Dead Sea minerals: New findings on skin and the biology beyond.

BACKGROUND: Therapeutic effects of Dead Sea (DS) minerals are well established, and their unique combination is analysed and reported. DS water (DSW) is a key source for DS minerals, and various studies report the capability of DSW to alleviate symptoms of different skin disorders and to contribute to skin maintenance. However, the biological mechanisms beyond reported effects are not fully understood yet. OBJECTIVE: To elucidate the effect of topically applied DSW via the expression of different skin biomarkers related to barrier function, homeostasis, inflammation and irritation. METHODS: In vitro skin equivalents and ex vivo human skin organ culture were used to assess the biological effects of DSW. Epidermal barrier protein expression and DSW ions transdermal penetration were analysed on skin equivalents. ß-endorphin secretion was tested on human skin organ culture. The capability of DSW to protect against skin inflammation and irritation was tested on ex vivo human skin organ culture by lipopolysaccharides and sodium dodecyl sulphate addition, respectively. RESULTS: Topical application of DSW encouraged the expression of the barrier-related proteins: filaggrin, involucrin and transglutaminase, while transdermal penetration of calcium ions was not detected. Additionally, DSW application had increased skin secretion of ß-endorphin and attenuated the expression of inflammatory and irritation-related cytokines. CONCLUSIONS: This study reports new findings of DSW effects on skin. Signalling pathway activation is proposed as a key step that may result in a vast range of proven biological activities following skin exposure to DS minerals, and specifically DSW.

Quantitative Flow Cytometry: Concerns and Recommendations in Clinic and Research.

BACKGROUND: Quantitative flow cytometry (QFCM) can be an important element within the developing toolbox for clinical diagnostics which relies on precise and rapid tests that provide a conclusive answer for physicians. The FC technology combines all of these features. Until recently, this imperative discipline was based on qualitative assessments of cell populations. However, due to the enormous advancement in FC technology, which allows the quantification of a number of antigens on cell surface and within the cells by units of median fluorescence intensity (MFI), this method becomes meaningful and fits the clinical needs. METHODS: On the basis of our experience in the field of quantitative FC, we wish to highlight some of the key concerns related to this methodology and suggest possible solutions for achieving uniform and standardized QFCM tests based on MFI. RESULTS: Several parameters are responsible for inter and intra laboratory variations. The standardization of quantitative FC relies on three major components; Samples and reagents handling, FC maintenance and data analysis. The use of specialized beads as a part of the routine calibration process lowers inter-test variability between different operators and different FC instruments. Similarly, the use of agreed biological controls contributes significantly to lowering test variability. CONCLUSIONS: The field of QFCM displays a significant part in the diagnostic clinical toolbox. We believe that the recommendations described herein can improve significantly the stability and accuracy of this method, thus assuring a more standardized cell analyses. © 2017 International Clinical Cytometry Society.

Clinical Significance of Circulating CD33+CD11b+HLA-DR- Myeloid Cells in Patients with Stage IV Melanoma Treated with Ipilimumab.

PURPOSE: High levels of circulating myeloid-derived suppressor cells (MDSCs) in various cancer types, including melanoma, were shown to correlate with poor survival. We investigated whether frequencies of circulating CD33+CD11b+HLA-DR- MDSCs could be used as immune system monitoring biomarkers to predict response and survival of patients with stage IV melanoma treated with anti-CTLA4 (ipilimumab) therapy. EXPERIMENTAL DESIGN: Peripheral blood samples from 56 patients and 50 healthy donors (HDs) were analyzed for CD33+CD11b+HLA-DR- MDSC percentage, NO-, and hROS levels by flow cytometry. We determined whether MDSC levels and suppressive features detected before anti-CTLA4 therapy correlate with the patients' response and overall survival (OS). RESULTS: Patients with melanoma had significantly higher levels of circulating CD33+CD11b+HLA-DR- MDSCs with suppressive phenotype when compared with HDs. Low levels of MDSCs before CTLA-4 therapy correlated with an objective clinical response, long-term survival, increased CD247 expression in T cells, and an improved clinical status. No predictive impact was observed for lactate dehydrogenase (LDH). Kaplan-Meier and log-rank tests performed on the 56 patients showed that the presence of more than 55.5% of circulating CD33+CD11b+ out of the HLA-DR- cells, were associated with significant short OS (P < 0.003), a median of 6.5 months, in comparison with the group showing lower MDSC frequencies, with a median survival of 15.6 months. CONCLUSIONS: Our study suggests the use of CD33+CD11b+HLA-DR- cells as a predictive and prognostic biomarker in patients with stage IV melanoma treated with anti-CTLA4 therapy. This monitoring system may aid in the development of combinatorial modalities, targeting the suppressive environment in conjunction with iplimumab, toward facilitating better disease outcomes. Clin Cancer Res; 22(23); 5661-72. ©2016 AACR.

