Monitoring Circulating CD207+CD1a+ Cells in Langerhans Cell Histiocytosis and Clinical Implications.

Langerhans cell histiocytosis (LCH) is a disorder characterized by an abnormal accumulation of CD207+ and CD1a+ cells in almost any tissue. Currently, there is a lack of prognostic markers to follow up patients and track disease reactivation or treatment response. Putative myeloid precursors CD207+ and CD1a+ cells were previously identified circulating in the blood. Therefore, we aim to develop a sensitive tracing method to monitor circulating CD207+ and CD1a+ cells in a drop of blood sample of patients with LCH. A total of 202 blood samples from patients with LCH and 23 controls were tested using flow cytometry. A standardized cellular score was defined by quantifying CD207+ and CD1a+ expression in monocytes and dendritic cells, based on CD11b, CD14, CD11c, and CD1c subpopulations, resulting in a unique value for each sample. The scoring system was validated by a receiver operating characteristic curve showing a reliable discriminatory capacity (area under the curve of 0.849) with a threshold value of 14, defining the presence of circulating CD207+ and CD1a+ cells. Interestingly, a fraction of patients with no evident clinical manifestation at the time of sampling also showed presence of these cells (29.6%). We also found a differential expression of CD207 and CD1a depending on the organ involvement, and a positive correlation between the cellular score and plasma inflammatory markers such as soluble CD40L, soluble IL-2Ra, and CXCL12. In conclusion, the analysis of circulating CD207 and CD1a cells in a small blood sample will allow setting a cellular score with minimal invasiveness, helping with prognostic accuracy, detecting early reactivation, and follow-up.

Pro-inflammatory monocyte profile in patients with major depressive disorder and suicide behaviour and how ketamine induces anti-inflammatory M2 macrophages by NMDAR and mTOR.

BACKGROUND: Depression is a highly prevalent disorder that is one of the leading causes of disability worldwide. Despite an unknown aetiology, evidence suggests that the innate and adaptive immune systems play a significant role in the development and maintenance of major depressive disorder (MDD). The non-competitive glutamatergic N-methyl-D-aspartate receptor (NMDAR) antagonist, (R,S)-ketamine (ketamine), has demonstrated rapid and robust efficacy as an antidepressant when administered at sub-anaesthetic doses. METHODS: Our goal was to characterize the pro-inflammatory profile of patients with MDD by measuring pro-inflammatory cytokines in plasma and circulating monocyte subsets and to understand how ketamine induces an anti-inflammatory program in monocyte and macrophages in vitro and vivo. FINDING: Our results show that patients with MDD without other comorbidities (N = 33) exhibited significantly higher levels of pro-inflammatory IL-12 and IL-6 in plasma and that these cytokines were associated with increased numbers of non-classical (CD11b+CD16brightCD14neg) monocytes and increased activation state (CD40+CD86+) of classical monocytes in circulation. Remarkably, we have demonstrated that sub-anaesthetic doses of ketamine programs human monocytes into M2c-like macrophages by inducing high levels of CD163 and MERTK with intermediate levels of CD64 and stimulating mTOR-associated gene expression in vitro. The NMDAR antagonist MK-801, but not the α-amino-3­hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) antagonist, NBQX, also polarizes macrophages to an M2c-like phenotype, but this phenotype disappears upon mTOR pathway inhibition. Sub-anaesthetic doses (10 mg/kg) of ketamine administration in mice both promote reduction of circulating classical pro-inflammatory monocytes and increase of alternative M2 macrophage subtypes in the spleen and CNS. INTERPRETATION: Our results suggest an anti-inflammatory property of ketamine that can skew macrophages to an M2-like phenotype, highlighting potential therapeutic implications not only for patients with MDD but also other inflammatory-based diseases. FUNDING: This study was supported by grants from the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) and Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT-FONCYT).

CD207+CD1a+ cells circulate in pediatric patients with active Langerhans cell histiocytosis.

