Thymic Stromal Chemokine TSLP Acts through Th2 Cytokine Production to Induce Cutaneous T-cell Lymphoma.

Thymic stromal lymphopoietin (TSLP) activates dendritic cells to induce Th2-mediated inflammation. Periostin, an extracellular matrix protein produced by fibroblasts, induces chronic inflammation by stimulating TSLP production. Recently, a reinforcing cycle linking Th2-type immune responses with periostin-induced keratinocyte activation has been proposed in atopic dermatitis pathogenesis. In this study, we investigated the role of TSLP and periostin in the development of cutaneous T-cell lymphoma (CTCL), where Th2 cytokines and chemokines are also dominant. TSLP and periostin mRNA expression levels were elevated in CTCL lesional skin, both of which correlated with IL4 expression levels. In vitro and ex vivo, IL4 or IL13 stimulated periostin expression by dermal fibroblasts, and fibroblasts from CTCL lesional skin expressed higher levels of periostin than those from control skin. Serum periostin levels of CTCL patients were also significantly higher than those of healthy individuals. Hut78 and MJ, CTCL cell lines, and peripheral blood mononuclear cells from leukemic CTCL patients expressed the TSLP receptor. TSLP induced production of IL4 and IL13 by Hut78 and MJ cells through the activation of STAT5. Moreover, TSLP induced proliferation of CTCL cells both in vitro and in vivo These data suggest that periostin-mediated TSLP production by keratinocytes directly stimulates CTCL tumor cell growth in addition to inducing a Th2-dominant tumor environment in CTCL. Cancer Res; 76(21); 6241-52. ©2016 AACR.

Mucosal-associated invariant T cell-rich congenic mouse strain allows functional evaluation.

Mucosal-associated invariant T cells (MAITs) have potent antimicrobial activity and are abundant in humans (5%-10% in blood). Despite strong evolutionary conservation of the invariant TCR-α chain and restricting molecule MR1, this population is rare in laboratory mouse strains (≈0.1% in lymphoid organs), and lack of an appropriate mouse model has hampered the study of MAIT biology. Herein, we show that MAITs are 20 times more frequent in clean wild-derived inbred CAST/EiJ mice than in C57BL/6J mice. Increased MAIT frequency was linked to one CAST genetic trait that mapped to the TCR-α locus and led to higher usage of the distal Vα segments, including Vα19. We generated a MAIThi congenic strain that was then crossed to a transgenic Rorcgt-GFP reporter strain. Using this tool, we characterized polyclonal mouse MAITs as memory (CD44+) CD4-CD8lo/neg T cells with tissue-homing properties (CCR6+CCR7-). Similar to human MAITs, mouse MAITs expressed the cytokine receptors IL-7R, IL-18Rα, and IL-12Rß and the transcription factors promyelocytic leukemia zinc finger (PLZF) and RAR-related orphan receptor γ (RORγt). Mouse MAITs produced Th1/2/17 cytokines upon TCR stimulation and recognized a bacterial compound in an MR1-dependent manner. During experimental urinary tract infection, MAITs migrated to the bladder and decreased bacterial load. Our study demonstrates that the MAIThi congenic strain allows phenotypic and functional characterization of naturally occurring mouse MAITs in health and disease.

Optimal cut-point definition in biomarkers: the case of censored failure time outcome.

BACKGROUND: Cut-point finding is a crucial step for clinical decision making when dealing with diagnostic (or prognostic) biomarkers. The extension of ROC-based cut-point finding methods to the case of censored failure time outcome is of interest when we are in the presence of a biomarker, measured at baseline, used to identify whether there will be the development, or not, of some disease condition within a given time point τ of clinical interest. METHODS: Three widely used cut-point finding methods, namely the Youden index, the concordance probability and the point closest to-(0,1) corner in the ROC plane, are extended to the case of censored failure time outcome resorting to non-parametric estimators of the sensitivity and specificity that account for censoring. The performance of these methods in finding the optimal cut-point is compared under Normal and Gamma distributions of the biomarker (in subjects developing or not the disease condition). Normality ensures that estimators point theoretically to the same cut-point. Two motivating examples are provided in the paper. RESULTS: The point closest-to-(0,1) corner approach has the best performance from simulations in terms of mean square error and relative bias. CONCLUSIONS: We discuss the use of the Youden index or concordance probability associated to the cut-point identified through the closest-to-(0,1) corner approach to ease interpretability of the classification performance of the dichotomized biomarker. In addition, the achieved performance of the dichotomized biomarker classification associated to the estimated cut-point can be represented through a confidence interval of the point on the ROC curve.