Impaired SNX9 Expression in Immune Cells during Chronic Inflammation: Prognostic and Diagnostic Implications.

Chronic inflammation is associated with immunosuppression and downregulated expression of the TCR CD247. In searching for new biomarkers that could validate the impaired host immune status under chronic inflammatory conditions, we discovered that sorting nexin 9 (SNX9), a protein that participates in early stages of clathrin-mediated endocytosis, is downregulated as well under such conditions. SNX9 expression was affected earlier than CD247 by the generated harmful environment, suggesting that it is a potential marker sensing the generated immunosuppressive condition. We found that myeloid-derived suppressor cells, which are elevated in the course of chronic inflammation, are responsible for the observed SNX9 reduced expression. Moreover, SNX9 downregulation is reversible, as its expression levels return to normal and immune functions are restored when the inflammatory response and/or myeloid-derived suppressor cells are neutralized. SNX9 downregulation was detected in numerous mouse models for pathologies characterized by chronic inflammation such as chronic infection (Leishmania donovani), cancer (melanoma and colorectal carcinoma), and an autoimmune disease (rheumatoid arthritis). Interestingly, reduced levels of SNX9 were also observed in blood samples from colorectal cancer patients, emphasizing the feasibility of its use as a diagnostic and prognostic biomarker sensing the host's immune status and inflammatory stage. Our new discovery of SNX9 as being regulated by chronic inflammation and its association with immunosuppression, in addition to the CD247 regulation under such conditions, show the global impact of chronic inflammation and the generated immune environment on different cellular pathways in a diverse spectrum of diseases.

Adverse immunoregulatory effects of 5FU and CPT11 chemotherapy on myeloid-derived suppressor cells and colorectal cancer outcomes.

Colorectal cancer is associated with chronic inflammation and immunosuppression mediated by myeloid-derived suppressor cells (MDSC). Although chemotherapy reduces tumor burden at early stages, it tends to have limited effect on a progressive disease, possibly due to adverse effects on the immune system in dictating disease outcome. Here, we show that patients with advanced colorectal cancer display enhanced MDSC levels and reduced CD247 expression and that some conventional colorectal cancer chemotherapy supports the immunosuppressive tumor microenvironment. A FOLFOX combined therapy reduced immunosuppression, whereas a FOLFIRI combined therapy enhanced immunosuppression. Mechanistic studies in a colorectal cancer mouse model revealed that FOLFIRI-like therapy including the drugs CPT11 and 5-fluorouracil (5FU) damaged host immunocompetence in a manner that limits treatment outcomes. CPT11 blocked MDSC apoptosis and myeloid cell differentiation, increasing MDSC immunosuppressive features and mouse mortality. In contrast, 5FU promoted immune recovery and tumor regression. Thus, CPT11 exhibited detrimental immunoregulatory effects that offset 5FU benefits when administered in combination. Our results highlight the importance of developing therapeutic regimens that can target both the immune system and tumor towards improved personalized treatments for colorectal cancer.

Unique ζ-chain motifs mediate a direct TCR-actin linkage critical for immunological synapse formation and T-cell activation.

TCR-mediated activation induces receptor microclusters that evolve to a defined immune synapse (IS). Many studies showed that actin polymerization and remodeling, which create a scaffold critical to IS formation and stabilization, are TCR mediated. However, the mechanisms controlling simultaneous TCR and actin dynamic rearrangement in the IS are yet not fully understood. Herein, we identify two novel TCR ζ-chain motifs, mediating the TCR's direct interaction with actin and inducing actin bundling. While T cells expressing the ζ-chain mutated in these motifs lack cytoskeleton (actin) associated (cska)-TCRs, they express normal levels of non-cska and surface TCRs as cells expressing wild-type ζ-chain. However, such mutant cells are unable to display activation-dependent TCR clustering, IS formation, expression of CD25/CD69 activation markers, or produce/secrete cytokine, effects also seen in the corresponding APCs. We are the first to show a direct TCR-actin linkage, providing the missing gap linking between TCR-mediated Ag recognition, specific cytoskeleton orientation toward the T-cell-APC interacting pole and long-lived IS maintenance.

