Does HTLV-1 Infection Show Phenotypes Found in Sjögren's Syndrome?

Viruses are a possible cause for Sjögren's syndrome (SS) as an environmental factor related to SS onset, which exhibits exocrine gland dysfunction and the emergence of autoantibodies. Although retroviruses may exhibit lymphocytic infiltration into exocrine glands, human T-cell leukemia virus type 1 (HTLV-1) has been postulated to be a causative agent for SS. Transgenic mice with HTLV-1 genes showed sialadenitis resembling SS, but their phenotypic symptoms differed based on the adopted region of HTLV-1 genes. The dominance of tax gene differed in labial salivary glands (LSGs) of SS patients with HTLV 1-associated myelopathy (HAM) and adult T-cell leukemia. Although HTLV-1 was transmitted to salivary gland epithelial cells (SGECs) by a biofilm-like structure, no viral synapse formation was observed. After infection to SGECs derived from SS patients, adhesion molecules and migration factors were time-dependently released from infected SGECs. The frequency of the appearance of autoantibodies including anti-Ro/SS-A, La/SS-B antibodies in SS patients complicated with HAM is unknown; the observation of less frequent ectopic germinal center formation in HTLV-1-seropositive SS patients was a breakthrough. In addition, HTLV-1 infected cells inhibited B-lymphocyte activating factor or C-X-C motif chemokine 13 through direct contact with established follicular dendritic cell-like cells. These findings show that HTLV-1 is directly involved in the pathogenesis of SS.

Utility of novel T-cell-specific extracellular vesicles in monitoring and evaluation of acute GVHD.

We have recently reported a new method for detecting T-cell-derived extracellular vesicles (EVs), CD3+CD4+EVs,CD3+CD8+EVs, and CD3+HLA-DR+EVs. In our previous study, CD3+HLA-DR+EVs were released profusely by CD8+T cells, only moderately by T helper1 (Th1) CD4+T cells, and very little from Th2 CD4+T cells in vitro. EVs were measured sequentially in patients undergoing hematopoietic stem cell transplantation (HSCT), and their relationship to GVHD was investigated in comparison with other conventional biomarkers. We analyzed peripheral blood samples from 20 patients (13 children and 7 adults) who underwent HSCT at Tokyo Medical and Dental University Hospital. CD3+CD4+EV and CD3+CD8+EV levels specifically correlated with the CD4+ and CD8+T lymphocyte counts, respectively. CD3+CD8+EVs and CD3+HLA-DR+EVs increased in GVHD and reflected the persistence of GVHD more specifically than soluble IL-2 receptor (sIL-2R). In engraftment syndrome, sIL-2R was markedly elevated, but CD3+HLA-DR+EVs were not. Furthermore, ferritin and sIL-2R markedly increased in hemophagocytic syndrome (HPS) that developed before engraftment; however, the change in CD3+HLA-DR+EVs was marginal. CD3+CD4+, CD3+CD8+, and CD3+HLA-DR+EVs efficiently reflect the cell-mediated immune response, and CD3+CD8+EVs and CD3+HLA-DR+EVs are more useful than other conventional biomarkers, such as sIL-2R, for monitoring and evaluation of acute GVHD.

Lipocalin-type prostaglandin D synthase regulates light-induced phase advance of the central circadian rhythm in mice.

We previously showed that mice lacking pituitary adenylate cyclase-activating polypeptide (PACAP) exhibit attenuated light-induced phase shift. To explore the underlying mechanisms, we performed gene expression analysis of laser capture microdissected suprachiasmatic nuclei (SCNs) and found that lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is involved in the impaired response to light stimulation in the late subjective night in PACAP-deficient mice. L-PGDS-deficient mice also showed impaired light-induced phase advance, but normal phase delay and nonvisual light responses. Then, we examined the receptors involved in the response and observed that mice deficient for type 2 PGD2 receptor DP2/CRTH2 (chemoattractant receptor homologous molecule expressed on Th2 cells) show impaired light-induced phase advance. Concordant results were observed using the selective DP2/CRTH2 antagonist CAY10471. These results indicate that L-PGDS is involved in a mechanism of light-induced phase advance via DP2/CRTH2 signaling.

