Distinctive phenotype for HLA-E- versus HLA-A2-restricted memory CD8 αßT cells in the course of HCMV infection discloses features shared with NKG2C+CD57+NK and δ2-γδT cell subsets.

The human cytomegalovirus (HCMV) triggers both innate and adaptive immune responses, including protective CD8+ αßT cells (CD8T) that contributes to the control of the infection. In addition to CD8T restricted by classical HLA class Ia molecules, HCMV also triggers CD8T recognizing peptides from the HCMV UL40 leader peptide and restricted by HLA-E molecules (HLA-EUL40 CD8T). This study investigated the frequency, phenotype and functions of HLA-EUL40 CD8T in comparison to the immunodominant HLA-A2pp65 CD8T upon acute (primary or secondary infection) or chronic infection in kidney transplant recipients (KTR) and in seropositive (HCMV+) healthy volunteer (HV) hosts. The frequency of hosts with detected HLA-EUL40 CD8T was similar after a primary infection (24%) and during viral latency in HCMV+ HV (26%) and equal to the frequency of HLA-A2pp65 CD8T cells in both conditions (29%). Both CD8T subsets vary from 0.1% to >30% of total circulating CD8T according to the host. Both HLA-EUL40 and HLA-A2pp65 CD8T display a phenotype specific of CD8+ TEMRA (CD45RA+/CCR7-) but HLA-EUL40 CD8T express distinctive level for CD3, CD8 and CD45RA. Tim3, Lag-3, 4-1BB, and to a lesser extend 2B4 are hallmarks for T cell priming post-primary infection while KLRG1 and Tigit are markers for restimulated and long lived HCMV-specific CD8T responses. These cell markers are equally expressed on HLA-EUL40 and HLA-A2pp65 CD8T. In contrast, CD56 and PD-1 are cell markers discriminating memory HLA-E- from HLA-A2-restricted CD8T. Long lived HLA-EUL40 display higher proliferation rate compared to HLA-A2pp65 CD8T consistent with elevated CD57 expression. Finally, a comparative immunoprofiling indicated that HLA-EUL40 CD8T, divergent from HLA-A2pp65 CD8T, share the expression of CD56, CD57, NKG2C, CD158 and the lack of PD-1 with NKG2C+CD57+ NK and δ2-γδT cells induced in response to HCMV and thus defines a common immunopattern for these subsets.

Targeting MHC Regulation Using Polycyclic Polyprenylated Acylphloroglucinols Isolated from Garcinia bancana.

Modulation of major histocompatibility complex (MHC) expression using drugs has been proposed to control immunity. Phytochemical investigations on Garcinia species have allowed the isolation of bioactive compounds such as polycyclic polyprenylated acylphloroglucinols (PPAPs). PPAPs such as guttiferone J (1), display anti-inflammatory and immunoregulatory activities while garcinol (4) is a histone acetyltransferases (HAT) p300 inhibitor. This study reports on the isolation, identification and biological characterization of two other PPAPs, i.e., xanthochymol (2) and guttiferone F (3) from Garcinia bancana, sharing structural analogy with guttiferone J (1) and garcinol (4). We show that PPAPs 1-4 efficiently downregulated the expression of several MHC molecules (HLA-class I, -class II, MICA/B and HLA-E) at the surface of human primary endothelial cells upon inflammation. Mechanistically, PPAPs 1-4 reduce MHC proteins by decreasing the expression and phosphorylation of the transcription factor STAT1 involved in MHC upregulation mediated by IFN-γ. Loss of STAT1 activity results from inhibition of HAT CBP/p300 activity reflected by a hypoacetylation state. The binding interactions to p300 were confirmed through molecular docking. Loss of STAT1 impairs the expression of CIITA and GATA2 but also TAP1 and Tapasin required for peptide loading and transport of MHC. Overall, we identified new PPAPs issued from Garcinia bancana with potential immunoregulatory properties.

Human CD8+ Tregs expressing a MHC-specific CAR display enhanced suppression of human skin rejection and GVHD in NSG mice.

