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.

Low CD34(+) cells, high neutrophils and the metabolic syndrome are associated with an increased risk of venous thromboembolism.

The relationship between MetS (metabolic syndrome), levels of circulating progenitor/immune cells and the risk of VTE (venous thromboembolism) has not yet been investigated. We studied 240 patients with previous VTE and 240 controls. The presence of MetS was identified according to NCEP ATP III guidelines and flow cytometry was used to quantify circulating CD34(+) cells. VTE patients showed higher BMI (body mass index), waist circumference, triacylglycerol (triglyceride) levels, blood glucose, hs-CRP (high-sensitivity C-reactive protein) and lower HDL-C (high-density lipoprotein cholesterol) levels. The prevalence of MetS was significantly higher in VTE (38.3%) than in control individuals (21.3%) with an adjusted OR (odds ratio) for VTE of 1.96 (P=0.002). VTE patients had higher circulating neutrophils (P<0.0001), while the CD34(+) cell count was significantly lower among patients with unprovoked VTE compared with both provoked VTE (P=0.004) and controls (P=0.003). Subjects were also grouped according to the presence/absence of MetS (MetS(+) or MetS(-)) and the level (high/low) of both CD34(+) cells and neutrophils. Very high adjusted ORs for VTE were observed among neutrophils_high/MetS(+) (OR, 3.58; P<0.0001) and CD34(+)_low/MetS(+) (OR, 3.98; P<0.0001) subjects as compared with the neutrophils_low/MetS(-) and CD34(+)_high/MetS(-) groups respectively. In conclusion, low CD34(+) blood cell count and high circulating neutrophils interplay with MetS in raising the risk for venous thromboembolic events.

New method to produce hemocomponents for regenerative use from peripheral blood: integration among platelet growth factors monocytes and stem cells.

Recent studies have shown the importance of monocytes/macrophageses and of CD34+ progenitors in tissue regeneration processes. These cells, obtained generally from bone marrow, are seen in damaged tissue. We have studied a method to collect from the peripheral blood, using a cell separator and without stimulation of the patient/donor, a leukocyte platelet concentrated hemocomponent (CLP) for regenerative use which contains platelets, monocytes/macrophages, fibrinogen and CD34+ cells. We appraised the composition and cell functionality of the final hemocomponent during production and cryoconservation. The results show a positive increase in concentration values, in comparison with the pre-collection, of the cells that were involved in regeneration; i.e. the platelets, monocytes and CD34+ cells. These concentrations were also maintained at an effective level during cryoconservation of the hemocomponent. The CLP also demonstrated positive clonogenic potential in culture, showing that the CD34+ progenitors involved in CFU formation are functional in the fresh and thawed product. In brief we have shown that it is possible to produce, in a simple way, a hemocomponent for regenerative use that is standardized, reliable, and is economically feasible.

Naïve/Effector CD4 T cell ratio as a useful predictive marker of immune reconstitution in late presenter HIV patients: A multicenter study.

A significant proportion of HIV-infected patients experiencing a late diagnosis highlights the need to define immunological protocols able to help the clinicians in identifying patients at higher risk for immunological failure. The aim of the study was to evaluate the feasibility of easy cytometric tests in defining the effect of antiretroviral treatment (cART) on immunological homeostasis and in identifying predictive markers of early immune recovery. Chronic HIV infected patients (n = 202) were enrolled in a prospective multicentric study, and their immunological profile was studied before (w0) and after 24 weeks (w24) of antiretroviral treatment (cART) using a standardized flow cytometric panel. Based on CD4 T cell count before treatment, patients were divided in late (LP: CD4 <350/mmc), intermediate (IP: 350/mmc500/mmc) presenters. In all groups, cART introduction increased CD4 and CD4/CD8 T cell ratio, naïve T cell (CD4 and CD8) and CD127-expressing CD4 T cells. In parallel, cART significantly reduced effector memory T cells (CD4 and CD8) and T cell activation (CD38+CD8 and CD95+CD4 T cells). Moreover, the frequency of Naïve and Effector CD4 T cells before treatment correlated with several immune parameters key associated with the pathogenesis of HIV, thus mirroring the health of immune system. Interestingly, we identified the Naïve/Effector CD4 T cell ratio (N/EM) at w0 as a marker able to predict early immune recovery. Specifically, in LP, N/EM ratio was significantly higher in immunological responder patients (CD4>500/mmc at w24) when compared to immunological non responder (CD4 T cells <500/mmc at w24). Finally, a multivariate analysis indicates that after 24w patients with N/EM ratio higher than 1.86 at w0 recovered 96 CD4 T cells more than those with N/EM ratio lower than 0.46. Altogether, our data define an easy protocol able to define reliable immunological markers useful for the characterization of immune profile in viremic HIV patients and identify the naïve/effector CD4 T cell ratio as a new tool able to predict an early immune reconstitution potential.

