Epigenetic modulation enhances immunotherapy for hepatocellular carcinoma.

BACKGROUND: Anti-PDL-1 immunotherapy for Hepatocellular Carcinoma (HCC) demonstrated a mixed response. Polycomb Repressor Complex 2(PRC2) contributes to the initiation and progression of HCC by suppressing tumor antigens and inhibiting an immune response. Two components of epigenetic modulation are Enhancer of Zeste Homolog 2 (EZH2, the catalytic component of PRC2) and DNA Methyltransferase 1 (DNMT1). We aim to investigate the potential role of epigenetic therapy targeting EZH2 and DNMT1 as a novel strategy to modulate immunotherapy response in HCC. METHODS: HepG2, Hep3B, and Hepa1-6 HCC cell lines were treated with EZH2 inhibitor (DZNep) and DNMT1 inhibitor (5-Azacytidine) with and without anti-PDL-1. Quantitative RT-PCR and immunohistochemistry were performed to evaluate the expression of tumor suppressors, tumor antigens, and Th1 chemokines. In-vivo C57/LJ immunocompetent mice model with subcutaneous tumor inoculation was performed with intraperitoneal drug injections. RESULTS: There was a significant upregulation of Th1 chemokines in HepG2 (CXCL9 5.5 ±â€¯0.2 relative fold change; CXCL10 1.44 × 103 ±â€¯37 relative fold change) and Hep3B (CXCL 9 6.85 × 103 ±â€¯1.3 × 103 relative fold change; CXCL 10 2.15 × 103 ±â€¯3.1 × 102 relative fold change). Additionally, there was a significant induction of cancer testis antigens NY-ESO-1 (3.6-3.7 ±â€¯0.3 relative fold change) and LAGE (8.3-11.7 ±â€¯1.9 relative fold change). In vivo model demonstrated statistically significant tumor regression in the combination treatment group (0.02 g ±â€¯0.02) compared to epigenetic therapy (0.63 g ±â€¯0.61) or immunotherapy alone (0.15 g ±â€¯0.21) with untreated control (2.4 g ±â€¯0.71). There was significantly increased trafficking of cytotoxic T- lymphocytes and associated apoptosis for the combination treatment group compared to epigenetic or immunotherapy alone. CONCLUSIONS: This study demonstrates that epigenetic modulation could be a novel potential strategy to augment immunotherapy for HCC by stimulating T cell trafficking into tumor microenvironment via activation of transcriptionally repressed chemokine genes responsible for T-cell trafficking, inducing previously silent neoantigens for immune targets, and allowing tumor regression as a result. A clinical trial of this feasible combination therapy of these clinically available agents is warranted.

Chronic inflammatory lesions of the placenta are associated with an up-regulation of amniotic fluid CXCR3: A marker of allograft rejection.

OBJECTIVE: The objective of this study is to determine whether the amniotic fluid (AF) concentration of soluble CXCR3 and its ligands CXCL9 and CXCL10 changes in patients whose placentas show evidence of chronic chorioamnionitis or other placental lesions consistent with maternal anti-fetal rejection. METHODS: This retrospective case-control study included 425 women with (1) preterm delivery (n=92); (2) term in labor (n=68); and (3) term not in labor (n=265). Amniotic fluid CXCR3, CXCL9 and CXCL10 concentrations were determined by ELISA. RESULTS: (1) Amniotic fluid concentrations of CXCR3 and its ligands CXCL9 and CXCL10 are higher in patients with preterm labor and maternal anti-fetal rejection lesions than in those without these lesions [CXCR3: preterm labor and delivery with maternal anti-fetal rejection placental lesions (median, 17.24 ng/mL; IQR, 6.79-26.68) vs. preterm labor and delivery without these placental lesions (median 8.79 ng/mL; IQR, 4.98-14.7; P=0.028)]; (2) patients with preterm labor and chronic chorioamnionitis had higher AF concentrations of CXCL9 and CXCL10, but not CXCR3, than those without this lesion [CXCR3: preterm labor with chronic chorioamnionitis (median, 17.02 ng/mL; IQR, 5.57-26.68) vs. preterm labor without chronic chorioamnionitis (median, 10.37 ng/mL; IQR 5.01-17.81; P=0.283)]; (3) patients with preterm labor had a significantly higher AF concentration of CXCR3 than those in labor at term regardless of the presence or absence of placental lesions. CONCLUSION: Our findings support a role for maternal anti-fetal rejection in a subset of patients with preterm labor.