Polyclonal CD8+CD45RClow/- Tregs are potent regulatory cells able to control solid organ transplantation rejection and even induce tolerance. However, donor major histocompatibility complex (MHC)-specific Tregs are more potent than polyclonal Tregs in suppressing T-cell responses and preventing acute as well as chronic rejection in rodent models. The difficulty of identifying disease-relevant antigens able to stimulate Tregs has reduced the possibility of obtaining antigen-specific Tregs. To bypass this requirement and gain the advantage of antigen specificity, and thus improve the therapeutic potential of CD8+ Tregs, we stably introduced a chimeric antigen receptor (CAR) derived from a HLA-A*02 antigen-specific antibody (A2-CAR) in human CD8+ Tregs and developed a clinically compatible protocol of transduction and expansion. We demonstrated that A2-CAR CD8+ Tregs were not phenotypically altered by the process, were specifically activated, and did not exhibit cytotoxic activity toward HLA-A*02+ kidney endothelial cells (ECs). We showed that A2-CAR CD8+ Tregs were more potent suppressors of immune responses induced by HLA-A*02 mismatch than control-CAR CD8+ Tregs, both in vitro and in vivo, in models of human skin graft rejection and graft-versus-host disease (GVHD) in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. We showed that integrity of human skin graft was preserved with A2-CAR CD8+ Tregs at least 100 days in vivo after administration, and that interaction between the A2-CAR CD8+ Tregs and HLA-A*02+ kidney ECs resulted in a fine-tuned and protolerogenic activation of the ECs without cytotoxicity. Together, our results demonstrated the relevance of the CAR engineering approach to develop antigen-specific CAR-CD8+ Tregs for clinical trials in transplantation, and potentially in other diseases.

IL-7 receptor influences anti-TNF responsiveness and T cell gut homing in inflammatory bowel disease.

It remains unknown what causes inflammatory bowel disease (IBD), including signaling networks perpetuating chronic gastrointestinal inflammation in Crohn's disease (CD) and ulcerative colitis (UC), in humans. According to an analysis of up to 500 patients with IBD and 100 controls, we report that key transcripts of the IL-7 receptor (IL-7R) pathway are accumulated in inflamed colon tissues of severe CD and UC patients not responding to either immunosuppressive/corticosteroid, anti-TNF, or anti-α4ß7 therapies. High expression of both IL7R and IL-7R signaling signature in the colon before treatment is strongly associated with nonresponsiveness to anti-TNF therapy. While in mice IL-7 is known to play a role in systemic inflammation, we found that in humans IL-7 also controlled α4ß7 integrin expression and imprinted gut-homing specificity on T cells. IL-7R blockade reduced human T cell homing to the gut and colonic inflammation in vivo in humanized mouse models, and altered effector T cells in colon explants from UC patients grown ex vivo. Our findings show that failure of current treatments for CD and UC is strongly associated with an overexpressed IL-7R signaling pathway and point to IL-7R as a relevant therapeutic target and potential biomarker to fill an unmet need in clinical IBD detection and treatment.

An early increase in endothelial protein C receptor is associated with excess mortality in pneumococcal pneumonia with septic shock in the ICU.

BACKGROUND: This study investigated changes in plasma level of soluble endothelial protein C receptor (sEPCR) in association with outcome in patients with septic shock. We explored sEPCR for early sepsis prognosis assessment and constructed a scoring system based on clinical and biological data, in order to discriminate between surviving at hospital discharge and non-surviving patients. METHODS: Clinical data and samples were extracted from the prospective "STREPTOGENE" cohort. We enrolled 278 patients, from 50 intensive care units (ICUs), with septic shock caused by pneumococcal pneumonia. Patients were divided into survivors (n = 194) and non-survivors (n = 84) based on in-hospital mortality. Soluble EPCR plasma levels were quantified at day 1 (D1) and day 2 (D2) by ELISA. The EPCR gene A3 haplotype was determined. Patients were followed up until hospital discharge. Univariate and multivariate analyses were performed. A scoring system was constructed using least absolute shrinkage and selection operator (lasso) logistic regression for selecting predictive variables. RESULTS: In-hospital mortality was 30.2% (n = 84). Plasma sEPCR level was significantly higher at D1 and D2 in non-surviving patients compared to patients surviving to hospital discharge (p = 0.0447 and 0.0047, respectively). Early increase in sEPCR at D2 was found in non-survivors while a decrease was observed in the survival group (p = 0.0268). EPCR A3 polymorphism was not associated with mortality. Baseline sEPCR level and its variation from D1 to D2 were independent predictors of in-hospital mortality. The scoring system including sEPCR predicted mortality with an AUC of 0.75. CONCLUSIONS: Our findings confirm that high plasma sEPCR and its increase at D2 are associated with poor outcome in sepsis and thus we propose sEPCR as a key player in the pathogenesis of sepsis and as a potential biomarker of sepsis outcome.