Langerhans cell histiocytosis (LCH) is a rare disease with an unknown etiology characterized by heterogeneous lesions containing CD207+CD1a+ cells that can arise in almost any tissue and cause significant morbidity and mortality. Precursors of pathological Langerhans cells have yet to be defined. Our aim was to identify circulating CD207+CD1a+ cells and their inducers in LCH. Expression of CD207 and CD1a in the blood myeloid compartment as well as thymic stromal lymphopoietin (TSLP) and transforming growth factor ß (TGF-ß) plasma levels were measured in 22 pediatric patients with active disease (AD) or nonactive disease (NAD). In patients with AD vs those with NAD, the myeloid compartment showed an increased CD11b (CD11bhigh plus CD11b+) fraction (39.7 ± 3.6 vs 18.6 ± 1.9), a higher percentage of circulating CD11bhighCD11c+CD207+ cells (44.5 ± 11.3 vs 3.2 ± 0.5), and the presence of CD11chighCD207+CD1a+ cells (25.0 ± 9.1 vs 2.3 ± 0.5). Blood CD207+CD1a+ cells were not observed in adult controls or umbilical cord. Increased TSLP and TGF-ß levels were detected in patients with AD. Interestingly, plasma from patients with AD induces CD207 expression on CD14+ monocytes. We conclude that CD207+CD1a+ cells are circulating in patients with active LCH, and TSLP and TGF-ß are potential drivers of Langerhans-like cells in vivo.

Involvement of Extracellular Signal-Regulated Kinase 5 in Kinin B1 Receptor Upregulation in Isolated Human Umbilical Veins.

The upregulated kinin B1 receptors exert a pivotal role in modulating inflammatory processes. In isolated human umbilical veins (HUVs), kinin B1 receptor is upregulated as a function of in vitro incubation time and proinflammatory stimuli. The aim of this study was to evaluate, using functional and biochemical methods, the involvement of extracellular signal-regulated kinase 5 (ERK5), p38 mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase 1/2 (ERK1/2) on the kinin B1 receptor upregulation process in HUV. Real-time polymerase chain reaction analysis revealed for the first time that kinin B1 receptor mRNA expression closely parallels the functional sensitization to kinin B1 receptor selective agonist des-Arg(10)-kallidin (DAKD) in HUV. Moreover, the selective inhibition of ERK5, p38 MAPK, and JNK, but not ERK1/2, produced a dose-dependent rightward shift of the concentration-response curves to DAKD after 5-hour incubation and a reduction in kinin B1 receptor mRNA expression. Biochemical analyses showed that ERK5, p38 MAPK, and JNK phosphorylation is maximal during the first 2 hours postisolation, followed by a significant reduction in the last 3 hours. None of the treatments modified the responses to serotonin, an unrelated agonist, suggesting a specific effect on kinin B1 receptor upregulation. The present work provides for the first time pharmacologic evidence indicating that ERK5 plays a significant role on kinin B1 receptor upregulation. Furthermore, we confirm the relevance of p38 MAPK and JNK as well as the lack of effect of ERK1/2 in this process. This study may contribute to a better understanding of MAPK involvement in inflammatory and immunologic diseases.

EV-077 in vitro inhibits platelet aggregation in type-2 diabetics on aspirin.