Circulating CD3+HLA-DR+ Extracellular Vesicles as a Marker for Th1/Tc1-Type Immune Responses.

Extracellular vesicles (EVs) are known to contain unique proteins that reflect the cells of origins. Activated T cells are reported to secrete various EVs. To establish T cell subset-specific biomarkers, we performed proteomic analysis with Th1- and Th2-derived EVs and identified HLA-DR as a Th1-dominated EV membrane protein. We designed a measurement system for CD3+CD4+, CD3+CD8+, and CD3+HLA-DR+ EVs to specifically determine EV subpopulations derived from CD4+, CD8+, and Th1-type T cells, respectively. In vitro analysis showed that CD3+CD4+ EVs and CD3+CD8+ EVs were selectively secreted from activated CD4+ and CD8+ T cells, respectively, and that CD3+HLA-DR+ EVs were actively secreted from not only Th1 but also activated CD8+ T (probably mostly Tc1) cells. To evaluate the clinical usefulness of these EVs, we measured the serum levels in patients with inflammatory diseases, including Epstein-Barr virus (EBV, n = 13) infection, atopic dermatitis (AD, n = 10), rheumatoid arthritis (RA, n = 20), and osteoarthritis (OA, n = 20) and compared the levels with those of healthy adults (n = 20). CD3 + CD4 + EVs were significantly higher in all of EBV infection, AD, RA, and OA while CD3+CD8+ EVs were higher in EBV infection, lower in RA, and not different in AD and OA relative to the control. The levels of CD3+HLA-DR+ EVs were markedly higher in EBV infection and significantly lower in AD. The results suggest that these EV subpopulations reflect in vivo activation status of total CD4+, total CD8+, and Th1/Tc1-type T cells, respectively, and may be helpful in T cell-related clinical settings, such as cancer immunotherapy and treatment of chronic infection, autoimmune diseases, and graft-versus-host disease.

Deficiency of CRTH2, a Prostaglandin D2 Receptor, Aggravates Bleomycin-induced Pulmonary Inflammation and Fibrosis.

Chemoattractant receptor homologous with T-helper cell type 2 cells (CRTH2), a receptor for prostaglandin D2, is preferentially expressed on T-helper cell type 2 lymphocytes, group 2 innate lymphoid cells, eosinophils, and basophils, and elicits the production of type 2 cytokines, including profibrotic IL-13. We hypothesized that lack of CRTH2 might protect against fibrotic lung disease, and we tested this hypothesis using a bleomycin-induced lung inflammation and fibrosis model in CRTH2-deficient (CRTH2-/-) or wild-type BALB/c mice. Compared with wild-type mice, CRTH2-/- mice treated with bleomycin exhibited significantly higher mortality, enhanced accumulation of inflammatory cells 14-21 days after bleomycin injection, reduced pulmonary compliance, and increased levels of collagen and total protein in the lungs. These phenotypes were associated with decreased levels of IFN-γ, IL-6, IL-10, and IL-17A in BAL fluid. Adoptive transfer of splenocytes from wild-type, but not CRTH2-/-, mice 2 days before injection of bleomycin resolved the sustained inflammation as well as the increased collagen and protein accumulation in the lungs of CRTH2-/- mice. We consider that the disease model is driven by γδT cells that express CRTH2; thus, the adoptive transfer of γδT cells could ameliorate bleomycin-induced alveolar inflammation and fibrosis.

Mast cells derived from human induced pluripotent stem cells are useful for allergen tests.

BACKGROUND: Several methods have been developed to detect allergen-specific IgE in sera. The passive IgE sensitization assay using human IgE receptor-expressing rat cell line RBL-2H3 is a powerful tool to detect biologically active allergen-specific IgE in serum samples. However, one disadvantage is that RBL-2H3 cells are vulnerable to high concentrations of human sera. Only a few human cultured cell lines are easily applicable to the passive IgE sensitization assay. However, the use of human induced pluripotent stem cells (iPSCs) to generate human mast cells (MCs) has not yet been reported. METHODS: The nuclear factor-kappa B (NF-κB)-responsive luciferase reporter gene was stably introduced into a human iPSC line 201B7, and the transfectants were induced to differentiate into MCs (iPSC-MCs). The iPSC-MCs were sensitized overnight with sera from subjects who were allergic to cedar pollen, ragweed pollen, mites, or house dust, and then stimulated with an extract of corresponding allergens. Activation of iPSC-MCs was evaluated by ß-hexosaminidase release, histamine release, or luciferase intensity. RESULTS: iPSCs-MCs stably expressed high-affinity IgE receptor and functionally responded to various allergens when sensitized with human sera from relevant allergic subjects. This passive IgE sensitization system, which we termed the induced mast cell activation test (iMAT), worked well even with undiluted human sera. CONCLUSIONS: iMAT may serve as a novel determining system for IgE/allergens in the clinical and research settings.

