Bone marrow donor-related variables associated with harvest outcome in HLA-haploidentical transplantation with postinfusion cyclophosphamide.

BACKGROUND AND OBJECTIVES: Several transplantation outcomes have been shown to be associated with the infused bone marrow cell dose/kg of the recipient's body weight. The donor bone marrow density is directly related to the infused cell dose. The aim of the present study was to identify donor-related variables that are associated with high donor bone marrow density. MATERIALS AND METHODS: We retrospectively analysed the predictive factors of high marrow density in 65 consecutive HLA-haploidentical bone marrow donors harvested at our centre between 2009 and 2013. RESULTS: Body mass index (BMI) and peripheral white blood cell (WBC) count were directly associated with bone marrow density (regression coefficient ß = 5·33 and ß = 2·93, respectively; P < 0·01). The likelihood of obtaining a collection with a high density was first predicted using BMI (BMI ≥30, mean density = 25·8 TNC/ml × 10(6) ). Second, donors with a BMI <30 were split into two groups according to peripheral WBC count (WBC <8 × 10(3) /mm(3) : mean density = 18·4 TNC/ml × 10(6) ; WBC ≥8 × 10(3) /mm(3) : mean density = 23·1 TNC/ml × 10(6) ). We also observed that the density of the first collected bag directly correlated with the overall density (R(2) = 0·69, P < 0·01). CONCLUSION: The donor-related features BMI and WBC count affect the cell quantity obtainable with the harvest and should be taken into account when choosing the donor.

Quality control on mononuclear cells collected for extracorporeal photochemotherapy: comparison between two UV-A irradiation devices.

Recently, MacoPharma released a new UV-A cell irradiator device (Macogenic G2) for extracorporeal photopheresis (ECP), smaller and lighter than the Macogenic G1 but with no integrated cooling system. We compared the two devices at different working temperatures (G1 at standard irradiation temperature - 21°C - and G2 set by purpose at 34°C) in patients affected with chronic graft-versus-host disease and chronic lung allograft dysfunction treated by ECP. We demonstrate that both G1 and G2 devices are efficient in inducing the inhibition of lymphocytic proliferation and mononuclear cells apoptosis after 48 h even when G2 is set at higher-than-standard temperature.

Autologous CD133+ cells augment the effect of portal embolization.

AIM: The standard to treat liver tumors is a resection. When the future liver remnant (FLRV) is below 30% (healthy livers) or 40% (cirrhotic livers or previous chemotherapy), surgery carries the risk of severe complications. Portal vein embolization (PVE) gained a worldwide diffusion as a tool to augment the FLRV. Cell therapies are recent players at the frontiers of medicine. This study presents a clinical experience to evaluate the synergistic effect of combined PVE and autologous CD133+ cells coadministration. METHODS: Sixteen patients have been enrolled in the study up today. Inclusion criteria were: primary or metastatic liver malignancy with a FLRV<30% or 40%. A baseline volumetric CT-scan was obtained. CD34+ were mobilized to the blood stream by G-CSF administration and collected by immunomagnetic separation. Simultaneously with PVE, cells were administered to the non occluded liver segments. Follow-up CT scans were taken at 30th post treatment day. RESULTS: The patients (N.=6) showed an increased volume gain (Mann-Whitney test P<0.001, two sided) compared to a set of cases whose treatment was PVE only (N.=10). DISCUSSION: The use of autologous stem cells as an augmenter of liver regeneration has a clinical potential to improve the resectability of liver tumors.

[Preliminary results of a multidisciplinary approach to gastroschisis]. / Resultados iniciales de un protocolo de manejo terapéutico de la gastrosquisis.

