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Patients with plasma cell type idiopathic multicentric Castleman disease (PC-iMCD) often show elevated serum IgG4 levels and IgG4-positive cell infiltration in tissues due to overproduction of interleukin-6, and may meet the diagnostic criteria for IgG4-related disease (IgG4-RD). Although PC-iMCD has been listed as a major exclusion disease for IgG4-RD, distinguishing between these diseases is challenging due to a lack of highly specific diagnostic biomarkers. In 2020, we proposed exclusion criteria of IgG4-RD mimickers. In this paper, we validated the accuracy of the criteria in excluding one of the mimickers, PC-iMCD, from IgG4-RD. Validation was performed on 57 PC-iMCD patients (39 presenting lymph node lesions and 19 with lung lesions) and 29 IgG4-RD patients (22 presenting lymph node lesions and seven with lung lesions). According to our results, 20.5% of the PC-iMCD patients with lymph node lesions and 42.1% of those with lung lesions met the diagnostic criteria for IgG4-RD. All these patients with PC-iMCD were excluded from a diagnosis of IgG4-RD by the proposed criteria. Additionally, 6.9% of IgG4-RD patients met the exclusion criteria. Thus, if the exclusion criteria are met, diagnosis should be made based on a combination of findings including organ distribution of disease, response to steroid therapy, and other pathological findings.

Sjögren's syndrome is a chronic autoimmune disorder whose pathogenesis is poorly understood and that lacks effective therapies. Detailed quantitative and spatial analyses of tissues affected by Sjögren's syndrome were undertaken, including the quantitation of the frequency of selected cell-cell interactions in the disease milieu. Quantitative analyses of CD4

Objective We reported previously that T-cell-specific RORγt-transgenic mice under human CD2 promoter (RORγt-Tg mice) developed severe spontaneous Sjogren's syndrome (SS)-like sialadenitis, induced by RORγt-overexpressing CD4+ T cells and reduced regulatory T cells. The purpose of this study was to clarify the effectiveness and mechanisms of action of A213, a RORγt antagonist, in RORγt-Tg mice with SS-like sialadenitis. Methods Six-week-old RORγt-Tg mice were administered orally of A213 or phosphate-buffered saline every 3 days for 2 weeks. We analyzed saliva volume, histopathology of salivary glands, populations of T cells in splenocytes and cervical lymph nodes (cLNs), and the protein expression levels of CD69 on CD4+ CD25+ Foxp3- and CD4+ CD25+ Foxp3+ cells in cLNs. We also investigated in vitro the potential immunomechanisms of action of A213. Results A213 significantly increased saliva volume, reduced mononuclear cell infiltration in salivary glands, and reduced the focus score of sialadenitis. Analysis of the immunomechanisms using cLNs showed A213 significantly reduced the proportion of CD4+ CD25+ /CD4+ T cells and the protein expression levels of CD69 on CD4+ CD25+ Foxp3- cells. In vitro experiments showed that A213 suppressed CD25 expression on CD4+ T cells and reduced IL-2 production from CD4+ T cells derived from RORγt-Tg mice. Conclusion A213 improves SS-like sialadenitis through the inhibition of CD4+ CD25+ cells in cLNs.

IgG4-related disease (IgG4-RD) is a fibro-inflammatory condition that can affect multiple organs. We previously demonstrated that TLR7-transgenic C57BL/6 mice showed elevated serum IgG1 levels and inflammation with fibrosis in the salivary glands (SGs), lungs, and pancreas. Moreover, we observed extensive Toll-like receptor 7 (TLR-7)-positive CD163+ M2 macrophage infiltration in SGs from IgG4-RD patients. We undertook this study to examine the fibrotic mechanism via the TLR-7 pathway.Gene expression in SGs from human TLR7-transgenic mice and IgG4-RD patients was analyzed using DNA microarrays. We extracted the common up-regulated TLR-7-related genes in SGs from TLR7-transgenic mice and IgG4-RD patients. Finally, we investigated the interaction between CD163+ M2 macrophages and fibroblasts before and after stimulation with the TLR-7 agonist loxoribine.In TLR7-transgenic mice and IgG4-RD patients, IRAK3 and IRAK4 were significantly overexpressed. Real-time polymerase chain reaction validated the up-regulation of only IRAK4 in IgG4-RD patients compared with the other groups (P 0.05). Interleukin-1 receptor-associated kinase 4 (IRAK4) was strongly detected in and around germinal centers in SGs from patients with IgG4-related dacryoadenitis and sialadenitis alone. Double immunofluorescence staining showed that IRAK4-positive cells were mainly colocalized with CD163+ M2 macrophages in SGs (P 0.05). After stimulation with loxoribine, CD163+ M2 macrophages exhibited significantly enhanced expression of IRAK4 and NF-κB and increased supernatant concentrations of fibrotic cytokines. Finally, we confirmed that the number of fibroblasts was increased by culture with the supernatant of CD163+ M2 macrophages following stimulation with loxoribine (P 0.05).CD163+ M2 macrophages promote fibrosis in IgG4-RD by increasing the production of fibrotic cytokines via TLR-7/IRAK4/NF-κB signaling.

Tumor-associated macrophages (TAMs) promote cancer cell proliferation and metastasis, as well as anti-tumor immune suppression. Recent studies have shown that tumors enhance the recruitment and differentiation of TAMs, but the detailed mechanisms have not been clarified. We thus examined the influence of cancer cells on the differentiation of monocytes to TAM subsets, including CD163+, CD204+, and CD206+ cells, in oral squamous cell carcinoma (OSCC) using immunohistochemistry, flow cytometry, and a cytokine array. Furthermore, we investigated the effect of OSCC cells (HSC-2, SQUU-A, and SQUU-B cells) on the differentiation of purified CD14+ cells to TAM subsets. The localization patterns of CD163+, CD204+, and CD206+ in OSCC sections were quite different. The expression of CD206 on CD14+ cells was significantly increased after the co-culture with OSCC cell lines, while the expressions of CD163 and CD204 on CD14+ cells showed no change. High concentrations of plasminogen activator inhibitor-1 (PAI-1) and interleukin-8 (IL-8) were detected in the conditioned medium of OSCC cell lines. PAI-1 and IL-8 stimulated CD14+ cells to express CD206. Moreover, there were positive correlations among the numbers of CD206+, PAI-1+, and IL-8+ cells in OSCC sections. These results suggest that PAI-1 and IL-8 produced by OSCC contribute to the differentiation of monocytes to CD206+ TAMs.

OBJECTIVE IgG4-related disease (IgG4-RD) is a unique inflammatory disorder in which Th2 cytokines promote IgG4 production. In addition, recent studies have implicated the Toll-like receptor (TLR) pathway. This study was undertaken to examine the expression of TLRs in salivary glands (SGs) from patients with IgG4-RD. METHODS SGs from 15 patients with IgG4-RD, 15 patients with Sjogren's syndrome (SS), 10 patients with chronic sialadenitis, and 10 healthy controls were examined histologically. TLR family gene expression (TLR-1 through TLR-10) was analyzed by DNA microarray in the submandibular glands (SMGs). Up-regulation of TLRs was confirmed in SGs from patients with IgG4-RD. Finally, the phenotype of human TLR-7 (huTLR-7)-transgenic C57BL/6 mice was assessed before and after stimulation with TLR agonist. RESULTS In patients with IgG4-RD, TLR-4, TLR-7, TLR-8, and TLR-9 were overexpressed. Polymerase chain reaction validated the up-regulation of TLR-7 in IgG4-RD compared with the other groups. Immunohistochemical analysis confirmed strong infiltration of TLR-7-positive cells in the SGs of patients with IgG4-RD. Double immunohistochemical staining showed that TLR-7 expression colocalized with CD163+ M2 macrophages. After in vitro stimulation with a TLR-7 agonist, CD163+ M2 macrophages produced higher levels of interleukin-33 (IL-33), which is a Th2-activating cytokine. In huTLR-7-transgenic mice, the focus and fibrosis scores in SMGs, pancreas, and lungs were significantly higher than those in wild-type mice (P < 0.05). Moreover, the concentration of serum IgG, IgG1, and IL-33 in huTLR-7-transgenic mice was distinctly increased upon stimulation with a TLR-7 agonist (P < 0.05). CONCLUSION TLR-7-expressing M2 macrophages may promote the activation of Th2 immune responses via IL-33 secretion in IgG4-RD.