HCMV triggers frequent and persistent UL40-specific unconventional HLA-E-restricted CD8 T-cell responses with potential autologous and allogeneic peptide recognition.

Immune response against human cytomegalovirus (HCMV) includes a set of persistent cytotoxic NK and CD8 T cells devoted to eliminate infected cells and to prevent reactivation. CD8 T cells against HCMV antigens (pp65, IE1) presented by HLA class-I molecules are well characterized and they associate with efficient virus control. HLA-E-restricted CD8 T cells targeting HCMV UL40 signal peptides (HLA-EUL40) have recently emerged as a non-conventional T-cell response also observed in some hosts. The occurrence, specificity and features of HLA-EUL40 CD8 T-cell responses remain mostly unknown. Here, we detected and quantified these responses in blood samples from healthy blood donors (n = 25) and kidney transplant recipients (n = 121) and we investigated the biological determinants involved in their occurrence. Longitudinal and phenotype ex vivo analyses were performed in comparison to HLA-A*02/pp65-specific CD8 T cells. Using a set of 11 HLA-E/UL40 peptide tetramers we demonstrated the presence of HLA-EUL40 CD8 αßT cells in up to 32% of seropositive HCMV+ hosts that may represent up to 38% of total circulating CD8 T-cells at a time point suggesting a strong expansion post-infection. Host's HLA-A*02 allele, HLA-E *01:01/*01:03 genotype and sequence of the UL40 peptide from the infecting strain are major factors affecting the incidence of HLA-EUL40 CD8 T cells. These cells are effector memory CD8 (CD45RAhighROlow, CCR7-, CD27-, CD28-) characterized by a low level of PD-1 expression. HLA-EUL40 responses appear early post-infection and display a broad, unbiased, Vß repertoire. Although induced in HCMV strain-dependent, UL4015-23-specific manner, HLA-EUL40 CD8 T cells are reactive toward a broader set of nonapeptides varying in 1-3 residues including most HLA-I signal peptides. Thus, HCMV induces strong and life-long lasting HLA-EUL40 CD8 T cells with potential allogeneic or/and autologous reactivity that take place selectively in at least a third of infections according to virus strain and host HLA concordance.

Notch signaling triggered via the ligand DLL4 impedes M2 macrophage differentiation and promotes their apoptosis.

BACKGROUND: Notch signaling controls many cellular processes, including cell fate determination, cell differentiation, proliferation and apoptosis. In mammals, four Notch receptors (Notch 1-4) can interact with five distinct ligands [Jagged1, Jagged2, Delta-like 1 (DLL1), DLL3, and DLL4]. We previously reported that Notch activation is modulated in endothelial cells and monocytes during inflammation and showed that inflammation upregulates DLL4 on endothelial cells. DLL4 promotes differentiation of blood monocytes into proinflammatory M1 macrophages. Here, we further investigated the ability of DLL4 to interfere with the polarization of blood monocytes into immunosuppressive M2 macrophages. METHODS: Human blood monocytes were differentiated in vitro into M0 macrophages and then polarized into M1 or M2 macrophages with LPS/IFNγ and IL-4, respectively. Polarization steps were performed in the presence of immobilized recombinant DLL4. Immune phenotype and apoptosis of macrophage subsets were analyzed and quantified by flow cytometry. Regulatory effects of DLL4 on gene expression, cell signaling and apoptotic pathways were investigated by QPCR and western blots. RESULTS: The phenotype of M2 macrophages was subject to specific alterations in the presence of recombinant DLL4. DLL4 inhibits the upregulation of IL-4 induced M2 markers such as CD11b, CD206, and CD200R. Survival of macrophages upon M2 polarization was also strongly reduced in the presence of DLL4. DLL4 induces a caspase3/7-dependent apoptosis during M2 but not M1 macrophage polarization. The Notch ligand DLL1 has no apoptotic effect. Both DLL4 signaling via Notch1 as well as DLL4-mediated apoptosis are Notch-dependent. Fully differentiated M2 macrophages became resistant to DLL4 action. Mechanistically, DLL4 selectively upregulates gene expression in macrophages upon M2 polarization, thereby affecting the Notch pattern (Notch1, 3, Jag1), activity (HES1), and transcription (IRF5, STAT1). The pro-apoptotic effectors Bax and Bak and the BH3-only proteins Bid and Bim seem to convey DLL4 apoptotic signal. CONCLUSION: Interplay between the DLL4/Notch and IL-4/IL-4R signaling pathways impairs M2 differentiation. Thus, DLL4 may drive a Notch-dependent selection process not only by promoting M1 macrophage differentiation but also by preventing M2 macrophage differentiation through inhibition of M2-specific gene expression and apoptotic cell death.