INTRODUCTION: In gastroschisis, premature birth may avoid the development of intestinal peel and favour the primary closure. We present the preliminary results obtained after following a multidisciplinary approach to gastroschisis. After prenatal ultrasound diagnosis, preterm caesarean delivery at 34-35 weeks of gestation is programmed. METHODS: Prospective design of a study, where we included all prenatal diagnosed gastrosquisis neonates, from July 2007 to January 2012. RESULTS: We followed 9 infants (3 male). Average weight at birth: 1,927 gr. (+/- 370). Primary closure was successfully accomplished in the first 3 hours of life all cases. We found two cases of slight peel. We found no associated intestinal malformations, except for one small bowel stenosis. No significant neonatal distress respiratory syndrome developed. Mean parenteral nutrition time was 13.9 days (+/- 3.8). 4 neonates developed central line associated infection. No surgical site infection developed. Enteral nutrition was started at day 8th (+/- 2.8). Enteral requirements were fulfilled at day 15th (+/- 3.6). Mean hospital stay was 31 days (+/- 10). Mean follow-up was 30 months. 4 cases developed a small (< 5 mm) umbilical hernia CONCLUSION: Programming premature cesarean section delivery at 34 weeks of gestation was beneficial to the neonates with gastroschisis, yet it avoided peel development, and rendered primary closure without serious difficulties possible. This diminishes hypoperistalsis time and allows rapid instauration of enteral feeding, so hospital stays may be shorter.

Characterization of mammary epithelial cell line HC11 using the NIA 15k gene array reveals potential regulators of the undifferentiated and differentiated phenotypes.

Differentiation of undifferentiated mammary epithelial stem and/or progenitor cells results in the production of luminal-ductal and myoepithelial cells in the young animal and upon pregnancy, the production of luminal alveolar cells. A few key regulators of differentiation have been identified, though it is not known yet how these proteins function together to achieve their well-orchestrated products. In an effort to identify regulators of early differentiation, we screened the NIA 15k gene array of 15,247 developmentally expressed genes using mouse mammary epithelial HC11 cells as a model of differentiation. We have confirmed a number of genes preferentially expressed in the undifferentiated cells (Lgals1, Ran, Jam-A and Bmpr1a) and in those induced to undergo differentiation (Id1, Nfkbiz, Trib1, Rps21, Ier3). Using antibodies to the proteins encoded by Lgals1, and Jam-A, we confirmed that their proteins levels were higher in the undifferentiated cells. Although the amounts of bone morphogenetic protein receptor-1A (BMPR1A) protein were present at all stages, we found the activity of its downstream signal transduction pathway, as measured by the presence of phosphorylated-SMAD1, -SMAD5, and -SMAD8, is elevated in undifferentiated cells and decreases in fully differentiated cells. This evidence supports that the BMPR1A pathway functions primarily in undifferentiated mammary epithelial cells. We have identified a number of genes, of known and unknown function, that are candidates for the maintenance of the undifferentiated phenotype and for early regulators of mammary alveolar cell differentiation.

Plasmapheresis for treatment of pulmonary alveolar proteinosis.

Whole lung lavage (WLL) is currently the standard therapy for pulmonary alveolar proteinosis (PAP). Nevertheless, some PAP patients respond poorly to WLL or require it frequently. The present paper reports a patient with autoimmune PAP with persistent disease despite three WLL treatments over 10 months. Plasmapheresis with ten 1.5-L plasma exchanges was performed, which lowered the serum granulocyte-macrophage colony-stimulating factor (GM-CSF) autoantibody level from 250 microg mL(-1) to 156 microg mL(-1) but did not improve respiratory impairment. Further WLL therapy was required and transiently effective. Serum GM-CSF autoantibody levels declined progressively, reaching a value of 56 microg mL(-1) 80 weeks after completion of plasmapheresis. However, this decrease was not accompanied by clinical improvement and the patient required additional WLL therapy. The results confirm that minor reductions in serum granulocyte-macrophage colony-stimulating factor autoantibody levels from plasmapheresis are not reflected in clinical improvement in the severity of lung disease in pulmonary alveolar proteinosis.