Tumor necrosis factor-α blocks differentiation and enhances suppressive activity of immature myeloid cells during chronic inflammation.

Elevated concentrations of tumor necrosis factor-α (TNF-α) are detected in pathologies characterized by chronic inflammation. Whether TNF-α plays a role in manipulating the host's immune system toward generating an immunosuppressive milieu, typical of ongoing chronic inflammation, is unclear. Here we showed that TNF-α exhibited a dual function during chronic inflammation: arresting differentiation of immature myeloid-derived suppressor cells (MDSCs) primarily via the S100A8 and S100A9 inflammatory proteins and their corresponding receptor (RAGE) and augmenting MDSC suppressive activity. These functions led to in vivo T and NK cell dysfunction accompanied by T cell antigen receptor ζ chain downregulation. Furthermore, administration of etanercept (TNF-α antagonist) during early chronic inflammatory stages reduced MDSCs' suppressive activity and enhanced their maturation into dendritic cells and macrophages, resulting in the restoration of in vivo immune functions and recovery of ζ chain expression. Thus, TNF has a fundamental role in promoting an immunosuppressive environment generated during chronic inflammation.

Nitric oxide and glucocorticoids synergize in inducing apoptosis of CD4⁺8⁺ thymocytes: implications for 'Death by Neglect' and T-cell function.

Thymic epithelial cells (TECs) play a central role in T-cell development by presenting self-antigens on MHC proteins. Double-positive (DP) thymocytes that fail to interact with TEC via their TCR die by 'Death by Neglect'. We demonstrated a role for TEC-derived glucocorticoids (GCs) in this process. In a previous study, we used an in vitro system recapitulating Death by Neglect, to demonstrate the involvement of nitric oxide (NO) and inducible NO synthase (iNOS) in this process. In this study, we show that NO synergizes with GCs to induce apoptosis of DP thymocytes in a fetal thymic organ culture. Also, DP thymocytes from iNOS⁻/⁻ mice are less sensitive to GC-induced apoptosis. Furthermore, the number of DP thymocytes in iNOS⁻/⁻ mice is higher than in wild-type mice, suggesting a role for NO in Death by Neglect. This phenomenon effects T-cell function profoundly: iNOS⁻/⁻ T cells do not respond to TCR-mediated activation signals, measured by up-regulation of CD69, IL-2R and IFNγ secretion. This failure to activate is a result of TCR incompetence because iNO⁻/⁻ T cells respond to TCR-independent stimuli (phorbol myristate acetate and calcium ionophore). This study suggests that NO and GCs synergize to execute TEC-induced death of DP thymocytes.

alpha2,6-Sialylation promotes binding of placental protein 14 via its Ca2+-dependent lectin activity: insights into differential effects on CD45RO and CD45RA T cells.

Placental protein 14 (PP14; glycodelin) is a pregnancy-associated immunoregulatory protein that is known to inhibit T cells via T-cell receptor desensitization. The recent demonstration of PP14 as lectin has provided insight into how it may mediate its CD45 glycoprotein-dependent T-cell inhibition. In this study, we have investigated PP14's lectin-binding properties in detail. Significantly, PP14 reacts with N-acetyllactosamine (LacNAc) as was also found for members of the galectin family, such as the potent immunoregulatory protein, galectin-1. However, in contrast to galectin-1, PP14's binding is significantly enhanced by alpha2,6-sialylation and also by the presence of cations. This was demonstrated by preferential binding to fetuin as compared with its desialylated variant asialofetuin (ASF) and by using free alpha2,6- versus alpha2,3-sialylated forms of LacNAc in competitive inhibition and direct solid-phase binding assays. Interestingly, from immunological point of view, PP14 also binds differentially to CD45 isoforms known to differ in their degree of sialylation. PP14 preferentially inhibits CD45RA+, as compared with CD45RO+ T cells, and preferentially co-capped this variant CD45 on the T-cell surface. Finally, we demonstrate that PP14 promotes CD45 dimerization and clustering, a phenomenon that may regulate CD45 activity.