CD69 controls the uptake of L-tryptophan through LAT1-CD98 and AhR-dependent secretion of IL-22 in psoriasis.

The activation marker CD69 is expressed by skin γδ T cells. Here we found that CD69 controlled the aryl hydrocarbon receptor (AhR)-dependent secretion of interleukin 22 (IL-22) by γδ T cells, which contributed to the development of psoriasis induced by IL-23. CD69 associated with the aromatic-amino-acid-transporter complex LAT1-CD98 and regulated its surface expression and uptake of L-tryptophan (L-Trp) and the intracellular quantity of L-Trp-derived activators of AhR. In vivo administration of L-Trp, an inhibitor of AhR or IL-22 abrogated the differences between CD69-deficient mice and wild-type mice in skin inflammation. We also observed LAT1-mediated regulation of AhR activation and IL-22 secretion in circulating Vγ9(+) γδ T cells of psoriatic patients. Thus, CD69 serves as a key mediator of the pathogenesis of psoriasis by controlling LAT1-CD98-mediated metabolic cues.

IL-23 signaling regulation of pro-inflammatory T-cell migration uncovered by phosphoproteomics.

Interleukin 23 (IL-23) triggers pathogenic features in pro-inflammatory, IL-17-secreting T cells (Th17 and Tγδ17) that play a key role in the development of inflammatory diseases. However, the IL-23 signaling cascade remains largely undefined. Here, we used quantitative phosphoproteomics to characterize IL-23 signaling in primary murine Th17 cells. We quantified 6,888 phosphorylation sites in Th17 cells and found 168 phosphorylations regulated upon IL-23 stimulation. IL-23 increased the phosphorylation of the myosin regulatory light chain (RLC), an actomyosin contractibility marker, in Th17 and Tγδ17 cells. IL-23-induced RLC phosphorylation required Janus kinase 2 (JAK2) and Rho-associated protein kinase (ROCK) catalytic activity, and further study of the IL-23/ROCK connection revealed an unexpected role of IL-23 in the migration of Tγδ17 and Th17 cells through ROCK activation. In addition, pharmacological inhibition of ROCK reduced Tγδ17 recruitment to inflamed skin upon challenge with inflammatory agent Imiquimod. This work (i) provides new insights into phosphorylation networks that control Th17 cells, (ii) widely expands the current knowledge on IL-23 signaling, and (iii) contributes to the increasing list of immune cells subsets characterized by global phosphoproteomic approaches.

Dissecting the complexity of γδ T-cell subsets in skin homeostasis, inflammation, and malignancy.

γδ T cells are much less common than αß T cells, accounting for 0.5% to 5% of all T lymphocytes in the peripheral blood and lymphoid tissues in mice and humans. However, they are the most abundant T-lymphocyte subset in some epithelial barriers such as mouse skin. γδ T cells are considered innate lymphocytes because of their non-MHC restricted antigen recognition, as well as because of their rapid response to cytokines, invading pathogens, and malignant cells. Exacerbated expansion and activation of γδ T cells in the skin is a common feature of acute and chronic skin inflammation such as psoriasis and contact or atopic dermatitis. Different γδ T-cell subsets showing differential developmental and functional features are found in mouse and human skin. This review discusses the state of the art of research and future perspectives about the role of the different subsets of γδ T-cells detected in the skin in steady-state, psoriasis, dermatitis, infection, and malignant skin diseases. Also, we highlight the differences between human and mouse γδ T cells in skin homeostasis and inflammation, as understanding the differential role of each subtype of skin γδ T cells will improve the discovery of new therapies.

Targeting L-type amino acid transporter 1 in innate and adaptive T cells efficiently controls skin inflammation.