INTRODUCTION: This study aimed to characterize the in vitro effect of EV-077, a compound that antagonises the binding of prostanoids and isoprostanes to the thromboxane receptor (TP) and inhibits the thromboxane synthase (TS), on platelet aggregation of patients with type-2 diabetes and coronary artery disease (CAD) on chronic aspirin treatment. The effect of EV-077 on 8-iso-PGE(2)-mediated TP receptor contraction of human arteries was also investigated. MATERIALS AND METHODS: Fifty-two type-2 diabetics with CAD on chronic aspirin (100 mg) treatment were studied. Arachidonic acid-induced platelet aggregation was measured by impedance aggregometry in platelet-rich plasma (PRP) and whole blood anticoagulated with hirudin, and by light transmission aggregometry in citrate-anticoagulated PRP following 10-min in vitro exposure to EV-077 (100 nmol/l) or control. The effect of EV-077 was measured on isometric contraction of 24 human umbilical arteries induced by isoprostane 8-iso-PGE(2). RESULTS: Arachidonic acid (1 mmol/l) induced substantial aggregation in hirudin-anticoagulated whole blood (63 ± 4 AU), which was significantly reduced by in vitro exposure to EV-077 (38 ± 3 AU, P<0.001). Virtually no arachidonic acid-induced aggregation in citrate-anticoagulated or hirudin-anticoagulated PRP was observed. EV-077 potently, competitively and reversibly inhibited TP mediated contraction of umbilical arteries by 8-iso-PGE(2) (P<0.01). CONCLUSIONS: Aspirin did not completely inhibit arachidonic acid-induced platelet aggregation in whole blood from type-2 diabetics with CAD. This aggregation is likely induced by prostanoids and/or isoprostanes produced by leukocytes, because it was significantly reduced by EV-077. The TP receptor-mediated contraction of human arteries induced by isoprostane 8-iso-PGE(2) was effectively inhibited by EV-077.

Endothelial angiotensin-converting enzyme and neutral endopeptidase in isolated human umbilical vein: an effective bradykinin inactivation pathway.

Kinins are metabolized by metallopeptidases present in different tissues. The aim of this study was to evaluate, employing functional studies in isolated human umbilical vein, the possible participation of angiotensin-converting enzyme, neutral endopeptidase and aminopeptidase P as an inactivation pathway of bradykinin, as well as assess if the endothelial layer is involved in this process. Concentration-response curves to bradykinin were constructed after 120 min incubation period on human umbilical vein rings with and without endothelium and enzymatic inhibitors were applied 30 min before construction of concentration-response curves. The presence of endothelium was confirmed by histological studies. Bradykinin-induced contractile responses were potentiated in human umbilical vein without endothelium when compared to intact tissues. Application of captopril 1 µM (angiotensin-converting enzyme inhibitor) or phosphoramidon 10 µM (neutral endopeptidase inhibitor) induced a leftward shift of bradykinin-elicited responses in human umbilical vein with endothelium while no effect was observed in tissues denuded of endothelium under the same treatment. Exposure to apstatin 10 µM (aminopeptidase P inhibitor) did not potentiate bradykinin-induced effects in intact human umbilical vein. When angiotensin-converting enzyme and neutral endopeptidase were concomitantly inhibited, there was a higher potentiation of bradykinin-elicited responses compared to the effects observed under individual inhibition of either enzyme. Moreover, concentration-response curves to FR190997, a non-peptidic bradykinin B(2) receptor agonist, were not modified under dual enzymatic inhibition. In conclusion, our results demonstrate for the first time the functional relevance of angiotensin-converting enzyme and neutral endopeptidase, localized on the endothelial layer, acting concurrently as a bradykinin inactivating pathway in isolated human umbilical vein.

Expression and functional evidence of the prostaglandin F2alpha receptor mediating contraction in human umbilical vein.

Our purposes were to perform the pharmacological characterization of PGF(2alpha) receptor (prostanoid FP-receptor) involved in human umbilical vein contraction and confirm its expression in this tissue. Umbilical cords from healthy patients after full-term deliveries were employed. The vein was dissected out of cords and used for either isolated organ bath or reverse transcription-polymerase chain reaction (RT-PCR) and Western blot assays. The natural prostanoid FP-receptor agonist, PGF(2alpha), and its selective analogues, latanoprost and bimatoprost free acids are full agonists (produce more than 80% of the maximal contractile response to 5-HT) in human umbilical vein. The agonist potency (pEC(50)) order was PGF(2alpha) (6.01+/-0.05)>latanoprost free acid (5.65+/-0.07)=bimatoprost free acid (5.59+/-0.08). The contractile effects of PGF(2alpha) and latanoprost free acid were blocked competitively by the prostanoid FP-receptor antagonist, AL-8810. The antagonist potencies (pK(B)) of AL-8810 vs. PGF(2alpha) (5.93+/-0.05) and vs. latanoprost free acid (6.40+/-0.08) in human umbilical vein are in good agreement with its ability to antagonize prostanoid FP receptors of rat, mouse and human cells. In all samples, clear signal was detected for cDNA amplification of prostanoid FP receptor and the specific prostanoid FP-receptor antibody recognized a protein of approximately 64 kDa. In conclusion, taking into account the obtained functional and biochemical data, we propose for the first time that human umbilical vein express prostanoid FP-receptors and these receptors could be involved in the vasoconstriction action of PGF(2alpha) in this tissue.

