Features, reason for testing, and changes with time of 583 paroxysmal nocturnal hemoglobinuria clones from 529 patients: a multicenter Italian study.

In this study, we aimed at disclosing the main features of paroxysmal nocturnal hemoglobinuria (PNH) clones, their association with presentation syndromes, and their changes during follow-up. A large-scale, cooperative collection (583 clones from 529 patients) of flow cytometric and clinical data was entered into a national repository. Reason for testing guidelines were provided to the 41 participating laboratories, which followed the 2010 technical recommendations for PNH testing by Borowitz. Subsequently, the 30 second-level laboratories adopted the 2012 guidelines for high-resolution PNH testing, both upon order by the local clinicians and as an independent laboratory initiative in selected cases. Type3 and Type2 PNH clones (total and partial absence of glycosyl-phosphatidyl-inositol-anchor, respectively) were simultaneously present in 54 patients. In these patients, Type3 component was sevenfold larger than Type2 (p < 0.001). Frequency distribution analysis of solitary Type3 clone size (N = 442) evidenced two discrete patterns: small (20% of peripheral neutrophils) and large (> 70%) clones. The first pattern was significantly associated with bone marrow failure and myelodysplastic syndromes, the second one with hemolysis, hemoglobinuria, and thrombosis. Pediatric patients (N = 34) showed significant preponderance of small clones and bone marrow failure. The majority of PNH clones involved neutrophils, monocytes, and erythrocytes. Nevertheless, we found clones made exclusively by white cells (N = 13) or erythrocytes (N = 3). Rare cases showed clonal white cells restricted only to monocytes (6 cases) or neutrophils (3 cases). Retesting over 1-year follow-up in 151 cases showed a marked clone size increase in 4 cases and a decrease in 13, demonstrating that early breaking-down of PNH clones is not a rare event (8.6% of cases). This collaborative nationwide study demonstrates a clear-cut difference in size between Type2 and Type3 clones, emphasizes the existence of just two classes of PNH presentations based on Type3 clone size, depicts an asymmetric cellular composition of PNH clones, and documents the possible occurrence of changes in clone size during the follow-up.

SerpinB3 induces dipeptidyl-peptidase IV/CD26 expression and its metabolic effects in hepatocellular carcinoma.

AIMS: In hepatocellular carcinoma (HCC), the regulatory protease Dipeptidyl-peptidase IV (DPPIV/CD26), that possesses pro-apoptotic properties, has been found abnormally regulated. The protease inhibitor SerpinB3, exerting anti-apoptotic activity, has also been described to be upregulated, especially in HCCs with poor prognosis. The aim of this study was to investigate the possible relationship between these two molecules in HCC patients and in experimental models. MATERIALS AND METHODS: DPPIV/CD26 and SerpinB3 expression was measured in liver specimens of 67 patients with HCC. HepG2 and Huh7 cells, stably transfected to overexpress SerpinB3, and respective control cells were used to assess biological and metabolic modifications of DPPIV/CD26 activity induced by this serpin. KEY FINDINGS: DPPIV/CD26 and SerpinB3 were localized in the same tumoral areas and both molecules were correlated with the grade of tumor differentiation, with the highest values detected in GI tumors. Cell lines over-expressing SerpinB3 displayed upregulation of DPPIV/CD26, likely as a feedback mechanism, due to the DPPIV/CD26 protease activity inhibition by SerpinB3, as confirmed by the similar behavior induced by the inhibitor Sitagliptin. Moreover, they exhibited lower glycogen storage and higher lipid accumulation, typical effects of DPPIV/CD26. SIGNIFICANCE: A close connection between SerpinB3 and DPPPIV has been identified, but further studies are required to better understand the mechanism by which these proteins communicate and exert metabolic effects in HCC.

STAT3 mutation impacts biological and clinical features of T-LGL leukemia.