Peripheral CD4(+)CD25(+) TREG cell counts and the response to extracorporeal photopheresis in lung transplant recipients.

Extracorporeal photopheresis (ECP) has been proposed as a possible alternative therapy for patients with bronchiolitis obliterans syndrome (BOS), with some evidence of efficacy. Although the mechanism by which ECP exerts its protective effects remains to be determined, two recent studies suggest that the modulation of transplant immune rejection may depend on the capacity to increase the number of peripheral T-regulatory (Treg) cells. We evaluated the effect of ECP treatment on the number of naturally occurring CD4(+)CD25(+) Treg cells in the peripheral blood of six lung transplant recipients: in five cases after failure of augmented or changed immunosuppression for BOS, and in one case owing to persistent acute rejection in a patient who contracted chronic hepatitis C viral infection after lung transplant. A functional stabilization was observed in three of our five patients with BOS, which was accompanied by a slight increase or stabilization of the number of peripheral blood CD4(+)CD25(high) cells with in vitro features of Treg cells. On the contrary, two patients with BOS who did not experience graft functional stabilization also showed a decline in the peripheral Treg subset. In the last patient Treg cell kinetics showed stabilization during the first 5 months of ECP treatment when lung function remained stable and graft histology normalized but showed a subsequent decrease, predating BOS diagnosis. In all, our results indicate that ECP may modulate peripheral Treg cell number but the time course of peripheral Treg cells varies according to graft function.

Long-term safety and efficacy of autologous platelet lysate drops for treatment of ocular GvHD.

Current ocular GvHD (oGvHD) treatments are suboptimal. We investigated the safety and efficacy of long-term continuous treatment with autologous platelet lysate (PL) drops in patients with oGvHD Dry Eye Syndrome (DES) score 2-3 refractory to topical conventional therapy. Ophthalmic evaluation was performed at 6 month intervals. Symptoms were assessed using the Glaucoma Symptom Scale (GSS). Patients were defined 'responders' when showing a reduction at least one grade on National Institutes of Health Eye Score from baseline at the 6 month visit. Thirty-one patients were included, and 16 (51%) completed 36 months of follow-up (range 6.5-72.7). At 6 months all patients were classified as responders: median GSS symptom score decreased from 70 to 41 (33 at 36 months), median GSS function score reduced from 68 to 46 (33 at 36 months) (all P<0.001). Median Tear Break Up Time improved from 3 to 6 s after 6 months and was maintained over time. All signs improved at 6 and 36 months (clinical and statistical significance). No severe adverse events occurred. Long-term treatment with PL drops is secure and effective for oGvHD and can be an efficient therapy option from initial stages of oGvHD to prevent permanent ocular impairment and improving quality of life.

Cord blood in vitro expanded CD41 cells: identification of novel components of megakaryocytopoiesis.

BACKGROUND: Megakaryopoiesis represents a multi-step, often unclear, process leading to commitment, differentiation, and maturation of megakaryocytes (MKs) that release platelets. AIM: To identify the novel genes that might help to clarify the molecular mechanisms of megakaryocytopoiesis and be regarded as potential candidates of inherited platelet defects, global gene expression of hematopoietic lineages was carried out. METHODS: Human cord blood was used to purify CD34+ stem cells and in vitro expand CD41+ cells and burst-forming unit-erythroid (BFU-E). We investigated the expression profiles of these three hematopoietic lineages in the Affymetrix system and selected genes specifically expressed in MKs by comparing transcripts of the different lineages using the dchip and pam algorithms. RESULTS: A detailed characterization of MK population showed that 99% of cells expressed the CD41 antigen whereas 73% were recognizable as terminally differentiated fetal MKs. The profile of these cells was compared with that of CD34+ cells and BFU-E allowing us to select 70 transcripts (MK-core), which represent not only the genes with a well-known function in MKs, but also novel genes never detected or characterized in these cells. Moreover, the specific expression was confirmed at both RNA and protein levels, thus validating the 'MK-core' isolated by informatics tools. CONCLUSIONS: This is a global gene expression that for the first time depicts a well-characterized population of cord blood-derived fetal MKs. Novel genes have been detected, such as those encoding components of the extracellular matrix and basal membrane, which have been found in the cytoplasm of Mks, suggesting that new physiological aspects of MKs should be studied.