Objectives: To determine the protein expression level, expressing cell types, and pathogenic roles of chemokine (C-C motif) ligand 18 (CCL18) and its receptor chemokine (C-C motif) receptor 8 (CCR8...

Toll-like receptor 8 (TLR8), a sensor for pathogen-derived single-stranded RNA (ssRNA), binds to uridine (Uri) and ssRNA to induce defense responses. We here show that cytidine (Cyd) with ssRNA also activated TLR8 in peripheral blood leukocytes (PBLs) and a myeloid cell line U937, but not in an embryonic kidney cell line 293T. Cyd deaminase (CDA), an enzyme highly expressed in leukocytes, deaminates Cyd to Uri. CDA expression enabled TLR8 response to Cyd and ssRNA in 293T cells. CDA deficiency and a CDA inhibitor both reduced TLR8 responses to Cyd and ssRNA in U937. The CDA inhibitor also reduced PBL response to Cyd and ssRNA. A Cyd analogue, azacytidine, is used for the therapy of myelodysplastic syndrome and acute myeloid leukemia. Azacytidine with ssRNA induced tumor necrosis factor-α expression in U937 and PBLs in a manner dependent on CDA and TLR8. These results suggest that CDA enables TLR8 activation by Cyd or its analogues with ssRNA through deaminating activity. Nucleoside metabolism might impact TLR8 responses in a variety of situations such as the treatment with nucleoside analogues.

BACKGROUND: IgG4-related disease (IgG4-RD) is an immune-mediated fibrotic disorder that has been linked to CD4+ cytotoxic T lymphocytes (CD4+CTLs). The effector phenotype of CD4+CTLs, the relevance of both CD8+ cytotoxic T lymphocytes (CD8+CTLs) and of apoptotic cell death remain undefined in IgG4-RD. OBJECTIVE: The goals of this study were to define CD4+CTL heterogeneity, characterize the CD8+CTL response in the blood and in lesions, and determine whether enhanced apoptosis may contribute to the pathogenesis of IgG4-RD. METHODS: Blood analyses were undertaken using flow cytometry, cell sorting, transcriptomic analyses at the population and single cell levels and next generation sequencing for the TCR repertoire. Tissues were interrogated using multi-color immunofluorescence. Results were correlated with clinical data. RESULTS: We establish that among circulating CD4+CTLs in IgG4-RD, CD27loCD28loCD57hi cells are the dominant effector subset, exhibit marked clonal-expansion and differentially express genes relevant to cytotoxicity, activation and enhanced metabolism. We also observed prominent infiltration of granzyme A-expressing CD8+CTLs in disease tissues and clonal-expansion in the blood of effector/memory CD8+ T cells with an activated and cytotoxic phenotype. Tissue studies revealed an abundance of cells undergoing apoptotic cell death disproportionately involving non-immune, non-endothelial cells of mesenchymal origin. Apoptotic cells showed significant upregulation of HLA-DR. CONCLUSION: CD4+CTLs and CD8+CTLs may induce apoptotic cell death in tissues of patients with IgG4-RD with preferential targeting of non-endothelial, non-immune cells of mesenchymal origin. CLINICAL IMPLICATION: T cell mediated apoptosis in disease lesions may contribute to the induction of fibrosis in IgG4-RD. Effector CD4+CTLs and CD8+CTLs correlate with disease severity in IgG4-RD.

Background Follicular helper CD4+ T (Tfh) cells have a critical role in IgG4 production by B cells in IgG4-related disease (IgG4-RD). Recent studies including ours showed that SLAMF7+CD4+ T cells are an important pathological driver of IgG4-RD. In this study, we have sought to elucidate a relationship between helper CD4+ T (Th), particularly Tfh, cells and SLAMF7+ CD4+ T cells in IgG4-RD. Results The patients with IgG4-RD enrolled in this study were aged 66 ± 12 years and their titers of serum IgG4 were 372 ± 336 mg/dl. Th1 cells, activated circulating Tfh1 (cTfh1), and activated cTfh2 cells increased in IgG4-RD. SLAMF7 was mainly expressed on Th1 and cTfh1, but not cTfh2, cells in the patients. SLAMF7+ cTfh1 cells were PD-1/CD28 double-positive, whereas SLAMF7+ Th1 cells were CD28 negative. Positive correlations were noted between serum IgG4 levels and the number of activated cTfh2 cells and SLAMF7+ cTfh1 cells, but not SLAMF7+ Th1 cells. Intriguingly, among cTfh1 cells, activated SLAMF7+ cTfh1 cells were high producers of IL-10 along with IL-21. Blimp-1, but not Bcl-6, mRNA was expressed at high levels in activated SLAMF7+ cTfh1 cells. In addition to CD4+ T cells, the frequency of SLAMF7+ fraction was higher in memory B cells than naïve B cells in patients with IgG4RD. Finally, upon stimulation via B-cell receptor and CD40, Tfh1-associated cytokines, IL-21 and IFN-γ, most significantly induced SLAMF7 expression in memory B cells. Conclusions Together, these results suggest that circulating SLAMF7+ Tfh1 cells, along with Tfh2 cells, play a pathologic role in IgG4 production in IgG4-RD.

Rationale: Kimura disease (KD) is a rare, chronic inflammatory disorder characterized by subcutaneous granuloma in the head and neck region, as well as increased eosinophil counts and high serum immunoglobulin E (IgE) levels. Kimura disease is suspected to be an IgE-mediated disease, associated with an allergic response, in which antigen-specific B cells are stimulated to undergo specific IgE class switching with disease-specific CD4+ T (Th) cells help. Thus, exploration of the Th cells in affected tissues with KD is a highly promising field of the investigation. However, there have been no reports with direct evidence to implicate Th cells in affected lesions with KD. Here we quantitatively demonstrate that CD4+ GATA3+ T cells and interleukin (IL)-4+ IgE+ c-kit+ mast cells prominently infiltrate in affected lesion with KD. Patient concerns: A 56-year-old Japanese man who exhibited painless swelling in the left parotid region. Diagnoses: Diagnosis of KD was made based on characteristic histopathologic findings, in conjunction with peripheral eosinophilia and elevated serum IgE levels. Interventions: The patient underwent corticosteroid therapy and had been followed for 2 years. Outcomes: We report a rare case of KD of the parotid region in a 56-year-old man, followed by corticosteroid therapy for 2 years. The mass decreased in size and skin itchiness decreased after therapy. He was discharged without any complications. Furthermore, we quantitatively demonstrate the dominance of CD4+ GATA3+ T cells in affected tissues of KD and detect IL-4+ IgE+ c-kit+ mast cells in lesions by multicolor staining approaches. Lessons: The findings from this case suggest that peripheral blood eosinophilia might serve as a marker of recurrent disease, long-term follow-up is necessary due to the possibility of recurrent. Interactions among expanded IgE+ B cells, CD4+ GATA3+ T cells, eosinophils, and activated mast cells might play a critical role in the pathogenesis of KD.

Immunoglobulin G4 (IgG4)-related disease (IgG4-RD) is a chronic, systemic, inflammatory condition of unknown etiology. Histopathologic examination is the key to diagnosis of IgG4-RD. The histopathologic features of IgG4-RD are lymphoplasmacytic infiltration, storiform fibrosis, and obliterative phlebitis. As for fewer than 15 years, IgG4-RD has been recognized as a unified diagnostic entity. CD4+ T and B cells, which likely cause organ damage and disabling tissue fibrosis, constitute the major inflammatory cell population in patients with IgG4-RD. Affected patients with active, untreated disease have a marked expansion of IgG4-secreting plasmablasts in the blood. Important mechanistic insights regarding the pathogenesis of IgG4-RD have been gradually disclosed in recent years. Exploring the role of interactions between these CD4+ T and B cells in patients with IgG4-RD is a highly promising field of investigation. In this review, we focus on CD4+ T cell subsets and the T-cell clones that are involved in the pathogenesis of IgG4-RD.