Prostaglandin D2 signaling mediated by the CRTH2 receptor is involved in MK-801-induced cognitive dysfunction.

Chemoattractant receptor-homologous molecule expressed on T helper type 2 cells (CRTH2), which is a second receptor for prostaglandin (PG) D2, is involved in inflammatory responses in peripheral tissue; however, its role in cognitive function remains unclear. Here, we demonstrate that CRTH2 is involved in cognitive function using a well-established animal model of cognitive dysfunction induced by MK-801, an N-methyl-d-aspartate receptor antagonist. Genetic deletion and pharmacological inhibition of CRTH2 suppressed MK-801-induced cognitive dysfunction. Pharmacological inhibition of cyclooxygenase-1, a rate-limiting enzyme in PG synthesis, also suppressed MK-801-induced cognitive dysfunction. Moreover, an MK-801-induced increase in c-Fos expression in the paraventricular nucleus (PVN) was abolished in the CRTH2-deficient mice. Together, these results suggest that PGD2-CRTH2 signaling is involved in both MK-801-induced cognitive dysfunction and neuronal activity regulation in the PVN. Furthermore, genetic association studies suggest that CRTH2 is weakly associated with cognitive function in humans. Our study provides evidence that PGD2-CRTH2 signaling is involved in cognitive function and may represent a potential therapeutic target for cognitive dysfunction in patients with psychiatric disorders.

Proteomic identification of autoantibodies in sera from patients with ovarian cancer as possible diagnostic biomarkers.

BACKGROUND/AIM: Accumulating evidence shows that various types of cancers induce a specific immune response resulting in the production of antibodies against self-components (autoantibodies). The aim of the present study was to identify antigens for autoantibodies in sera from patients with ovarian cancer, especially clear cell carcinoma (CCC), as novel diagnostic markers for the disease. MATERIALS AND METHODS: The reactivity of individual sera from patients was examined by two-dimensional (2-D) immunoblotting using lysates of CCC cell lines, ES-2 and RMG-1, as antigens to identify autoantigens. ELISA was established to quantitatively measure autoantibody titer of patients' sera. RESULTS: Autoantibodies against RhoGDI were induced in sera of ovarian cancer patients. Elevated levels of autoantibodies against heterogeneous nuclear ribonucleoprotein L (hnRNPL) and a mitochondrial protein, dihydrolipoamide dehydrogenase (DLD), were detected in patients with CCC. CONCLUSION: Autoantibodies against RhoGDI and hnRNPL and DLD may serve as novel diagnostic markers for ovarian cancer and CCC, respectively.

CRTH2, a prostaglandin D2 receptor, mediates depression-related behavior in mice.

Depression is a complex neuropsychiatric disorder with an unclear molecular etiology. Inflammatory cytokines and molecular intermediates (including prostaglandins) are suggested to be involved in depression; however, the roles of prostaglandins and their respective receptors are largely unknown in depression. Using genetic and pharmacological approaches, we show here that chemoattractant receptor-homologous molecule expressed on T helper type 2 cells (CRTH2), a second receptor for prostaglandin D2 (PGD2), mediates depression-related behavior in mice. CRTH2-deficient (CRTH2(-/-)) mice showed antidepressant-like activity in a chronic corticosterone treatment-induced depression. Consistent with this observation, the pharmacological inhibition of CRTH2 via the clinically available drug ramatroban also rescued abnormal social interaction and depression-related behavior in well-established models, including chronic corticosterone-, lipopolysaccharide-, and tumor-induced pathologically relevant depression models. Importantly, chronic stress via corticosterone treatment increased mRNA levels in PGD2-producing enzymes, such as cyclooxygenase-2 and lipocalin-type PGD2 synthase, in the brain. Furthermore, the activity of the hippocampal noradrenergic system but not the dopaminergic or serotonergic systems was increased in CRTH2(-/-) mice. Together with the observation that untreated CRTH2(-/-) mice showed antidepressant-like activity in the forced swim test, these results provide evidence that central CRTH2-mediated signaling is critically involved in depression-related behavior.

