Autologous bone marrow concentrate combined with platelet-rich plasma enhance bone allograft potential to induce spinal fusion.

Bone marrow cells concentrate (BMCs) is a source of osteoprogenitor cells and platelet-rich plasma (PRP) is a source of growth factors. The objective of the study was to determine whether BMC and PRP could increase the potential of bone allograft to induce posterolateral-lumbar spinal fusion compared to the bone allograft alone. A prospective nonrandomized radiographic study has been conduced on 10 patients with posterolateral instrumented fusion for degenerative lumbar disease with 1-year follow-up using CT scan. A fresh frozen bone allograft alone and bone allograft with a mixture of autologous BMC and PRP blended with thrombin were apposed in the right and left posterolateral side, respectively. CT showed good right fusion masses (allograft alone) in 4 patients and poor in 6; good left masses (BMC and PRP plus allograft) in 9 patients and poor in 1. The differences detected between right-side and left-side masses show an advantage in adding BMC and PRP to the bone allograft to increase spinal fusion rate.

A climatology of 7Be in surface air in European Union.

This study presents a European-wide analysis of the spatial and temporal distribution of the cosmogenic isotope (7)Be in surface air. This is the first time that a long term database of 34 sampling sites that regularly provide data to the Radioactivity Environmental Monitoring (REM) network, managed by the Joint Research Centre (JRC) in Ispra, is used. While temporal coverage varies between stations, some of them have delivered data more or less continuously from 1984 to 2011. The station locations were considerably heterogeneous, both in terms of latitude and altitude, a range which should ensure a high degree of representativeness of the results. The mean values of (7)Be activity concentration presented a spatial distribution value ranging from 2.0 to 5.4 mBq/m(3) over the European Union. The results of the ANOVA analysis of all (7)Be data available indicated that its temporal and spatial distributions were mainly explained by the location and characteristic of the sampling sites rather than its temporal distribution (yearly, seasonal and monthly). Higher (7)Be concentrations were registered at the middle, compared to high-latitude, regions. However, there was no correlation with altitude, since all stations are sited within the atmospheric boundary layer. In addition, the total and yearly analyses of the data indicated a dynamic range of (7)Be activity for each solar cycle and phase (maximum or minimum), different impact on stations having been observed according to their location. Finally, the results indicated a significant seasonal and monthly variation for (7)Be activity concentration across the European Union, with maximum concentrations occurring in the summer and minimum in the winter, although with differences in the values reached. The knowledge of the horizontal and vertical distribution of this natural radionuclide in the atmosphere is a key parameter for modelling studies of atmospheric processes, which are important phenomena to be taken into account in the case of a nuclear accident.

Toxoplasma gondii and Neospora caninum infections in goat abortions from Argentina.

The aims of this study were to identify the occurrence of Toxoplasma gondii and Neospora caninum abortions in goats from Argentina by serological, macroscopical and microscopical examination and bioassay, and to characterize the obtained isolates by molecular techniques. For this purpose, 25 caprine fetal fluids, 18 caprine fetal brains and 10 caprine placentas from 8 dairy/meat goat farms from Argentina were analyzed. Gestational age of the aborted fetuses was determined in 18 cases. Protozoal infections were detected by at least one of the applied diagnostic techniques in 44% (11/25) of examined fetuses; specifically, 24% (6/25) were positive to T. gondii, 8% (2/25) were positive to N. caninum and 12% (3/25) were positive to both parasites. In this study IFAT titers were similarly distributed in younger and older fetuses. Macroscopical and microscopical examination of one placenta revealed chalky nodules in the fetal cotyledons and normal intercotyledonary areas, as well as necrosis and calcification of mesenchymal cells in villi. Tachyzoites were observed in peritoneal wash from 2 mice inoculated with brain and a pool of brain and placenta of two fetuses. Cell culture growth of tachyzoites was achieved from one inoculated mouse, and confirmed as T. gondii by PCR. The T. gondii isolate was identified as atypical or non-canonical by nested-PCR-RFLP. This is the first study that investigated the involvement of N. caninum and T. gondii in cases of goat abortion in Argentina.

Abuse-related and abuse-limiting effects of methcathinone and the synthetic "bath salts" cathinone analogs methylenedioxypyrovalerone (MDPV), methylone and mephedrone on intracranial self-stimulation in rats.

