Lazzaro Di Mauro

Predictive parameters for mobilized peripheral blood CD34+ progenitor cell collection in patients with hematological malignancies

In order to investigate what is the best single parameter to predict the leukapheretic yield of circulating CD34+ progenitor cells, we retrospectively analyzed data from 68 patients with hematological malignancies who underwent mobilizing therapy. Three main parameters were monitored: total white blood cell (WBC), CD34+ cells, and monocyte counts in peripheral blood (PB) at the same day and at the preceding day of the apheretic procedure. Linear regression analysis revealed a strong correlation between CD34+ cell value in PB just before harvest and the number of CD34+ cells collected (P < 0.0001), but not at the preceding day. Monocyte PB concentration and absolute WBC count did not correlate with CD34+ cells harvested, at the preceding day of leukapheresis as well as at the same day of the procedure. The number of CD34+ cells in mobilized PB at the same day of harvest evidenced a very good capacity of predicting the value of harvested CD34+ cell number after collection, while WBC and monocyte count displayed quite a wide dispersion of results. In particular, an amount greater than 50/μL of circulating CD34+ cells ensured the best collections. Finally, CD34+ and CFU‐GM content evaluated for each apheresis showed a strong reciprocal correlation (r 0.78; P < 0.0001). We conclude that the absolute number of CD34+ cells at the day of leukapheresis is the only parameter for identifying the exact timing for apheresis and predicting the amount of peripheral blood progenitor cells (PBPCs) that will be collected. In this setting, WBC and monocyte counts, at the day of collection or at the preceding day, are not useful tools. Am. J. Hematol. 58:255–262, 1998.

MiRNAs and piRNAs from bone marrow mesenchymal stem cell extracellular vesicles induce cell survival and inhibit cell differentiation of cord blood hematopoietic stem cells: a new insight in transplantation

Hematopoietic stem cells (HSC), including umbilical cord blood CD34+ stem cells (UCB-CD34+), are used for the treatment of several diseases. Although different studies suggest that bone marrow mesenchymal stem cells (BM-MSC) support hematopoiesis, the exact mechanism remains unclear. Recently, extracellular vesicles (EVs) have been described as a novel avenue of cell communication, which may mediate BM-MSC effect on HSC. In this work, we studied the interaction between UCB-CD34+ cells and BM-MSC derived EVs. First, by sequencing EV derived miRNAs and piRNAs we found that EVs contain RNAs able to influence UCB-CD34+ cell fate. Accordingly, a gene expression profile of UCB-CD34+ cells treated with EVs, identified about 100 down-regulated genes among those targeted by EV-derived miRNAs and piRNAs (e.g. miR-27b/MPL, miR-21/ANXA1, miR-181/EGR2), indicating that EV content was able to modify gene expression profile of receiving cells. Moreover, we demonstrated that UCB-CD34+ cells, exposed to EVs, significantly changed different biological functions, becoming more viable and less differentiated. UCB-CD34+ gene expression profile also identified 103 up-regulated genes, most of them codifying for chemokines, cytokines and their receptors, involved in chemotaxis of different BM cells, an essential function of hematopoietic reconstitution. Finally, the exposure of UCB-CD34+ cells to EVs caused an increased expression CXCR4, paralleled by an in vivo augmented migration from peripheral blood to BM niche in NSG mice. This study demonstrates the existence of a powerful cross talk between BM-MSC and UCB-CD34+ cells, mediated by EVs, providing new insight in the biology of cord blood transplantation.

Treatment optimization and prediction of HCV clearance in patients with acute HCV infection