BACKGROUND: Psoriasis is a frequent inflammatory skin disease that is mainly mediated by IL-23, IL-1ß, and IL-17 cytokines. Although psoriasis is a hyperproliferative skin disorder, the possible role of amino acid transporters has remained unexplored. OBJECTIVE: We sought to investigate the role of the essential amino acid transporter L-type amino acid transporter (LAT) 1 (SLC7A5) in psoriasis. METHODS: LAT1 floxed mice were crossed to Cre-expressing mouse strains under the control of keratin 5, CD4, and retinoic acid receptor-related orphan receptor γ. We produced models of skin inflammation induced by imiquimod (IMQ) and IL-23 and tested the effect of inhibiting LAT1 (JPH203) and mammalian target of rapamycin (mTOR [rapamycin]). RESULTS: LAT1 expression is increased in keratinocytes and skin-infiltrating lymphocytes of psoriatic lesions in human subjects and mice. LAT1 deletion in keratinocytes does not dampen the inflammatory response or their proliferation, which could be maintained by increased expression of the alternative amino acid transporters LAT2 and LAT3. Specific deletion of LAT1 in γδ and CD4 T cells controls the inflammatory response induced by IMQ. LAT1 deletion or inhibition blocks expansion of IL-17-secreting γ4+δ4+ and CD4 T cells and dampens the release of IL-1ß, IL-17, and IL-22 in the IMQ-induced model. Moreover, inhibition of LAT1 blocks expansion of human γδ T cells and IL-17 secretion by human CD4 T cells. IL-23 and IL-1ß stimulation upregulates LAT1 expression and induces mTOR activation in IL-17+ γδ and TH17 cells. Deletion or inhibition of LAT1 efficiently controls IL-23- and IL-1ß-induced phosphatidylinositol 3-kinase/AKT/mTOR activation independent of T-cell receptor signaling. CONCLUSION: Targeting LAT1-mediated amino acid uptake is a potentially useful immunosuppressive strategy to control skin inflammation mediated by the IL-23/IL-1ß/IL-17 axis.

HDAC6 controls innate immune and autophagy responses to TLR-mediated signalling by the intracellular bacteria Listeria monocytogenes.

Recent evidence on HDAC6 function underlines its role as a key protein in the innate immune response to viral infection. However, whether HDAC6 regulates innate immunity during bacterial infection remains unexplored. To assess the role of HDAC6 in the regulation of defence mechanisms against intracellular bacteria, we used the Listeria monocytogenes (Lm) infection model. Our data show that Hdac6-/- bone marrow-derived dendritic cells (BMDCs) have a higher bacterial load than Hdac6+/+ cells, correlating with weaker induction of IFN-related genes, pro-inflammatory cytokines and nitrite production after bacterial infection. Hdac6-/- BMDCs have a weakened phosphorylation of MAPK signalling in response to Lm infection, suggesting altered Toll-like receptor signalling (TLR). Compared with Hdac6+/+ counterparts, Hdac6-/- GM-CSF-derived and FLT3L-derived dendritic cells show weaker pro-inflammatory cytokine secretion in response to various TLR agonists. Moreover, HDAC6 associates with the TLR-adaptor molecule Myeloid differentiation primary response gene 88 (MyD88), and the absence of HDAC6 seems to diminish the NF-κB induction after TLR stimuli. Hdac6-/- mice display low serum levels of inflammatory cytokine IL-6 and correspondingly an increased survival to a systemic infection with Lm. The impaired bacterial clearance in the absence of HDAC6 appears to be caused by a defect in autophagy. Hence, Hdac6-/- BMDCs accumulate higher levels of the autophagy marker p62 and show defective phagosome-lysosome fusion. These data underline the important function of HDAC6 in dendritic cells not only in bacterial autophagy, but also in the proper activation of TLR signalling. These results thus demonstrate an important regulatory role for HDAC6 in the innate immune response to intracellular bacterial infection.

CD69: from activation marker to metabolic gatekeeper.

CD69 is a membrane-bound, type II C-lectin receptor. It is a classical early marker of lymphocyte activation due to its rapid appearance on the surface of the plasma membrane after stimulation. CD69 is expressed by several subsets of tissue resident immune cells, including resident memory T (TRM) cells and gamma delta (γδ) T cells, and is therefore considered a marker of tissue retention. Recent evidence has revealed that CD69 regulates some specific functions of selected T-cell subsets, determining the migration-retention ratio as well as the acquisition of effector or regulatory phenotypes. Specifically, CD69 regulates the differentiation of regulatory T (Treg) cells as well as the secretion of IFN-γ, IL-17, and IL-22. The identification of putative CD69 ligands, such as Galectin-1 (Gal-1), suggests that CD69-induced signaling can be regulated not only during cognate contacts between T cells and antigen-presenting cells in lymphoid organs, but also in the periphery, where cytokines and other metabolites control the final outcome of the immune response. Here, we will discuss new aspects of the molecular signaling mediated by CD69 and its involvement in the metabolic reprogramming regulating TH-effector lineages.