STAT3 mutations have been described in 30-40% of T-large granular lymphocyte (T-LGL) leukemia patients, leading to STAT3 pathway activation. Considering the heterogeneity of the disease and the several immunophenotypes that LGL clone may express, the aim of this work was to evaluate whether STAT3 mutations might be associated with a distinctive LGL immunophenotype and/or might be indicative for specific clinical features. Our series of cases included a pilot cohort of 101 T-LGL leukemia patients (68 CD8+/CD4- and 33 CD4+/CD8±) from Padua Hematology Unit (Italy) and a validation cohort of additional 20 patients from Rennes Hematology Unit (France). Our results indicate that i) CD8+ T-LGL leukemia patients with CD16+/CD56- immunophenotype identify a subset of patients characterized by the presence of STAT3 mutations and neutropenia, ii) CD4+/CD8± T-LGL leukemia are devoid of STAT3 mutations but characterized by STAT5b mutations, and iii) a correlation exists between STAT3 activation and presence of Fas ligand, this molecule resulting highly expressed in CD8+/CD16+/CD56- patients. Experiments with stimulation and inhibition of STAT3 phosphorylation confirmed this relationship. In conclusion, our data show that T-LGL leukemia with specific molecular and phenotypic patterns is associated with discrete clinical features contributing to get insights into molecular bases accounting for the development of Fas ligand-mediated neutropenia.

Integrated CLL Scoring System, a New and Simple Index to Predict Time to Treatment and Overall Survival in Patients With Chronic Lymphocytic Leukemia.

INTRODUCTION: Several prognostic factors have been identified to predict the outcome of patients with chronic lymphocytic leukemia (CLL), but only a few studies analyzed more markers together. PATIENTS AND METHODS: Taking advantage of a population of 608 patients, we identified the strongest prognostic markers of survival and, subsequently, in a cohort of 212 patients we integrated data of cytogenetic lesions, IGHV mutational status, and CD38 expression in a new and easy scoring system we called the integrated CLL scoring system (ICSS). ICSS defines 3 groups of risk: (1) low risk (patients with 13q(-) or normal fluorescence in-situ hybridization analysis results, mutated IGHV, and CD38) (2) high risk (all 11q(-) or 17p(-) patients and/or all unmutated IGHV and CD38(+) patients); and (3) intermediate risk (all remaining patients). RESULTS: Using only these 3 already available prognostic factors, we were able to properly redefine patients and better predict the clinical course of the disease. CONCLUSION: ICSS could become a useful tool for CLL patients' management.

Reduced levels of circulating progenitor cells in juvenile idiopathic arthritis are counteracted by anti TNF-α therapy.

BACKGROUND: Endothelial progenitor cells (EPC) promote angiogenesis and vascular repair. Though reduced EPC levels have been shown in rheumatoid arthritis, no study has so far evaluated EPCs in children with juvenile idiopathic arthritis (JIA). We aimed to study circulating EPCs in children with JIA, their relation to disease activity, and effects of anti TNF-α treatment. METHODS: Circulating EPCs were quantified by flow cytometry based on CD34, CD133 and KDR expression in peripheral blood of 22 patients with oligoarticular JIA and 29 age-matched controls. EPCs were re-assessed in children with methotrexate-resistant oligo-extended JIA before and up to 12 month after initiation of anti-TNF-alpha therapy. Plasma concentrations of inflammatory and EPC-regulating factors were measured using a multiplex array. Confocal immunofluorescence was used to demonstrate EPCs in synovial tissues. RESULTS: Children with active JIA showed a significant reduction of relative and absolute counts of circulating progenitor cells and EPCs compared to age-matched healthy controls. CD34(+) cell levels were modestly and inversely correlated to disease activity. A strong inverse correlation was found between serum TNF-α and EPC levels. In 8 patients treated with anti TNF-α agents, the number of EPCs rose to values similar to healthy controls. CD34(+)KDR(+) EPCs were found in the synovial tissue of JIA children, but not in control. CONCLUSIONS: Children with JIA have reduced levels of the vasculoprotective and proangiogenic EPCs. While EPCs may contribute to synovial tissue remodelling, EPC pauperization may indicate an excess cardiovascular risk if projected later in life.

Endothelial progenitor cells are reduced in acromegalic patients and can be restored by treatment with somatostatin analogs.