Effect of orthodontic forces on cytokine and receptor levels in gingival crevicular fluid: a systematic review.

This systematic review aimed to generate evidence on role of potent markers of inflammation [cytokines, chemokines, their associated receptors and antagonists] following the application of orthodontic forces. Subsequent to registration with PROSPERO, literature search followed a predetermined search strategy to key databases along with hand search (HS). Seventy-seven articles from PubMed (P), 637 from Scopus (S), 51 from Embase (E), and 3 from hand search (HS) were identified. A total of 39 articles were shortlisted that met strict inclusion and exclusion criteria and quality assessment. Each study was evaluated for participant characteristics, study design, oral hygiene regimen, and gingival crevicular fluid (GCF) handling. Among these studies, biomarkers in the order of frequency were interleukin (IL)-1ß (N=21), tumor necrosis factor (TNF)-α (N=10), IL-8,IL-6(N=8), receptor activator of nuclear factor kappa-B ligand (RANKL) (N=7), monocyte chemoattractant protein (MCP)-1 (N=3), IL-2 (N=4), IL-4, IL-10, RANTES (N=2), IL-1, IL-5, IL-1α, IP-10, osteopontin (OPN) (N=1) and receptors and their antagonists in the order of osteoprotegerin (OPG) (N=8), IL-1RA (N=5), and RANK (N=1). Results revealed an immediate release of inflammatory bone-resorptive mediators, IL-1ß and TNF-α, where IL-1ß increased as early as 1 min to 1 h reaching peak at 24 h while TNF-α increased at 1 h or 1 day. This was accompanied by a fall in bone-protective mediator (OPG) levels at 1 h and 24 h after orthodontic force application. Continuous forces were accompanied by a decrease in mediator levels after attaining peak levels (most commonly at 24 h) while repeated activations in interrupted force upregulated their secretion. Significant correlations of IL-1ß levels with pain intensity, rate of orthodontic tooth movement (OTM) and of activity index (AI) (IL-1ß/IL-1RA) with velocity of tooth movement and growth status of individuals have also been deduced. A greater AI and RANKL/OPG ratio was seen in juveniles as compared to adults or non-growers that were associated with faster rate of OTM in juveniles. None of the studies addressed the effect of estrous cycle in female subjects. Lack of homogeneity in several parameters calls for a better controlled research on the biology of OTM.

Infection with human rhinovirus 16 promotes enhanced IgE responsiveness in basophils of atopic asthmatics.

BACKGROUND: Rhinovirus and IgE act in concert to promote asthma exacerbations. While basophils are the principal cell type in the blood that is activated by IgE, their role in virus-induced asthma episodes remains elusive. OBJECTIVE: To monitor IgE responsiveness in circulating basophils of rhinovirus-infected atopic asthmatics during acute infection and convalescence. METHODS: The capacity for basophils to respond to IgE was assessed by testing the effects of allergen, or cross-linking anti-FcεRI and anti-IgE antibodies, on surface TSLP receptor in 24-hour PBMC cultures. Activation profiles of basophils from atopic asthmatics challenged intranasally with human rhinovirus 16 were monitored directly ex vivo or else in 24-hour cultures, at baseline (day 0), and then at days 4 and 21 post-challenge. RESULTS: Basophils in atopic asthmatics, but not in non-atopic controls, upregulated TSLP receptor upon IgE receptor ligation. The magnitude of this response was correlated with the proportion of serum total IgE that was allergen-specific (r = 0.615, P < 0.05). Following rhinovirus infection, all subjects developed nasal symptoms that peaked 3-5 days after viral challenge. Basophils displayed maximal IgE responsiveness 3 weeks post-challenge as judged by TSLP receptor levels in 24-hour cultures. No significant change in total IgE or specific IgE antibodies was detected during rhinovirus infection. By contrast, levels of IgE receptor-associated spleen tyrosine kinase, Syk, were increased on day 4 (P < 0.05), and elevated levels were also detected three weeks post-challenge. CONCLUSIONS AND CLINICAL RELEVANCE: Circulating basophils display increased IgE responsiveness 3 weeks after rhinovirus infection in atopic asthmatics. This observation, coupled with increased expression of Syk, implicates basophils in promoting, or else prolonging, rhinovirus-induced inflammation in atopic asthmatics.