Growth hormone releasing peptide-6 (GHRP-6) prevents doxorubicin-induced myocardial and extra-myocardial damages by activating prosurvival mechanisms.

Introduction: Dilated cardiomyopathy (DCM) is a fatal myocardial condition with ventricular structural changes and functional deficits, leading to systolic dysfunction and heart failure (HF). DCM is a frequent complication in oncologic patients receiving Doxorubicin (Dox). Dox is a highly cardiotoxic drug, whereas its damaging spectrum affects most of the organs by multiple pathogenic cascades. Experimentally reproduced DCM/HF through Dox administrations has shed light on the pathogenic drivers of cardiotoxicity. Growth hormone (GH) releasing peptide 6 (GHRP-6) is a GH secretagogue with expanding and promising cardioprotective pharmacological properties. Here we examined whether GHRP-6 administration concomitant to Dox prevented the onset of DCM/HF and multiple organs damages in otherwise healthy rats. Methods: Myocardial changes were sequentially evaluated by transthoracic echocardiography. Autopsy was conducted at the end of the administration period when ventricular dilation was established. Semiquantitative histopathologic study included heart and other internal organs samples. Myocardial tissue fragments were also addressed for electron microscopy study, and characterization of the transcriptional expression ratio between Bcl-2 and Bax. Serum samples were destined for REDOX system balance assessment. Results and discussion: GHRP-6 administration in parallel to Dox prevented myocardial fibers consumption and ventricular dilation, accounting for an effective preservation of the LV systolic function. GHRP-6 also attenuated extracardiac toxicity preserving epithelial organs integrity, inhibiting interstitial fibrosis, and ultimately reducing morbidity and mortality. Mechanistically, GHRP-6 proved to sustain cellular antioxidant defense, upregulate prosurvival gene Bcl-2, and preserve cardiomyocyte mitochondrial integrity. These evidences contribute to pave potential avenues for the clinical use of GHRP-6 in Dox-treated subjects.

Psoriasis in humans is associated with down-regulation of galectins in dendritic cells.

We have investigated the expression and role of galectin-1 and other galectins in psoriasis and in the Th1/Th17 effector and dendritic cell responses associated with this chronic inflammatory skin condition. To determine differences between psoriasis patients and healthy donors, expression of galectins was analysed by RT-PCR in skin samples and on epidermal and peripheral blood dendritic cells by immunofluorescence and flow cytometry. In the skin of healthy donors, galectin-1, -3 and -9 were expressed in a high proportion of Langerhans cells. Also, galectins were differentially expressed in peripheral blood dendritic cell subsets; galectin-1 and galectin-9 were highly expressed in peripheral myeloid dendritic cells compared with plasmacytoid dendritic cells. We found that non-lesional as well as lesional skin samples from psoriasis patients had low levels of galectin-1 at the mRNA and protein levels, in parallel with low levels of IL-10 mRNA compared with skin from healthy patients. However, only lesional skin samples expressed high levels of Th1/Th17 cytokines. The analysis of galectin-1 expression showed that this protein was down-regulated in Langerhans cells and dermal dendritic cells as well as in peripheral blood CD11c(+) DCs from psoriasis patients. Expression of galectin-1 correlated with IL-17 and IL-10 expression and with the psoriasis area and index activity. Addition of galectin-1 to co-cultures of human monocyte-derived dendritic cells with autologous T lymphocytes from psoriasis patients attenuated the Th1 response. Conversely, blockade of galectin binding increased IFNγ production and inhibited IL-10 secretion in co-cultures of monocyte-derived dendritic cells with CD4(+) T cells. Our results suggest a model in which galectin-1 down-regulation contributes to the exacerbation of the Th1/Th17 effector response in psoriasis patients.