Functional reprogramming of human prostate cancer to promote local attraction of effector CD8(+) T cells.

BACKGROUND: Local infiltration of CD8(+) T cells (CTLs) in tumor lesions predicts overall clinical outcomes and the clinical benefit of cancer patients from immune checkpoint blockade. In the current study, we evaluated local production of different classes of chemokines in prostate cancer lesions, and the feasibility of their modulation to promote selective entry of CTLs into prostate tumors. METHODS: Chemokine expression in prostate cancer lesion was analyzed by TaqMan-based quantitative PCR, confocal fluorescence microscopy and ELISA. For ex vivo chemokine modulation analysis, prostate tumor explants from patients undergoing primary prostate cancer resections were cultured for 24 hr, in the absence or presence of the combination of poly-I:C, IFNα, and celecoxib (PAC). The numbers of cells producing defined chemokines in the tissues were analyzed by confocal microscopy. Chemotaxis of effector CD8(+) T cells towards the untreated and PAC-treated tumor explant supernatants were evaluated in a standard in vitro migration assays, using 24 well trans-well plates. The number of effector cells that migrated was enumerated by flow cytometry. Pearson (r) correlation was used for analyzing correlations between chemokines and immune filtrate, while paired two tailed students t-test was used for comparison between treatment groups. RESULTS: Prostate tumors showed uniformly low levels of CTL/NK/Th1-recruiting chemokines (CCL5, CXCL9, CXCL10) but expressed high levels of chemokines implicated in the attraction of myeloid derived suppressor cells (MDSC) and regulatory T cells (Treg ): CCL2, CCL22, and CXCL12. Strong positive correlations were observed between CXCL9 and CXCL10 and local CD8 expression. Tumor expression levels of CCL2, CCL22, and CXCL12 were correlated with intratumoral expression of MDSC/Treg markers: FOXP3, CD33, and NCF2. Treatment with PAC suppressed intratumoral production of the Treg -attractant CCL22 and Treg /MDSC-attractant, CXCL12, while increasing the production of the CTL attractant, CXCL10. These changes in local chemokine production were accompanied by the reduced ability of the ex vivo-treated tumors to attract CD4(+) FOXP3(+) Treg cells, and strongly enhanced attraction of the CD8(+) Granzyme B(+) CTLs. CONCLUSIONS: Our data demonstrate that the chemokine environment in prostate cancer can be reprogrammed to selectively enhance the attraction of type-1 effector immune cells and reduce local attraction of MDSCs and Tregs . Prostate 76:1095-1105, 2016. © 2016 Wiley Periodicals, Inc.

Cathelicidin related antimicrobial peptide, laminin, Toll-like receptors and chemokines levels in experimental hypersensitivity pneumonitis in mice.

INTRODUCTION: Hypersensitivity pneumonitis (HP) is an interstitial lung disease caused by unresolved inflammation and tissue repair pathologies triggered by repeated organic dust exposure. The aim of the study was to investigate changes in levels of the cathelicidin related antimicrobial peptide (CRAMP), laminin (LAM-A1), selected Toll-like receptors (TLR) and chemokines in experimental HP in mice. MATERIALS AND METHODS: Three and 18-month-old female C57BL/6J mice underwent inhalations of the saline extract of Pantoea agglomerans cells, Gram-negative bacterium common in organic dust and known for its pathogenic impact. The inhalations were repeated daily (28 days). ELISA was used for measuring in lung tissue homogenates concentration of CRAMP, LAM-A1, TLR2, TLR4, TLR8, CXCL9 (chemokine [C-X-C motif] ligand) and CXCL10. RESULTS: Levels of TLR2, TLR4 and CXCL9 were significantly higher in both young and old mice lungs already after 7 days of inhalations, while significant increase of LAM-A1 and CXCL10 was noted after 28 days, compared to untreated samples. TLR8 level was significantly augmented only in young mice. Only CRAMP level significantly declined. Significantly higher TLR8 and CXCL9 concentration in untreated samples were noted in old animals compared to young ones. CONCLUSION: Significant alterations of the examined factors levels indicate their role in HP pathogenesis.