CONTEXT: Acromegaly increases cardiovascular risk, possibly due to the high prevalence of classical risk factors. However, in vitro studies show a protective role of GH/IGF-1 on the endothelium. OBJECTIVE: The objective of the study was to investigate circulating endothelial progenitor cells (EPCs), a marker of vascular regeneration, in acromegalic patients and how they are affected by acromegaly treatment. DESIGN: This was a cross-sectional case-control and observational study. SETTING: The study was conducted at a tertiary ambulatory referral endocrinology center. PATIENTS: Forty-three acromegalic patients (26 active; 17 inactive) and 43 control subjects matched by age, gender, and degree of glucose tolerance participated in the study. INTERVENTION: Circulating EPCs were quantified by flow cytometry based on the expression of CD34, CD133, and kinase insert domain-containing receptor (KDR). Nine patients with active acromegaly were reevaluated after 24 weeks of treatment with somatostatin analogs (SSAs). MAIN OUTCOME MEASURE: Differences in EPC levels between patients and controls were measured. RESULTS: Acromegalic patients showed a significant reduction of the total CD34(+)KDR(+) EPC population compared with controls, which was more evident in patients without diabetes or hypertension. More definite CD34(+)CD133(+)KDR(+) EPCs were reduced in patients with active compared with those with inactive acromegaly and compared with controls. The number of CD34(+)CD133(+)KDR(+) EPCs correlated with IGF-1 levels (r = -0.45; P < .001), fasting plasma glucose (r = -0.40; P = .004), and the homeostasis model assessment index of insulin resistance (r = -0.32; P = .026). CD34(+)CD133(+)KDR(+) EPCs increased 2-fold after SSA treatment. CONCLUSIONS: Acromegalic patients have a reduced endothelial regenerative capacity, possibly due to activation of the GH/IGF-1, rather than concomitant risk factors. Treatment with SSAs can restore immature EPCs to normal levels.

Clinical significance of LAIR1 (CD305) as assessed by flow cytometry in a prospective series of patients with chronic lymphocytic leukemia.

Most patients affected by chronic lymphocytic leukemia are diagnosed by flow cytometry. Several immunophenotypic markers have been identified as significant and independent prognostic variables, especially from retrospective cohorts. However, while attractive because their detection is inexpensive and feasible in most laboratories, only few have been validated by independent series. The expression of leukocyte-associated immunoglobulin-like receptor-1 (also known as LAIR1, LAIR-1 or CD305), an inhibitor of B-cell receptor-mediated signaling, has been reported to be lacking in high-risk chronic lymphocytic leukemia. However, its correlation with biological variables and its prognostic significance remain unknown. We investigated 311 consecutive patients, prospectively enrolled since 2007. Methods for studying patients were standardized and included clinical assessment, immunophenotype, fluorescence in situ hybridization, and status of immunoglobulin heavy chain variable region genes. Overall, 22.1% of patients had Binet stage B or C disease, 38.5% had unmutated immunoglobulin genes, 15.1% had high-risk cytogenetic abnormalities, 23.4% were CD38(+), 37.8% CD49d(+), and 59.8% LAIR1(+). Expression of LAIR1 was inversely related to that of CD38 (P=0.0005), but was not associated with CD49d expression (P=0.96). A significantly lower expression of LAIR1 was observed in patients with Binet stage B or C disease (P=0.023), and in the presence of high-risk cytogenetic abnormalities (P=0.048) or unmutated immunoglobulin heavy chain variable region genes (P<0.0001). At univariate analysis LAIR1(+) was significantly associated with longer time to first treatment (P=0.0002). This favorable effect of LAIR1(+) was confirmed by multivariate analysis (hazard ratio=2.1, P=0.03 for LAIR1). Our results indicate that LAIR1 expression is a reliable and inexpensive marker capable of independently predicting time to first treatment in newly diagnosed unselected patients with chronic lymphocytic leukemia.

Effect of olmesartan medoxomil on number and survival of circulating endothelial progenitor cells and calcitonin gene related peptide in hypertensive patients.