Programmed cell death ligand 1 (PD-L1) and its receptor PD-1 are immune checkpoint molecules that attenuate the immune response. Blockade of PD-L1 enhances the immune response in a variety of tumours and thus serves as an effective anti-cancer treatment. However, the biological and prognostic roles of PD-L1/PD-1 signalling in oral squamous cell carcinoma (OSCC) remain to be elucidated. The purpose of this study was to examine the correlation of PD-L1/PD-1 signalling with the prognosis of OSCC patients to assess its potential therapeutic relevance. The expression of PD-L1 and of PD-1 was determined immunohistochemically in 97 patients with OSCC and the association of this expression with clinicopathological characteristics was examined. Increased expression of PD-L1 was found in 64.9% of OSCC cases and increased expression of PD-1 was found in 61.9%. Univariate and multivariate analysis revealed that increased expression of PD-L1 and PD-1 positively correlated with cervical lymph node metastasis. The expression of CD25, an activated T-cell marker, was negatively correlated with the labelling index of PD-L1 and PD-1. Moreover, the patient group with PD-L1-positive and PD-1-positive expression showed a more unfavourable prognosis than the group with PD-L1-negative and PD-1-negative expression. These data suggest that increased PD-L1 and PD-1 expression is predictive of nodal metastasis and a poor prognosis and is possibly involved in cancer progression via attenuating the immune response.

The objective of this study is to develop clinical practice guideline (CPG) for Sjögren's syndrome (SS) based on recently available clinical and therapeutic evidences.The CPG committee for SS was organized by the Research Team for Autoimmune Diseases, Research Program for Intractable Disease of the Ministry of Health, Labor and Welfare (MHLW), Japan. The committee completed a systematic review of evidences for several clinical questions and developed CPG for SS 2017 according to the procedure proposed by the Medical Information Network Distribution Service (Minds). The recommendations and their strength were checked by the modified Delphi method. The CPG for SS 2017 has been officially approved by both Japan College of Rheumatology and the Japanese Society for SS.The CPG committee set 38 clinical questions for clinical symptoms, signs, treatment, and management of SS in pediatric, adult and pregnant patients, using the PICO (P: patients, problem, population, I: interventions, C: comparisons, controls, comparators, O: outcomes) format. A summary of evidence, development of recommendation, recommendation, and strength for these 38 clinical questions are presented in the CPG.The CPG for SS 2017 should contribute to improvement and standardization of diagnosis and treatment of SS.

Long-term methotrexate (MTX) treatment can cause MTX-related lymphoproliferative disorder (MTX-LPD). We experienced a case of MTX-LPD that was associated with severe osteonecrosis of the jaw mimicking medication-related osteonecrosis of the jaw. The patient was an 81-year-old woman with rheumatoid arthritis (RA) who was treated with MTX and bisphosphonate. After 7 years, she was referred to our department for the assessment of giant ulcer and exposure of the alveolar bone of the left maxilla. Histopathological and immunological analyses confirmed a diagnosis of MTX-LPD. At seven months after the cessation of MTX treatment, the ulcerative and necrotic lesions had markedly decreased in size. A 1-year follow-up examination showed no evidence of recurrence and good RA control.

Tumor-associated macrophages (TAMs) promote tumor progression and inhibit anti-tumor immune response by producing various mediators and preferentially express CD163, CD204, and CD206. However, the role of these TAM subsets in oral squamous cell carcinoma (OSCC) remains unclear. Here we investigated the expression and function of TAM subsets in OSCC, especially in cancer cell proliferation. Biopsy sample from 44 patients with OSCC were examined for the expression of TAM markers and EGF by immunohistochemistry. EGF production of TAM subsets isolated from OSCC patients was assessed by flow cytometry. We also examined the effect of conditioned medium from TAM subsets on the proliferation of OSCC cells. CD163+ cells were detected diffusely all over the tumor and connective tissue area, while CD204+ and CD206+ cells were mainly detected in/around the tumors. Flow cytometric analysis found that CD206+ TAMs strongly produced EGF compared with CD163+ and CD204+ TAMs. Cell proliferation and invasion of OSCC cells cultured with conditioned medium of CD206+ TAMs were strongly enhanced and inhibited by anti-EGFR. The number of CD206+ TAMs positively correlated with worse clinical prognosis. Our results revealed differences in localization and EGF production among these TAM subsets. CD206+ TAMs might play a critical role in the proliferation of OSCC via EGF production.

Objectives: In this study, we investigated the diagnostic utility of submandibular gland (SMG) sonography and labial salivary gland (LSG) biopsy as a less invasive procedure for diagnosing ...

ObjectivesIgG4-related disease (IgG4-RD) is a chronic, systemic, inflammatory condition of unknown aetiology. We have recently described clonally expanded circulating CD4+ cytotoxic T lymphocytes (CTLs) in IgG4-RD that infiltrate affected tissues where they secrete interleukin (IL)-1β and transforming growth factor -β1 (TGF-β1). In this study, we sought to examine the role of CD4+ CTLs in the pathogenesis of IgG4-related dacryoadenitis and sialoadenitis (IgG4-DS) and to determine whether these cells secrete interferon-gamma (IFN-γ) at lesional sites.MethodsSalivary glands of 25 patients with IgG4-DS, 22 patients with Sjögren's syndrome (SS), 12 patients with chronic sialoadenitis (CS) and 12 healthy controls were analysed in this study. Gene expression analysis was performed on submandibular glands (SMGs) from five patients with IgG4-DS, three with CS and three healthy controls. Infiltrating CD4+ CTLs were examined by quantitative multicolour imaging in tissue samples from 20 patients with IgG4-DS, 22 patients with SS, 9 patients with CS and 9 healthy controls.ResultsIn IgG4-DS tissues, nine genes associated with CD4+ CTLs were overexpressed. The expression of granzyme A (GZMA) mRNA was significantly higher in samples from patients with IgG4-RD compared with corresponding tissues from SS and healthy controls. Quantitative imaging showed that infiltrating CD4+ GZMA+ CTLs were more abundant in patients with IgG4-DS than in the other groups. The ratio of CD4+GZMA+ CTLs in SMGs from patients with IgG4-DS correlated with serum IgG4 concentrations and the number of affected organs. A large fraction of CD4+GZMA+ CTLs in SMGs from patients with IgG4-DS secreted IFN-γ.ConclusionsThe pathogenesis of IgG4-DS is associated with tissue infiltration by CD4+GZMA+ CTLs that secrete IFN-γ.

For the definitive diagnosis of IgG4-related disease (IgG4-RD), biopsies of local lesions are recommended so as to exclude other diseases, including lymphoma and cancer. However, performing biopsies of underlying organs is technically difficult. In this study, we examined the diagnostic utility of labial salivary gland (LSG) biopsy as a less invasive procedure.Sixty-six patients with suspected IgG4-RD by clinical findings or high serum IgG4 underwent LSG biopsy. We examined the relationship between the number of IgG4-positive plasma cells in LSG and clinical findings.The final diagnosis was 45 patients with IgG4-RD, 12 with Sjögren's syndrome, four with suspected Sjögren's syndrome, three with malignant lymphoma, one with systemic lupus erythematosus, and one with Warthin's tumor. The sensitivity, specificity, and accuracy of LSG biopsy were 55.6%, 100.0%, and 70.0%, respectively. Forty-five IgG4-RD patients were divided into two groups: 1) 25 with lesions of salivary glands (IgG4-RD S+) and 2) 20 without these lesions (IgG4-RD S-). Seventeen of 25 (68.0%) IgG4-RD S + and 8 of 20 (40.0%) IgG4-RD S - patients were positive for LSG biopsy. In the IgG4-RD S - patients, the mean number of affected organs and serum IgG4 in the positive cases for LSG biopsy were significantly higher than in the negative cases.A solo LSG biopsy is insufficient for the diagnosis of IgG4-RD because of its low sensitivity. However, LSG biopsy combined with clinical findings, including serum IgG4 and number of affected organs, may contribute towards a diagnosis of IgG4-RD patients with affected underlying organs.

