Although HIV-1 can be successfully eradicated from the circulating blood of HIV-1-infected individualsusing anti-retroviral therapy (ART), HIV-1 virions emerge immediately after the interruptionof ART. This study was aimed to investigate the origin of the emerged HIV-1. After obtaininginformed consent, blood samples from nine HIV-1-infected individuals and endoscopic ileum samplesfrom five of the individuals were obtained. Purified peripheral mononuclear cells (PBMCs)and ileum cells were analyzed by flow-cytometry, and the V3 loop sequences of the HIV-1 envelopeprotein were determined. By comparing the V3 loop sequences of the samples, we confirmedthat the provirus hidden in the CD4+ PBMCs was not the source of the HIV-1 that emerged afterthe interruption of ART. Although free virus and HIV-1-p24 antigen (p24)-positive cells were notseen in the blood of patients receiving ART, proviral DNA and p24 could be detected in the ileumfrom the same patient. Among the HIV-1-infected CD4+ cells in the ileum samples, Vα24+ naturalkiller T (NKT) cells were the major p24-positive cells. These results suggest that the innate NKTcells in the mucosal compartment are the most likely candidates for the origin of the HIV-1 thatemerged after ART was interrupted.
Melanoma differentiation-associated gene 5 (MDA-5) and retinoic acid-inducible gene-I (RIG-I)are members of DExH family of proteins, and known to play important roles in antiviral responsesto induce type I interferons (IFNs). MDA-5 has been thought to sense RNA virus with long(>1 kb) double-stranded RNA. However, MDA-5 is also induced by type II IFN that is involvedin acquired immunity, suggesting that role of MDA-5 remains to be elucidated. In addition, nostudy regarding MDA-5 in oral region has been performed. Here we investigated the role ofMDA-5 in HCS-3 squamous carcinoma cells derived from oral epithelial cells. Treatment of HCS-3 cells with IFN-α2b or IFN-γ significantly induced MDA-5 as well as RIG-I. IFN-α2b exertedanti-proliferative effect in HSC-3 cells while no such effect was observed in the cells treated withIFN-γ. MDA-5 is known to be associated with tumor cell growth in melanoma. However, overexpressionof MDA-5 did not alter the proliferation in HSC-3 cells, indicating that MDA-5 is unrelatedto the cell growth in this type of cells. We conclude that MDA-5 is induced by both typeI- and type II-IFNs in HSC-3 cells, and this suggests MDA-5 may play a role in immune responsesin oral cavity.
We examined the inhibitory effects of loxoprofen, a cyclooxygenase inhibitor, and glycine, a majorinhibitory neurotransmitter, on the micturition reflex in conscious rats and hypothesized thatthese drugs would interact synergistically to inhibit micturition. Voiding behaviors were assessedusing a metabolic cage. Oral loxoprofen decreased the urinary frequency, and only a high dose(10 mg/kg) significantly reduced the voided volume. With cystometry, intravenous loxoprofen(0.1–3 mg/kg) and glycine (30 and 100 mg/kg) prolonged the intercontraction intervals (ICI) in adose-dependent manner, but did not change the maximum voiding pressure (MVP) in consciousrats. The combination of loxoprofen (3 mg/kg) and glycine (100 mg/kg) strongly prolonged theICI more than with either drug alone. The lowest dose of loxoprofen (0.1 mg/kg) and glycine(30 mg/kg) did not affect either the ICI or the MVP, but their combination resulted in a significantincrease in the ICI. These results suggest that the combined administration of loxoprofen and glycineproduced a synergistic inhibitory effect on the micturition reflex.