OBJECTIVE: Injury of vascular endothelium, crucial in vascular disease, is repaired via circulating endothelial progenitor cells (cEPCs). In hypertension, cEPCs number is reduced and function impaired adding further risk for cardiovascular (CV) events. Angiotensin II (Ang II)-induced oxidative stress (OxSt), accelerates cEPCs senescence. Calcitonin gene-related peptide (CGRP), able to prevent and reverse Ang II-induced cEPCs senescence, is reduced in hypertension and stimulated by the antioxidant and anti-inflammatory heme oxygenase (HO)-1. In essential hypertensive patients olmesartan reduced OxSt and markers of CV remodeling and increased HO-1. This study reports in essential hypertensive patients the effect of 6 months treatment with olmesartan on plasma level of CGRP and number and survival of cEPCs. METHODS AND RESULTS: In 20 essential hypertensive patients treated with olmesartan medoxomil (20  mg per day for 6 months), cEPCs (CD34(+)KDR(+), CD133(+)KDR(+) and CD34(+)CD133(+)KDR(+)) (direct 3-color flow cytometry analysis), apoptosis of cEPCs (CD133(+)KDR(+) cells with Annexin V expression), CGRP determination (ELISA) and HO-1 protein level (western blot) were assessed at baseline and after 3 and 6 months of treatments. Olmesartan normalized blood pressure (P < 0.001), increased cEPCs from baseline (CD34(+)KDR(+): P < 0.003; CD133(+)KDR(+): P < 0.0002; CD34(+)CD133(+)KDR(+): P = 0.0008), reduced cEPCs apoptosis (P < 0.001) and increased CGRP (P < 0.013) and HO-1 (P = 0.039). CONCLUSION: These results provide a mechanistic rationale for the olmesartan's antioxidant and anti-inflammatory potential translation toward antiatherosclerotic and antiremodeling effects reported on clinical ground.

Myeloid calcifying cells promote atherosclerotic calcification via paracrine activity and allograft inflammatory factor-1 overexpression.

Several cell types contribute to atherosclerotic calcification. Myeloid calcifying cells (MCCs) are monocytes expressing osteocalcin (OC) and bone alkaline phosphatase (BAP). Herein, we tested whether MCCs promote atherosclerotic calcification in vivo. We show that the murine spleen contains OC(+)BAP(+) cells with a phenotype similar to human MCCs, a high expression of adhesion molecules and CD11b, and capacity to calcify in vitro and in vivo. Injection of GFP(+) OC(+)BAP(+) cells into 8- or 40-week ApoE(-/-) mice led to more extensive calcifications in atherosclerotic areas after 24 or 4 weeks, respectively, compared to control OC(-)BAP(-) cells. Despite that OC(+)BAP(+) cells had a selective transendothelial migration capacity, tracking of the GFP signal revealed that presence of injected cells within atherosclerotic areas was an extremely rare event and so GFP mRNA was undetectable by qPCR of lesion extracts. By converse, injected OC(+)BAP(+) cells persisted in the bloodstream and bone marrow up to 24 weeks, suggesting a paracrine effect. Indeed, OC(+)BAP(+) cell-conditioned medium (CM) promoted calcification by cultured vascular smooth muscle cells (VSMC) more than CM from OC(-)BAP(-) cells. A genomic and proteomic investigation of MCCs identified allograft inflammatory factor (AIF)-1 as a potential candidate of this paracrine activity. AIF-1 stimulated VSMC calcification in vitro and monocyte-specific (CD11b-driven) AIF-1 overexpression in ApoE(-/-) mice increased calcium content in atherosclerotic areas. In conclusion, we show that murine OC(+)BAP(+) cells correspond to human MCCs and promote atherosclerotic calcification in ApoE(-/-) mice, through paracrine activity and modulation of resident cells by AIF-1 overexpression.

NK cells and their receptors in naive and rituximab-treated patients with anti-MAG polyneuropathy.

BACKGROUND: Natural killer (NK) cells can bridge innate and acquired immunity, and play a role in autoimmunity. A few studies evaluated the distribution of NK cells and the expression of their receptors in chronic immune-mediated demyelinating polyneuropathies. We investigated NK cell distribution and NK cell receptor expression in 20 naïve patients with anti-MAG polyneuropathy (MAG-PN). METHODS: Using flow cytometry, we analysed NK cells and a series of NK cell receptors in the peripheral blood of patients with MAG-PN, and, as controls, in patients with chronic inflammatory demyelinating peripheral polyradiculoneuropathy (CIDP) and in healthy subjects. Six MAG-PN patients were also tested after rituximab treatment. RESULTS: At baseline the percentage of NK cells did not differ among the groups. KIR2DL2 receptor expression in MAG-PN patients was higher, andCD94/NKG2A receptor expression in both MAG-PN and CIDP patients was lower than in healthy controls. These abnormalities did not correlate with any clinical or demographic variable. No modification was found after rituximab therapy. CONCLUSIONS: The data suggest that MAG-PN shows abnormalities in NK cell receptors that characterise other autoimmune diseases, and cannot help in differential diagnosis with CIDP. The impairment of the relevant CD94/NKG2A inhibitory pathway, which might play a central role in the development and perpetuation of MAG-PN, warrants further functional investigations.