Adrenomedullin and endothelin-1 stimulate in vitro expansion of cord blood hematopoietic stem cells.

The improvement of techniques for in vitro expansion of cord blood (CB) hematopoietic stem cells (SCs) is, at present, one main task of tissue engineering. Hence, we investigated whether endothelin-1 (ET-1) and adrenomedullin (AM), two regulatory peptides exerting growth promoting action on several cell systems, favor the in vitro expansion of CB SCs in liquid culture. CB hematopoietic cell middle-term expansion was carried out in a stroma-free liquid culture medium in the presence of ET-1, AM and three different cytokine combinations. After two weeks of incubation, aliquots of expanded-cell suspension were seeded on semisolid medium and clonogenic tests were carried out by counting the number of colony forming units (CFUs) after 14 days of culture. Neither ET-1 nor AM (2.5 x 10(-8) M) were per se able to significantly increase the CFU number, but both peptides magnified the pro-expansive effects of some cytokine cocktails. In light of these findings, we conclude that ET-1 and AM are to be considered novel promising molecules that, in association with cytokines, can be utilized as pro-expansive factors of CB SCs in prevision of their clinical use in allogeneic transplantation.

Effect of cryopreservation on in vitro clonal growth of cordonal blood cells.

Cordonal blood (CB) is today recognized as a potentially important source of hematopoietic stem cells (SCs) for allogeneic transplantation, and to this task it would be of extreme importance to have the possibility of using cryopreserved CB units. Hence we investigated whether freezing and thawing alter the viability of CB hematopoietic cells. Mononuclear cells, recovered from fresh CB units by density-gradient centrifugation, were partly frozen and then thawed and partly immediately utilized for clonogenic tests. The cells were cultured in H4330 (C group) or H4434 (H4330 added with SC clonogenic growth factors) (C+ group) semisolid media added or not with serum-free Dulbecco modified Eagle medium (DMEM/sf). Cells seeded on H4330 were exposed to the conditioned supernatants from two human-embryo liver cell lines, which have been previously found to stimulate clonal growth of fresh CB hematopoietic cells. As expected, the number of colony-forming units (CFU) was higher in C+ than C group, and was not influenced by the addition of DMEM/sf. CFU number was higher in cryopreserved than fresh cells in both C and C+ culture groups. Conditioned supernatants from both cell lines stimulated clonal growth in both fresh and cryopreserved cell cultures. These findings indicate that cryopreservation and thawing do not alter the viability of CB SCs, but, on the contrary, improve their basal and cytokine-stimulated clonal growth, probably by negatively selecting SCs among the mononuclear CB cell population.

New experimental models for the in vitro reconstitution of human bladder mucosa.

This study describes two experimental models for the in vitro reconstitution of the human bladder mucosa (neo-bladder): human urothelial stabilized cell lines were cultured on three-dimensional matrices, collagen or platelet-fibrin gels, containing murine fibroblast 3T3-J2 cells. Low-density seeding (2x10(4) cells/ml) of both normal (TCA-48) and neoplastic cell lines (TCA-47) on collagen matrix gave rise to isolated papillar colonies, while high-density seeding (3.75x10(6) cells/ml) led to the formation of wide pluristratified epithelial sheets, resembling the normal transitional epithelium. In contrast, high-density seeding (5x10(5) cells/ml) on platelet-fibrin matrix did not allow the formation of epithelial sheets: only isolated voluminous colonies of normal TCA-48 cells, and sparse and small colonies of neoplastic TCA-47 could be observed. Growth assays and cytotoxicity reduction tests showed that the growth inhibitory effect of platelet-fibrin gel on urothelial cells was probably due to the aspecific activation of the complement contained in the plasmatic fraction, whose precipitation forms fibrin-glue. Collectively, these findings allow us to draw the following conclusions: i) neobladders obtained by culturing urothelial cells on collagen matrix reproduce normal bladder mucosa and could be utilized in pharmacological studies; and ii) platelet-fibrin gels, that specifically inhibit neoplastic urothelial cell growth, could be used as scaffolds in surgical bladder reconstitution.