CXCL9:SPP1 macrophage polarity identifies a network of cellular programs that control human cancers.

Tumor microenvironments (TMEs) influence cancer progression but are complex and often differ between patients. Considering that microenvironment variations may reveal rules governing intratumoral cellular programs and disease outcome, we focused on tumor-to-tumor variation to examine 52 head and neck squamous cell carcinomas. We found that macrophage polarity-defined by CXCL9 and SPP1 (CS) expression but not by conventional M1 and M2 markers-had a noticeably strong prognostic association. CS macrophage polarity also identified a highly coordinated network of either pro- or antitumor variables, which involved each tumor-associated cell type and was spatially organized. We extended these findings to other cancer indications. Overall, these results suggest that, despite their complexity, TMEs coordinate coherent responses that control human cancers and for which CS macrophage polarity is a relevant yet simple variable.

CXC ligand 9 response to malaria during pregnancy is associated with low-birth-weight deliveries.

Placental infection with Plasmodium falciparum is associated with increased levels of proinflammatory cytokines, including tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-γ), and previous studies have associated increased levels of these cytokines with low birth weight (LBW), especially for malaria-infected primigravidae. To define the contribution of TNF-α and IFN-γ networks to placental-malaria-associated LBW, we measured chemokines induced by TNF-α and IFN-γ and related them to birth weight in a birth cohort of 782 mother-infant pairs residing in an area of P. falciparum holoendemicity in Tanzania. Among primigravidae, levels of CCL2, CXC ligand 9 (CXCL9), and CXCL13 were significantly higher during malaria infection in both the placenta and peripheral blood. Placental CXCL9 and CXCL13 levels were also higher in placental blood from secundigravidae and multigravidae. In multivariate analyses adjusted for known predictors of birth weight, malaria-infected primigravidae with placental CXCL9 levels in the lowest tertile gave birth to babies who weighed 610 g more than babies born to mothers with high CXCL9 levels. CXCL9 expression is induced by IFN-γ, and the strong association between birth weight and placental CXCL9 is consistent with previous observations relating IFN-γ to poor pregnancy outcomes.

CXCL9, CXCL10, and CXCL11; biomarkers of pulmonary inflammation associated with autoimmunity in patients with collagen vascular diseases-associated interstitial lung disease and interstitial pneumonia with autoimmune features.

INTRODUCTION: Interstitial lung disease (ILD) is a heterogeneous group of diseases characterized by varying degrees of lung inflammation and/or fibrosis. We investigated biomarkers to infer whether patients with collagen vascular diseases associated ILD (CVD-ILD) and interstitial pneumonia with autoimmune features (IPAF) benefit from immunosuppressive therapy. MATERIALS AND METHODS: We retrospectively investigated patients with CVD-ILD, IPAF, and idiopathic pulmonary fibrosis (IPF) between June 2013 and May 2017 at our department. First, we assessed differences in serum and bronchoalveolar lavage fluid (BALF) levels of cytokines between groups. Second, we assessed the associations of patient's clinical variables with serum and BALF levels of those cytokines that were different between groups. Finally, we assessed the associations of diagnosis and response to immunosuppressive therapy with serum levels of those cytokines that were different between groups. RESULTS: We included 102 patients (51 with IPF, 35 with IPAF, and 16 with CVD-ILD). Serum and BALF levels of CXCL9, CXCL10, and CXCL11 were significantly elevated in patients with IPAF or CVD-ILD compared with those in patients with IPF. BALF levels of CXCL9 and CXCL10 were correlated with the percentages of lymphocytes and macrophages in BALF. Serum levels of CXCL9 and CXCL10 were correlated with BALF levels. Serum levels of CXCL9, CXCL10, and CXCL11 were correlated C-reactive protein, percent predicted forced vital capacity, alveolar-arterial oxygen difference, and the percentages of lymphocytes and macrophages in BALF. Serum levels of CXCL9, CXCL10, and CXCL11 showed moderate accuracy to distinguish patients with CVD-ILD from those with IPAF and IPF. Pre-treatment serum levels of CXCL9 and CXCL11 showed strong positive correlations with the annual forced vital capacity changes in patients with IPAF and CVD-ILD treated with immunosuppressive drugs. CONCLUSIONS: Serum CXCL9, CXCL10, and CXCL11 are potential biomarkers for autoimmune inflammation and predictors of the immunosuppressive therapy responses in ILD with background autoimmunity.