Intrinsic and extrinsic mechanisms contribute to maintain the JAK/STAT pathway aberrantly activated in T-type large granular lymphocyte leukemia.

The JAK/STAT pathway is altered in T-cell large granular lymphocytic leukemia. In all patients, leukemic LGLs display upregulation of phosphorylated STAT3 (P-STAT3) that activates expression of many antiapoptotic genes. To investigate the mechanisms maintaining STAT3 aberrantly phosphorylated using transcriptional protein and functional assays, we analyzed interleukin (IL)-6 and suppressor of cytokine signaling-3 (SOCS3), 2 key factors of the JAK/STAT pathway that induce and inhibit STAT3 activation, respectively. We showed that IL-6 was highly expressed and released by the patients' peripheral blood LGL-depleted population, accounting for a trans-signaling process. By neutralizing IL-6 or its specific receptor with specific antibodies, a significant reduction of P-STAT3 levels and, consequently, LGL survival was demonstrated. In addition, we found that SOCS3 was down-modulated in LGL and unresponsive to IL-6 stimulation. By treating neoplastic LGLs with a demethylating agent, IL-6-mediated SOCS3 expression was restored with consequent P-STAT3 and myeloid cell leukemia-1 down-modulation. Methylation in the SOCS3 promoter was not detectable, suggesting that an epigenetic inhibition mechanism occurs at a different site. Our data indicate that loss of the inhibitor SOCS3 cooperates with IL-6 to maintain JAK/STAT pathway activation, thus contributing to leukemic LGL survival, and suggest a role of demethylating agents in the treatment of this disorder.

Diabetes impairs stem cell and proangiogenic cell mobilization in humans.

OBJECTIVE: Diabetes mellitus (DM) increases cardiovascular risk, at least in part, through shortage of vascular regenerative cells derived from the bone marrow (BM). In experimental models, DM causes morphological and functional BM alterations, but information on BM function in human DM is missing. Herein, we sought to assay mobilization of stem and proangiogenic cells in subjects with and without DM. RESEARCH DESIGN AND METHODS: In a prospective trial (NCT01102699), we tested BM responsiveness to 5 µg/kg human recombinant granulocyte colony-stimulating factor (hrG-CSF) in 24 individuals with DM (10 type 1 and 14 type 2) and 14 individuals without DM. Before and 24 h after hrG-CSF, we quantified circulating stem/progenitor cells and total and differential white blood cell counts. We also evaluated in vivo the proangiogenic capacity of peripheral blood mononuclear cells using the Matrigel plug assay. RESULTS: In response to hrG-CSF, levels of CD34(+) cells and other progenitor cell phenotypes increased in subjects without DM. Patients with DM had significantly impaired mobilization of CD34(+), CD133(+), and CD34(+)CD133(+) hematopoietic stem cells and CD133(+)KDR(+) endothelial progenitors, independently of potential confounders. The in vivo angiogenic capacity of peripheral blood mononuclear cells significantly increased after hrG-CSF in control subjects without DM, but not in patients with DM. DM was also associated with the inability to upregulate CD26/DPP-4 on CD34(+) cells, which is required for the mobilizing effect of granulocyte colony-stimulating factor. CONCLUSIONS: Stem and proangiogenic cell mobilization in response to hrG-CSF is impaired in DM, possibly because of maladaptive CD26/DPP-4 regulation. These alterations may hamper tissue repair and favor the development of cardiovascular complications.

Stem cell compartmentalization in diabetes and high cardiovascular risk reveals the role of DPP-4 in diabetic stem cell mobilopathy.