Proteomic identification of dihydrolipoamide dehydrogenase as a target of autoantibodies in patients with endometrial cancer.

BACKGROUND/AIM: Accumulating evidence shows that various types of cancers induce a specific immune response, resulting in the production of antibodies against self-components (autoantibodies). The aim of the present study was to identify antigens for autoantibodies in sera from endometrial cancer patients as novel diagnostic markers for the disease. MATERIALS AND METHODS: The reactivity of individual sera from patients was examined by 2-dimensional (2-D) immunoblotting using HeLa cell lysates as antigens to identify autoantigens. ELISA was established to quantitatively measure autoantibody titer of patients' sera. RESULTS: A mitochondrial protein, dihydrolipoamide dehydrogenase (DLD), was identified as an autoantigen specific to endometrial cancer patients. The levels of immunoglobulin (Ig)A but not IgG autoantibody to DLD were significantly increased in the sera of endometrial cancer patients. CONCLUSION: IgA autoantibody against DLD could be a novel diagnostic marker for endometrial cancer.

Prostaglandin D2 acts through the Dp2 receptor to influence male germ cell differentiation in the foetal mouse testis.

Through intercellular signalling, the somatic compartment of the foetal testis is able to program primordial germ cells to undergo spermatogenesis. Fibroblast growth factor 9 and several members of the transforming growth factor ß superfamily are involved in this process in the foetal testis, counteracting the induction of meiosis by retinoic acid and activating germinal mitotic arrest. Here, using in vitro and in vivo approaches, we show that prostaglandin D2 (PGD2), which is produced through both L-Pgds and H-Pgds enzymatic activities in the somatic and germ cell compartments of the foetal testis, plays a role in mitotic arrest in male germ cells by activating the expression and nuclear localization of the CDK inhibitor p21(Cip1) and by repressing pluripotency markers. We show that PGD2 acts through its Dp2 receptor, at least in part through direct effects in germ cells, and contributes to the proper differentiation of male germ cells through the upregulation of the master gene Nanos2. Our data identify PGD2 signalling as an early pathway that acts in both paracrine and autocrine manners, and contributes to the differentiation of germ cells in the foetal testis.

Central CRTH2, a second prostaglandin D2 receptor, mediates emotional impairment in the lipopolysaccharide and tumor-induced sickness behavior model.

Chemoattractant receptor-homologous molecule expressed on T helper type 2 cells (CRTH2) is a second prostaglandin D2 receptor involved in mediating the allergic response; however, its central function is not yet known. Here, we demonstrate that central CRTH2 mediates emotional impairment. Lipopolysaccharide (LPS)-induced decreases in social interaction and novel exploratory behavior were observed in wild-type (CRTH2(+/+)) mice but not CRTH2-deficient (CRTH2(-/-)) mice, but both genotypes showed hypolocomotion and anorexia following LPS injection. Tumor (colon 26) inoculation, a more pathologically relevant model, induced decreases in social interaction and novel exploratory behavior in CRTH2(+/+), but not CRTH2(-/-) mice. In addition, the CRTH2 antagonists including clinically available ramatroban reversed impaired social interaction and novel exploratory behavior after either LPS or tumor inoculation in CRTH2(+/+) mice. Finally, LPS-induced c-Fos expression in the hypothalamic paraventricular nucleus (PVN) and central amygdala (CeA) was selectively abolished in CRTH2(-/-) mice. These results show that CRTH2 participates in LPS-induced emotional changes and activation in the PVN and CeA. Our study provides the first evidence that central CRTH2 regulates specific emotional behaviors, and that CRTH2 antagonism has potential as a therapeutic target for behavioral symptoms associated with tumors and infectious diseases.

Chemotactic responses of peripheral blood eosinophils to prostaglandin D2 in atopic keratoconjunctivitis.