The Role of CXCL13 and CXCL9 in Early Breast Cancer.

BACKGROUND: Chemokines, cytokines in the immune microenvironment of tumors, may be associated with patient outcome. We assessed the impact of CXCL13 and CXCL9 on disease-free (DFS) and overall survival (OS), in an attempt to retrospectively evaluate both T and B cell function in the microenvironment of primary tumors from patients with breast cancer. MATERIALS AND METHODS: Formalin-fixed paraffin-embedded tissue blocks from patients with intermediate/high-risk, early breast cancer, treated with sequential adjuvant epirubicin, paclitaxel, and cyclophosphamide methotrexate fluorouracil within a randomized trial, were tested for CXCL13 and CXCL9 messenger RNA expression; 557 patients with adequate tissue were eligible for the analysis. RESULTS: CXCL13 was correlated with CXCL9 (rho = 0.52; P < .001). High-expressing CXL13 and CXCL9 tumors had higher Ki67 and tumor infiltrating lymphocyte density (P-values < .001). High CXCL9 expression was an unfavorable prognosticator for OS among all patients (hazard ratio [HR], 1.73; P = .021), whereas it showed favorable significance for both DFS and OS in patients with triple negative disease (HR, 0.29; P = .027 and HR, 0.32; P = .045). High CXCL13 conferred longer DFS and OS among patients with luminal-human epidermal growth factor receptor 2 disease (HR, 0.31; P = .013 and HR, 0.25; P = .005). Patients with low CXCL13 and high CXCL9 expression had shorter DFS and OS compared with those with high expression of both chemokines (HR, 1.63; P = .006 and HR, 1.61; P = .016). CONCLUSIONS: Both biomarkers were associated with poor prognosis characteristics and with tumor infiltrating lymphocyte density. High CXCL9 conferred an improved prognosis in the triple negative subtype, whereas high CXCL13 was associated with improved outcome in the luminal-human epidermal growth factor receptor 2 subtype. Chemokines can be associated with breast cancer subtype and outcome. These data should be evaluated prospectively.

CXCR3 and CCR5 chemokines in induced sputum from patients with COPD.

BACKGROUND: COPD is associated with increased numbers of CD4(+) and CD8(+) lymphocytes and macrophages in the small airways and lung parenchyma. The chemokines regulating T-cell recruitment into the lung are unknown but may involve CXCR3 and CCR5 chemoattractants. The aims of this study were to determine the concentrations of CXCR3 chemokines CXCL9, CXCL10, CXCL11, and the CCR5 chemokine CCL5 in induced sputum from patients with COPD, smokers, and nonsmokers, and to examine the relationship between chemokine expression, inflammatory cells, and airway obstruction. METHODS: Differential cell counts were performed and concentrations of CXCL9, CXCL10, CXCL11, and CCL5 were measured in induced sputum from nonsmokers (n = 18), smokers (n = 20), and COPD patients (n = 35) using an enzyme-linked immunosorbent assay. RESULTS: Concentrations of CXCL9, CXCL10, CXCL11, and CCL5 were significantly increased in the sputum of patients with COPD when compared with nonsmokers but not smokers without obstruction: CXCL9 (median, 14.3 pg/mL; interquartile range [IQR], 6.5 to 99.3; vs median, 1.4 pg/mL; IQR, 0 to 10.4 [p < 0.001]; vs 8.5 pg/mL; IQR, 0 to 16.0, respectively); CXCL10 (16.9 pg/mL; IQR, 6.2 to 148.8; vs 3.7 pg/mL; IQR, 0 to 18.8 [p < 0.05]; vs 11.3 pg/mL; IQR, 3.7 to 46.7); CXCL11 (58.1 pg/mL; IQR, 34.5 to 85.3; vs 33.5 pg/mL; IQR, 23.2 to 49.7 [p < 0.05]; vs 49.8 pg/mL; IQR, 32.6 to 105.6); and CCL5 (59.9 pg/mL; IQR, 57.1 to 67.8; vs 33.5 pg/mL; IQR, 31.6 to 36.9 [p < 0.001]). CCL5 in sputum from smokers was also significantly increased compared with that from nonsmokers (median, 63.0 pg/mL; IQR, 60.8 to70.2; p < 0.001). There was a negative correlation between FEV(1) percentage of predicted, FEV(1)/FVC ratio, and percentage of macrophages, and all the chemokines analyzed. Neutrophil numbers correlated positively with the concentrations of chemokines. CONCLUSIONS: CXCR3 chemokines and CCL5 are increased in sputum from COPD patients compared with nonsmokers, and may be important in COPD pathogenesis.