Alternative Splice Transcripts for MHC Class I-like MICA Encode Novel NKG2D Ligands with Agonist or Antagonist Functions.

MHC class I chain-related proteins A and B (MICA and MICB) and UL16-binding proteins are ligands of the activating NKG2D receptor involved in cancer and immune surveillance of infection. Structurally, MICA/B proteins contain an α3 domain, whereas UL16-binding proteins do not. We identified novel alternative splice transcripts for MICA encoding five novel MICA isoforms: MICA-A, -B1, -B2, -C, and -D. Alternative splicing associates with MICA*015 and *017 and results from a point deletion (G) in the 5' splice donor site of MICA intron 4 leading to exon 3 and exon 4 skipping and/or deletions. These changes delete the α3 domain in all isoforms, and the α2 domain in the majority of isoforms (A, B1, C, and D). Endothelial and hematopoietic cells contained endogenous alternative splice transcripts and isoforms. MICA-B1, -B2, and -D bound NKG2D by surface plasmon resonance and were expressed at the cell surface. Functionally, MICA-B2 contains two extracellular domains (α1 and α2) and is a novel potent agonist ligand for NKG2D. We found that MICA-D is a new truncated form of MICA with weak affinity for NKG2D despite lacking α2 and α3 domains. MICA-D may functionally impair NKG2D activation by competing with full-length MICA or MICA-B2 for NKG2D engagement. Our study established NKG2D binding for recombinant MICA-B1 but found no function for this isoform. New truncated MICA isoforms exhibit a range of functions that may drive unexpected immune mechanisms and provide new tools for immunotherapy.

MICA Mutant A5.1 Influences BK Polyomavirus Reactivation and Associated Nephropathy After Kidney Transplantation.

BACKGROUND: BK polyomavirus (BKPyV) frequently reactivates in kidney transplant recipients during immunosuppressive therapy and triggers BKPyV-associated nephropathy and graft rejection. Determining effective risk factors for BKPyV reactivation is required to achieve efficient prevention. METHODS: This study investigated the role of major histocompatibility complex (MHC) class I-related chain A (MICA) in BKPyV reactivation in a cohort of 144 transplant donor/recipient pairs, including recipients with no reactivation (controllers) and those with mild (virurics) or severe (viremics) BKPyV reactivation after graft receipt. RESULTS: We show that, in the kidney, MICA is predominantly expressed in tubule epithelial cells, the natural targets of BKPyV, questioning a role for MICA in the immune control of BKPyV infection. Focusing on MICA genotype, we found a lower incidence of BKPyV reactivation in recipients of a renal graft from a donor carrying the MICA A5.1 mutant, which encodes a truncated nonconventional MICA. We established that a mismatch for MICA A5.1 between transplant donor and recipient is critical for BKPyV reactivation and BKPyV-associated nephropathy. Functionally, we found that a low prevalence of BKPyV reactivation was associated with elevated anti-MICA sensitization and reduced plasma level of soluble MICA in recipients, 2 potential effector mechanisms. DISCUSSIONS: These findings identify the MHC-related MICA as an immunogenetic factor that may functionally influence anti-BKPyV immune responses and infection outcomes.

Cytomegalovirus-Infected Primary Endothelial Cells Trigger NKG2C+ Natural Killer Cells.