BACKGROUND: Eosinophils appear to be key cells in the pathogenesis of conjunctival inflammation in atopic keratoconjunctivitis (AKC). Chemoattractant receptor homologous molecule expressed on TH2 cells (CRTH2) mediates prostaglandin D2 (PGD2)-dependent migration of eosinophils. However, it is unclear whether CRTH2/PGD2-dependent eosinophil migration is upregulated in allergic diseases. OBJECTIVE: To compare the chemotactic responses of peripheral blood eosinophils to prostaglandin D2 in patients with severe AKC and healthy individuals. METHODS: We used an enzyme immunoassay system to measure PGD2 levels in tears and blood samples from healthy individuals and patients with AKC. CRTH2 expression on peripheral blood eosinophils was determined using reverse-transcriptase polymerase chain reaction (RT-PCR), flow cytometry, and an oligonucleotide array system. Chemotaxis experiments were performed using a modified Boyden chamber technique and an optical assay system. RESULTS: The PGD2 concentrations were higher in tears from patients with severe AKC compared with healthy individuals. RT-PCR (severe and mild cases), flow cytometry (mild cases), and GeneChip analyses revealed a significantly higher expression of CRTH2 on peripheral blood eosinophils in patients with AKC than in healthy individuals. PGD2 and its stable metabolite 13,14-dihydro-15-keto-PGD2, a CRTH2 agonist, induced chemotaxis of eosinophils from patients with AKC; chemotaxis was significantly enhanced in eosinophils from patients with severe AKC compared with those from healthy individuals. CONCLUSION: CRTH2 is more abundantly expressed on eosinophils from patients with AKC and promoted PGD2-dependent migration to a greater extent than in healthy individuals.

Serum antibodies against the 70k polypeptides of the U1 ribonucleoprotein complex are associated with psychiatric syndromes in systemic lupus erythematosus: a retrospective study.

OBJECTIVES: We assessed the association between serum autoantibodies against the 70-kDa polypeptide of the U1-ribonucleoprotein (RNP) complex (U1-70k) and the central nervous system (CNS) syndromes in systemic lupus erythematosus (SLE) patients. METHODS: We studied 106 hospitalized patients with active SLE, comparing those with (n = 32) and without (n = 74) CNS syndromes. CNS syndromes were further classified into neurologic (n = 21) and psychiatric (n = 15) disorders. Immunoglobulin G (IgG) anti-U1-70k antibodies were measured by enzyme-linked immunosorbent assay (ELISA) using recombinant antigens. IgG antibodies against whole U1-RNP were measured using commercial ELISA kits. RESULTS: Although there was no significant difference in the levels of serum anti-U1-70k antibodies in SLE patients with or without CNS syndromes (p = 0.83), the levels were significantly elevated in SLE patients compared with patients without psychiatric syndromes (p = 0.030). In contrast, no significant difference was observed in the levels of serum anti-U1-RNP antibodies in SLE patients with or without psychiatric syndromes (p = 0.555). CONCLUSIONS: These results indicate that serum anti-U1-70k antibodies are associated with psychiatric syndromes in SLE but that they are not associated with CNS syndromes as a whole or with neurologic syndromes. The anti-U1-70k antibodies might be involved in the pathological mechanisms of psychiatric syndromes in SLE.

CRTH2 is a critical regulator of neutrophil migration and resistance to polymicrobial sepsis.

Although arachidonic acid cascade has been shown to be involved in sepsis, little is known about the role of PGD(2) and its newly found receptor, chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2), on the septic response. Severe sepsis is associated with the failure of neutrophil migration. To investigate whether CRTH2 influences neutrophil recruitment and the lethality during sepsis, sepsis was induced by cecal ligation and puncture (CLP) surgery in mice. CRTH2 knockout (CRTH2(-/-)) mice were highly resistant to CLP-induced sepsis, which was associated with lower bacterial load and lower production of TNF-α, IL-6, and CCL3. IL-10, an anti-inflammatory cytokine, was higher in CRTH2(-/-) mice, blunting CLP-induced lethality in CRTH2(-/-) mice. Neutrophil accumulation in the peritoneum was more pronounced after CLP in CRTH2(-/-) mice, which was associated with higher CXCR2 levels in circulating neutrophils. Furthermore, sepsis caused a decrease in the level of acetylation of histone H3, an activation mark, at the CXCR2 promoter in wild-type neutrophils, suggesting that CXCR2 expression levels are epigenetically regulated. Finally, both pharmacological depletion of neutrophils and inhibition of CXCR2 abrogated the survival benefit in CRTH2(-/-) mice. These results demonstrate that genetic ablation of CRTH2 improved impaired neutrophil migration and survival during severe sepsis, which was mechanistically associated with epigenetic-mediated CXCR2 expression. Thus, CRTH2 is a potential therapeutic target for polymicrobial sepsis.