Induction of Th1 chemokine secretion in dermal fibroblasts by vanadium pentoxide.

Vanadium is a soft, silvery­grey metal with a number of different oxidation states. The most common commercial form of vanadium is vanadium pentoxide (V2O5). All vanadium compounds are considered toxic. An increase in skin rashes has been observed in certain vanadium workers, including the development of atopic dermatitis. However, to the best of our knowledge, no prior in vivo or in vitro studies have evaluated the effect of vanadium exposure in human dermal fibroblasts. The present study evaluated the effect of V2O5 on proliferation and chemokine secretion in dermal fibroblasts. The results revealed that V2O5 had no significant effect on the viability or proliferation of fibroblasts, however it was able to induce the secretion of T­helper (Th)1 chemokines from dermal fibroblasts, synergistically increasing the effect of important Th1 cytokines, including interferon­Î³ and tumor necrosis factor­α. Through these processes, V2O5 may lead to the induction and perpetuation of an inflammatory reaction in dermal tissue. The induction and perpetuation of inflammation in the dermis and the variety of involved candidate genes may be at the base of V2O5­induced effects following occupational and environmental exposures. Further studies are necessary to evaluate dermal integrity and manifestations in subjects who are occupationally exposed, or living in polluted areas.

A fine-needle aspiration-based protein signature discriminates benign from malignant breast lesions.

There are increasing demands for informative cancer biomarkers, accessible via minimally invasive procedures, both for initial diagnostics and to follow-up personalized cancer therapy. Fine-needle aspiration (FNA) biopsy provides ready access to relevant tissues; however, the minute sample amounts require sensitive multiplex molecular analysis to achieve clinical utility. We have applied proximity extension assays (PEA) and NanoString (NS) technology for analyses of proteins and of RNA, respectively, in FNA samples. Using samples from patients with breast cancer (BC, n = 25) or benign lesions (n = 33), we demonstrate that these FNA-based molecular analyses (a) can offer high sensitivity and reproducibility, (b) may provide correct diagnosis in shorter time and at a lower cost than current practice, (c) correlate with results from routine analysis (i.e., benchmarking against immunohistochemistry tests for ER, PR, HER2, and Ki67), and (d) may also help identify new markers related to immunotherapy. A specific 11-protein signature, including FGF binding protein 1, decorin, and furin, distinguished all cancer patient samples from all benign lesions in our main cohort and in smaller replication cohort. Due to the minimally traumatic sampling and rich molecular information, this combined proteomics and transcriptomic methodology is promising for diagnostics and evaluation of treatment efficacy in BC.

Keratinocyte-Derived Chemokines Orchestrate T-Cell Positioning in the Epidermis during Vitiligo and May Serve as Biomarkers of Disease.

Vitiligo is an autoimmune disease of the skin that results in the destruction of melanocytes and the clinical appearance of white spots. Disease pathogenesis depends on IFN-γ and IFN-γ-induced chemokines to promote T-cell recruitment to the epidermis where melanocytes reside. The skin is a complex organ, with a variety of resident cell types. We sought to better define the microenvironment and distinct cellular contributions during autoimmunity in vitiligo, and we found that the epidermis is a chemokine-high niche in both a mouse model and human vitiligo. Analysis of chemokine expression in mouse skin showed that CXCL9 and CXCL10 expression strongly correlate with disease activity, whereas CXCL10 alone correlates with severity, supporting them as potential biomarkers for following disease progression. Further studies in both our mouse model and human patients showed that keratinocytes were the major chemokine producers throughout the course of disease, and functional studies using a conditional signal transducer and activator of transcription (STAT)-1 knockout mouse showed that IFN-γ signaling in keratinocytes was critical for disease progression and proper autoreactive T-cell homing to the epidermis. In contrast, epidermal immune cell populations including endogenous T cells, Langerhans cells, and γδ T cells were not required. These results have important clinical implications, because topical therapies that target IFN-γ signaling in keratinocytes could be safe and effective new treatments, and skin expression of these chemokines could be used to monitor disease activity and treatment responses.