Among innate cells, natural killer (NK) cells play a crucial role in the defense against cytomegalovirus (CMV). In some individuals, CMV infection induces the expansion of NKG2C+ NK cells that persist after control of the infection. We have previously shown that KIR2DL+ NK cells, in contrast to NKG2C+ NK cells, contribute to controlling CMV infection using a CMV-infected monocyte-derived dendritic cell (MDDC) model. However, the nature of CMV-infected cells contributing to the expansion of the NKG2C+ NK cell subset remains unclear. To gain more insight into this question, we investigated the contribution of NKG2C+ NK cell activation by CMV-infected primary human aortic endothelial cells (EC) isolated from kidney transplant donors, which constitutively express the human leukocyte antigen (HLA)-E molecule. Here, we show that, although classic HLA class I expression was drastically downregulated, nonclassic HLA-E expression was maintained in CMV-infected EC. By comparing HLA expression patterns in CMV-infected EC, fibroblasts and MDDC, we demonstrate a cell-dependent modulation of HLA-E expression by CMV infection. NKG2C+ NK cell degranulation was significantly triggered by CMV-infected EC regardless of the nature of the HLA-E allele product. EC, predominantly present in vessels, may constitute a privileged site for CMV infection that drives a 'memory' NKG2C+ NK cell subset.

Notch signaling mediates crosstalk between endothelial cells and macrophages via Dll4 and IL6 in cardiac microvascular inflammation.

Although short-term outcomes have improved with modern era immunosuppression, little progress has been made in long-term graft survival in cardiac transplantation. Antibody-mediated rejection (AMR) is one of the leading causes of graft failure and contributes significantly to poor long-term outcomes. Endothelial cell (EC) injury, intravascular macrophage infiltrate and microvascular inflammation are the histological features of AMR. Nevertheless, mechanisms of AMR remain unclear and treatment is still limited. Here, we investigated the mechanisms underlying vascular and inflammatory cell network involved in AMR at endothelial and macrophage levels, using endomyocardial transplant biopsies and EC/monocyte cocultures. First, we found that AMR associates with changes in Notch signaling at endothelium/monocyte interface including loss of endothelial Notch4 and the acquisition of the Notch ligand Dll4 in both cell types. We showed that endothelial Dll4 induces macrophage polarization into a pro-inflammatory fate (CD40(high)CD64(high)CD200R(low) HLA-DR(low)CD11b(low)) eliciting the production of IL-6. Dll4 and IL-6 are both Notch-dependent and are required for macrophage polarization through selective down and upregulation of M2- and M1-type markers, respectively. Overall, these findings highlight the impact of the graft's endothelium on macrophage recruitment and differentiation upon AMR via Notch signaling. We identified Dll4 and IL-6 as coregulators of vascular inflammation in cardiac transplantation and as potential targets for immunotherapy.

The disintegrin and metalloproteinase ADAM10 mediates a canonical Notch-dependent regulation of IL-6 through Dll4 in human endothelial cells.

Although the involvement of the disintegrin and metalloproteinase ADAM10 in several areas of vascular biology is now clearly established, its role in vascular inflammation and in Notch signaling at the endothelial level remains unclear. In this study, we demonstrated that ADAM10 specifically localizes in the CD31(+) endothelial cells (ECs) in normal human cardiac tissues and in cultured primary arterial ECs. In vitro, ADAM10 drives a specific regulation of the Notch pathway in vascular ECs. Using an ADAM10 gain and loss of function approach we show an ADAM10-dependent regulation of Dll1 and Dll4 expression in association with changes in Hes1 and Hey1 expression. We also identified IL-6, IL-8, MCP-1 and sVCAM-1 as novel targets of ADAM10 upon inflammation. Although Notch pathway does not seem to be required for the production of IL-8, MCP-1 and sVCAM-1, the release of IL-6 by ECs occurred through ADAM10 and a canonical Notch signaling pathway, dependent of γ-secretase activity. Moreover, sustained expression of Dll4 mediated by ADAM10 elicits an increased release of IL-6 suggesting a strong implication of the specific Dll4 signaling in this mechanism. Modulation of IL-6 mediated by ADAM10/Notch signaling required PI3K activity. Thus, our findings suggest that ADAM10/Dll4 signaling is a major signaling pathway in ECs driving inflammatory events involved in inflammation and immune cell recruitment.