In situ expression and serum level of thymic stromal lymphopoietin in oral lichen planus.

BACKGROUND: Oral lichen planus (OLP) is a chronic inflammatory disease of oral mucosa in which the CD8(+) T cell-mediated cytotoxicity is regarded as a major mechanism of pathogenesis. The main objective of this study is to investigate in situ expression and secretion of thymic stromal lymphopoietin (TSLP) in specimens and sera from patients with oral lichen planus. METHODS: Thirty-six patients with OLP and 35 donors enrolled in specimen and serum collection. Immunohistochemical method and immunofluorescence double-staining method were used to detect the expression of thymic stromal lymphopoietin and its receptor (TSLPR) together with CD8 in OLP specimens. Enzyme-linked immunosorbent assay (ELISA) was used to detect TSLP secretion. RESULTS: More TSLP- or TSLPR-positive cells showed in OLP specimens than in normal controls, and TSLP-positive cells were mainly in the epithelium, while TSLPR-positive cells mainly in the lamina propria. Furthermore, the number of TSLP-positive cells in the stratum basal was associated with the amount of mononuclear cells infiltrating in the lamina propria of OLP specimens. Among infiltrating mononuclear cells in the lamina propria, some CD8-positive cells also expressed TSLPR. The TSLP serum level of patients with OLP was significantly higher than of healthy donors, but there was no statistically difference between two clinical subtypes of OLP. CONCLUSIONS: Our findings provided the first evidence that TSLP may enroll in the pathology of OLP and the TSLP-TSLPR interaction may play an important role in it.

Modulation of conjunctival goblet cell function by inflammatory cytokines.

Ocular surface inflammation associated with Sjögren's syndrome is characterized by a loss of secretory function and alteration in numbers of mucin secreting goblet cells. Such changes are a prominent feature of ocular surface inflammatory diseases and are attributed to inflammation; however, the exact effect of the inflammatory cytokines on conjunctival goblet cell function remains largely unknown. In this study, we developed a primary culture of mouse goblet cells from conjunctival tissue and evaluated the effects on their function by inflammatory cytokines detected in the conjunctiva of mouse model of Sjögren's syndrome (Thrombospondin-1 deficient mice). We found that apoptosis of goblet cells was primarily induced by TNF-α and IFN-γ. These two cytokines also inhibited mucin secretion by goblet cells in response to cholinergic stimulation, whereas IL-6 enhanced such secretion. No changes in secretory response were detected in the presence of IL-13 or IL-17. Goblet cells proliferated to varying degrees in response to all the tested cytokines with the greatest response to IL-13 followed by IL-6. Our results therefore reveal that inflammatory cytokines expressed in the conjunctiva during an ocular surface disease directly disrupt conjunctival goblet cell functions, compromising the protective function of tears, thereby contributing to ocular surface damage.

Co-expression of multiple cytokines and their receptors in primary clear cell sarcoma of the pubic bone with features of a small round cell tumor.

We present the case of an 81-year-old man with primary clear cell sarcoma (CCS) of the pubic bone with an associated aggressive clinical course. The patient's laboratory tests showed marked leukocytosis, elevated levels of C-reactive protein and multiple cytokines, including interleukin-6 (IL-6) and granulocyte colony-stimulating factor (G-CSF). Histological examination showed monomorphic small cells predominantly arranged as a diffuse sheet with morphological features of a small round cell tumor (SRCT). Immunohistochemical staining indicated that the tumor cells were positive for HMB45, S100, Melan A, IL-6, IL-6 receptor, G-CSF, and G-CSF receptor and negative for cytokeratin (AE1/AE3) and epithelial membrane antigen. To the best of our knowledge, this is the first case report of aggressive primary CCS of the pubic bone with features of SRCT showing the production and co-expression of multiple cytokines and their receptors. Thus, we suggest that proliferation through an IL-6- and G-CSF-associated autocrine mechanism may play an important role in the aggressive clinical course and poor prognosis of some CCSs showing features of SRCT.