Unraveling CD69 signaling pathways, ligands and laterally associated molecules.

CD69 is an early leukocyte activation marker involved in the regulation of the immune response. Initial in vitro studies evaluated its function using monoclonal antibodies until knock-out mice were developed. Subsequently, four ligands for CD69 have been identified, namely galectin-1, S100A8/S100A9 complex, myosin light chains 9 and 12, and oxidized low-density lipoproteins. In addition, several molecules are laterally associated with and regulated by CD69, including calreticulin and two transmembrane receptors, sphingosine-1-phosphate receptor (S1P1) and the heterodimeric amino acid transporter complex SLC7A5-SLC3A2 (LAT1-CD98). Recently, CD69 engagement has been shown to induce the expression of the immunoregulatory receptor programmed cell death-1 (PD-1) in T cells. The molecular signaling induced by CD69 has been explored in different scenarios and cell types. This review provides a perspective on the molecular pathways, ligands and cellular functions known to be regulated by CD69.

Erythroid SLC7A5/SLC3A2 amino acid carrier controls red blood cell size and maturation.

Inhibition of the heterodimeric amino acid carrier SLC7A5/SLC3A2 (LAT1/CD98) has been widely studied in tumor biology but its role in physiological conditions remains largely unknown. Here we show that the SLC7A5/SLC3A2 heterodimer is constitutively present at different stages of erythroid differentiation but absent in mature erythrocytes. Administration of erythropoietin (EPO) further induces SLC7A5/SLC3A2 expression in circulating reticulocytes, as it also occurs in anemic conditions. Although Slc7a5 gene inactivation in the erythrocyte lineage does not compromise the total number of circulating red blood cells (RBCs), their size and hemoglobin content are significantly reduced accompanied by a diminished erythroblast mTORC1 activity. Furthermore circulating Slc7a5-deficient reticulocytes are characterized by lower transferrin receptor (CD71) expression as well as mitochondrial activity, suggesting a premature transition to mature RBCs. These data reveal that SLC7A5/SLC3A2 ensures adequate maturation of reticulocytes as well as the proper size and hemoglobin content of circulating RBCs.

Role of AHR Ligands in Skin Homeostasis and Cutaneous Inflammation.

Aryl hydrocarbon receptor (AHR) is an important regulator of skin barrier function. It also controls immune-mediated skin responses. The AHR modulates various physiological functions by acting as a sensor that mediates environment-cell interactions, particularly during immune and inflammatory responses. Diverse experimental systems have been used to assess the AHR's role in skin inflammation, including in vitro assays of keratinocyte stimulation and murine models of psoriasis and atopic dermatitis. Similar approaches have addressed the role of AHR ligands, e.g., TCDD, FICZ, and microbiota-derived metabolites, in skin homeostasis and pathology. Tapinarof is a novel AHR-modulating agent that inhibits skin inflammation and enhances skin barrier function. The topical application of tapinarof is being evaluated in clinical trials to treat psoriasis and atopic dermatitis. In the present review, we summarize the effects of natural and synthetic AHR ligands in keratinocytes and inflammatory cells, and their relevance in normal skin homeostasis and cutaneous inflammatory diseases.

Galectin-1 Expression in CD8+ T Lymphocytes Controls Inflammation in Contact Hypersensitivity.

Allergic contact dermatitis, also known as contact hypersensitivity, is a frequent T-cell‒mediated inflammatory skin disease characterized by red, itchy, swollen, and cracked skin. It is caused by the direct contact with an allergen and/or irritant hapten. Galectin-1 (Gal-1) is a ß-galactoside‒binding lectin, which is highly expressed in several types of immune cells. The role of endogenous Gal-1 in contact hypersensitivity is not known. We found that Gal-1‒deficient mice display more sustained and prolonged skin inflammation than wild-type mice after oxazolone treatment. Gal-1‒deficient mice have increased CD8+ T cells and neutrophilic infiltration in the skin. After the sensitization phase, Gal-1‒depleted mice showed an increased frequency of central memory CD8+ T cells and IFN-γ secretion by CD8+ T cells. The absence of Gal-1 does not affect the migration of transferred CD4+ and CD8+ T cells from the blood to the lymph nodes or to the skin. The depletion of CD4+ T lymphocytes as well as adoptive transfer experiments demonstrated that endogenous expression of Gal-1 on CD8+ T lymphocytes exerts a major role in the control of contact hypersensitivity model. These data underscore the protective role of endogenous Gal-1 in CD8+ but not CD4+ T cells in the development of allergic contact dermatitis.