Small animal PET imaging of the type 1 cannabinoid receptor in a rodent model for anorexia nervosa.

PURPOSE: Several lines of evidence strongly implicate a dysfunctional endocannabinoid system (ECS) in eating disorders. Using [(18)F]MK-9470 and small animal positron emission tomography (PET), we investigated for the first time cerebral changes in type 1 cannabinoid (CB1) receptor binding in vivo in the activity-based rat model of anorexia (ABA), in comparison to distinct motor- and food-related control conditions and in relation to gender and behavioural variables. METHODS: In total, experiments were conducted on 80 Wistar rats (23 male and 57 female). Male rats were assigned to the cross-sectional conditions: ABA (n = 12) and CONTROL (n = 11), whereas female rats were divided between two settings: (1) a cross-sectional design using ABA (n = 13), CONTROL (n = 9), and two extra control conditions for each of the variables manipulated in ABA, i.e. DIET (n = 8) and WHEEL (n = 9), and (2) a longitudinal one using ABA (n = 10) and CONTROL (n = 8) studied at baseline, during the model and upon recovery. The ABA group was subjected to food restriction in the presence of a running wheel, the DIET group to food restriction without wheel, the WHEEL group to a normal diet with wheel and CONTROL animals had a normal diet and no running wheel. Parametric CB1 receptor images of each group were spatially normalized to Paxinos space and analysed voxel-wise. RESULTS: In the ABA model, absolute [(18)F]MK-9470 binding was significantly increased in all cortical and subcortical brain areas as compared to control conditions (male +67 %; female >51%, all p cluster < 6.3×10(-6)) that normalized towards baseline values after weight gain. Additionally, relative [(18)F]MK-9470 binding was increased in the hippocampus, inferior colliculus and entorhinal cortex of female ABA (+4.6%; p cluster < 1.3×10(-6)), whereas no regional differences were observed in male subjects. Again, relative [(18)F]MK-9470 binding values normalized upon weight gain. CONCLUSION: These data point to a widespread transient disturbance of the endocannabinoid transmission, specifically for CB1 receptors in the ABA model. Our data also suggest (1) gender effects on regional CB1 receptor binding in the hippocampus and (2) add further proof to the validity of the ABA model to mimic aspects of human disease.

MICA variant promotes allosensitization after kidney transplantation.

MHC class I-related chain A (MICA) antigens are surface glycoproteins strongly implicated in innate immunity, and the MICA gene is highly polymorphic. Clinical observations suggest a role for donor MICA antigens expressed on transplant endothelial cells in the alloimmune response, but the effect of MICA genotype is not well understood. Here, we investigated the immunologic effect of the A5.1 mutation, related to the common MICA*008 allele. Compared with wild-type endothelial cells (ECs), homozygosity for MICA A5.1 associated with an endothelial phenotype characterized by 7- to 10-fold higher levels of MICA mRNA and MICA proteins at the cell surface, as well as exclusive release in exosomes instead of enzymatic cleavage. Mechanistically, we did not detect quantitative changes in regulatory microRNAs. Functionally, A5.1 ECs enhanced NKG2D interaction and natural killer cell activation, promoting NKG2D-dependent lysis of ECs. In kidney transplant recipients, polyreactive anti-MICA sera bound preferentially to ECs from MICA A5.1 donors, suggesting that MICA*008(A5.1) molecules are the preferential antigenic determinants on ECs of grafts. Furthermore, the incidence of MICA A5.1 mismatch revealed a statistically significant association between donor MICA A5.1 and both anti-MICA sensitization and increased proteinuria in kidney recipients. Taken together, these results identify the A5.1 mutation as an immunodominant factor and a potential risk factor for transplant survival.

Molecular spectrum of ß-thalassemia mutations in the admixed Venezuelan population, and their linkage to ß-globin gene haplotypes.