Treatment outcome of CRLF2-rearranged childhood acute lymphoblastic leukaemia: a comparative analysis of the AIEOP-BFM and UK NCRI-CCLG study groups.

The prognostic relevance of CRLF2 -rearrangements in childhood acute B-cell precursor lymphoblastic leukaemia (ALL), was assessed by a comparative analysis of 114 non-Down-syndrome patients (99 P2RY8-CRLF2+ , 15 IGH@-CRLF2+ ), 76 from the AIEOP-BFM ALL 2000 and 38 from the MRC ALL97 trials. The 6-year cumulative relapse incidence of P2RY8-CRLF2+ patients treated on the two trials was not statistically different: 0·37 ± 0·06 vs. 0·25 ± 0·08 (P = 0·194). In contrast, 0/9 IGH@-CRLF2+ AIEOP-BFM, but 5/6 ALL97 patients relapsed. Conclusively, P2RY8-CRLF2+ patients had an intermediate protocol-independent outcome while the different prognosis of IGH@-CRLF2+ patients could be related to the different structures of the applied treatment protocols.

Molecular cloning and characterization of canine fractalkine and its receptor CX3CR1.

Fractalkine, also known as CX(3)CL1, is a unique chemokine that mediates inflammatory responses and is involved in the pathogenesis of several inflammatory disorders, including inflammatory bowel disease (IBD) in humans. In this study, we isolated cDNAs encoding canine fractalkine and its receptor CX(3)CR1, and assessed the biological activity of these molecules. The deduced amino acid sequence of the canine fractalkine cDNA showed 66% and 57% identity to human and mouse homologs, respectively. The N-terminal chemokine domain of the canine fractalkine showed 68% and 65% identity to human and mouse counterparts, respectively. The canine CX(3)CR1 amino acid sequence showed close homology to its human (83% identity) and mouse (81% identity) counterparts. Fractalkine and CX(3)CR1 mRNA were detected in all tissues in this study. Relatively higher expression levels of fractalkine mRNA were observed in the brain, medulla spinalis, small intestine, and mesenteric lymph nodes (MLNs), whereas higher expression levels of CX(3)CR1 mRNA were observed in the medulla spinalis, brain, liver, small intestine, and MLNs. The cross-reactivities of anti-human fractalkine antibody and anti-rat CX(3)CR1 antibody to canine proteins were confirmed using recombinant canine fractalkine and a cell line overexpressing canine CX(3)CR1, respectively. A transwell chemotaxis assay showed that the recombinant canine fractalkine induced migration in canine lymphoid cells expressing CX(3)CR1. The present study will be useful in understanding the canine immune system and the immunopathogenesis of canine inflammatory diseases.

Antitumor activity of type III interferon alone or in combination with type I interferon against human non-small cell lung cancer.

The antitumor activities of type III interferon (IFN) (interleukin [IL]-28 and IL-29) and the combination of type III IFN and type I IFN (IFN-α) were evaluated using human non-small cell lung cancer (NSCLC). The expression of type III and type I receptor complexes was detected in NSCLC lines. IL-29 significantly inhibited the in vitro growth of a wide range of NSCLC lines in a dose-dependent fashion. To a lesser degree, IL-28A also displayed growth inhibitory activity. Antitumor activity of type III IFN is associated with cell cycle arrest at the G1 phase and apoptosis. IL-29 upregulated cyclin-dependent kinase inhibitor p21Waf1/Cip1 in cells sensitive, but not insensitive, to antiproliferative activity, and knockdown of p21 with small interfering RNA largely attenuated the antiproliferative effect. Intratumoral and systemic administration of IL-29 inhibited OBA-LK1 and LK-1, but not A549, tumor growth in severe combined immunodeficiency mice. Immunohistochemical analyses demonstrated marked upregulated p21 and downregulated Ki-67 expression in tumors treated with IL-29. The interferon combination of IL-29 and IFN-α displayed a more effective antiproliferative effect and a more intense p21 expression than each reagent alone in vitro. Furthermore, interferon combination therapy suppressed in vivo NSCLC growth more effectively than interferon monotherapy. These findings demonstrate that type III IFN can mediate direct antitumor activities via increased p21 expression and induction of apoptosis and cooperate with type I IFN to elicit more efficient direct antitumor activities, and suggest the possibility that type III IFN might improve the efficacy and reduce the side-effects of type I IFN cancer therapy.