BACKGROUND & AIMS The lack of consensus on the optimal timing, regimen, and duration of treatment, in patients with acute HCV infection, stimulates the research on both favourable outcome predictors and individualized treatment regimens. This study aimed at investigating the impact of IL28B SNP rs12979860 alone or in combination with HLA class II alleles in both predicting spontaneous viral clearance and individualizing treatment strategies for patients with HCV persistence, after acute HCV exposure. METHODS 178 patients with AHC, consecutively treated with interferon alone or in combination with ribavirin, starting within or after 48 weeks from the diagnosis of AHC, were tested for IL28B SNPs and HLA class II alleles. RESULTS Spontaneous viral clearance was achieved in 28% of 169 patients available for genetic testing. Factors associated with HCV elimination were jaundice (OR 2.75, 95% CI 1.31-5.77) and IL28B CC (OR 3.87, CI 1.71-8.51), but not HLA alleles. In CT/TT patients without jaundice, NPV for virus persistence was 98%. In patients with IL28B CT/TT, starting treatment 48 weeks after the onset was significantly associated with lower rates of response (28% vs. 100%, p=0.027). By contrast, no significant differences in the rate of SVR were observed for CC carriers who started treatment later (65% vs. 85%, p=1.0). CONCLUSIONS In patients with acute HCV hepatitis, lack of viral clearance may be predicted by absence of jaundice and IL28B CT/TT genotype; in patients with these characteristics, treatment needs to be started immediately.

GALNT2 Expression Is Reduced in Patients with Type 2 Diabetes: Possible Role of Hyperglycemia

Impaired insulin action plays a major role in the pathogenesis of type 2 diabetes, a chronic metabolic disorder which imposes a tremendous burden to morbidity and mortality worldwide. Unraveling the molecular mechanisms underlying insulin resistance would improve setting up preventive and treatment strategies of type 2 diabetes. Down-regulation of GALNT2, an UDPN-acetyl-alpha-D-galactosamine polypeptideN-acetylgalactosaminyltransferase-2 (ppGalNAc-T2), causes impaired insulin signaling and action in cultured human liver cells. In addition, GALNT2 mRNA levels are down-regulated in liver of spontaneously insulin resistant, diabetic Goto-Kakizaki rats. To investigate the role of GALNT2 in human hyperglycemia, we measured GALNT2 mRNA expression levels in peripheral whole blood cells of 84 non-obese and 46 obese non-diabetic individuals as well as of 98 obese patients with type 2 diabetes. We also measured GALNT2 mRNA expression in human U937 cells cultured under different glucose concentrations. In vivo studies indicated that GALNT2 mRNA levels were significantly reduced from non obese control to obese non diabetic and to obese diabetic individuals (p<0.001). In vitro studies showed that GALNT2 mRNA levels was reduced in U937 cells exposed to high glucose concentrations (i.e. 25 mmol/l glucose) as compared to cells exposed to low glucose concentration (i.e. 5.5 mmol/l glucose +19.5 mmol/l mannitol). In conclusion, our data indicate that GALNT2 is down-regulated in patients with type 2 diabetes and suggest that this association is, at least partly, secondary to hyperglycemia. Further studies are needed to understand whether GALNT2 down-regulation plays a pathogenic role in maintaining and/or aggravating the metabolic abnormalities of diabetic milieu.

IL28B CC-genotype association with HLA-DQB1*0301 allele increases the prediction of spontaneous HCV RNA clearance in thalassaemic HCV-infected patients.

BACKGROUND A single nucleotide polymorphism (SNP), upstream of the IL28B gene has been recently associated with natural clearance of HCV. In a well-characterized cohort of patients with thalassaemia major exposed to the risk of acquiring HCV infection by blood transfusions, we aimed to replicate this finding and to evaluate whether combining the IL28B genotype and HLA class II alleles allow viral clearance to be accurately predicted. METHODS Of 168 patients, 130 with complete clinical history were included in the analysis. According with their HCV antibodies status 13 were defined HCV resistant, and 117 infected. Infected patients were subdivided, giving 49 with self-limiting and 68 with ongoing infection. RESULTS IL28B CC-genotype was observed in 32 patients with self-limiting and in 23 with ongoing infection (64% versus 34%; P=0.004). HLA DQB1*0301 allele was associated with viral clearance in 36 cases (73%; P<0.0001). Both DQB1*0301 and IL28B CC-genotype were found to be independent predictors of HCV clearance (OR=5.64, 95% CI 1.52-20.9 and OR=5.76, 95% CI 2.16-15.33, respectively). With the addition of DQB1*0301, the accuracy of the prediction increased from 63% to 69%. CONCLUSIONS In addition to IL28B CC-genotype, HLA DQB1*0301 helps in predicting natural clearance of HCV after acute infection.