In order to establish the spectrum of ß-thalassemia (ß-thal) mutations in the Venezuelan population for the first time, 127 unrelated subjects either with a suspicion of ß-thal trait or with a clinically recognized ß-thal syndrome of different degrees of severity, were studied. DNA from these subjects was analyzed by a polymerase chain reaction (PCR)-based reverse dot-blot method or amplification refractory mutation system (ARMS). Prototype ß-globin gene sequencing of relevant DNA was performed to confirm the mutations. Fifteen different mutations were identified accounting for 92.0% of the mutant alleles explored, revealing a significant genetic heterogeneity at the ß-globin gene locus in this population. The most frequent mutations were codon 39 (C >T) 34.1%, IVS-I-1 (G >A) 11.1%, IVS-I-6 (T > C) 6.6%, IVS-I-110 (G >A) 6.6%, IVS-II-849 (A >G) 6.6%, -88 (C >T) 6.0%, -29 (A >G) 5.2%, followed by the less common IVS-I-5 (G >A) 3.7%, the 1,393 bp deletion 3.0%, IVS-II-1 (G >A) 3.0%, -86 (C >G) 2.2%, IVS-II-1 (G >T) 1.5%, codons 41/42 (-TCTT) 1.5%, IVS-II-745 (C >G) 0.7% and deletional δß-thal 0.7%. Overall, these data demonstrate that the major sources of ß-thal alleles in Venezuela, as expected, are of Mediterranean and African origins. This is the first large study defining the molecular spectrum of ß-thal in the highly admixed population of Venezuela and lays the foundation for genetic counseling as well as implementing comprehensive clinical care programs. Diversity of haplotypes associated with some of the ß-thal mutations can be explained by in situ recombination events in Venezuela.

LNK (SH2B3) is a key regulator of integrin signaling in endothelial cells and targets α-parvin to control cell adhesion and migration.

Focal adhesion (FA) formation and disassembly play an essential role in adherence and migration of endothelial cells. These processes are highly regulated and involve various signaling molecules that are not yet completely identified. Lnk [Src homology 2-B3 (SH2B3)] belongs to a family of SH2-containing proteins with important adaptor functions. In this study, we showed that Lnk distribution follows that of vinculin, localizing Lnk in FAs. Inhibition of Lnk by RNA interference resulted in decreased spreading, whereas sustained expression dramatically increases the number of focal and cell-matrix adhesions. We demonstrated that Lnk expression impairs FA turnover and cell migration and regulates ß1-integrin-mediated signaling via Akt and GSK3ß phosphorylation. Moreover, the α-parvin protein was identified as one of the molecular targets of Lnk responsible for impaired FA dynamics and cell migration. Finally, we established the ILK protein as a new molecular partner for Lnk and proposed a model in which Lnk regulates α-parvin expression through its interaction with ILK. Collectively, our results underline the adaptor Lnk as a novel and effective key regulator of integrin-mediated signaling controlling endothelial cell adhesion and migration.

Profiling posttransplant circulating antibodies in kidney transplantation using donor endothelial cells.

BACKGROUND: Pathogenesis of antibody (Ab) responses to transplant are yet not well defined. This study aimed to detect and to analyze posttransplant circulating allo-Abs reacting toward graft endothelial cells (ECs) using primary EC cultures prospectively isolated from the transplant donor at the time of transplantation. METHODS: This study shows a retrospective analysis performed using a dedicated EC crossmatch (ECXM) assay that we developed for the experimental assessment of donor-specific EC-reactive Abs. Donor-specific ECXM was performed by flow cytometry on posttransplant sera (n=256) from an historical cohort of 22 kidney allograft recipients. RESULTS: In this study, we show that 27.3% (6/22) of recipients have a positive ECXM that strictly correlates (100%, 6/6) with the presence of anti-human leukocyte antigen (HLA) Abs posttransplantation. ECXM identifies both donor-specific Abs (DSA; 50%) and non-DSA (50%) reactive to EC. DSA and non-DSA are mostly IgG1 and exhibit peak titers ranging from 1/8 to 1/1024. ECXM indicates that DSA correspond to anti-HLA class II Abs; this immunization is late (M3-M60) but persistent (still detected at M60). In contrast, non-DSA are non-HLA-type Abs reacting with third-party EC and reflecting an early but transient immunization (ended at M3-M12). Our findings demonstrate selective regulatory pathways initiated by anti-HLA class II and non-DSA in graft EC reflected by CCR4 and interleukin 1ß up-regulation, respectively. CONCLUSIONS: We provide evidence that circulating Abs in HLA-sensitized transplant recipients include both DSA and non-HLA/non-DSA able to bind to graft EC and induce specific gene transcription.