Exploration of target molecules for molecular imaging of inflammatory bowel disease.

Molecular imaging technology is a powerful tool for the diagnosis of inflammatory bowel disease (IBD) and the efficacy evaluation of various drug therapies for it. However, it is difficult to elucidate directly the relationships between the responsible molecules and IBD using existing probes. Therefore, the development of an alternative probe that is able to elucidate the pathogenic mechanism and provide information on the appropriate guidelines for treatment is earnestly awaited. In this study, we investigated pathognomonic molecules in the intestines of model mice. The accumulation of fluorine-18 fluorodeoxyglucose ((18)F-FDG) in the inflamed area of the intestines of dextran sulfate sodium (DSS)- or indomethacin (IND)-induced IBD model mice was measured by positron emission tomography (PET) and autoradiography to confirm the inflamed area. The results suggested that the inflammation was selectively induced in the colons of mice by the administration of DSS, whereas it was induced mainly in the ilea and the proximal colons of mice by the administration of IND. To explore attractive target molecules for the molecular imaging of IBD, we evaluated the gene expression levels of cytokines and cytokine receptors in the inflamed area of the intestines of both model mice. We found that the expression levels of cytokines and cytokine receptors were significantly increased during the progression of IBD, whereas the expression levels were decreased as the mucosa began to heal. In particular, the expression levels of these molecules had already changed before the symptoms of IBD appeared. In addition, the alterations of cytokine and cytokine receptor expression levels indicated differences in the expression pattern depending on the pathogenic mechanism or the region of inflammation (e.g., TNF-α). Our results suggest that these cytokines or cytokine receptors participate in the pathogenesis of IBD and are valuable biomarkers for the detection of the different circumstances underlying inflammation by the molecular imaging method. Finally, the development of an imaging probe for our target molecules is expected to improve our understanding of the inflammatory conditions of IBD.

Asociación entre el polimorfismo genético de la interleucina-1Beta (3953 T/C) y la hernia discal lumbar sintomática / Association between the genetic polymorphism of interleukin-1Beta (3953 T/C) and symptomatic lumbar herniated disc

Objetivo. Evaluar la asociación entre la presencia en el genotipo de determinados polimorfismos genéticos (PG) de las citocinas y del óxido nítrico sintasa (NOS) y el desarrollo de la hernia discal lumbar (HDL) sintomática.Material y método. Se revisaron 179 pacientes en un estudio retrospectivo de casos y controles. El grupo de casos estaba formado por 50 pacientes con HDL confirmada mediante resonancia magnética. El grupo control lo componían pacientes ingresados para cirugía protésica de la cadera o de la rodilla que no presentaban ni habían presentado nunca clínica compatible con HDL. Se realizó una extracción de sangre a todos los participantes del estudio. Se genotiparon los PG de las citocinas que pretendíamos estudiar: interleucina (IL)-1 (IL-1alfa [−889 C/T] e IL-1Beta [+3953 T/C]) y factor de necrosis tumoral-alfa (TNF-alfa´ [−308 G/A] y TNF-alfa´ [−238 G/A]). Resultados. El genotipo CC y la frecuencia del alelo C del PG IL-1Beta (+3953 T/C) fueron significativamente mayores en el grupo de pacientes con HDL respecto a la población control. Por el contrario, los pacientes del grupo control portaban los PG de NOS endotelial (−768 T/C) y de NOS inducible 22 G/A con mayor frecuencia que el grupo de pacientes con HDL, esta diferencia es estadísticamente significativa para ambos polimorfismos. Conclusiones. Encontramos que ser portador del alelo C del PG IL-1Beta (+3953 T/C) puede ser un factor de predisposición para desarrollar una HDL. Por otro lado, ser portador del PG NOS endotelial (−768 T/C) y del NOS inducible 22 G/A parece comportarse como un factor protector frente al desarrollo de esta enfermedad (AU)