Diagnostic value of procalcitonin measurement in febrile patients with systemic autoimmune diseases.

OBJECTIVE: To determine the usefulness of plasma procalcitonin (PCT) measurement to suspect infectious etiology in febrile patients with systemic autoimmune disease. METHODS: PCT, C-Reactive protein (CRP), erythrocyte sedimentation rate (ESR) and white blood cell count (WBC) were measured in 44 consecutive inpatients with a diagnosis of systemic autoimmune disease and fever >38 masculine C. After careful microbiologic screening no obvious infection was demonstrated in 24 patients (Group A) while an infectious bacterial complication was diagnosed in 20 cases (Group B). RESULTS: Median PCT levels were significantly higher in the group B (1.11 vs 0.24 ng/ml; p = 0.0007), whereas the differences for CRP, WBC and ESR did not reach statistical significance. PCT also exhibited a good sensitivity and specificity (75%) in differentiating patients with infection from those with disease flare. With respect to positive and negative predictive values (71.4% and 78.2%), PCT markedly exceeded the other variables. By analyzing PCT values by disease we identified a false positive subgroup of patients suffering from adult onset Still's disease (AOSD), showing markedly elevated PCT levels in absence of infection. By excluding these patients, PCT showed a very good sensitivity and specificity (73.6% and 89.4%) and the area under receiver operating characteristics (ROC) curve rose from 0.801 to 0.904. CONCLUSION: Our data indicate that elevated PCT concentrations offer good sensitivity and specificity for the diagnosis of systemic bacterial infection in febrile patients with systemic autoimmune diseases. However, in fever associated with AOSD PCT may be elevated even in the absence of infectious complication.

Concurrent chemo-radiotherapy in elderly patients: tolerance and compliance in a series of 137 patients.

INTRODUCTION: Aggressive cancer treatment is a challenge in elderly patients. The present study aims to assess tolerance in terms of acute toxicity and compliance of concurrent chemo-radiotherapy (cCRT) in a series of patients aged ≥70 years. MATERIALS AND METHODS: Clinical records of patients aged ≥70 years who underwent cCRT between January 2005 and December 2013 were reviewed. Concurrent CRT had curative intent in 134 patients (97.8 %) and palliative intent in 3 patients (2.2 %). Chemotherapy (CT) drugs and schedule were selected according to tumor histology. Radiotherapy median dose was 45.0 Gy (range 11-70 Gy) for curative purposes and 54 Gy (range 40-56 Gy) for palliative purposes. Incidence of acute toxicity and compliance to cCRT were analyzed and correlated with age, Karnofsky Performance Status (KPS), and Charlson Comorbidity Index (CCI). RESULTS: Overall, 137 patients, 82 males (60 %) and 55 females (40 %), median age 74 years (range 70-90 years) were analyzed. Concurrent CRT schedule was completed by 132 patients (96.4 %). Thirty-one of these patients (23.5 %) temporarily interrupted treatment. Hematological toxicity with grade ≥1 was observed in 25 patients (18.2 %), gastrointestinal toxicity in 55 (40.1 %), and genitourinary in 13 (9.5 %). Mucositis with grade ≥1 was recorded in 19 patients (13.9 %). No statistical significant correlation between KPS, CCI, and toxicity was found. A correlation trend between mucositis and patient age (p = 0.05) was observed. CONCLUSION: Concurrent CRT for elderly was feasible and quite well tolerated. Great attention in prescribing CT dose should be paid to limit acute toxicity.

Factors influencing post-transfusional platelet increment in pediatric patients given hematopoietic stem cell transplantation.

Patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT) always require platelet transfusions, but the increase in platelet count is often less than expected. Since factors responsible for poor response to platelet transfusions in this clinical setting are largely unknown, we performed a prospective study in 87 consecutive children transplanted in a single institution. The mean 16-h corrected count increment (CCI) of 598 platelet transfusions was 5.76 +/- 8.32 x 10(9)/l. Both before and after HSCT, 13.8% of patients had antibodies against HLA and/or platelet-specific antigens. Univariate analysis identified 12 factors significantly associated with a lower post-transfusion CCI, but only four reached statistical significance in the multivariate analysis. These four factors were concomitant therapy with vancomycin, alloimmunization, use of an Autopheresis cell separator for preparation of platelet concentrates and cytomegalovirus infection. We, therefore, suggest that a better response to platelet transfusions could be obtained by choosing a suitable cell separator, by avoiding the use of vancomycin and by adopting measures that reduce alloimmunization and CMV infection. Moreover, screening patients for HLA and platelet-specific antibodies before HSCT would identify the majority of subjects who will develop alloimmune refractoriness after transplantation and would allow the search for a compatible donor in advance.

A cross-sectional study on vision-related quality of life in patients with ocular GvHD.

Ocular GvHD affects about 40-60% of patients receiving bone marrow transplantation. Ocular complaints worsen quality of life (QoL), which, besides survival time, is a primary end point in a patient's follow-up. The aim of our study was to assess the ocular surface status and vision-related QoL (VRQoL) and explore the potential determinants in VRQoL in patients with chronic GvHD with ocular involvement. In this cross-sectional study, we investigated 40 patients with ocular GvHD after allogeneic hematopoietic stem cell transplantation assessing ocular symptoms and signs, VRQoL and ophthalmologic parameters. The median age was 52.1 years; 32.5% were females. Most of them presented a multiple organ involvement. Ophthalmological parameter examinations were on average abnormal. Corneal staining was severe/very severe in 25%; conjunctival staining in 10% of subjects. The worse QoL scores were on 'general vision', 'ocular pain', 'vision-specific mental health' and 'vision-specific role difficulties'. Both symptoms and sign scores indicate poor VRQoL. A lower VRQoL was related to schooling level, job position, underlying disease and extracorporeal photopheresis. Corneal staining, Schirmer and tear film breakup time were negatively associated to visual function-related subscales. An accurate ophthalmological and VRQoL assessment should be mandatory for a long time to promptly recognize early signs of ocular suffering, and to prevent irreversible ocular complications.

Impact of leukapheresis cell composition on immunomagnetic cell selection with the Baxter Isolex 300i device: a statistical analysis.

Immunomagnetic cell selection (ICS) of CD34(+) cells is increasingly adopted in allogeneic and autologous transplant settings. Because many variables can affect the final results of ICS, we focused our study toward the influence exerted by the leukapheresis (LKF) cell composition on recovery, purity, and log of T and B depletion of the immunoselected cells. A total of 39 consecutive CD34(+) ICS were performed with the Isolex 300i (Baxter) device on 39 LKF from 9 HLA haploidentical donors and 20 patients. Flow cytometric analysis was performed both on the leukapheresis content and on the immunoselected cells. The statistical analysis was performed utilizing the Pearson's correlation test and the Mann-Whitney U test. The median purity and recovery of the immunoselected CD34(+) cells were 95.3% (IR: 93.0-99.0) and 55.1% (IR: 41.8-68.2), respectively. The median log of T and B depletion were 3.87 (IR: 3.5-4.3) and 2.9 (IR: 2.5-3.5), respectively. Our data indicate that not only the CD34(+) cell load but also the ratio among the cells belonging to the starting fraction can influence the results of ICS. LKF collection protocols have to be addressed to collect an high number of CD34(+) cells (>500 x 10(6)) without taking care of the contaminating cells when the Baxter Isolex 300i device is employed.