RATIONALE: Abuse of synthetic cathinones, popularized as "bath salts," has increased dramatically in the USA since their debut in 2010. Preclinical behavioral studies may clarify determinants of the abuse-related effects produced by these compounds. OBJECTIVES: This study examined behavioral effects of (±)-methcathinone, (±)-3,4-methylenedioxypyrovalerone (MDPV), (±)-3,4-methylenedioxymethcathinone (methylone), and (±)-4-methylmethcathinone (mephedrone) in rats using intracranial self-stimulation (ICSS). METHODS: Male Sprague-Dawley rats (n = 18) with electrodes targeting the medial forebrain bundle responded for multiple frequencies of brain stimulation and were tested in two phases. First, dose-effect curves for methcathinone (0.1-1.0 mg/kg), MDPV (0.32-3.2 mg/kg), methylone (1.0-10 mg/kg), and mephedrone (1.0-10 mg/kg) were determined. Second, time courses were determined for effects produced by the highest dose of each compound. RESULTS: Methcathinone produced dose- and time-dependent facilitation of ICSS. MDPV, methylone, and mephedrone produced dose- and time-dependent increases in low rates of ICSS maintained by low brain stimulation frequencies, but also produced abuse-limiting depression of high ICSS rates maintained by high brain stimulation frequencies. Efficacies to facilitate ICSS were methcathinone ≥ MDPV ≥ methylone > mephedrone. Methcathinone was the most potent compound, and MDPV was the longest acting compound. CONCLUSIONS: All compounds facilitated ICSS at some doses and pretreatment times, which is consistent with abuse liability for each of these compounds. However, efficacies of compounds to facilitate ICSS varied, with methcathinone displaying the highest efficacy and mephedrone displaying the lowest efficacy to facilitate ICSS.

Radioactivity from Fukushima Dai-ichi in air over Europe; part 1: spatio-temporal analysis.

Radionuclides emitted from the Fukushima I nuclear power plant have been detected in air all over Europe. Concentrations remained far below levels which could have caused radiological concern: probably the committed thyroid dose due to inhalation remained below about 1 µSv (for 10 y children), within the investigated region. They provided, however, a spatio-temporal signal which could be used to develop and test tools to provide additional information on the large-scale situation (Europe-wide, in this case) during a nuclear emergency. In this part we discuss the spatial distribution of the contaminated air masses over Europe. Using (131)I as an example, we present a method to construct maps of the time-cumulated (131)I concentration in air and of the peak concentrations. Procedures to deal with the statistical limitations of a data set stemming from different monitoring schemes are discussed. As over all results, the mean (over the investigated region) cumulated concentration of particular (131)I is estimated about 9 mBq d/m(3), with observed maximum of about 23 mBq d/m(3). The probability that much higher concentrations occurred at unsampled locations, than have been observed anywhere, is assessed low, e.g. about 2.5% for the cumulated (131)I(part.) concentration to exceed 30 mBq d/m(3). Our method can be used in nuclear emergencies for providing spatial analyses if radionuclide concentrations of health concern are detected by atmospheric monitoring stations. We suggest considering such methods of data harmonization if synoptic assessment based on heterogeneous datasets is attempted.

Pre-transplant prognostic factors for patients with high-risk leukemia undergoing an unrelated cord blood transplantation.

From July 1995 to December 2001, 42 patients with leukemia aged 1-42 years underwent cord blood transplant (CBT) from unrelated, < or = 2 antigen HLA mismatched donors. In all, 26 patients were in < or = 2nd complete remission and 16 in more advanced phase. Conditioning regimens, graft-versus-host disease (GVHD) prophylaxis and supportive policy were uniform for all patients. The cumulative incidence of engraftment was 90% (95% CI: 0.78-0.91). The cumulative incidence of III-IV grade acute- and chronic-GVHD was 9% (95% CI: 0.04-0.24) and 35% (95% CI: 0.21-0.60), respectively. The 4-year cumulative incidence of transplant-related mortality (TRM) and relapse was 28% (95% CI: 0.17-0.47) and 25% (95% CI: 0.14-0.45), respectively. The 4-year overall survival (OS), leukemia-free survival (LFS) and event-free survival (EFS) were 45% (95% CI: 0.27-0.63), 47% (95% CI: 0.30-0.64) and 46% (95% CI: 0.30-0.62), respectively. In multivariate analysis, the most important factor affecting outcomes was the CFU-GM dose, associated with CMV serology (P=0.003 and 0.04, respectively) in influencing OS and with patient sex (P=0.008 and 0.03, respectively) in influencing LFS. Finally, CFU-GM dose was the only factor that affected EFS significantly (P=0.02). In conclusion, the infused cell dose expressed as in vitro progenitor cell growth is highly predictive of outcomes after an unrelated CBT and should be considered the main parameter in selecting cord blood units for transplant.