Growth arrest and DNA damage-inducible proteins (GADD45) in psoriasis.

The interplay between T cells, dendritic cells and keratinocytes is crucial for the development and maintenance of inflammation in psoriasis. GADD45 proteins mediate DNA repair in different cells including keratinocytes. In the immune system, GADD45a and GADD45b regulate the function and activation of both T lymphocytes and dendritic cells and GADD45a links DNA repair and epigenetic regulation through its demethylase activity. Here, we analyzed the expression of GADD45a and GADD45b in the skin, dendritic cells and circulating T cells in a cohort of psoriasis patients and their regulation by inflammatory signals. Thirty patients (17 male/13 female) with plaque psoriasis and 15 controls subjects (7 male/8 female), were enrolled. Psoriasis patients exhibited a lower expression of GADD45a at the epidermis but a higher expression in dermal infiltrating T cells in lesional skin. The expression of GADD45a and GADD45b was also higher in peripheral T cells from psoriasis patients, although no differences were observed in p38 activation. The expression and methylation state of the GADD45a target UCHL1 were evaluated, revealing a hypermethylation of its promoter in lesional skin compared to controls. Furthermore, reduced levels of GADD45a correlated with a lower expression UCHL1 in lesional skin. We propose that the demethylase function of GADD45a may account for its pleiotropic effects, and the complex and heterogeneous pattern of expression observed in psoriatic disease.

Metabolic Pathways That Control Skin Homeostasis and Inflammation.

Keratinocytes and skin immune cells are actively metabolizing nutrients present in their microenvironment. This is particularly important in common chronic inflammatory skin diseases such as psoriasis and atopic dermatitis, characterized by hyperproliferation of keratinocytes and expansion of inflammatory cells, thus suggesting increased cell nutritional requirements. Proliferating inflammatory cells and keratinocytes express high levels of glucose transporter (GLUT)1, l-type amino acid transporter (LAT)1, and cationic amino acid transporters (CATs). Main metabolic regulators such as hypoxia-inducible factor (HIF)-1α, MYC, and mechanistic target of rapamycin (mTOR) control immune cell activation, proliferation, and cytokine release. Here, we provide an updated perspective regarding the potential role of nutrient transporters and metabolic pathways that could be common to immune cells and keratinocytes, to control psoriasis and atopic dermatitis.

CD13 as a new tumor target for antibody-drug conjugates: validation with the conjugate MI130110.

BACKGROUND: In the search for novel antibody-drug conjugates (ADCs) with therapeutic potential, it is imperative to identify novel targets to direct the antibody moiety. CD13 seems an attractive ADC target as it shows a differential pattern of expression in a variety of tumors and cell lines and it is internalized upon engagement with a suitable monoclonal antibody. PM050489 is a marine cytotoxic compound tightly binding tubulin and impairing microtubule dynamics which is currently undergoing clinical trials for solid tumors. METHODS: Anti-CD13 monoclonal antibody (mAb) TEA1/8 has been used to prepare a novel ADC, MI130110, by conjugation to the marine compound PM050489. In vitro and in vivo experiments have been carried out to demonstrate the activity and specificity of MI130110. RESULTS: CD13 is readily internalized upon TEA1/8 mAb binding, and the conjugation with PM050489 did not have any effect on the binding or the internalization of the antibody. MI130110 showed remarkable activity and selectivity in vitro on CD13-expressing tumor cells causing the same effects than those described for PM050489, including cell cycle arrest at G2, mitosis with disarrayed and often multipolar spindles consistent with an arrest at metaphase, and induction of cell death. In contrast, none of these toxic effects were observed in CD13-null cell lines incubated with MI130110. Furthermore, in vivo studies showed that MI130110 exhibited excellent antitumor activity in a CD13-positive fibrosarcoma xenograft murine model, with total remissions in a significant number of the treated animals. Mitotic catastrophes, typical of the payload mechanism of action, were also observed in the tumor cells isolated from mice treated with MI130110. In contrast, MI130110 failed to show any activity in a xenograft mouse model of myeloma cells not expressing CD13, thereby corroborating the selectivity of the ADC to its target and its stability in circulation. CONCLUSION: Our results show that MI130110 ADC combines the antitumor potential of the PM050489 payload with the selectivity of the TEA1/8 monoclonal anti-CD13 antibody and confirm the correct intracellular processing of the ADC. These results demonstrate the suitability of CD13 as a novel ADC target and the effectiveness of MI130110 as a promising antitumor therapeutic agent.