Potentiation of adrenaline vasoconstrictor response by sub-threshold concentrations of U-46619 in human umbilical vein: involvement of smooth muscle prostanoid TP(alpha) receptor isoform.

Considering the potential physiological, pharmacological and therapeutic relevance of synergistic interaction of thromboxane A(2) with adrenaline at postjunctional receptor sites, we examined whether sub-threshold concentrations of thromboxane A(2) mimetic U-46619 (9,11-dideoxy-9alpha, 11alpha-methanoepoxy prostaglandin F(2alpha)) could amplify adrenaline-induced contraction in human umbilical vein. The receptor involved in U-46619-induced potentiation of adrenaline contractility was also investigated. Umbilical cords (n=125) from healthy patients after full-term vaginal or caesarean deliveries were employed. The vein was dissected out of cords and rings used for isolated organ bath experiments or reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. Presence of endothelium did not modify U-46619-induced contraction in human umbilical vein. Prostanoid TP-selective receptor antagonist, SQ-29548 (7-[3-[[2-[(phenylamino)carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-[1S(1alpha,2alpha(Z),3alpha,4alpha)]-5-Heptenoic acid), inhibited U-46619-induced contraction (pA(2)=8.22+/-0.11). U-46619 sub-threshold concentrations (0.1-0.3 nM) potentiated adrenaline-vasoconstriction response in a concentration-dependent manner. SQ-29548 (0.1 microM) abolished this potentiation. Using RT-PCR, we found that human umbilical vein rings with or without endothelium express the prostanoid TP(alpha), but not the prostanoid TP(beta) receptor isoform. Western blot allowed the identification of proteins with an electrophoretic mobility (47- and 55-kDa) indistinguishable from human platelet prostanoid TP receptor, a rich source of prostanoid TP(alpha) receptor isoform. Collectively, present results demonstrate that prostanoid TP(alpha) is the major receptor isoform localized on smooth muscle cells which participate in both direct vasoconstriction and potentiating effects of U-46619 on adrenaline contractions in human umbilical vein. These results suggest that thromboxane A(2) may interact synergistically with adrenaline in pathophysiological situations that lead to an increase of its umbilical venous levels (e.g. preeclampsia associated with fetal distress) raising the possibility of vasoconstriction affecting fetal blood flow.

Functional evidence of des-Arg10-kallidin enzymatic inactivating pathway in isolated human umbilical vein.

It has been known for many years that plasma and tissues contain a variety of enzymes capable of metabolizing kinins. The aim of the present study was to evaluate, by means of functional studies in a capacitance vessel such as the human umbilical vein (HUV), the possible role played by the metallopeptidases angiotensin-converting enzyme (ACE), neutral endopeptidase (NEP), and aminopeptidase M (APM) as an inactivating pathway of the B(1) receptor endogenous agonist des-Arg(10)-kallidin (DAKD). In HUV rings with and without endothelium, concentration-response curves (CRCs) to DAKD were determined after a 300-min incubation period, and enzymatic inhibitors were added to the organ baths 30 min before construction of the CRC. Presence of endothelial layer was confirmed by histological studies. There was a significant leftward shift observed in control HUV rings devoid of endothelium compared with intact tissues. Exposure to 1 microM captopril (ACE inhibitor) potentiated DAKD-elicited vasoconstrictor responses in HUV rings with endothelium while no such effect was observed in tissues devoid of endothelium. Application of 10 microM amastatin (APM inhibitor) induced a leftward shift of DAKD-elicited contractile responses in HUV with and without endothelium. On the other hand, 10 microM phosphoramidon (NEP inhibitor) showed no potentiating effect in HUV rings either with or without endothelium. However, under concurrent inhibition of ACE, NEP and APM, there was a higher potentiation of DAKD-elicited contractile responses compared with the effect observed with combined inhibition of ACE and APM. Moreover, when we evaluated contractile responses induced by Sar(0)-D-Phe(8)-des-Arg(9)-BK (a metabolically protected B(1) receptor agonist), no potentiating effect was observed under triple enzymatic inhibition. In conclusion, in the present study for the first time, we demonstrated in a capacitance vessel, HUV, that metallopeptidases ACE, NEP and APM represent a relevant functional inactivation pathway of DAKD.