Interictal type 1 cannabinoid receptor binding is increased in female migraine patients.

OBJECTIVE: To compare binding of the type 1 cannabinoid receptor (CB1R) between migraine patients and healthy volunteers. BACKGROUND: It has been suggested that endocannabinoid deficiency may play a role in the pathophysiology of migraine. Nonetheless, biochemical studies substantiating this idea remain scarce and are faced with methodological shortcomings partly because of the difficulty to perform measurements of endocannabinoids within the central nervous system itself. METHODS: An observational cross-sectional study was conducted in 20 female migraine patients and 18 healthy women matched for age and body mass index. Positron emission tomography acquisition was performed 90 minutes after intravenous injection of the radioligand [(18)F]MK-9470 to assess binding of [(18)F]MK-9470 to CB1R. RESULTS: Binding of CB1 R was globally increased in migraine patients vs healthy controls (average gray matter difference +16%; P = .009, 2-sample 2-sided Student's t-test). There were no correlations between CB1R binding and any predefined migraine characteristics. Increases in CB1R binding were most pronounced in the anterior cingulate, mesial temporal, prefrontal, and superior frontal cortices. CONCLUSION: The increased interictal CB1R binding, especially in brain regions that exert top-down influences to modulate pain, supports the idea that endocannibinoid deficiency is present in female patients suffering from episodic migraine.

Brain type 1 cannabinoid receptor availability in patients with anorexia and bulimia nervosa.

BACKGROUND: The endocannabinoid system is a possible target in the treatment of eating disorders. We used positron emission tomography to investigate the type 1 cannabinoid receptor (CB1R) in bulimic and anorectic patients. METHODS: We investigated 16 female bulimia nervosa patients (BN) (age = 23.8 ± 7.1 years) and 14 female anorexia nervosa patients (AN) (age = 20.5 ± 3.6 years) using the selective CB1R ligand [(18)F]MK-9470. The control group consisted of 19 age-matched women (age = 25.2 ± 8.5 years). Statistical parametric mapping (p(family-wise error) < .05) and volume-of-interest analyses of CB1R availability were performed. RESULTS: Global CB1R availability was significantly increased in cortical and subcortical brain areas in AN patients compared with healthy control subjects (+24.5%, p = .0003). Regionally, CB1R availability was increased in the insula in both AN and BN patients (p = .01 and p = .0004) and the inferior frontal and temporal cortex in AN patients only (p = .02). CONCLUSIONS: Global CB1R upregulation in AN patients is a possible long-term compensatory mechanism to an underactive endocannabinoid system in anorectic conditions. There is a similarity in CB1R dysregulation both in AN and BN in the insular cortex, which is involved in the integration of interoceptive information, gustatory information, reward, and emotion processing.

Early rise in circulating endothelial protein C receptor correlates with poor outcome in severe sepsis.

PURPOSE: The endothelial protein C receptor (EPCR) negatively regulates the coagulopathy and inflammatory response in sepsis. Mechanisms controlling the expression of cell-bound and circulating soluble EPCR (sEPCR) are still unclear. Moreover, the clinical impact of EPCR shedding and its potential value to predict sepsis progression and outcome remain to be established. METHODS: We investigated the time course of plasma sEPCR over the 5 first days (D) of severe sepsis in 40 patients. RESULTS: No significant difference was observed when comparing sEPCR at admission (D1) to healthy volunteers and to patients with vasculitis. We report that the kinetics profile of plasma sEPCR in patients was almost stable at the onset of sepsis with no change from D1 to D4 and then a significant decrease at D5. This pattern of release was consistently observed whatever the level of sEPCR at D1. Characteristics of patients or of infections (except Gram negative) had no or little critical influence on the sEPCR profile. However, we found that sEPCR kinetics was clearly associated with patient's outcome (D28 survival). We demonstrate that a significant but moderate (<15% of basal level) and transient increase in sEPCR level at D2 is associated with poor outcome at D28. CONCLUSION: Severe sepsis, at the onset, only triggers moderate quantitative changes in plasma sEPCR levels. Our findings suggest that in severe sepsis, an early (at D2), transient but significant increase in circulating sEPCR may be detrimental suggesting that sEPCR could provide an early biological marker of sepsis outcome.