Glucocorticoids (GC) are effective for treating IgG4-related disease (IgG4-RD); however, relapse is often observed. We conducted a retrospective multicentre study to investigate risk factors in GC regimens associated with relapses of IgG4-RD. Data on 166 patients with definitive IgG4-RD diagnosis were collected from 12 institutions. Comprehensive surveillance of clinical backgrounds and GC regimens as well as multivariate analysis of factors associated with treatment responses and relapses was performed. To determine the initial maximal GC dose, the patients were stratified into three groups depending on the initial prednisolone (PSL) dosage: 0.7 mg/kg/day. The multivariate analysis extracted the disease duration and reduction speed of initial GC dose. Patients treated with initial GC 0.7 mg/kg/day of PSL showed higher relapse rates than those treated with 0.4–0.69 mg/kg/day. The relapse rates were significantly higher in patients with fast reduction of the initial dose (>0.4 mg/day) than in patients with slow reduction (

Recent studies have revealed that cancer cells are exacerbated by chronic inflammation. The present study examined the immunohistochemical expression for interleukin-6 (IL-6), a pleiotropic inflammatory cytokine, in oral squamous cell carcinoma (OSCC) to elucidate the association of IL-6 expression with tumor progression, chemoresistance and prognosis. Seventy-eight patients with primary OSCC were analyzed by immunohistochemical staining for IL-6. These labeling indexes (LIs) were calculated and evaluated in association with the clinicopathologic characteristics and prognosis in the OSCC patients. The patients were divided into three groups as follows: negative group = LI

Summary To examine genes expressed specifically in labial salivary glands (LSGs) of patients with Sjögren's syndrome (SS) in comparison with those of patients with immunoglobulin (Ig)G4-related disease (IgG4-RD), and to identify the genes involved in the pathogenesis of SS. Gene expression in LSGs of SS patients, IgG4-RD patients and healthy controls (HC) was analysed by cDNA microarray. Quantitative polymerase chain reaction (qPCR) was used to validate the up-regulation of differentially expressed genes (DEGs) in SS. Protein production of the validated gene in LSGs was examined by immunofluorescence (IF) assay. The association of molecular functions of the gene with the pathological conditions in SS was examined using peripheral blood lymphocytes. Among 1320 DEGs up-regulated in SS, qPCR confirmed the up-regulation of NR4A2 in LSGs of SS compared with IgG4-RD. IF staining showed higher production of NR4A2 in nuclei of CD4+ T cells and interleukin (IL)-17-producing cells in LSGs of SS, compared with IgG4-RD. Over-expression of NR4A2 mRNA was observed in peripheral CD4+ T cells of SS patients, compared with HC. Nuclear NR4A2 expression in T helper type 17 (Th17)-polarized CD4+ T cells determined by cellular IF was significantly higher in SS than in HC. Importazole, an inhibitor of importin-β, inhibited nuclear transport of NR4A2 and Th17 polarization along with IL-21 expression in naive CD4+ T cells under Th17-polarizing conditions, but did not alter retinoic acid receptor-related orphan receptor C (RORC) expression. NR4A2 seems to promote Th17 polarization via increased expression and intranuclear localization in CD4+ T cells of SS patients, which could play a critical role in the pathogenesis of SS.

Tumor-associated macrophages (TAMs) promote cancer cell proliferation, invasion, and metastasis by producing various mediators. Although preclinical studies demonstrated that TAMs preferentially express CD163 and CD204, the TAM subsets in oral squamous cell carcinoma (OSCC) remain unknown. In this study, we examined the expression and role of TAM subsets in OSCC. Forty-six patients with OSCC were analyzed for expression of TAMs in biopsy samples by immunohistochemistry. We examined TAM subsets and their production of immune suppressive molecules (IL-10 and PD-L1) in peripheral blood mononuclear cells from three OSCC patients by flow cytometry. CD163 was detected around the tumor or connective tissue, while CD204 was detected in/around the tumors. Flow cytometric analysis revealed that CD163+CD204+ TAMs strongly produced IL-10 and PD-L1 in comparison with CD163+CD204− and CD163−CD204+ TAMs. Furthermore, the number of activated CD3+ T cells after co-culture with CD163+CD204+ TAMs was significantly lower than that after co-culture with other TAM subsets. In clinical findings, the number of CD163+CD204+ TAMs was negatively correlated with that of CD25+ cells and 5-year progression-free survival. These results suggest that CD163+CD204+ TAMs possibly play a key role in the invasion and metastasis of OSCC by T-cell regulation via IL-10 and PD-L1 production.

IgG4-related disease (IgG4-RD) is characterized by elevated serum IgG4 and marked infiltration of IgG4-positive cells in multiple organs. Interleukin-33 (IL-33) is a recently described cytokine that is secreted by damaged epithelial cells, macrophages, and dendritic cells, and potently activates helper T type 2 (Th2) immune responses, which have been suggested to play a major role in IgG4 production of IgG4-RD. Here, we assessed the expression of IL-33 and related molecules in the salivary glands (SGs) of patients with IgG4-RD versus that in patients with Sjögren’s syndrome (SS) and controls. Expression of IL-33 and its receptor (ST2) was strongly detected around ectopic germinal centers (GCs) in the SGs from patients with IgG4-RD, whereas IL-33 was expressed only in epithelial cells in patients with SS and controls. Moreover, IL-33 and CD68+/CD163+ macrophages were mainly distributed around ectopic GCs in patients with IgG4-RD. Double immunofluorescence staining showed that IL-33 expression co-localized with CD68+/CD163+ macrophages. Finally, mRNA expression levels of IL-33 showed a positive correlation to those of Th2 cytokines (IL-4 and IL-13) in patients with IgG4-RD. Our data suggest that IL-33 produced by M2 macrophages might contribute to the pathogenesis of IgG4-RD via aberrant activation of Th2 immune responses.

IgG4-related dacryoadenitis and sialoadenitis (IgG4-DS) is characterized by serum IgG4 elevation and the infiltration of IgG4-positive plasma cells in glandular tissues. For definitive diagnosis of IgG4-DS, biopsies of local lesions are recommended to exclude Sjögren's syndrome (SS), malignant tumours, and similar disorders. In this study, we examined the diagnostic utility of submandibular gland (SMG) and labial salivary gland (LSG) biopsies in IgG4-DS. Fourteen patients presenting with swelling of the SMG (eight females and six males) underwent both SMG and LSG biopsies. The sensitivity, specificity, and accuracy of SMG biopsies were all 100.0%. In contrast, those of LSG biopsies were 69.2%, 100.0%, and 71.4%, respectively. Thirty-three out of 61 LSG biopsies (54.1%) from all 14 patients were positive for the diagnostic criteria of IgG4-DS (IgG4-positive/IgG-positive plasma cells >0.4). None of the patients experienced complications such as facial nerve palsy, sialocele, or hyposalivation. The IgG4/IgG ratio showed no significant correlation between the LSG and SMG. The final diagnosis was IgG4-DS in 13 patients and marginal zone B-cell lymphoma (MZL) in one. These results suggest that incisional biopsy of the SMG is useful and appropriate for the definitive diagnosis of IgG4-DS, while diagnosis by LSG biopsy alone requires more caution.

IgG4-related disease (IgG4-RD) is a systemic disease characterized by the elevation of serum IgG4 and infiltration of IgG4-positive plasma cells in multiple target organs, including the pancreas, kidney, biliary tract and salivary glands. In contrast, Mikulicz's disease (MD) has been considered a subtype of Sjögren's syndrome (SS) based on histopathological similarities. However, it is now recognized that MD is an IgG4-RD distinguishable from SS and called as IgG4-related dacryoadenitis and sialoadenitis (IgG4-DS). Regarding immunological aspects, it is generally accepted that CD4+ T helper (Th) cells play a crucial role in the pathogenesis of SS. Since it is well known that IgG4 is induced by Th2 cytokines such as interleukin (IL)-4 and IL-13, IgG4-DS is speculated to be a unique inflammatory disorder characterized by Th2 immune reactions. However, the involvement of Th cells in the pathogenesis of IgG4-DS remains to be clarified. Exploring the role of Th cell subsets in IgG4-DS is a highly promising field of investigation. In this review, we focus on the selective localization and respective functions of Th cell subsets and discuss the differences between SS and IgG4-DS to clarify the pathogenic mechanisms of these diseases.