Neutral endopeptidase up-regulation in isolated human umbilical artery: involvement in desensitization of bradykinin-induced vasoconstrictor effects.

Previous reports show that bradykinin B(2) receptors mediate contractile responses induced by bradykinin (BK) in human umbilical artery (HUA). However, although it has been reported that BK-induced responses can desensitize in several inflammatory models, the effects of prolonged in vitro incubation on BK-induced vasoconstriction in HUA have not been studied. In isolated HUA rings, BK-induced responses after a 5-h in vitro incubation showed a marked desensitization compared with responses at 2 h. Inhibition of either angiotensin-converting enzyme (ACE) or neutral endopeptidase (NEP), both BK-inactivating enzymes, failed to modify responses to BK at 2 h. After 5 h, ACE inhibition produced only a slight potentiation of BK-induced responses. In contrast, BK-induced vasoconstriction at 5 h was markedly potentiated by NEP inhibition. Moreover, NEP activity, measured by hydrolysis of its synthetic substrate (Z-Ala-Ala-Leu-p-nitroanilide), showed a 2.4-fold increase in 5-h incubated versus 2-h incubated tissues, which was completely reversed by cycloheximide (CHX) treatment. Furthermore, CHX significantly potentiated BK-induced responses, suggesting that NEP-mediated kininase activity increase at 5 h depends on de novo protein synthesis. In addition, under NEP inhibition, CHX treatment failed to produce an additional potentiation of BK-induced vasoconstriction. Still, NEP up-regulation was confirmed by Western blot, showing a 2.1-fold increase in immunoreactive NEP in 5-h incubated versus 2-h incubated HUA. In summary, the present study provides strong pharmacological evidence that NEP is up-regulated and plays a key role in desensitization of BK-induced vasoconstriction after prolonged in vitro incubation in HUA. Our results provide new insights into the possible mechanisms involved in BK-induced response desensitization during sustained inflammatory conditions.

Involvement of endothelial thromboxane A2 in the vasoconstrictor response induced by 15-E2t-isoprostane in isolated human umbilical vein.