Secreted proteins play essential roles in the process of tumorigenesis, and the analysis of tumorsecretedproteins has been suggested as a promising strategy for identifying cancer biomarkers. Inthis study, we performed proteomic analysis to identify proteins secreted from bladder cancer celllines that are recognized by autoantibodies in sera from patients with bladder cancer. In addition,autoantibodies against the identified proteins were validated using a dot-blot array with sera frompatients with bladder cancer and normal controls. As the results, we detected twenty-five and thirty-two immunoreactive spots in sera from patients with high- and low-grade bladder cancer, respectively.In addition, validation analysis revealed that serum IgG levels of anti-calreticulin andmatrix metalloproteinase-2 (MMP2) autoantibodies were significantly higher in bladder cancer patientsthan in normal controls (both P < 0.05). Furthermore, the serum IgG level of anti-MMP2autoantibody was significantly higher in patients with high- compared to low-grade bladder cancer(P < 0.05). On multivariate analysis, the serum IgG level of anti-MMP2 autoantibody was an independentpredictor of cancer-specific survival (P < 0.05). Based on these findings, serum IgG levelsof anti-calreticulin and MMP2 autoantibodies may be novel biomarker candidates for bladdercancer and its clinical outcome.
Immunohistochemistry using whole mount preparations of the murine mesentery revealed twotypes of LYVE-1-immunoreactive cells with dendritic morphology other than F4/80+ typical macrophages.The two types of LYVE-1+ cells were regularly distributed with constant intervalsthroughout the mesentery and appeared to possess their own territory. Both types of LYVE-1+ cells were weakly or moderately immunopositive for F4/80 antibody, a marker of macrophages,while F4/80+ round macrophages were absolutely free from the LYVE-1 immunoreactivity. Only macrophages could ingest latex particles of 20 nm in diameter 3 h after a peritoneal injection.Peritoneal administration of lipopolysaccharide (LPS) induced a rapid reduction of LYVE-1 immunoreactivityin the cells with dendritic morphology followed by an increased immunoreactivityto F4/80 antibody, and simultaneously by dynamic changes in their shape. Under normal conditions,F4/80+ macrophages in various connective tissues expressed LYVE-1, in contrast to lack ofLYVE-1 in F4/80+ macrophages within the parenchyma of visceral organs and macrophages residingin hepatic sinusoids and pulmonary alveoli. LYVE-1 may play a role in cell adhesion and migrationof macrophagic cells within connective tissues rich in hyaluronan, and loss of LYVE-1becomes a reliable sign of activated conditions in inflammation.
Membrane progestin receptors (mPRs) are key mediators of rapid, nongenomic actions of progestinson plasma membranes. We established a procedure for the expression and purification of recombinantgoldfish mPRα using the methylotropic yeast Pichia pastoris. In P. pastoris, therecombinant protein, which carried C-terminal histidine and c-Myc tags, was expressed in an activeform as the receptor for maturation-inducing steroids of fish. Expressed proteins were boundreversibly with a high affinity (Kd = 9.4 nM) at a single binding site that could be saturated. Aftersolubilization of mPRα with n-dodecyl-β-D-maltoside (DDM) from yeast membranes, the recombinantprotein was purified using three different columns: first it was affinity-purified over nickelnitrilotriaceticacid (Ni-NTA), then bound to a cellulose resin with free amino groups and finallyto a column with affinity for the c-Myc epitope. The identity of the purified protein was verifiedby MALDI-TOF/MS analysis and its capacity to bind progestin remained. Expression and purificationof mPRα protein in its functional form will enable the screening of ligands and the determinationof its three dimensional structure.
Phosphoinositide metabolism is intimately involved in cellular signal transduction. In response toextracellular stimuli, it generates diacylglycerol (DG), which serves as a lipid second messengermolecule to activate various proteins in various organs under pathophysiological conditions. Diacylglycerolkinase (DGK) constitutes an enzyme family that catalyzes conversion of DG to phosphatidicacid. It is therefore regarded as a regulator of the DG signal. Previous studies haverevealed the critical role of α and ζ types of DGK in T cell functions. Nevertheless, little is knownabout the expression patterns of the DGK family in immune cells of various kinds. After examinationof the expression profile of DGK isozymes in immune cells that are isolated from humanblood, we investigated whether their mRNA expression levels would be changed during an inflammatoryreaction. Results showed that DGK isozyme mRNAs are widely expressed in immunecells, except for DGKβ and DGKι. During an inflammatory reaction, DGKε mRNA was increasedtransiently in the initial phase (20–40 min) of stimulation with both LPS and IL-2 in T cellderivedHUT-102 cells and macrophage-derived RAW264 cells. At the organismal level, an intraperitonealinjection of LPS also induced upregulation of DGKε mRNA in the spleen in a similar,but not identical, manner. These results suggest that DGKε is involved in inflammatory processesof the cellular immune system.