Serotonin and norepinephrine transporters: possible relationship between oligomeric structure and channel modes of conduction.

In the past several years there has been significant progress made on the biophysics of neurotransmitter transporters, leading to the proposal of new models of substrate and ion permeation across membranes. Questions arising from these studies are as follows: How are substrate uptake and substrate-induced current related? Where and how does substrate-ion coupling occur? What is the functional significance of the coupled and uncoupled currents? Because of a long-standing interest and collaboration, and because of their importance for normal function and disease, the authors have focused on the properties of human norepinephrine and serotonin transporters, using other clones and mutations as specific needs arise. It has been know for decades that hNETs (human norepinephrine transporters) clear NE+ (norepinephrine) following its release in peripheral sympathetic and central noradrenergic synapses. Neuronal activity influences NE+ uptake, so one is also interested in the acute regulation of hNET. To study these problems, hNET-expressing cells have been developed that are suitable for patch clamp, radioligand uptake, biochemistry, and transiently expressed clones for structure-function analysis, and new protocols have been designed combining patch-clamp, microamperometry, Ca2+ imaging, and native catecholamine transporter preparations to study transporters in whole cells and isolated patches. Using these methods, Na-dependent, NE+-induced hNET currents that are blocked by cocaine and antidepressants, channel modes of NE+ conduction, voltage-dependent uptake coupled to NE+-induced ion channel activity, PKC (phosphokinase C) regulation of NE+ uptake, and transporter modulation by [Ca2+]i have all been discovered. There is also provocative new data on other transporters in this family, such as Li/Na mole fraction experiments in the Drosophila serotonin transporters and sided enkephalin block in proline transporters. These studies have led one to postulate the existence of a narrow pore within transporters through which the substrate (NE+ or serotonin, 5HT+) and other ions (principally Na+) pass. It is hypothesized that the pore resides in an oligomeric structure and that separate gene products of hNET or hSERT (human serotonin transporters) come together to form a channel.

[Retinoic acid syndrome. Severe respiratory insufficiency treated with CPAP]. / Sindrome da acido retinoico: grave insufficienza respiratoria trattata con ventilazione in maschera con CPAP.

Retinoic acid syndrome is a potentially life-threatening complication of therapy for acute promyelocytic leukemia (APL) with all-transretinoic acid (ATRA). The case of a 55-year old male patient admitted to the hospital because of a bleeding diathesis is reported. APL was diagnosed and he underwent treatment with idarubicin and ATRA (GIMEMA protocol); 24 hrs after ATRA treatment he developed retinoic acid syndrome and was admitted to the Intensive Care Unit because of severe respiratory insufficiency (dyspnoea, tachypnea and severe hypoxemia (SpO2 75%). Pulmonary insufficiency was treated non-invasively with CPAP and the patient recovered from pulmonary distress one week later.

Idarubicin intensified BUCY2 regimen in allogeneic unmanipulated transplant for high-risk hematological malignancies.

Twenty-nine consecutive patients with high-risk hematological malignancy aged from 3 to 58 years underwent an unmanipulated graft from an HLA-identical sibling after an irradiation-free preparative regimen consisting of idarubicin (IDA), 21 mg/m2/day administered by continuous infusion on days -12 and -11, followed by busulphan (BU), 4 mg/kg/day orally from day -7 to -4, and cyclophosphamide (CY), 60 mg/kg/day intravenously on days -3 and -2 (IDA-BUCY2). Most clinically relevant extra-hematological regimen-related toxicities consisted of stomatitis observed in all subjects and hemorrhagic cystitis occurred in five cases (17%) within 100 days after transplant. Six patients (21%) developed a grade 2 acute graft-versus-host disease (GVHD) and three (10%) a grade 3 or 4; extensive chronic GVHD was assessed in nine of 22 (41%) evaluable patients. So far, 12 patients have died and 17 are alive, 16 of whom disease-free, 5-41 months after transplant (median, 15 months). The causes of death were related to GVHD in three patients, to sepsis in one and to disease recurrence in the remaining eight. At present, only one of nine relapsed patients is alive. For all patients the actuarial probability of survival (OS) at 1 and 2 years +/- standard error (s.e.) was 63 +/- 9% and 52 +/- 10%, respectively. The actuarial probabilities of disease-free survival (DFS), relapse and transplant-related mortality (TRM) at both 1 and 2 years +/- s.e. were 53 +/- 9%, 35 +/- 9% and 16 +/- 7%, respectively. These results are encouraging but not substantially different from those obtained in 28 patients with malignancy in advanced phase transplanted after the standard BUCY2 regimen, who had an actuarial probability of OS, DFS, relapse and TRM projected at 10 years +/- s.e. of 54 +/- 10%, 57 +/- 9%, 36 +/- 9% and 11 +/- 6%, respectively. Although the retrospective comparison between the two groups does not seem to show any advantage in the use of the IDA intensified regimen, only a prospective randomized trial could answer this question.