Dual functions of prostaglandin D2 in murine contact hypersensitivity via DP and CRTH2.

Prostaglandin D2 (PGD2) exerts its effects through two distinct receptors: the chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) and the D prostanoid (DP) receptor. Our previous study demonstrated that CRTH2 mediates contact hypersensitivity (CHS) in mice. However, the function of DP receptor remains to be fully established. In this study, we examine the pathophysiological roles of PGD2 using DP-deficient (DP(-/-)) and CRTH2/DP-deficient (CRTH2(-/-)/DP(-/-)) mice to elucidate receptor-mediated PGD2 action in CHS. We observed profound exacerbation of CHS in DP(-/-) mice. CRTH2(-/-)/DP(-/-) mice showed similar exacerbation, but to a lesser extent. These symptoms were accompanied by increased production of interferon-γ and IL-17. The increase in IL-17 producing γδ T cells was marked and presumably contributed to the enhanced CHS. DP deficiency promoted the in vivo migration of dendritic cells to regional lymph nodes. A DP agonist added to DCs in vitro was able to inhibit production of IL-12 and IL-1ß. Interestingly, production of IL-10 in dendritic cells was elevated via the DP pathway, but it was lowered by the CRTH2 pathway. Collectively, PGD2 signals through CRTH2 to mediate CHS inflammation, and conversely, DP signals to exert inhibitory effects on CHS. Thus, we report opposing functions for PGD2 that depend on receptor usage in allergic reactions.

First trimester pregnancy decidual natural killer cells contain and spontaneously release high quantities of granulysin.

PROBLEM: Granulysin (GNLY) is a novel cytolytic protein lytic against a variety of tumor cells and microbes. The role of GNLY during pregnancy has not been extensively explored. The aim of this study is to examine GNLY expression and distribution in the first trimester pregnancy peripheral blood (PB) and decidua, the ability of decidual and PB natural killer (NK) cells to secrete GNLY spontaneously, and the role of antigen-presenting cells (APC) in the regulation of GNLY expression in decidual NK cells. METHOD OF STUDY: GNLY expression was analyzed using cell permeabilization method, flow cytometry, and immunohistochemistry. GNLY secretion by purified NK cells was detected by ELISA method. RESULTS: GNLY is abundantly expressed at the maternal-fetal interface in the first trimester pregnancy. Decidual T lymphocytes express significantly higher levels of GNLY (58%) then PB T lymphocytes (11%). Over 85% of decidual CD56(+) cells express GNLY and when cultured spontaneously release high quantities of GNLY. Decidual APC participate in the control of GNLY expression in CD56(+) cells. CONCLUSION: Abundant expression of GNLY in the decidual immunocompetent cells and the capacity of decidual CD56(+) cells to spontaneously secrete high quantities of GNLY point to important protective and immunomodulatory role that this molecule could play at the maternal-fetal interface.

Role of prostaglandin D2 receptor CRTH2 in sustained eosinophil accumulation in the airways of mice with chronic asthma.