Salivary and Serum Markers Related to Innate Immunity in Generalized Aggressive Periodontitis.

BACKGROUND: Host inflammatory and immune responses play an important role in aggressive periodontitis (AgP). Thus, this study aims to evaluate levels of the innate immunity-related markers calprotectin, colony-stimulating factor (CSF)-1, macrophage migration inhibitory factor (MIF), monokine induced by interferon-γ (MIG), and matrix metalloproteinase (MMP)-8 in serum and saliva from patients with generalized AgP and those with gingivitis or a healthy periodontium. METHODS: This study enrolled 40 individuals (17 males and 23 females; mean age 33.30 ± 9.31 years), 15 with generalized AgP, 15 with gingivitis, and 10 who were periodontally healthy. Full-mouth periodontal examinations were performed, and serum and saliva were collected. Levels of calprotectin, CSF-1, MIF, MIG, and MMP-8 were measured using enzyme-linked immunosorbent assays. RESULTS: In serum, mean levels of calprotectin were 2.06-fold higher in patients with AgP than in healthy patients (P = 0.01). Serum levels of MMP-8 were significantly elevated in patients with AgP compared with both healthy patients and those with gingivitis, by 2.60-fold and 2.77-fold, respectively (P = 0.03 and P = 0.009, respectively). In saliva, levels of MMP-8 were 5.66-fold higher in patients with AgP than in healthy patients (P = 0.02). CSF-1, MIF, and MIG levels in both serum and saliva did not differ significantly among the groups. CONCLUSIONS: Serum levels of calprotectin and MMP-8 are elevated in patients with AgP. MMP-8 levels are also increased in saliva from patients with AgP. These results support involvement of innate immune response in the pathogenesis of AgP.

Direct label-free electrical immunodetection of transplant rejection protein biomarker in physiological buffer using floating gate AlGaN/GaN high electron mobility transistors.

Monokine induced by interferon gamma (MIG/CXCL9) is used as an immune biomarker for early monitoring of transplant or allograft rejection. This paper demonstrates a direct electrical, label-free detection method of recombinant human MIG with anti-MIG IgG molecules in physiologically relevant buffer environment. The sensor platform used is a biologically modified GaN-based high electron mobility transistor (HEMT) device. Biomolecular recognition capability was provided by using high affinity anti-MIG monoclonal antibody to form molecular affinity interface receptors on short N-hydroxysuccinimide-ester functionalized disulphide (DSP) self-assembled monolayers (SAMs) on the gold sensing gate of the HEMT device. A floating gate configuration has been adopted to eliminate the influences of external gate voltage. Preliminary test results with the proposed chemically treated GaN HEMT biosensor show that MIG can be detected for a wide range of concentration varying from 5 ng/mL to 500 ng/mL.

Effects of resolvin D1 on cell survival and cytokine expression of human gingival fibroblasts.

BACKGROUND: Tissue breakdown in periodontitis is initiated by bacteria, such as Porphyromonas gingivalis, and is caused largely by host responses. Resolvins protect the host against acute inflammation by blocking the migration of polymorphonuclear neutrophils to initiate resolution. The effects of resolvins on human gingival fibroblasts (HGFs) are unknown. This study examines the effects of resolvin D1 on HGF survival and cytokine expression when treated with or without P. gingivalis supernatant. METHODS: Cytotoxicity of resolvin D1 on HGFs with or without a toxic level of P. gingivalis supernatant was measured with lactate dehydrogenase assays. Cytokine arrays were performed on HGF-conditioned media treated with or without resolvin D1 and with or without P. gingivalis supernatant. RESULTS: Resolvin D1 had no cytotoxic effects on HGFs at concentrations between 1 and 1,000 nM (all P > 0.05). Resolvin D1 (1,000 nM) significantly inhibited the toxic effects of 13.5% (v/v) P. gingivalis supernatant on HGFs (P = 0.002). Resolvin D1 significantly reduced the expression of interleukin (IL)-6 (P = 0.010) and monocyte chemoattractant protein (MCP)-1 (P = 0.04) in untreated fibroblasts. P. gingivalis (10%) supernatant significantly increased the expression levels of granulocyte-macrophage colony-stimulating factor (CSF), granulocyte CSF, growth-regulated oncogene (GRO), IL-5, IL-6, IL-7, IL-8, IL-10, MCP-1, MCP-2, MCP-3, and monokine induced by γ-interferon. Resolvin D1 significantly reduced the expression of GRO (P = 0.04), marginally reduced the levels of MCP-1 (P = 0.10), and marginally increased the levels of transforming growth factor (TGF)-ß1 (P = 0.07) from HGFs treated with P. gingivalis supernatant. CONCLUSIONS: Resolvin D1 altered the cytotoxicity of P. gingivalis supernatant on HGFs. Resolvin D1 significantly reduced GRO, marginally reduced MCP-1, and marginally increased TGF-ß1 from P. gingivalis-treated HGFs, which could alter the ability of P. gingivalis to induce inflammation.