Transplantation of peripheral blood stem cells mobilized by haematopoietic growth factors in childhood.

Peripheral blood stem cells (PBSC), mobilised by means of haematopoietic growth factors (HGF) with or without chemotherapy, are being used routinely for autologous rescue after high-dose chemo-radiotherapy in paediatric patients with lymphoma and selected solid tumours because of the ease of collection and the accelerated kinetics of neutrophil and platelet engraftment as compared with bone marrow cells. Recent reports indicated that HGF-mobilised PBSC can also be employed in childhood as an alternative to bone marrow allograft when the donor is an adult or with the aim of reversing graft failure in patients who were previously given a marrow allograft. Notwithstanding this wide use of PBSC, several biological and clinical questions of crucial relevance are still unsolved. In this article, we will analyse: (1) the optimal timing for PBSC collection after cytokine-based mobilising regimens; (2) the variables affecting the yield of peripheral blood progenitors; (3) the minimum threshold and the optimal number of PBSC that should be infused for autologous and allogeneic transplant, respectively; (4) the biological mechanisms underlying mobilisation of haematopoietic stem cells into circulation; (5) the incidence of graft-versus-host disease and the biological characteristics of donor lymphocytes in patients given allogeneic transplant of PBSC; and (6) the most relevant peculiarities in the kinetics of immune recovery of patients given allogeneic transplant of PBSC, as compared to bone marrow transplant recipients.

Intensive chemotherapy followed by donor PBSC in ANLL relapsed after allogeneic BMT.

We describe a 42-year-old man with ANLL-M4 in relapse after allogeneic BMT, in whom a new CR was obtained by conventional chemotherapy followed by the infusion of his female donor PBSC. At the time of BMT he was in CR. Six months later a full hematological relapse occurred an a three drug 5-day regimen was started. Two days after the end of chemotherapy he received donor PBSC collected by two leukaphereses after mobilization with G-CSF, given subcutaneously at 5 micrograms/kg/day for 7 days. The mononuclear PBSC were 4.2 x 10(8)/kg; the CD34 positive cells were 8.2 x 10(6)/kg and the CFU-GM were 14 x 10(4)/kg. Two days after PBSC infusion the patient received G-CSF at a dose of 5 micrograms/kg/day. Hemopoietic recovery occurred promptly on day + 13 and Y-FISH revealed 14% of Y-spot positive cells in the marrow. On day +20 hematological and cytogenetic remission was documented. The percentage of recipient cells decreased from day +36 onwards following the occurrence of a grade II GVHD, from which the patient recovered 1 week later with oral cyclosporin A and intravenous high-dose steroids. At present (day +200 from relapse) the patient is still in CR with 3% of Y-spot positive cells.

Epithelial tumour cell detection and the unsolved problems of nested RT-PCR: a new sensitive one step method without false positive results.

Sensitive detection of circulating epithelial cancer cells might have important therapeutic and prognostic implications in patients with breast cancer (BC) receiving high-dose chemotherapy and PBSC support. We have compared the specificity and sensitivity of the recently developed 'one tube' reverse transcriptase PCR (RT-PCR) assay with the more widely used nested RT-PCR method for detection of cytokeratin 19 (CK19)-positive cells. The analysis of 30 control samples provides evidence that one tube RT-PCR is highly specific in contrast to the nested method which showed 23% false positive results. The sensitivity of both techniques to detect tumour contamination was 10(-6). PBSC harvests from 45 BC patients were tested with both RT-PCR methods and the results were compared with immunocytochemistry (ICC). The five samples found positive by ICC were also positive by one tube RT-PCR; in addition, 11 more samples were positive by one tube RT-PCR analysis. The greater number of PBSC found positive by one tube RT-PCR might be due to the larger number of cells analysed. We conclude that one tube RT-PCR is sensitive and reveals no false positive results. This method is less time consuming than the nested one, technically simpler and should be considered for tumour cell detection.