Objective. To evaluate the association between the presence of the genotype of certain genetic polymorphisms (GP) of the cytokine and oxide nitric synthase (NOS) and the development of lumbar herniated disc (LHD). Materials and methods. We reviewed 179 patients in a retrospective case-control study. The case group was made up of 50 patients with confirmed lumbar herniated disc diagnosed by Magnetic Resonance Imaging (MRI). The control group was made up of patients admitted for hip and knee prosthetic surgery who did not have or had not had any symptoms consistent with LHD. Blood was drawn from all of the study participants. The genotypes of the GP were obtained of the cytokines to be studied: Interleukin-1 [IL-1alpha(−889 C/T), IL-1Beta(+3953 T/C)], Tumor Necrosis Factor-alpha [TNF-alpha (−308 G/A) and (−238G/A)]. Results. The CC genotype and C allele frequency of the IL-1Betaβ PG (+3953T/C) polymorphism were significantly more frequent in patients with LDH compared to the controls. On the contrary, the control group patients carried eNos GPs (−768 T/C) and iNOS22 G/A polymorphisms more frequently than the LHD group, this difference being statistically different for both polymorphisms. Conclusions. We found that individuals who were carriers of the CC genotype of the IL-1b(+3953T/C) polymorphism showed higher susceptibility to suffer lumbar disc herniation. Furthermore, being a carrier of ENOS (−786 T/C) and iNOS (22 G/A) polymorphisms suggests that this could behave as a protection factor against disc herniation (AU)

Cytokine expression and accelerated tooth movement.

It has been shown that inhibiting the expression of certain cytokines decreases the rate of tooth movement. Here, we hypothesized that stimulating the expression of inflammatory cytokines, through small perforations of cortical bone, increases the rate of bone remodeling and tooth movement. Forty-eight rats were divided into 4 groups: 50-cN force applied to the maxillary first molar (O), force application plus soft tissue flap (OF), force application plus flap plus 3 small perforations of the cortical plate (OFP), and a control group (C). From the 92 cytokines studied, the expression of 37 cytokines increased significantly in all experimental groups, with 21 cytokines showing the highest levels in the OFP group. After 28 days, micro-computed tomography, light and fluorescent microscopy, and immunohistochemistry demonstrated higher numbers of osteoclasts and bone remodeling activity in the OFP group, accompanied by generalized osteoporosity and increased rate of tooth movement.

Increased levels of soluble cytokine receptors in the synovial fluid of temporomandibular joint disorders in relation to joint effusion on magnetic resonance images.

PURPOSE: The purpose of this study is to clarify the significance of joint effusion (JE) on T2-weighted magnetic resonance images of the temporomandibular joint (TMJ) in comparison to various soluble cytokine receptors in the synovial fluid of patients with temporomandibular disorders (TMDs). PATIENTS AND METHODS: Magnetic resonance imaging of 55 TMJs of 55 patients with TMD was performed, and synovial fluid samples were obtained on the same day. The grade of JE was evaluated on a scale from 0 to 3, with grade 0 indicating the absence of JE and grades 1 to 3 indicating the presence of JE. Correlations were measured between JE and the concentrations of soluble tumor necrosis factor receptors I and II, interleukin (IL) 6 soluble receptor, IL-1 soluble receptor type II, and IL-1 receptor antagonist and protein in the synovial fluid samples. RESULTS: The mean concentrations of cytokine receptors in the synovial fluid were significantly higher in the 30 joints with JE than in the 25 joints without JE. There were no correlations between the JE grade and the level of any mediators. CONCLUSION: Increased levels of cytokine receptors are likely to influence the expression of JE and may play important roles in the pathogenesis of TMD. These results also suggest that JE may reflect synovial inflammation of the TMJ.