The present study was undertaken to evaluate the contractile response of several E- and F-ring isoprostanes (IsoP) in human umbilical vein (HUV) and to investigate the role of the endothelium on the effect of 15-E2t-IsoP, the most potent vasoconstrictor isoprostane, in human vessels. HUV rings with or without endothelium were suspended in an organ bath for recording the isometric tension in response to different agonists. The inhibitors to be evaluated were applied 30 min before the addition of the agonist. All of the compounds tested produced concentration-dependent contractions when tested on HUV rings with endothelium. Although these compounds were equieffective, significant differences were observed in their potency, with U46619 being the most potent followed by 15-E2t-IsoP > 15-E1t-IsoP = 15-F2t-IsoP > 15-F1t-IsoP = 9-epi-15-F2t-IsoP in descending rank order of potency. 15-E2t-IsoP was the most potent of the isoprostanes evaluated and, therefore, the one employed in the present study. When intact endothelium HUV rings were used, 15-E2t-IsoP-induced contraction was unaffected by the endothelin-converting enzyme inhibitor, phosphoramidon (10 microM), suggesting that short-term endothelin-1 release is not involved in this response. However, the non-selective cyclooxygenase (COX) inhibitor, indomethacin (10 and 30 microM), and the COX-2 selective inhibitor, NS-398 (3, 10 and 30 microM) produced inhibitory effects on 15-E2t-IsoP-induced contraction of HUV rings with endothelium. These results indicate that COX-derived contractile prostanoids are involved in this effect. Furthermore, the apparent pKb values estimated for indomethacin (5.5) and NS-398 (5.4) suggest that the prostanoids involved are derived from the COX-2 isoenzyme pathway. On HUV rings with endothelium, the phospholipase A2 inhibitor, oleyloxyethyl phosphorylcholine (30 and 100 microM), induced an inhibitory effect on 15-E2t-IsoP-induced contraction, suggesting that the phospholipase A2 pathway is also involved in this effect. In addition, the thromboxane A2 synthase inhibitor furegrelate (10 and 30 microM) also inhibited 15-E2t-IsoP-induced contraction of HUV rings with endothelium, indicating that thromboxane A2 is one of the contractile prostanoids involved in this response. Endothelium denudation clearly diminished the vasoconstrictor potency of 15-E2t-IsoP, demonstrating that the endothelium releases a vasoconstrictor factor in response to 15-E2t-IsoP. The absence of an inhibitory effect at the highest concentration of furegrelate (30 microM) on 15-E2t-IsoP-induced contraction of HUV rings without endothelium suggested that endothelium is the source of thromboxane A2. We conclude that prostanoids derived from the COX-2 isoenzyme pathway participate in 15-E2t-IsoP-induced vasoconstriction of isolated HUV rings. Our results also indicate that endothelial thromboxane A2 is one of the prostanoids involved in this effect.

Characterization of 5-HT receptor subtypes mediating contraction in human umbilical vein. 1. Evidence of involvement of 5-HT2A receptors using functional and radioligand binding assays.

This study attempted to characterize pharmacologically the involvement of 5-HT(2A) receptors in 5-HT-induced contractile responses in human umbilical vein (HUV) rings employing functional and radioligand binding assays. In HUV rings, prazosin 1 micro M did not affect contractile responses elicited by 5-HT, ruling out the involvement of alpha(1)-adrenoceptors in contractile responses to 5-HT. 5-HT-induced contractions were competitively blocked by ketanserin, a 5-HT(2A)-selective antagonist. The apparent pA(2) value was 9.8 and the Schild slope significantly less than unity, suggesting that 5-HT-induced responses are mediated by a heterogeneous receptor population. Alpha-methyl-5-HT, a selective 5-HT(2) receptor agonist, induced contractions that were antagonized in a competitive manner by ketanserin. The slope regression was not significantly different from unity and the pA(2) value was 8.8. The selective 5-HT(2A) ligand spiperone produced a parallel rightward shift on 5-HT CRCs in HUV rings. The calculated pA(2) was 9.0, which is in accord for an interaction with the 5-HT(2A) receptor subtype. Alpha-methyl-5-HT CRCs were competitively blocked by spiperone treatment. The Schild analysis yielded a pA(2) of 9.1 with a slope not significantly different from unity. The 5-HT(2C/2A) antagonist mesulergine 10 nM did not affect 5-HT CRCs, suggesting that 5-HT(2C) receptors are not involved in 5-HT-elicited contractions. Higher concentrations of mesulergine showed a parallel rightward shift on 5-HT responses. The calculated pA(2) was 7.44, which suggests an interaction with the 5-HT(2A) receptor subtype. In addition, mesulergine competitively blocked alpha-methyl-5-HT CRCs. The Schild slope was not significantly different from unity and the p A(2) value was 7.98. The binding of [(3)H]ketanserin to HUV membranes was saturable and of high affinity. Ketanserin displayed a monophasic curve which was best fit with a single component of binding. Nonlinear least squares analysis of the binding curves revealed a high affinity K(d) of 0.30 nM and a B(max) of 134 fmol/mg protein. These findings provide strong pharmacological evidence of the involvement of 5-HT(2A) receptors in 5-HT-induced vasoconstriction in HUV. In addition, the contribution of another receptor population cannot be excluded. The results also suggest that this receptor population is neither an alpha(1)-adrenoceptor nor a 5-HT(2C) receptor subtype.