Bone marrow (BM) derived stem and progenitor cells contribute to cardiovascular homeostasis and are affected by cardiovascular risk factors. We devised a clinical data-driven approach to test candidate stem cell mobilizing mechanisms in pre-clinical models. We found that PB and BM CD34+ cell counts were directly correlated, and that most circulating CD34+ cells were viable, non-proliferating and derived from the BM. Thus, we analyzed PB and BM CD34+ cell levels as a two-compartment model in 72 patients with or without cardiovascular disease. Self-organizing maps showed that disturbed compartmentalization of CD34+ cells was associated with aging and cardiovascular risk factors especially diabetes. High activity of DPP-4, a regulator of the mobilizing chemokine SDF-1α, was associated with altered stem cell compartmentalization. For validation of these findings, we assessed the role of DPP-4 in the BM mobilization response of diabetic rats. Diabetes differentially affected DPP-4 activity in PB and BM and impaired stem/progenitor cell mobilization after ischemia or G-CSF administration. DPP-4 activity in the BM was required for the mobilizing effect of G-CSF, while in PB it blunted ischemia-induced mobilization. Indeed, DPP-4 deficiency restored ischemia (but not G-CSF)-induced stem cell mobilization and improved vascular recovery in diabetic animals. In conclusion, the analysis of stem cell compartmentalization in humans led us to discover mechanisms of BM unresponsiveness in diabetes determined by tissue-specific DPP-4 dysregulation.

Increased tissue endothelial progenitor cells in end-stage lung diseases with pulmonary hypertension.

BACKGROUND: Diffuse lung diseases promote the development of vascular changes and pulmonary hypertension (PH). Endothelial progenitor cells (EPCs) seem to be involved in pulmonary vascular remodeling. We evaluated circulating and intra-pulmonary EPCs in end-stage lung diseases in relation to pulmonary arterial pressure (PAP). METHODS: The study included 19 patients affected by different end-stage lung diseases, with or without PH. Six lung donors were considered as control group. EPCs were measured in blood samples taken at the time of transplant from pulmonary arteries and veins (by flow cytometry) as well as in lung specimen sections (by confocal microscopy) and expressed as percentage of total number of cells. RESULTS: The amount of EPC in lung specimens was significantly different according to type of disease (p = 0.001). Specifically, a higher number of EPCs was detected in idiopathic pulmonary hypertension and idiopathic pulmonary fibrosis with high (> 25 mm Hg) mean PAP (p = 0.03 for both) compared with chronic obstructive pulmonary disease and control group. There was a direct correlation between intrapulmonary EPCs and PAP. According to receiver operating characteristic curve analysis, the presence of > 3% EPCs had a 91% sensitivity and 93% specificity in identifying high mean PAP. There were no differences in circulating arterial or venous EPCs among groups. CONCLUSIONS: Intra-pulmonary EPCs are increased in lung diseases with high PAP, suggesting that EPCs may contribute to vascular remodeling in end-stage pulmonary disease.

Procalcific phenotypic drift of circulating progenitor cells in type 2 diabetes with coronary artery disease.

Diabetes mellitus (DM) alters circulating progenitor cells relevant for the pathophysiology of coronary artery disease (CAD). While endothelial progenitor cells (EPCs) are reduced, there is no data on procalcific polarization of circulating progenitors, which may contribute to vascular calcification in these patients. In a cohort of 107 subjects with and without DM and CAD, we analyzed the pro-calcific versus endothelial differentiation status of circulating CD34+ progenitor cells. Endothelial commitment was determined by expression of VEGFR-2 (KDR) and pro-calcific polarization by expression of osteocalcin (OC) and bone alkaline phosphatase (BAP). We found that DM patients had significantly higher expression of OC and BAP on circulating CD34+ cells than control subjects, especially in the presence of CAD. In patients with DM and CAD, the ratio of OC/KDR, BAP/KDR, and OC+BAP/KDR was about 3-fold increased than in other groups. EPCs cultured from DM patients with CAD occasionally formed structures highly suggestive of calcified nodules, and the expression of osteogenic markers by EPCs from control subjects was significantly increased in response to the toll-like receptor agonist LPS. In conclusion, circulating progenitor cells of diabetic patients show a phenotypic drift toward a pro-calcific phenotype that may be driven by inflammatory signals.

Endothelial progenitor cells, bronchopulmonary dysplasia and other short-term outcomes of extremely preterm birth.