ObjectivesTo compare the performance of the new 2016 American College of Rheumatology (ACR)-European League Against Rheumatism (EULAR) classification criteria for primary Sjögren's syndrome (SS) with 1999 revised Japanese Ministry of Health criteria for diagnosis of SS (JPN), 2002 American-European Consensus Group classification criteria for SS (AECG) and 2012 ACR classification criteria for SS (ACR) in Japanese patients.MethodsThe study subjects were 499 patients with primary SS (pSS) or suspected pSS who were followed up in June 2012 at 10 hospitals in Japan. All patients had been assessed for all four criteria of JPN (pathology, oral, ocular, anti-SS-A/SS-B antibodies). The clinical diagnosis by the physician in charge was set as the ‘gold standard’.ResultspSS was diagnosed in 302 patients and ruled out in 197 patients by the physician in charge. The sensitivity of the ACR-EULAR criteria in the diagnosis of pSS (95.4%) was higher than those of the JPN, AECG and ACR (82.1%, 89.4% and 79.1%, respectively), while the specificity of the ACR-EULAR (72.1%) was lower than those of the three sets (90.9%, 84.3% and 84.8%, respectively). The differences of sensitivities and specificities between the ACR-EULAR and other three sets of criteria were statistically significant (pConclusionsThe ACR-EULAR criteria had significantly higher sensitivity and lower specificity in diagnosis of pSS, compared with the currently available three sets of criteria.

IgG4-related disease (IgG4-RD) is a systemic disease characterized by elevated serum IgG4 levels and a strong infiltration of IgG4-positive plasma cells in various organs. IgG4-RD patients also frequently suffer from allergic diseases, including asthma and atopic dermatitis. It is well known that T helper type 2 (Th2) cells have an important role in the initiation of allergic diseases, and Th2 cytokines such as interleukin (IL)-4 and IL-13 promote class switching to IgG4. Therefore, IgG4-RD is considered to be a Th2-predominant disease. However, other Th subsets, including regulatory T cells and T follicular helper cells, have recently received increasing attention with regard to the pathogenesis of IgG4-RD. Exploring the interconnected network of Th subsets in IgG4-RD is a highly promising field of investigation. In this review, we focus on the localization and functions of individual Th subsets to clarify the involvement of these cells in the pathogenesis of IgG4-RD.

Oral candidiasis is closely associated with changes in oral fungal biodiversity and is caused primarily by Candida albicans. However, the widespread use of empiric and prophylactic antifungal drugs has caused a shift in fungal biodiversity towards other Candida or yeast species. Recently, next-generation sequencing (NGS) has provided an improvement over conventional culture techniques, allowing rapid comprehensive analysis of oral fungal biodiversity. In this study, we used NGS to examine the oral fungal biodiversity of 27 patients with pseudomembranous oral candidiasis (POC) and 66 healthy controls. The total number of fungal species in patients with POC and healthy controls was 67 and 86, respectively. The copy number of total PCR products and the proportion of non-C. albicans, especially C. dubliniensis, in patients with POC, were higher than those in healthy controls. The detection patterns in patients with POC were similar to those in controls after antifungal treatment. Interestingly, the number of fungal species and the copy number of total PCR products in healthy controls increased with aging. These results suggest that high fungal biodiversity and aging might be involved in the pathogenesis of oral candidiasis. We therefore conclude that NGS is a useful technique for investigating oral candida infections.

Chronic GVHD (cGVHD) after allogeneic hematopoietic SCT (HSCT) is characterized by an infiltration of T cells into target organs including the oral mucosa and salivary glands. This study was designed to clarify the molecular mechanism of the local accumulation of pathogenic T cells in cGVHD. The expression of cytokines, chemokines and chemokine receptors in the buccal mucosa (BM), labial salivary glands (LSG) and PBMC from 16 patients with cGVHD after allogeneic HSCT was examined. The mRNA expression of T helper 1 (Th1) and Th2 cytokines, and several chemokines and chemokine receptors was significantly increased in the BM and LSG from cGVHD patients, in comparison with both those in the BM and LSG from controls, respectively, and also with those in the PBMC from cGVHD patients. Furthermore, the mRNA expression of Th2 cytokines, macrophage-derived chemokine and CC chemokine receptor 4 was closely associated with a strong T-cell infiltration in the BM and LSG from cGVHD patients. These results suggest that cGVHD might be initiated and/or maintained by Th1/Th0 cells and thereafter progresses in association with Th2 cell accumulation via the interaction of particular chemokine and chemokine receptors.

Summary To investigate the pathogenesis of localized autoimmune damage in Sjögren's syndrome (SS) by examining the expression patterns of cytokines, chemokines and chemokine receptors at sites of autoimmune damage. mRNA expression of these molecules in the labial salivary glands (LSGs) and peripheral blood mononuclear cells (PBMCs) from 36 SS patients was examined using a real-time polymerase chain reaction-based method. Subsets of the infiltrating lymphocytes and chemokines/chemokine receptors expression in the LSG specimens were examined by immunohistochemistry. Cytokines/chemokine concentrations in the saliva were analysed using flow cytometry or enzyme-linked immunosorbent assay. mRNA expression of T helper type 1 (Th1) cytokines, chemokines and chemokine receptors was higher in LSGs than in PBMCs. In contrast, mRNA expression of Th2 cytokines, chemokines [thymus and activation-regulated chemokine (TARC/CCL17), macrophage-derived chemokine (MDC/CCL22)] and chemokine receptor (CCR4) was associated closely with strong lymphocytic accumulation in LSGs. Furthermore, TARC and MDC were detected immunohistochemically in/around the ductal epithelial cells in LSGs, whereas CCR4 was detected on infiltrating lymphocytes. The concentrations of these cytokines/chemokines were significantly higher in the saliva from SS patients than those from controls, and the concentrations of Th2 cytokines/chemokines were associated closely with strong lymphocytic accumulation in LSGs. These results suggest that SS might be initiated and/or maintained by Th1 and Th17 cells and progress in association with Th2 cells via the interaction between particular chemokines/chemokine receptors. Furthermore, the measurement of cytokines/chemokines in saliva is suggested to be useful for diagnosis and also to reveal disease status.

Objective The aim was to investigate the diagnostic imaging characteristics of Mikulicz disease (MD), especially sonographic ones, and to clarify the differences between them and those in Sjogren syndrome (SS), based on new criteria of MD. Study design The sonographic and sialographic images, as well as clinical, histopathologic, and serologic findings of 9 patients satisfying the new criteria of MD were analyzed and compared with those in SS. Results All swollen submandibular glands showed bilateral nodal hypoechoic areas with high vascularization on sonograms and a parenchymal defect on sialograms, whereas parotid glands showed normal or slight change on both images. Nodal areas in submandibular gland sonograms were unclear on computerized tomography and on magnetic resonance imaging, but showed accumulation on gallium scintigraphy. Conclusion Mikulicz disease showed a high rate of bilateral nodal change in submandibular glands, which was completely different from SS. For detection and follow-up of these changes, sonography may be the best imaging modality.

Mikulicz’s disease (MD) is a unique IgG4-related systemic disease indicated by enlargement of the lachrymal and salivary glands and which differs substantially from Sjogren’s syndrome. A male patient with pleural effusion, swelling of the submandibular glands, and swelling of the paraaortic, mediastinal, and pararenal lymph nodes was diagnosed with MD. Analysis of peripheral CD4+ T cells from the patient revealed deviation of the Th1/Th2 balance to Th2. Prednisolone therapy ameliorated the disease and corrected the Th1/Th2 imbalance.