Tumor-associated lymphocytes as an independent predictor of response to neoadjuvant chemotherapy in breast cancer.

PURPOSE Preclinical data suggest a contribution of the immune system to chemotherapy response. In this study, we investigated the prespecified hypothesis that the presence of a lymphocytic infiltrate in cancer tissue predicts the response to neoadjuvant chemotherapy. METHODS We investigated intratumoral and stromal lymphocytes in a total of 1,058 pretherapeutic breast cancer core biopsies from two neoadjuvant anthracycline/taxane-based studies (GeparDuo, n = 218, training cohort; and GeparTrio, n = 840, validation cohort). Molecular parameters of lymphocyte recruitment and activation were evaluated by kinetic polymerase chain reaction in 134 formalin-fixed, paraffin-embedded tumor samples. Results In a multivariate regression analysis including all known predictive clinicopathologic factors, the percentage of intratumoral lymphocytes was a significant independent parameter for pathologic complete response (pCR) in both cohorts (training cohort: P = .012; validation cohort: P = .001). Lymphocyte-predominant breast cancer responded, with pCR rates of 42% (training cohort) and 40% (validation cohort). In contrast, those tumors without any infiltrating lymphocytes had pCR rates of 3% (training cohort) and 7% (validation cohort). The expression of inflammatory marker genes and proteins was linked to the histopathologic infiltrate, and logistic regression showed a significant association of the T-cell-related markers CD3D and CXCL9 with pCR. CONCLUSION The presence of tumor-associated lymphocytes in breast cancer is a new independent predictor of response to anthracycline/taxane neoadjuvant chemotherapy and provides useful information for oncologists to identify a subgroup of patients with a high benefit from this type of chemotherapy.

Strong overexpression of CXCR3 axis components in childhood inflammatory bowel disease.

BACKGROUND: Inflammatory bowel disease (IBD) is a polygenetic disorder. Our group previously showed that a variant within the CXCL9 gene is associated with pediatric Crohn's disease. As CXCL9, CXCL10, and CXCL11 are the 3 ligands to the receptor CXCR3, the aim of this study was to investigate the colonic transcriptional activity of the CXCR3 axis and to perform SNP genotyping of a CXCL11 polymorphism in a large pediatric and adult IBD cohort. METHODS: mRNA expression of CXCR3, CXCL9, CXCL10, CXCL11, and IL8 was analyzed in colonic biopsies using real-time PCR. CXCL11 rs6817952 nucleotide substitution was determined in 501 German individuals with IBD (336 CD, 165 UC) including 258 children and 243 adults as well as in 231 controls by a TaqMan SNP genotyping assay. RESULTS: CXCR3 axis genes were significantly overexpressed in inflamed colonic tissue of pediatric CD and UC patients. The prevalence of hetero- and homozygous variants of the rs6817952 genotype was higher in pediatric but not in adult CD patients compared with that in controls (P = 0.04). Moreover, carriers of the hetero- and homozygous genotype variants of rs6817952 were at increased risk for UC in all age groups (P = 0.009). CONCLUSIONS: Our study provides evidence of the significant overexpression of the CXCR3 axis in active IBD, suggesting it has a role in IBD pathogenesis. The rs6817952 A variant is a risk allele for pediatric CD and UC in all age groups. Therapeutic studies will have to show whether the blockade of chemokine receptors such as CXCR3 can modulate intestinal inflammation in a clinical application.

Activation of the interferon-gamma signaling pathway in systemic lupus erythematosus peripheral blood mononuclear cells.