AIM: To evaluate the impact of endothelial progenitor cells (EPCs), a subset of committed circulatory stem cells, on the development of bronchopulmonary dysplasia (BPD) and other short term outcomes in a cohort of extremely premature newborns. METHODS: Progenitor cells were quantified by flow cytometry at birth in 36 neonates born <=28 weeks of gestation and at 36 postmenstrual weeks in 18 of them. Cells expressing the stemness markers CD34, CD133, or both were defined as circulating progenitor cells (CPCs). EPCs were defined as CPCs co-expressing the endothelial marker KDR. RESULTS: Mean (SD) gestational age and birth weight of the infants studied were 26.2(1.5) weeks and 761.6(171.8) grams, respectively. EPC levels at birth did not differ between infants who subsequently developed BPD (n=9) and those who did not (n=24) [CD34(+)KDR(+) EPCs: 81(34-41) vs 80(56-110), p=0.7] and were not correlated with the duration of mechanical ventilation or O2-dependence, nor with the need of surfactant replacement. Infants with a hemodynamically significant patent ductus arteriosus (PDA) (n=22) had significantly lower EPC levels at birth than those with no PDA (n=11) [CD34(+)KDR(+) cells: 47(34-92) vs 142(84.5-221), p=0.008]. Data from the 18 infants studied both at birth and at 36 postmenstrual weeks showed that, while CPCs sharply decline over time, levels of all EPCs phenotypes are preserved after delivery. CONCLUSIONS: Levels of EPCs at birth did not affect the risk of developing BPD in our group of extremely premature neonates. However, the association between low EPC counts at birth and PDA may be clinically relevant, and deserves further studies.

Widespread increase in myeloid calcifying cells contributes to ectopic vascular calcification in type 2 diabetes.

RATIONALE: Acquisition of a procalcific phenotype by resident or circulating cells is important for calcification of atherosclerotic plaques, which is common in diabetes. OBJECTIVE: We aim to identify and characterize circulating calcifying cells, and to delineate a pathophysiological role for these cells in type 2 diabetes. METHODS AND RESULTS: We demonstrate for the first time that a distinct subpopulation of circulating cells expressing osteocalcin and bone alkaline phosphatase (OC(+)BAP(+)) has procalcific activity in vitro and in vivo. The study of naïve patients with chronic myeloid leukemia indicated that OC(+)BAP(+) cells have a myeloid origin. Myeloid calcifying OC(+)BAP(+) cells (MCCs) could be differentiated from peripheral blood mononuclear cells, and generation of MCCs was closely associated with expression of the osteogenic transcription factor Runx2. In gender-mismatched bone marrow-transplanted humans, circulating MCCs had a much longer half-life compared with OC(-)BAP(-) cells, suggesting they belong to a stable cell repertoire. The percentage of MCCs was higher in peripheral blood and bone marrow of type 2 diabetic patients compared with controls but was lowered toward normal levels by optimization of glycemic control. Furthermore, diabetic carotid endoarterectomy specimens showed higher degree of calcification and amounts of cells expressing OC and BAP in the α-smooth muscle actin-negative areas surrounding calcified nodules, where CD68(+) macrophages colocalize. High glucose increased calcification by MCCs in vitro, and hypoxia may regulate MCC generation in vitro and in vivo. CONCLUSIONS: These data identify a novel type of blood-derived procalcific cells potentially involved in atherosclerotic calcification of diabetic patients.

KIR/HLA-I mismatching and risk of relapse in paediatric patients undergoing non-haploidentical allogeneic haematopoietic stem cell transplantation.

In HSCT setting, KIR-driven alloreactivity might be better predicted if the donor KIR genotype is considered in addition to the recipient HLA genotype. The prediction of NK cell alloreactivity relies on the missing ligand in the recipient, a scenario that can be found in HLA-identical and non-identical allotransplants. The aim of this study was to investigate at genetic level the prognostic impact of recipient HLA-I lacking for donor KIR on allotransplanted patients outcome. We analysed donors KIR genotype and HLA genotype of 60 paediatric patients who received related (n=15) or unrelated (n=45) transplantation. When patients were grouped based on the KIR gene type involved in the KIR/HLA-I mismatch, we did not observe any relapse in the group of patients characterized by mismatches involving only inhibitory KIR. On the contrary, all relapses were observed in patients showing at least one activating gene involved in the mismatch (p<0.05). Although the biological mechanism accounting for this putative genetic rule is still to be clarified, we suggest that a careful survey of KIR/HLA-I mismatching should be taken into account in the selection of donor in related and unrelated HSCT.