New human embryo liver cell lines obtained by stabilization and immortalization enhance in vitro clonal growth of cordonal blood cells.

We developed two new cell lines derived from embryo liver and tested their inductive capacity on in vitro clonal growth of cordonal blood (CB) hematopoietic cells. One line was stabilized and named BAEP2-WILD (W), and the other one was immortalized by retroviral transduction with SV40 Large T antigen and called BAEP2-SV40. Southern blot analysis demonstrated the integration of the Large T antigen gene in the BAEP2-SV40 cell genome, but this line did not display the expected growth arrest at the non-permissive temperature of 39 degrees C. Immunocytochemistry showed that BAEP2-SV40 cell line was positive for several cytokeratins and stromal markers (vimentin, desmin and laminin), as well as for epidermal growth factor (EGF), fibroblast growth factor (FGF) and their receptors (Rs). In contrast, BAEP2-W evidenced positivity only for cytokeratin-7 and laminin, and low positivity to EGF, EGF-R, FGF and FGF-R. BAEP2-SV40 cell line, but not BAEP2-W, expressed interleukin (IL)-1, IL-6, granulocyte macrophage-colony stimulating factor (GM-CSF), granulocyte-colony stimulating factor, stem cell factor and vascular-cell adhesion molecule-1 mRNAs, and secreted IL-6 and GM-CSF. Taken together, these findings could suggest that BAEP2-W cell line possesses the phenotype of fetal hepatocytes, while BAEP2-SV40 cell line has that of stromal cells. The supernatants conditioned by both cell lines stimulated the clonal growth of CB hematopoietic cells cultured on semisolid media deprived of growth factors and cytokines, the inductive capacity of the BAEP2-SV40 cell line being markedly higher than that of its wild counterpart, conceivably due to its ability to produce cytokines. Our study indicates that these two new cell lines, and especially BAEP2-SV40 one could be used in co-culture systems as feeder-layers for hematopoietic CB SC expansion in vitro.

Adrenomedullin is expressed in cord blood hematopoietic cells and stimulates their clonal growth.

Adrenomedullin (AM) is a hypotensive peptide, which originates from the proteolytic cleavage of pro(p)AM and acts via AM22-52-sensitive receptors. Reverse transcription polymerase chain reaction allowed the detection of the specific mRNAs of pAM in the mononuclear hematopoietic cells of the cord blood and immunocytochemistry demonstrated their abundant AM-immunoreactivity. AM (10(-8) M) markedly enhanced clonal growth of cord blood hematopoietic cells cultured on semisolid media added with stem cell-growth promoting cytokines, and this effect was abolished by AM22-52 (10(-6) M). Collectively, these findings indicate that AM is expressed in and stimulates the proliferation of cord blood hematopoietic stem cells. Cord blood has been proposed as a source of stem cells alternative to bone marrow for allogeneic transplantation, and our study suggests that AM may be used, in addition to the classic cytokines, to expand in vitro cord blood stem cells in advance of their clinical use.

Prognostic significance of circulating and endothelial progenitor cell markers in type 2 diabetic foot.

Objective. We studied circulating precursor cells (CPC) in type 2 diabetes mellitus (T2DM) with neuropathic foot lesions with or without critical limb ischemia and relationships between endothelial precursor cells (EPC) and peripheral neuropathy. Methods and Subjects. We measured peripheral blood CD34, CD133, and CD45 markers for CPC and KDR, CD31 markers for EPC by citofluorimetry and systemic neural nociceptor CGRP (calcitonin gene related protein) by ELISA in 8 healthy controls (C) and 62 T2DM patients: 14 with neuropathy (N), 20 with neuropathic foot lesions (N1), and 28 with neuroischemic recent revascularized (N2) foot lesions. Timing of lesions was: acute (until 6 weeks), healed, and not healed. Results. CD34+ and CD133+ were reduced in N, N1, and N2 versus C, and CD34+ were lower in N2 versus N1 (P = 0.03). In N2 CD34+KDR+ remain elevated in healed versus chronic lesions and, in N1 CD133+31+ were elevated in acute lesions. CGRP was reduced in N2 and N1 versus C (P < 0.04 versus C 26 ± 2 pg/mL). CD34+KDR+ correlated in N2 with oximetry and negatively in N1 with CGRP. Conclusions. CD34+ CPC are reduced in diabetes with advanced complications and diabetic foot. CD34+KDR+ and CD31+133+ EPC differentiation could have a prognostic and therapeutic significance in the healing process of neuropathic and neuroischemic lesions.