence the size of the medulla. Deleting Stat3 expression selectively in thymic epithelia precludes the postnatal enlargement of the medulla retaining a neonatal architecture of small separate medullary islets. In contrast, loss of Stat3 expression in cortical TEC neither affects the cellularity or organization of the epithelia. Activation of Stat3 is mainly positioned downstream of EGF-R as its ablation in TEC phenocopies the loss of Stat3 expression in these cells. These results indicate that Stat3 meditated signal via EGF-R is required for the postnatal development of thymic medullary regions.     

Imaginal induction of diapause in several 'adult-female diapausing' spider mites

Abstract. The possibility of imaginal induction of diapause in adult female spider mites is suggested in Stigmaeopsis miscanthi. Here, several spider mites are studied to investigate the generality of this phenomenon. The existence of imaginal induction of reproductive diapause is established in Stigmaeopsis longus, two forms of S. miscanthi and Tetranychus kanzawai adult females. The females that develop under diapause-averting (long days and 18 °C) conditions are induced to diapause under short-daylength conditions at 18 °C after moulting to the adult stage. However, the adult females of Tetranychus urticae never diapause under these conditions.     

Growth and differentiation in vitro of the accumulating Lyt-2-/L3T4- subset in lpr mice

Mice bearing the recessive gene lpr develop an autoimmune syndrome associated with a massive lymphadenopathy, both of which are age and thymus dependent. The predominant accumulating cells in lymphoid tissue of lpr/lpr mice are Thy-1+ but express neither of the mature T cell markers, Lyt-2 or L3T4. We have purified this Lyt-2-/L3T4- subset and examined its phenotype. These cells are not actively cycling, do not express interleukin-2 (IL 2) receptors nor significant levels of antigen receptor, but do express the B cell marker B220. In vitro growth conditions were examined for the lpr Lyt-2-/L3T4- subset. By using a combination of phorbol ester and IL 2, these cells acquired transient expression of IL 2 receptors and grew in an IL 2-dependent manner. Furthermore, these proliferating cells underwent differentiation to a more mature T cell phenotype, with loss of cell surface B220 and acquisition, by a portion, of antigen receptor and Lyt-2. The possible parallels with normal T cell maturation are discussed.     

Igh-C allotype-linked control of anti-DNA production and clonotype expression in mice infected with Plasmodium yoelii

The infection of nonlethal strain of Plasmodium yoelii induces the formation of IgG anti-DNA antibodies as a result of polyclonal B cell activation. By using various nonautoimmune strains of mice including H-2 or Igh congenic or recombinant mice, the levels and clonotypes of anti-DNA antibodies elicited by the malaria infection were analyzed in relation to the expression of the MHC or Igh gene. Our results showed there were little, if any, differences in serum anti-DNA levels and their clonotypes among B10 and B6 H-2 congenic mice. In contrast, malaria-induced IgG anti-DNA responses markedly differed quantitatively and clonotypically between murine strains bearing the Ighb allotype and those bearing the Igha, Ighj, Ighd, or Ighn allotype. The latter group of mice produced approximately 5 to 10 times more IgG anti-DNA antibodies than the former group of mice. Clonotypically, mice bearing the Ighb allotype developed high alkaline anti-DNA antibodies of pH 8.0 to 8.5, whereas non-Ighb mice failed to express such alkaline anti-DNA spectrotypes, and exhibited more neutral spectrotypes (pH 7.0 to 8.0). Studies on the Igh recombinant mice indicated that the observed quantitative and clonotypical differences in IgG anti-DNA production was not associated with the variable region, but with the constant region of the Igh gene complex. Our results have suggested that IgG anti-DNA responses occurring as a result of polyclonal B cell activation during the course of malaria infection markedly differs quantitatively and clonotypically among murine strains and appear to be controlled at least in part by the Igh-C gene or gene(s) closely linked to it.     

Suppression of B cell development and antibody responses in mice with polyclonal rabbit and monoclonal rat anti-IgM antibodies. I. Characterization of the suppressed state

Mice treated from birth with polyclonal, crude or affinity purified rabbit or monoclonal rat anti-mouse IgM antibodies [b-7-6 and C-2-23: Eur. J. Immunol. 14: 753-757, 1984] were found to be heavily suppressed with respect to B-cell activities. Crude or affinity purified rabbit or monoclonal rat anti-mouse IgM gave comparable results as follows: serum IgM was below detectable levels; serum IgG was reduced to about 1-3% of normal levels; free anti-IgM was always detectable; IgM and/or kappa-light-chain positive cells as well as IgM-secreting cells were absent in various lymphoid organs; the B-cell mitogen lipopolysaccharide was unable to induce proliferative responses; primary antibody responses could not be induced against sheep red blood cells and phosphorylcholine; lymphoid organs were reduced in size and B-cell areas were not populated with lymphocytes; besides a 40% reduction in absolute lymphocyte numbers in the blood, we found increased platelet counts and a 10% eosinophilia in anti-IgM-treated mice.     

Microglia Endocytose Amyloid β Through the Binding of Transglutaminase 2 and Milk Fat Globule EGF Factor 8 Protein

Activation of glial cells has been observed in neurodegenerative diseases including Alzheimer’s disease (AD). Aggregation of amyloid β (Aβ) is profusely observed as characteristic pathology in AD brain. In our previous study using microglial cell line BV-2, tissue-type transglutaminase (TG2) was found to be involved in phagocytosis (Kawabe et al., in Neuroimmunomodulation 22(4):243–249, 2015; Kawabe et al., Neurochem Res 2017). In the present study, we examined whether TG2 and milk fat globule EGF factor 8 protein (MFG-E8), an adaptor protein promotes macrophage to engulf apoptotic cells, were involved in Aβ endocytosis. When the neuronal/glial mixed culture was stimulated freshly prepared Aβ_1?42 for 3 days, the incorporation of Aβ was observed by immunofluorescence staining technique in Iba-1-positive microglia. Cystamine, a broad competitive inhibitor of TGs, suppressed it. When aggregated Aβ was added to the mixed culture, the immunoreactivity of MFG-E8 surrounding Aβ was observed, and then followed by microglial endocytosis. Using western blotting technique, MFG-E8 was detected in cell lysate of astrocyte culture, and was also detected in the medium. When microglia culture was incubated with astrocyte conditioned medium, MFG-E8 levels in microglia tended to increase. It is likely that microglia might utilize MFG-E8 released from astrocytes as well as that expressed in themselves in order to endocytose Aβ aggregation. Furthermore, we confirmed that MFG-E8 could bind with TG2 in microglia culture by immunoprecipitate technique. These results suggest that microglia might uptake Aβ as a complex of aggregated Aβ/MFG-E8/TG2.