Time related variations in stem cell harvesting of umbilical cord blood

Umbilical cord blood (UCB) contains hematopoietic stem cells and multipotent mesenchymal cells useful for treatment in malignant/nonmalignant hematologic-immunologic diseases and regenerative medicine. Transplantation outcome is correlated with cord blood volume (CBV), number of total nucleated cells (TNC), CD34+ progenitor cells and colony forming units in UCB donations. Several studies have addressed the role of maternal/neonatal factors associated with the hematopoietic reconstruction potential of UCB, including: gestational age, maternal parity, newborn sex and birth weight, placental weight, labor duration and mode of delivery. Few data exist regarding as to how time influences UCB collection and banking patterns. We retrospectively analyzed 17.936 cord blood donations collected from 1999 to 2011 from Tuscany and Apulia Cord Blood Banks. Results from generalized multivariable linear mixed models showed that CBV, TNC and CD34+ cell were associated with known obstetric and neonatal parameters and showed rhythmic patterns in different time domains and frequency ranges. The present findings confirm that volume, total nucleated cells and stem cells of the UCB donations are hallmarked by rhythmic patterns in different time domains and frequency ranges and suggest that temporal rhythms in addition to known obstetric and neonatal parameters influence CBV, TNC and CD34+ cell content in UBC units.

Role of CYP2D6 Polymorphisms in the Outcome of Postoperative Pain Treatment.

OBJECTIVE To investigate the role of CYP2D6 phenotype in the outcome of postoperative (PO) pain (POP) treatment. DESIGN Longitudinal cohort study. Open-label trial with post hoc analysis. SETTING General Hospital Surgery and Recovery Units. PATIENTS Ninety unrelated Caucasians submitted to abdominal/thoracic surgery. INTERVENTIONS Standard multimodal POP treatment including opioids (tramadol) and nonsteroidal anti-inflammatory drugs (ketoprofen) at different dosages and infusion rates according to the predicted mild, moderate, or severe POP. OUTCOME MEASURES Pain (Numeric Rating Scale-NRS) and sedation (Ramsay Sedation Scale-RSS) up to 24 hours after surgery. By genotyping 16 CYP2D6 alleles, the four CYP2D6 phenotypes poor metabolizer (PM), intermediate metabolizers (IM), extensive metabolizers (EM) and ultrarapid metabolizers (UM) were predicted. RESULTS As compared with the CYP2D6-EM phenotype, in the early PO time (30 min) a higher RSS mean score in IM was observed (P = 0.035). A suggestion towards higher mean score in PM (P = 0.091) and a minor mean score in UM (P = 0.091) was also detected. No difference in the outcome of pain across the CYP2D6 phenotypes was observed. CONCLUSIONS In respect to the normal CYP2D6 phenotype, our results suggested that slowly metabolizers (IMs and PMs) might have a major sedation, whereas more rapid metabolizers (UM) a minor sedation, in the early time after surgery. A minor role of CYP2D6 phenotype in PO analgesia may be suggested.

Normoalbuminuric renal impairment and all-cause mortality in type 2 diabetes mellitus

A growing body of evidences indicate that, in patients with type 2 diabetes mellitus (T2DM), reduced glomerular filtration rate (GFR) is a strong predictor not only of endstage renal disease but also of all-cause mortality [1]. The relationship between low GFR and mortality rate in diabetic patients with normoalbuminuria, a condition known as normoalbuminuric renal impairment [2], is still debated [1, 3] and has been here investigated. We studied two previously described cohorts of patients with T2DM, followed-up for all-cause mortality [4]: the ‘‘Gargano Mortality Study’’ (GMS, n = 786) and the ‘‘Foggia Mortality Study’’ (FMS, n = 972). Estimated GFR (eGFR) from serum creatinine by Epidemiology Chronic Kidney Disease formula was defined as low when \60 ml/min/1.73 m. Albuminuria (albumin/creatinine ratio, ACR) was defined as normal when\2.5/3.5 mg/ mmol in males/females. In GMS (follow-up = 7.4 years) and in FMS (followup = 4.0 years), 156 and 135 patients died with a similar age adjusted annual incidence rate of 2.0 and 2.1 %, respectively. The proportion of patients with low eGFR (i.e., eGFR \60/ml/min/1.73 m) was 20.1 % in the GMS and 22.2 % in the FMS. To increase statistical power, the two samples were pooled (n = 1,758) and stratified in four groups on the basis of the presence/absence of albuminuria and low eGFR. While patients with no albuminuria and no low eGFR had the lowest mortality rate (1.6 % personyear), those with both albuminuria and low eGFR had the highest one (8.4 % person-year), with the other two groups being at intermediate risk (Table 1). Of note in the specific context of our primary aim, as compared to individuals with normal eGFR, those with low eGFR had a similarly increased mortality risk in patients with normoalbuminuria and in those with albuminuria (HR = 1.74, P = 0.002 and 1.67, P = 0.006, after adjusting for sex, age, HbA1c,