Background IgG4-related dacryoadenitis and sialoadenitis (IgG4-DS), so-called Mikulicz’s disease, is characterized by elevated serum IgG4 and infiltration of IgG4-positive plasma cells in glandular tissues. Recently, several studies reported both malignant lymphoma developed on the background of IgG4-associated conditions and IgG4-producing malignant lymphoma (non-IgG4-related disease). Case presentation We report on the case of a 70-year-old man who was strongly suspected IgG4-DS because of high serum IgG4 concentration (215 mg/dl) and bilateral swelling of parotid and submandibular glands. Biopsies of cervical lymph node and a portion of submandibular gland were performed. These histopathological findings subsequently confirmed a diagnosis of marginal zone B cell lymphoma. Conclusion Differential diagnosis of IgG4-DS is necessary from other disorders, including Sjögren’s syndrome, sarcoidosis, Castleman’s disease, Wegener’s granulomatosis, lymphoma, and cancer. We suggest that biopsy of swollen lesions is important for a definitive diagnosis of IgG4-DS and discuss the mechanism of development in this case.

Oral lichen planus (OLP) is a chronic inflammatory disease characterized by subepithelial T-cell infiltration. Recent studies reported that specific T helper (Th) subsets, especially Th2 cells, are involved in the pathogenesis of OLP. Thymic stromal lymphopoietin (TSLP) is mainly secreted by epithelial cells and potently activates myeloid dendritic cells (mDCs) to induce Th2-mediated inflammation. Here, we investigated the expression of TSLP and related molecules in OLP. Buccal mucosa specimens from patients with OLP, hyperkeratosis, and ulcer were analyzed by immunohistochemistry for expression of TSLP, its receptor (TSLPR), and inflammatory cells. TSLP was detected in/around the epithelium of patients with OLP and hyperkeratosis, whereas TSLPR, CD11c (mDC), and GATA3 (Th2) were strongly expressed in the subepithelial layer only in OLP patients. Double immunofluorescence staining showed that TSLPR expression mainly co-localized with CD11c. Moreover, the number of CD11c- and GATA-3 positive cells was correlated in OLP patients. In lesions selectively extracted by laser microdissection, the mRNA expression of Th2 (IL-4, MDC, TARC, GATA3)- and Th17 (IL-17, RORγt)-related molecules in OLP patients was significantly higher than in other groups. These results suggest that CD11c+ mDCs expressing TSLPR contribute to aberrant Th2 immune responses and the pathogenesis of OLP via TSLP stimulation.

Objectives Kuttner tumour (KT), so-called chronic sclerosing sialoadenitis, is characterised by concomitant swelling of the submandibular glands secondary to strong lymphocytic infiltration and fibrosis independent of sialolith formation. However, recent studies have indicated that some patients with KT develop high serum levels of IgG4 and infiltration of IgG4-positive plasma cells, namely IgG4-related dacryoadenitis and sialoadenitis (IgG4-DS), so-called Mikulicz's disease. The aim of this study was to clarify the clinical and pathological associations between KT and IgG4-DS. Materials and Methods Fifty-four patients pathologically diagnosed with KT or chronic sialoadenitis were divided into two groups according to the presence or absence of sialolith (KT-S (+) or KT-S (−), respectively). Results There were no significant differences in the clinical findings, including the mean age, sex and disease duration, between the two groups. All patients in the KT-S (+) group showed unilateral swelling without infiltration of IgG4-positive plasma cells or a history of other IgG4-related diseases (IgG4-RD), while those in the KT-S (−) group showed bilateral swelling (37.5%), strong infiltration of IgG4-positive plasma cells (87.5%) and a history of other IgG4-RD (12.5%). Conclusions These results suggest an association between the pathogeneses of KT-S (−) and IgG4-DS, but not KT-S (+).

Oral candidiasis is closely associated with changes in the oral fungal flora and is caused primarily by Candida albicans. Conventional methods of fungal culture are time-consuming and not always conclusive. However, molecular genetic analysis of internal transcribed spacer (ITS) regions of fungal rRNA is rapid, reproducible and simple to perform. In this study we examined the fungal flora in patients with oral candidiasis and investigated changes in the flora after antifungal treatment using length heterogeneity-polymerization chain reaction (LH-PCR) analysis of ITS regions. Fifty-two patients with pseudomembranous oral candidiasis (POC) and 30 healthy controls were included in the study. Fungal DNA from oral rinse was examined for fungal species diversity by LH-PCR. Fungal populations were quantified by real-time PCR and previously-unidentified signals were confirmed by nucleotide sequencing. Relationships between the oral fungal flora and treatment-resistant factors were also examined. POC patients showed significantly more fungal species and a greater density of fungi than control individuals. Sixteen fungi were newly identified. The fungal populations from both groups were composed predominantly of C. albicans, though the ratio of C. dubliniensis was significantly higher in POC patients than in controls. The diversity and density of fungi were significantly reduced after treatment. Furthermore, fungal diversity and the proportion of C. dubliniensis were positively correlated with treatment duration. These results suggest that C. dubliniensis and high fungal flora diversity might be involved in the pathogenesis of oral candidiasis. We therefore conclude that LH-PCR is a useful technique for diagnosing and assessing the severity of oral candidal infection.

To characterize the epidemiology of Sjögren's syndrome (SS), including prevalence, disease type, extra-glandular involvement, satisfaction of diagnostic criteria sets, and treatment used in Japan.The Research Team for Autoimmune Diseases, the Research Program for Intractable Disease by the Ministry of Health, Labor and Welfare conducted primary and secondary surveys on epidemiology of SS in 2011. The primary survey covered 4,729 out of 14,095 Japan-wide Hospital Departments to investigate the prevalence of SS. The secondary survey encompassed 214 Hospital Departments that agreed to the survey, to characterize disease type, extra-glandular involvement, satisfaction of diagnostic criteria sets, and treatments.The number of patients with SS in Japan estimated by the primary survey was 68,483. The secondary survey involving data collected from 2,195 SS patients from 98 Hospital Departments showed that the mean age of patients was 60.8 ± 15.2 years, male/female ratio was 1/17.4, primary/secondary SS was about 60%/40% and glandular/extra-glandular form in primary SS was about 70%/25%. The satisfaction rate was 53.8% for the 1999 revised Japanese Ministry of Health criteria for the diagnosis of SS, 47.7% for the 2002 American-European Consensus Group classification criteria for SS and 49.6% for 2012 American College of Rheumatology classification criteria for SS. Corticosteroids were used by 752 of 2,195 patients (34%), immunosuppressants by 358 patients (16%), biologics by 68 patients (3%) and secretagogues by 695 patients (32%).The surveys provided valuable information on the epidemiology of SS including prevalence, disease type, extra-glandular involvement, satisfaction of diagnostic criteria sets and treatments used today in Japan.

To compare gene expression in labial salivary glands (LSGs) from patients with IgG4-related disease with that in LSGs from patients with Sjögren's syndrome (SS).Gene expression was analyzed by DNA microarray in LSG samples from 5 patients with IgG4-related disease, 5 SS patients, and 3 healthy controls. Genes differentially expressed in IgG4-related disease and SS were identified, and gene annotation enrichment analysis of these differentially expressed genes was performed using Gene Ontology (GO) annotation. Validation of the results was performed by quantitative polymerase chain reaction (PCR) using LSG samples from 9 patients with IgG4-related disease, 10 SS patients, and 4 controls.Gene expression patterns in patients with IgG4-related disease, SS patients, and healthy controls were quite different from each other in hierarchical clustering as well as in principal components analysis. In IgG4-related disease compared with SS, a total of 1,771 probe sets (corresponding to 1,321 genes) were identified as up-regulated, and 1,785 probe sets (corresponding to 1,320 genes) were identified as down-regulated (false discovery rate of5%). GO term analysis indicated that the up-regulated set of differentially expressed genes in IgG4-related disease encoded proteins that function in cell proliferation, extracellular matrix organization, and organ development. PCR validated significantly higher expression of lactotransferrin in patients with IgG4-related disease than in SS patients (P0.05) and significantly higher expression of CCL18 in patients with IgG4-related disease than in SS patients and controls (P0.05).The results clearly showed that the gene expression pattern in LSGs from patients with IgG4-related disease is different from that in LSGs from SS patients.