The prostaglandin D(2) (PGD(2))/CRTH2 pathway is important for eosinophil trafficking in vitro; however, genetic deficiency of CRTH2 does not suppress in vivo eosinophilic airway inflammation in acute models of asthma, and the role of CRTH2 in the pathogenesis of asthma is still ambiguous. Therefore, in the present study we explored whether the PGD(2)/CRTH2 pathway could affect the phenotypes of chronic asthma. Either CRTH2-deficient (CRTH2-/-) or wild-type mice were sensitized and exposed to ovalbumin (OVA) for 3 days (acute model) or 6 weeks (chronic model). While the magnitude of the acute eosinophilic inflammation was equivalent between CRTH2-/- and wild-type mice, the number of inflammatory cells and eosinophils in bronchoalveolar lavage fluid after chronic OVA exposure was significantly reduced in CRTH2-/- mice (18.0 ± 2.6 × 10(4) cells and 2.0 ± 0.5 × 10(4) cells) compared to wild-type mice (27.9 ± 2.5 × 10(4) cells and 6.8 ± 1.1 × 10(4) cells, p < 0.001). On the contrary, no difference was observed between CRTH2-/- and wild-type mice in terms of airway hyperresponsiveness or remodeling (goblet cell hyperplasia) in the chronic model of asthma. In conclusion, CRTH2 that mediates PGD(2) activity is essential for sustained eosinophilic inflammation in the airways, and its antagonists could exert an anti-inflammatory effect in chronic asthma.

Identification of three new autoantibodies associated with systemic lupus erythematosus using two proteomic approaches.

Our objective was to identify new serum autoantibodies associated with systemic lupus erythematosus (SLE), focusing on those found in patients with central nervous system (CNS) syndromes. Autoantigens in human brain proteins were screened by multiple proteomic analyses: two-dimensional polyacrylamide gel electrophoresis/Western blots followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis and immunoprecipitation followed by liquid chromatography-tandem mass spectrometry shotgun analysis. The presence of serum IgG autoantibodies against 11 selected recombinant antigens was assessed by Western blot and enzyme-linked immunosorbent assay (ELISA) in the sera of 106 SLE patients and 100 normal healthy controls. The O.D. values in sera from SLE patients were significantly higher than those of controls for the antigens crystallin αB (p = 0.0002), esterase D (p = 0.0002), APEX nuclease 1 (p < 0.0001), ribosomal protein P0 (p < 0.0001), and PA28γ (p = 0.0005); the first three are newly reported. The anti-esterase D antibody levels were significantly higher in the CNS group than in the non-CNS group (p = 0.016). Moreover, when the SLE patients were categorized using CNS manifestations indicating neurologic or psychiatric disorders, the anti-APEX nuclease 1 antibody levels were significantly elevated in SLE patients with psychiatric disorders (p = 0.037). In conclusion, the association of SLE with several new and previously reported autoantibodies has been demonstrated. Statistically significant associations between anti-esterase D antibodies and CNS syndromes as well as between anti-APEX nuclease 1 antibodies and psychiatric disorders in SLE were also demonstrated. The combined immunoproteomic approaches used in this study are reliable and effective methods for identifying SLE autoantigens.

Reevaluation of quantitative flow cytometric analysis for TLR2 on monocytes using F(ab')2 fragments of monoclonal antibodies.

In patients with refractory infections, reliable markers that monitor the severity and healing process are needed. The expression level of toll-like receptor 2 (TLR2) on monocytes is such candidate. In the conventional assay system, the whole IgG (wIgG) form of anti-TLR2 mAb has been used with control IgG, which blocks nonantigen-specific bindings. However, the competitive reactions against Fcγ receptors (FcγRs) between labeled anti-TLR2 mAbs and control IgG should be considered. Our goal was to precisely quantify TLR2 expression level on monocytes by flow cytometry (FCM). In this study, we prepared anti-TLR2 mAbs, D45 (IgG2a), and D29 (IgG1), as well as their fragment antigen-binding [F(ab')(2) ] fragments to avoid nonantigen-specific binding to FcγRs. And then, we determined TLR2 expression levels on monocytes by using these mAbs/fragments and our calibration system using recombinant TLR2 beads. The binding of PE-labeled D45 wIgG to monocytes was completely blocked with unlabeled D45 wIgG, but not with unlabeled D45 F(ab')(2) fragment. Although the nonantigen-specific binding of D29 wIgG to nonstimulated monocytes was negligible, it was enhanced in interleukin-10-stimulated monocytes. It proved difficult to completely block nonantigen-specific binding of D45 and D29 wIgGs by treatment with control IgG. It was demonstrated that the use of fluorescent-labeled antigen-binding region lacking the fragment crystallizable portion of anti-TLR2 mAb [such as the PE-labeled F(ab')(2) fragment] is indispensible for quantification of TLR2 levels on monocytes in flow cytometry. .