The circadecadal rhythm of oscillation of umbilical cord blood parameters correlates with geomagnetic activity – An analysis of long-term measurements (1999–2011)

ABSTRACT Recently, we have shown that the contents of total nucleated cells (TNCs) and CD34+ hematopoietic stem and progenitor cells (CD34+ HSPCs) as well as the cord blood volume (CBV) in umbilical cord blood (UCB) show a circadecadal (~10 years) rhythm of oscillation. This observation was based on an analysis of 17,936 cord blood donations collected during 1999–2011. The aim of the present study was to investigate whether this circadecadal rhythm of oscillation in TNCs, CD34+ HSPCs and CBV is related to geomagnetic activity. For the analysis, the yearly averages of TNCs, CD34+ HSPCs and CBV in UCB were correlated with geomagnetic activity (Dcx index). Our analysis revealed that (i) all three UCB parameters were statistically significantly correlated with the level of geomagnetic activity, (ii) CBV showed a linear correlation with the Dcx index (r = 0.5290), (iii) the number of TNCs and CD34+ HSPCs were quadratic inversely correlated with the Dcx index (r = −0.5343 and r = −0.7749, respectively). Furthermore, (iv) CBV and the number of TNCs were not statistically significantly correlated with the number of either modest or intense geomagnetic storms per year, but (v) the number of CD34+ HSPCs was statistically significantly correlated with the number of modest (r = 0.9253) as well as intense (r = 0.8683) geomagnetic storms per year. In conclusion, our study suggests that UCB parameters correlate with the state of the geomagnetic field (GMF) modulated by solar activity. Possible biophysical mechanisms underlying this observation, as well as the outcome of these findings, are discussed.

Multicentre standardisation of a clinical grade procedure for the preparation of allogeneic platelet concentrates from umbilical cord blood.

BACKGROUND In addition to a largely prevalent use for bleeding prophylaxis, platelet concentrates from adult blood have also been used for many years to prepare platelet gels for the repair of topical skin ulcers. Platelet gel can be obtained by activation of fresh, cryopreserved, autologous or allogeneic platelet concentrates with calcium gluconate, thrombin and/or batroxobin. The high content of tissue regenerative factors in cord blood platelets and the widespread availability of allogeneic cord blood units generously donated for haematopoietic transplant but unsuitable for this use solely because of low haematopoietic stem cell content prompted us to develop a national programme to standardise the production of allogeneic cryopreserved cord blood platelet concentrates (CBPC) suitable for later preparation of clinical-grade cord blood platelet gel. MATERIALS AND METHODS Cord blood units collected at public banks with total nucleated cell counts <1.5×10(9), platelet count >150×10(9)/L and volume >50 mL, underwent soft centrifugation within 48 hours of collection. Platelet-rich plasma was centrifuged at high speed to obtain a CBPC with target platelet concentration of 800-1,200×10(9)/L, which was cryopreserved, without cryoprotectant, below -40 °C. RESULTS During 14 months, 13 banks produced 1,080 CBPC with mean (± standard deviation) volume of 11.4±4.4 mL and platelet concentration of 1,003±229×10(9)/L. Total platelet count per CBPC was 11.3±4.9×10(9). Platelet recovery from cord blood was 47.7±17.8%. About one-third of cord blood units donated for haematopoietic transplant could meet the requirements for preparation of CBPC. The cost of preparation was € 160.92/CBPC. About 2 hours were needed for one technician to prepare four CBPC. DISCUSSION This study yielded valuable scientific and operational information regarding the development of clinical trials using allogeneic CBPC.