Expression of miR-135b in Psoriatic Skin and Its Association with Disease Improvement.

miRNAs have been associated with psoriasis since just over a decade. However, we are far from a complete understanding of their role during the development of this disease. Our objective was to characterize the cutaneous expression of miRNAs not previously described in psoriasis, the changes induced following the treatment with biologicals and their association with disease improvement. Next generation sequencing was performed from five skin samples from psoriasis patients (lesional and non-lesional skin) and five controls, and from this cohort, 12 microRNAs were selected to be analyzed in skin samples from 44 patients with plaque psoriasis. In 15 patients, an additional sample was obtained after three months of biological treatment. MiR-9-5p, miR-133a-3p and miR-375 were downregulated in the lesional skin of psoriasis patients. After treatment, expression of miR-133a-3p, miR-375, miR-378a and miR-135b in residual lesions returned towards the levels observed in non-lesional skin. The decrease in miR-135b levels after treatment with biologics was associated with both the improvement of patients evaluated through Psoriasis Area and Severity Index score and the decrease in local inflammatory response. Moreover, basal expression of miR-135b along with age was associated with the improvement of psoriasis, suggesting its possible usefulness as a prognostic biomarker.

Thrombospondin-1/CD47 Interaction Regulates Th17 and Treg Differentiation in Psoriasis.

Accumulating evidence on the role of Thrombospondin-1 (TSP-1) in the immune response has emerged during the last years. In spite of the importance of TSP-1 not only as anti-angiogenic factor but also as an immunomodulatory molecule, studies on the role of TSP-1 in psoriasis have been neglected. TSP-1 and CD47 expression were analyzed in skin samples from psoriasis patients and control subjects using RT-PCR and immunofluorescence. Expression of these molecules was also evaluated in peripheral blood CD4+ T cells, moDCs, and circulating primary DCs. The functional role of TSP-1/CD47 signaling axis in psoriasis was assessed in Th17 and Treg differentiation assays. Additionally, small interfering RNA assays specific to TSP-1 were performed in CD4+ T cells and monocyte derived DC to specifically evaluate the function of this protein. Lesional skin of psoriasis patients expressed lower TSP-1 and CD47 mRNA levels compared to non-lesional skin or skin from controls. Immunofluorescence staining revealed decreased expression of CD47 in CD45+ dermal cells from psoriasis samples compared to control subjects. Peripheral CD4+ T cells and circulating primary DCs from psoriasis also expressed lower levels of CD47 compared to controls. Although no significant differences were detected in TSP-1 expression in CD4+ T cells and moDCs between patients and controls, TSP-1 expression in psoriasis patients inversely correlated with disease activity evaluated by the Psoriasis Area and Index Activity. Furthermore, exogenous TSP-1 inhibited Th17 differentiation and stimulated the differentiation of CD4+ T cells toward Treg cells. Furthermore, RNA interference specific for TSP-1 confirmed the role of this molecule as a negative regulator of T cell activation. Because of the impact of TSP-1/CD47 signaling axis in Th17 and Treg differentiation, a dysregulated expression of these molecules in the immune cells from psoriasis patients may favor the exacerbated inflammatory response in this disease.