To validate the revised Japanese Ministry of Health criteria for the diagnosis of Sjögren's syndrome (SS) (JPN) (1999), The American-European Consensus Group classification criteria for SS (AECG) (2002), and American College of Rheumatology classification criteria for SS (ACR) (2012).The study subjects were 694 patients with SS or suspected SS who were followed-up in June 2012 at ten hospitals that form part of the Research Team for Autoimmune Diseases, The Research Program for Intractable Disease by the Ministry of Health, Labor and Welfare (MHLW). All patients had been checked for all four criteria of the JPN (pathology, oral, ocular, anti-SS-A/SS-B antibodies). We studied the clinical diagnosis made by the physician in charge and the satisfaction of the above criteria.Of the 694 patients, 499 patients did not have other connective tissue diseases (CTDs). SS was diagnosed in 476 patients (primary SS in 302, secondary SS in 174), whereas non-SS was diagnosed in 218 patients (without other CTDs in 197, with other CTDs in 21) by the physician in charge. The sensitivities of JPN, AECG, and ACR in the diagnosis of all forms of SS (both primary and secondary SS) were 79.6, 78.6, and 77.5 %, respectively, with respective specificities of 90.4, 90.4, and 83.5 %. The sensitivities of the same systems in the diagnosis of primary SS were 82.1, 83.1, and 79.1 %, respectively, with specificities of 90.9, 90.9, and 84.8 %, respectively. The sensitivities of the same systems in the diagnosis of secondary SS were 75.3, 70.7, and 74.7 %, respectively, with specificities of 85.7, 85.7, and 71.4 %, respectively.The sensitivity of JPN to all forms of SS and secondary SS, the sensitivity of AECG to primary SS, and the specificities of JPN and AECG for all forms of SS, primary SS, and secondary SS were highest in the diagnosis of SS in Japanese patients. These results indicate that the JPN criteria for the diagnosis of SS in Japanese patients are superior to ACR and AECG.

Summary The aim of this study was to investigate the initiation and progression of autoimmune damage in the lesions of labial salivary glands (LSGs) from primary Sjögren's syndrome (SS) patients by examining the selective localization of T helper (Th) subsets such as Th1, Th2, Th17 regulatory T cells (Tregs) and follicular T helper cells (Tfh). The expression of cytokines and transcription factors associated with these Th subsets in the LSGs from 54 SS patients and 16 healthy controls was examined using real-time polymerase chain reaction (PCR) and immunostaining. Additionally, infiltrating lymphocytes without germinal centre (GC-) and with GC (GC+) in the LSGs specimens from eight SS patients were extracted selectively by laser capture microdissection (LCM). The mRNA expression of these molecules was compared between the two sample groups of GC- and GC+ by real-time PCR. The mRNA expression of cytokines and transcription factors of all T helper (Th) subsets in the LSGs from the SS patients was increased significantly in comparison with controls. In LSGs from the SS patients, Th2 and Tfh was associated closely with strong lymphocytic infiltration; however, Th1, Th17 and Tregs was not. In the selectively extracted lesions of LSGs, Th1 and Th17-related molecules were detected strongly in the GC-, while Th2 and Tfh-related molecules were detected in the GC+. In contrast, no significant association with strong lymphocytic infiltration was observed in Treg-related molecules. These results indicate that SS has selective localization of Th subsets such as Th1, Th2, Th17 and Tfh in the LSGs, which is associated closely with disease severity and/or status. SS might be initiated by Th1 and Th17 cells, and then progressed by Th2 and Tfh cells via GC formation.

Objectives Interleukin (IL)-21 is mainly produced by CD4 T helper (Th) cells including Th2, Th17 and follicular helper T (Tfh) cells. Recent studies have reported that IL-21 is involved in the formation of germinal centres (GCs) and class switching of IgG4. It has been suggested that IgG4-related dacryoadenitis and sialoadenitis (IgG4-DS), so-called Mikulicz9s disease (MD), is distinct from Sjogren9s syndrome (SS) and shows a high frequency of GC formation in salivary glands. In this study the expression of IL-21 in IgG4-DS and SS patients was examined. Methods Twelve patients with IgG4-DS, 15 with SS and 15 healthy subjects were screened for (1) ectopic GC formation in formalin-fixed labial salivary gland (LSG) biopsy samples; (2) expression of IL-21, Th2-, Th17- and Tfh-related molecules (cytokines, chemokine receptors and transcription factors) in LSGs; (3) relationship between IgG4/IgG ratio and mRNA expression of IL-21 in LSGs. Results mRNA expression of IL-21 and Bcl-6 in LSGs from patients with IgG4-DS was significantly higher than in patients with SS and controls. IL-21 and CXCR5 were detected by immunohistochemistry in or around GC in patients with SS and those with IgG4-DS. IL-21 was detected in infiltrating lymphocytes outside GC only in patients with IgG4-DS. Expression of IL-21 was consistent with that of Th2-related molecules while IL-17 was rarely seen in IgG4-DS. Furthermore, the expression of IL-21 in LSGs was correlated with the number of GC formations and the IgG4/IgG ratio in patients with IgG4-DS. Conclusions These results suggest that overexpression of IL-21 by Th2 cells might play a key role in GC formation and IgG4 production in IgG4-DS.

Mikulicz’s disease (MD) was considered to be a subtype of Sjogren’s syndrome (SS), based on histopathological similarities. However, recent studies have indicated that patients with MD show high serum IgG4 concentration, and suggested that MD is one of “IgG4-related disease” and distinguishable from SS. Therefore, we clinically and histopathologically examined the disease states of MD and SS in detail. Twenty patients with Mikulicz’s disease and 18 with SS were comparatively studied to determine clinical characteristics in MD patients. Sialography in MD patients did not show the “apple-tree sign” typically seen in SS. Serologically, high serum IgG4 levels but not anti-SS-A or anti-SS-B antibodies were observed in MD. SS showed lymphocytic infiltration of various subsets with atrophy or severe destruction of the acini, while MD showed selective infiltration of IgG4+ plasma cells with hyperplastic germinal centers and mild acini destruction. Corticosteroid treatment of MD reduced IgG and IgG4 levels and improved salivary function. A negative correlation between disease duration and increasing rate of salivary flow was observed in MD. These results suggested that the pathogenesis of MD might be different from those of SS. Clinical Relevance: early diagnosis and treatment of MD is important for the improvement of salivary function.

Objective Mikulicz disease has been considered to be a subtype of Sjogren's syndrome (SS). However, recent studies have suggested that Mikulicz disease is an IgG4-related disease and is distinguishable from SS. In addition, it has been reported that both interleukin-4 (IL-4) and IL-10 induce IgG4 production and inhibit IgE. This study was undertaken to examine the expression of these cytokines in patients with Mikulicz disease and patients with SS. Methods Labial salivary gland (LSG) sections from 15 patients with Mikulicz disease and 18 patients with SS were examined for subsets of the infiltrating lymphocytes, expression patterns of messenger RNA (mRNA) for cytokines/chemokines, and relationships between the IgG4:IgG ratio and the expression of mRNA for IL-4 or IL-10. Results Immunohistochemical analysis showed lymphocyte infiltration of various subsets in the LSGs of SS patients, and the selective infiltration of IgG4-positive plasma cells and Treg cells in the LSGs of Mikulicz disease patients. The levels of mRNA for both Th1 and Th2 cytokines and chemokines in LSGs from patients with SS were significantly higher than in controls, while the expression of both Th2 and Treg cells was significantly higher in the patients with Mikulicz disease than in controls. Furthermore, the expression of IL-4 or IL-10 in the LSGs was correlated with the IgG4:IgG ratio. Conclusion These results suggest that the pathogenesis of Mikulicz disease is different from that of SS. Mikulicz disease is a unique inflammatory disorder characterized by Th2 and regulatory immune reactions that might play key roles in IgG4 production.

Background IgG4-related tubulointerstitial nephritis (TIN) shows characteristic serum IgG4 elevation and increased IgG4-positive plasma cells in the renal interstitium, and inclusion of TIN as an IgG4-related systemic disease has been suggested. IgG4 is the rarest IgG subclass and is a Th2-dependent isotype with low affinity for target antigen. Although the pathogenesis of this disease has not been elucidated, positive serum immune complex and hypocomplementemia in some patients with this disease suggest that immune complex mechanisms are involved in the causation of this disease. Method We selected 20 cases of histological diagnosed TIN. These cases were etiologically different and included 4 cases of IgG4-related TIN. We extracted RNA from paraffin embedded biopsied kidney and evaluated expression levels of various cytokines for each case by real time PCR. Results Comparison of cytokine production patterns among different disease-associated TINs revealed that IgG4-related TIN exhibited a quite distinct pattern. On the one hand, there was no expression of IL-2, IFN-gamma IL-17 and IL-6, whereas production of IL-4, IL-10 and TGF-beta was, on the other hand, remarkably increased in IgG4-related TIN. Conclusion Based on these cytokine production results, Th2 and Treg appear to play a central role in IgG4-related TIN.