Flt3LP3nduces the ex-vivo amplification of umbilical cord blood committed progenitors and early stem cells in short-term cultures.

Umbilical cord blood (UCB) has been successfully used for haemopoietic stem cell transplantation, although its use has been cautiously limited to paediatric patients because of the reduced volume produced. The clinical results have confirmed that either engraftment or survival significantly correlate with cell dose infused. We have standardized a culture method providing in a short time a significant amplification of both committed progenitors and primitive stem cells for clinical use. Eight-day culture of UCB cells with flt3L/SCF/PIXY 321 induced a 10-fold amplification of CD34+ cells and the expansion of multipotent (CFU-GEMM) and committed (CFU-GM, BFU-E) progenitors respectively of 5-, 7- and 9-fold over input cells. As to the early stem cell pool, the primitive CD34+Thy-1+ cell fraction increased 6-fold and the LTC-IC were amplified 17-fold. Furthermore, the in vitro proliferation was detected by the gradual loss of fluorescence of the CD34+ cells tracked at day 0 with the dye PKH26. After 8 d of amplification >6% of the CD34+ cells remained intensely fluorescent. This subpopulation represents a deeply quiescent cell fraction unresponsive to cytokines and very enriched of primitive stem cells. These cells are most likely to be responsible for long-term reconstitution after transplant.

Umbilical cord blood transplant from unrelated HLA-mismatched donors in children with high risk leukemia.

In the last 3 years, 14 children with high-risk leukemia (11 ALL, 2 AML and 1 CML) underwent cord blood transplantation from unrelated HLA-mismatched donors at a median of 99 days from the start of search. Eight patients were transplanted in second CR, one in accelerated phase, three at relapse and two patients in first CR. Conditioning regimen (fractionated TBI, etoposide, CY and anti-lymphocyte serum) and prophylaxis of GVHD (CsA and 6-methylprednisolone) were identical for all patients. Neutrophils >0.5x10(9)/l were reached at a median of 33 days from transplant, but in four cases we observed an autologous hematopoietic reconstitution (three spontaneous, one after autologous BM rescue). Acute and chronic GVHD were observed in 10/14 and 3/8 evaluable cases, respectively. Three patients died of transplant-related toxicity and three patients relapsed. The probabilities of event-free, disease-free and overall survival were 50, 53 and 64%, respectively. Cord blood transplant from HLA-mismatched unrelated donor is a valid option for the treatment of children with high-risk leukemia. With our eligibility criteria, conditioning regimen and prophylaxis of graft-versus-host disease, the main obstacles to successful transplant were represented by graft failure and fatal acute GVHD.

A prospective molecular study of chimaerism in patients with haematological malignancies receiving unrelated cord blood or bone marrow transplants: detection of mixed chimaerism predicts graft failure with or without early autologous reconstitution in cord blood recipients.

We prospectively studied the chimaerism status in the bone marrow (BM) and peripheral blood (PB) of 23 patients receiving umbilical cord (UCB, 14 cases) or BM (nine cases) transplants from unrelated donors by PCR amplification of four individual-specific VNTR genetic loci. Haematological engraftment, with persistent full donor pattern. was observed in 10/14 (72%) patients receiving UCB and in 9/9 (100%) patients transplanted with marrow from an unrelated donor (MUD). In contrast, the remaining four patients converted to an autologous pattern. Three out of these four patients had an early autologous haematological reconstitution reaching a neutrophil level >0.5 x 10(9)/l at days 27, 33 and 37 after transplant, respectively. In all three of these patients, chimaerism analysis demonstrated an early appearance of donor cells (i.e. within 35 d after UCB transplant) showing a transient full donor (one case) or mixed chimaerism condition (two cases). Despite the early autologous haemopoietic reconstitution, one of the three patients died of GVHD at day 60, which was explained by the demonstration of low levels of donor lymphoid cells. In the MUD group all nine patients converted to a persistent full donor pattern with haematological reconstitution, accompanied in two of them by transient mixed chimaerism lasting to days 60 and 270 after transplant. Our data show that monitoring of chimaerism may predict graft failure with or without early autologous haemopoietic reconstitution in patients receiving unrelated UCB transplants. Furthermore, chimaerism analysis may identify, in patients with autologous reconstitution, those at risk of severe GVHD in whom immunosuppressive therapy should not be discontinued.