OBJECTIVE: To investigate interferon-gamma (IFNgamma) signaling in peripheral blood mononuclear cells (PBMCs) from patients with systemic lupus erythematosus (SLE) by analyzing IFNgamma receptor (IFNgammaR) expression, STAT-1 expression and phosphorylation, and the regulation of IFNgamma-inducible genes. METHODS: Fluorocytometry was used to investigate expression of STAT-1, pSTAT-1, CD95, HLA-DR, class I major histocompatibility complex (MHC), IFNgamma-inducible 10-kd protein (IP-10), monokine induced by IFNgamma (Mig), and IFNgammaR in PBMCs from SLE patients and healthy individuals. STAT-1 phosphorylation was determined by fluorocytometry and Western blotting after stimulation with IFNalpha or IFNgamma. Quantitative polymerase chain reaction was used to assess messenger RNA (mRNA) expression of the IFNgamma-inducible genes IP-10 and Mig shortly after preparation or after stimulation with IFNgamma in monocytes. RESULTS: STAT-1 expression was increased in PBMCs from SLE patients and correlated significantly with disease activity and with the IFN-inducible expression of CD95 and HLA-DR. STAT-1 expression also showed a trend toward association with class I MHC expression. In addition, the expression of other IFNgamma-inducible genes, such as IP-10 or Mig, was increased in SLE monocytes. While STAT-1 phosphorylation in SLE PBMCs and PBMCs from healthy individuals was similar after IFNalpha stimulation, incubation with IFNgamma induced STAT-1 phosphorylation only in SLE lymphocytes. Moreover, SLE monocytes showed a considerably higher increase in pSTAT-1 expression upon IFNgamma stimulation than monocytes from healthy individuals. Increased responsiveness of SLE monocytes to IFNgamma was also confirmed on the mRNA level, where expression of the IFN-inducible, STAT-1-dependent genes IP-10 and Mig was more efficiently increased in SLE cells. However, IFNgammaR was similarly expressed on SLE lymphocytes and monocytes and those from healthy individuals. CONCLUSION: In addition to supporting the role of IFNs in SLE immunopathogenesis in general, the findings of the present study support a role of IFNgamma in this disease.

Detection of clinically relevant levels of protein analyte under physiologic buffer using planar field effect transistors.

Electrochemical detection of protein binding at physiological salt concentration by planar field effect transistor platforms has yet to be documented convincingly. Here we report detection of streptavidin and clinically relevant levels of biotinylated monokine induced by interferon gamma (MIG) at physiological salt concentrations with AlGaN heterojunction field effect transistors (HFETs). The AlGaN HFETs are functionalized with a silane linker and analyte-specific affinity elements. Polarity of sensor responses is as expected from n-type HFETs to negatively and positively charged analytes. Sensitivity of the HFET sensors increases when salt concentration decreases, and the devices also exhibit dose-dependent responses to analyte. Detection of clinically relevant MIG concentrations at physiological salt levels demonstrates the potential for AlGaN devices to be used in development of in vivo biosensors.

Gene expression profiling of lichen planus reflects CXCL9+-mediated inflammation and distinguishes this disease from atopic dermatitis and psoriasis.

Here, we present data of a gene expression profiling approach to apply the diagnostic value and pathological significance of this method in different inflammatory skin diseases, using whole skin biopsies. Initially, SAGE was performed to identify frequent tags differentially expressed in various skin diseases. On the basis of these results, a new skin pathology-oriented PIQOR microarray was designed. Lichen planus (LP) was chosen as a model disease to evaluate this system. Controls included healthy skin, atopic dermatitis (AD), and psoriasis (Pso). Gene expression analyses using the topic-defined microarray followed by unclassified clustering was able to discriminate LP from AD and Pso. Genes significantly expressed in LP included type I IFN inducible genes and a specific chemokine expression pattern. The CXCR3 ligand, CXCL9, was the most significant marker for LP. In situ hybridization and immunohistochemistry confirmed the results and revealed that keratinocytes are type I IFN producers in LP skin lesions. Our results show that gene expression profiling using a skin-specific microarray is a reliable method to identify patients with LP in the chosen context and reflect recent models concerning the pathogenesis of this disease. Gene expression profiling might complement the diagnostic spectrum in dermatology and may provide new pathogenetic insights.