We described and analyzed the pathogenic difference between Good syndrome (GS) and oral lichen planus (OLP) in oral mucosa. Good syndrome (GS) is a rare disease characterized by B and T cell immunodeficiency associated with hypogammaglobulinemia and thymoma. GS patients frequently develop oral lichenoid lesions with lymphocytic infiltration beneath the basal layer. Oral lichen planus (OLP) is a chronic inflammatory disease of the oral mucosa characterized by destruction of basal cells by Langerhans cells, macrophages, and T lymphocytes. Although the histological features of the lesions of both diseases are very similar, the pathogenesis of GS in the oral mucosa remains unknown. In this study, we thus investigated the expression of infiltrating lymphocyte subsets (CD3, CD20, CD4, and CD8) and T helper (Th) cytokines including interferon (IFN)-γ (Th1 type), interleukin (IL)-4 (Th2 type), IL-17 (Th17 type), and IL-10 (regulatory T cell type) by immunohistochemistry in buccal mucosa specimens from 2 GS patients compared with 15 OLP patients. All patients showed a predominance of CD3+ T cells over CD20+ B cells, and CD4+ Th cells over CD8+ cytotoxic T cells. This polarization was especially prominent in GS. IFN-γ and IL-10 were strongly detected in the infiltrating lymphocytes of all patients. However, IL-4 and IL-17 were detected in OLP patients only. These results suggest that the pathogenesis of GS is different from that of OLP. GS is a unique inflammatory disorder characterized by dysfunction of Th2 and Th17 immune reactions via abnormal T–B cell interaction.

Background: RCAS1 (receptor-binding cancer antigen expressed on SiSo cells) is known to induce apoptosis in its receptor-positive cells. The authors investigated RCAS1 expression in oral squamous cell carcinoma (SCC) and its association with the apoptosis of tumor-infiltrating lymphocytes (TILs). Methods: In 130 patients with oral SCC, the expression of RCAS1 in tumor cells was immunohistochemically examined and the apoptosis of TILs was examined by Terminal Deoxynucleotidyltransferase-mediated dUTP Nick End Labeling (TUNEL) staining. Results: RCAS1 was detected both on the cytoplasm and the membrane of tumor cells in 41 of 130 cases (31.5%). Focusing on the expression at the invasive front interacting with host immune cells, RCAS1 was detected in 22 of 130 cases (16.9%). The percentage of TUNEL-positive TILs in cases with RCAS1-positive SCCs was significantly higher than in cases with RCAS1-negative SCCs (P

Introduction Immunoglobulin G4 (IgG4)-related disease (IgG4-RD) is a new disease entity characterized by high serum IgG4 levels, IgG4-positive plasmacytic infiltration, and fibrosis in various organs. The purpose of this study was to determine the mechanism of upregulation of IgG4 class switch recombination in IgG4-RD. Methods We extracted RNA from peripheral blood mononuclear cells (PBMCs) of patients with IgG4-RD (n = 6), Sjögren syndrome (SS) (n = 6), and healthy controls (n = 8), from CD3-positive T cells and CD20-positive B cells sorted from PBMCs of patients with IgG4-RD (n = 3), SS (n = 4), and healthy controls (n = 4), as well as from labial salivary glands (LSGs) of patients with IgG4-RD (n = 11), SS (n = 13), and healthy controls (n = 3). The mRNA expression levels of IgG4-specific class switch-related molecules, such as Th2 cytokines (IL-4 and IL-13), Treg cytokines (IL-10 and TGF-β), and transcriptional factors (GATA3 and Foxp3) were examined with quantitative polymerase chain reaction (PCR). IgG4-nonspecific class switch-related molecules, such as CD40, CD154, BAFF, APRIL, IRF4, and AID, were also examined. Results The expression levels of Treg cytokines (IL-10 and TGF-β) and AID were significantly higher in LSGs of IgG4-RD than in SS and the controls (P < 0.05, each). In contrast, those of CD40 and CD154 were significantly lower in PBMCs of IgG4-RD than in SS (P < 0.05, each), whereas CD40 in CD20-positive B cells and CD154 in CD3-positive T cells were comparable in the three groups. Conclusion Overexpression of IL-10, TGF-β, and AID in LSGs might play important roles in the pathogenesis of IgG4-RD, such as IgG4-specific class-switch recombination and fibrosis. IgG4 class-switch recombination seems to be mainly upregulated in affected organs.

IgG4-related dacryoadenitis and sialoadenitis (IgG4-DS) is characterized by bilateral swelling of glandular tissues with extensive fibrosis, and is immunologically considered a Th2-predominant disease. Recent studies reported that alternatively activated (M2) macrophages enhanced Th2 immune responses and fibrosis by production of pro-fibrotic factors (IL-10, IL-13 and CCL18). Therefore, we examined the association between M2 macrophages and fibrosis in submandibular glands from 7 patients with IgG4-DS, 10 patients with chronic sialoadenitis, 10 patients with Sjögren's syndrome, and 10 healthy subjects. The number of M2 macrophages in SMGs from patients with IgG4-DS was also significantly higher than in the other groups. Double immunofluorescence staining showed that IL-10 and CCL18 expression co-localized with M2 macrophage-marker (CD163). Furthermore, the SMG fibrosis score was positively correlated with the frequency of M2 macrophages in only IgG4-DS. These results indicate that IL-10 and CCL18 secreted by preferential M2 macrophages possibly play a key role in the development of severe fibrosis in IgG4-DS.

Introduction The aim of this study was to clarify the effectiveness of various imaging modalities and characteristic imaging features in the screening of IgG4-related dacryoadenitis and sialadenitis (IgG4-DS), and to show the differences in the imaging features between IgG4-DS and Sjögren’s syndrome (SS). Methods Thirty-nine patients with IgG4-DS, 51 with SS and 36 with normal salivary glands were enrolled. Images of the parotid and submandibular glands obtained using sonography, 2-[18F]-fluoro-2-deoxy-D-glucose positron emission tomography/computed tomography (FDG-PET/CT), computed tomography (CT) and magnetic resonance imaging (MRI) were retrospectively analyzed. Six oral and maxillofacial radiologists randomly reviewed the arranged image sets under blinded conditions. Each observer scored the confidence rating regarding the presence of the characteristic imaging findings using a 5-grade rating system. After scoring various findings, diagnosis was made as normal, IgG4-DS or SS, considering all findings for each case. Results On sonography, multiple hypoechoic areas and hyperechoic lines and/or spots in the parotid glands and obscuration of submandibular gland configuration were detected mainly in patients with SS (median scores 4, 4 and 3, respectively). Reticular and nodal patterns were observed primarily in patients with IgG4-DS (median score 5). FDG-PET/CT revealed a tendency for abnormal 18F-FDG accumulation and swelling of both the parotid and submandibular glands in patients with IgG4-DS, particularly in the submandibular glands. On MRI, SS had a high score regarding the findings of a salt-and-pepper appearance and/or multiple cystic areas in the parotid glands (median score 4.5). Sonography showed the highest values among the four imaging modalities for sensitivity, specificity and accuracy. There were significant differences between sonography and CT (p = 0.0001) and between sonography and FDG-PET/CT (p = 0.0058) concerning accuracy. Conclusions Changes in the submandibular glands affected by IgG4-DS could be easily detected using sonography (characteristic bilateral nodal/reticular change) and FDG-PET/CT (abnormal 18F-FDG accumulation). Even inexperienced observers could detect these findings. In addition, sonography could also differentiate SS. Consequently, we recommend sonography as a modality for the screening of IgG4-DS, because it is easy to use, involves no radiation exposure and is an effective imaging modality.