The periosteum supplies osteoblasts and nutrients for bone metabolism and is important for osteoblastdifferentiation and osteogenesis. Recently, periosteum-derived cells have been used for orofacialbone regeneration therapy. However, little is known about the function of the periosteum inphysiological bone remodeling. On our hypothesis that the periosteum senses a mechanical stressto induce bone remodeling, we subjected human jaw bone periosteum cells (HJBPCs) to uniaxialstretching for 24 h and characterized their gene expression profiles by microarray analysis. Of62,976 genes detected, 550 genes related to bone metabolism were extracted, and 76 of thesegenes with large changes in gene expression were short-listed. The results indicated that mechanicalstretch in HJBPCs regulated the expression levels of genes involved in the Wingless-typeMMTV integration (Wnt) site, bone morphogenetic protein (BMP) signaling pathways, and inflammatorycytokines. We propose that periosteum-derived cells sense mechanical stress and thenactivate and regulate signals for osteoblast differentiation and osteogenesis.
The inferior colliculus (IC) transmits the ascending auditory signal to the thalamic medial geniculatenucleus. Previous studies have reported that serotonergic input originating from the raphe nucleihas a strong influence on signal processing within the central nucleus of the IC. To identifythe cellular target for the serotonergic modulation in the IC, we examined the effect of serotoninas well as selective serotonin reuptake inhibitor (SSRI) fluvoxamine on spontaneous GABAergicand glycinergic inhibitory postsynaptic currents (sIPSCs) recorded with whole-cell recordings.Consistent with earlier studies, we confirmed that serotonin robustly enhanced the frequency, butnot amplitude, of GABAergic sIPSCs. It should be noted that the application of fluvoxamine alonemarginally increased the frequency of GABAergic sIPSCs. These findings suggest that serotonin isendogenously released even in slice preparations, and it negatively modulates the tone of activityof inhibitory neurons within IC. We also examined the effect of serotonin and fluvoxamine on glycinergicsIPSCs and found that serotonin has a significantly weaker effect on glycinergic sIPSCsthan on GABAergic sIPSCs. The differential sensitivity of the GABAergic and glycinergic sIPSCsto serotonin implies that serotonergic input plays a specific role in auditory information processing.Moreover, it suggests that the serotonergic input may contribute to pathological conditionssuch as tinnitus.
The monocarboxylate transporter (MCT)-1 plays an important role in the transfer of monocarboxylatemetabolites such as lactate, ketone bodies, and acetic acid. The present study revealed theselective localization of MCT1 in reticular cells of the murine lymph node. An intense MCT1 immunoreactivitywas found in the reticular cells forming a cellular network together with sinusliningcells in the medullary sinuses and in cells covering the inside of subcapsular sinuses.Electron-microscopically, MCT1 was localized along the plasma membrane of the reticular cells.The medullary reticular cells vigorously ingested carboxylate-modified latex particles, but any reticularcells within the cortical lymphoid follicles and medullary cords neither expressed MCT1nor incorporated latex particles. MCT1-immunoreactive reticular cells also expressed LYVE-1,which is a hyaluronan receptor abundant in both the lymphatic endothelium and hepatic sinusoidalepithelium. The selective localization of MCT1 and LYVE-1 suggests a high level of activity forlymphoid reticular cells in the uptake of carboxylate-modified and hyaluronate waste substancescirculating in the body.