Growth-hormone-releasing peptide 6 (GHRP6) prevents oxidant cytotoxicity and reduces myocardial necrosis in a model of acute myocardial infarction.

Therapies aimed at enhancing cardiomyocyte survival following myocardial injury are urgently required. As GHRP6 [GH (growth hormone)-releasing peptide 6] has been shown to stimulate GH secretion and has beneficial cardiovascular effects, the aim of the present study was to determine whether GHRP6 administration reduces myocardial infarct size following acute coronary occlusion in vivo. Female Cuban Creole pigs were anaesthetized, monitored and instrumented to ensure a complete sudden left circumflex artery occlusion for 1 h, followed by a 72 h reperfusion/survival period. Animals were screened clinically before surgery and assigned randomly to receive either GHRP6 (400 microg/kg of body weight) or normal saline. Hearts were processed, and the area at risk and the infarct size were determined. CK-MB (creatine kinase MB) and CRP (C-reactive protein) levels and pathological Q-wave-affected leads were analysed and compared. Evaluation of the myocardial effect of GHRP6 also included quantitative histopathology, local IGF-I (insulin-growth factor-I) expression and oxidative stress markers. GHRP6 treatment did not have any influence on mortality during surgery associated with rhythm and conductance disturbances during ischaemia. Infarct mass and thickness were reduced by 78% and 50% respectively, by GHRP6 compared with saline (P<0.01). More than 50% of the GHRP6-treated pigs did not exhibit pathogological Q waves in any of the ECG leads. Quantitative histopathology and CK-MB and CRP serum levels confirmed the reduction in GHRP6-mediated necrosis (all P<0.05). Levels of oxidative stress markers suggested that GHRP6 prevented myocardial injury via a decrease in reactive oxygen species and by the preservation of antioxidant defence systems (all P<0.05). Myocardial IGF-I transcription was not amplified by GHRP6 treatment compared with the increase induced by the ischaemic episode in relation to expression in intact hearts (P<0.01). In conclusion, GHRP6 exhibits antioxidant effects which may partially contribute to reduce myocardial ischaemic damage.

Tetraspanin CD9 Limits Mucosal Healing in Experimental Colitis.

Tetraspanins are a family of proteins with four transmembrane domains that associate between themselves and cluster with other partner proteins, conforming a distinct class of membrane domains, the tetraspanin-enriched microdomains (TEMs). These TEMs constitute macromolecular signaling platforms that regulate key processes in several cellular settings controlling signaling thresholds and avidity of receptors. In this study, we investigated the role of CD9, a tetraspanin that regulates major biological processes such as cell migration and immunological responses, in two mouse models of colitis that have been used to study the pathogenesis of inflammatory bowel disease (IBD). Previous in vitro studies revealed an important role in the interaction of leukocytes with inflamed endothelium, but in vivo evidence of the involvement of CD9 in inflammatory diseases is scarce. Here, we studied the role of CD9 in the pathogenesis of colitis in vivo. Colitis was induced by administration of dextran sodium sulfate (DSS), a chemical colitogen that causes epithelial disruption and intestinal inflammation. CD9-/- mice showed less severe colitis than wild-type counterparts upon exposure to DSS (2% solution) and enhanced survival in response to a lethal DSS dose (4%). Decreased neutrophil and macrophage cell infiltration was observed in colonic tissue from CD9-/- animals, in accordance with their lower serum levels of TNF-α, IL-6, and other proinflammatory cytokines in the colon. The specific role of CD9 in IBD was further dissected by transfer of CD4+ CD45RBhi naive T cells into the Rag1-/- mouse colitis model. However, no significant differences were observed in these settings between both groups, ruling out a role for CD9 in IBD in the lymphoid compartment. Experiments with bone marrow chimeras revealed that CD9 in the non-hematopoietic compartment is involved in colon injury and limits the proliferation of epithelial cells. Our data indicate that CD9 in non-hematopoietic cells plays an important role in colitis by limiting epithelial cell proliferation. Future strategies to repress CD9 expression may be of therapeutic benefit in the treatment of IBD.