Mechanisms of osteoclast dysfunction in human osteopetrosis: abnormal osteoclastogenesis and lack of osteoclast-specific adhesion structures.

Osteoclasts from a patient affected by osteopetrosis were examined in vivo and in vitro. Iliac crest biopsy revealed an osteosclerotic pattern, with prominent numbers of osteoclasts noted for hypernuclearity and incomplete adherence to the bone surface. A population comprising tartrate-resistant acid phosphatase (TRAP)-positive, multinucleated and mononuclear cells, and alkaline phosphatase-positive stromal fibroblasts was obtained in vitro from bone marrow. Mononuclear TRAP-positive precursors spontaneously fused in culture to form giant osteoclast-like cells. These cells expressed the osteoclast marker MMP-9 and calcitonin receptor, and lacked the macrophage marker, Fc receptor. Expression and distribution of c-src, c-fms, and CD68, and response to steroid hormones relevant to osteoclast differentiation and function were apparently normal, whereas cell retraction in response to calcitonin was impaired. TRAP-positive multinucleated cells did not form osteoclast-specific adhesion structures (clear zone, podosomes, or actin rings). Bone resorption rate was severely reduced in vitro. Focal adhesions and stress fibers were observed en lieu of podosomes and actin rings. Adhesion structures contained low levels of immunoreactive vitronectin receptor, most of this integrin being retained in cytoplasmic vesicles. These data provide the first characterization of abnormal differentiation and function of human osteopetrotic osteoclast-like cells.

Umbilical cord blood transplant from HLA-mismatched unrelated donor in high-risk leukemia.

Twelve consecutive children with high-risk leukemia have been submitted to UCB transplant from unrelated 1 or 2 loci HLA-mismatched donor. All patients received an identical regimen for conditioning and GVHD prophylaxis. The median dose of viable nucleated cells infused was 2.8 x 10(7)/kg bw (range 1.4-7.9). Of 11 patients evaluable for engraftment, the hematopoiesis was of full donor origin in seven patients and autologous in four. The probability of disease-free survival at 1 and 2 years from UCB transplant is 60 and 42%, respectively.

Flt3L enhances the early stem cell compartment after ex vivo amplification of umbilical cord blood CD34+ cells.

Umbilical cord blood (UCB) transplant represents a promising therapeutic approach, nevertheless this procedure has been so far almost exclusively used in pediatric patients because of the reduced volume of UCB units. The availability of larger numbers of early and late hematopoietic progenitors by ex vivo amplification procedure may allow the use of UCB in adults and improve the rate and time to engraftment. We describe a stroma-free liquid culture system that induces a 10-fold increase of CD34+ cells and hematopoietic progenitors after 8 days in vitro amplification. The presence of flt3L is essential to preserve and amplify the early stem cell compartment identified by the phenotype CD34+Thy-1+CD45RO+.

Umbilical cord blood (UCB) transplant from unrelated mismatched donor in patients with high risk (HR) leukemia.

Ten consecutive children with high risk leukemia have been submitted to UCB transplant from unrelated HLA mismatched donors. All patients received an identical regimen for conditioning and GVHD prophylaxis. The median dose of viable nucleated cells infused was 2.6 x 10(6)/kg b.w. Among the nine patients evaluable for engraftment the hematopoiesis was of full donor origin in six patients and autologous in three. At a median follow-up of 9 months, six of nine (67%) patients are alive in CR.

Intravenous immune globulin in lysinuric protein intolerance.

In addition to systemic manifestations with skeletal, pulmonary, renal, and haematological signs, lysinuric protein intolerance (LPI), a membrane transport defect of cationic amino acids, is often complicated by severe life-threatening immunological manifestations. A 10-year-old boy with LPI who exhibited a severe systemic immunohaematological disease is described here. This patient showed cutaneous lesions similar to the subacute form of systemic lupus erythematosus, severe anaemia and dysproteinaemia, and a marked reduction of circulating T lymphocytes, mainly the CD4+ cells. In vitro bone marrow cell culture studies showed that addition of patient's serum induced macrophage proliferation and inhibited erythroid progenitor cell growth. Treatment with high-dose intravenous immune globulin resolved most of the clinical and laboratory abnormalities.

Stem cell factor and PIXY-321 in acute lymphoblastic leukemia: in vitro study on proliferative effects and apoptosis.

Management of acute lymphoblastic leukemia (ALL) patients may include growth factors (GFs) to reduce post-chemotherapy aplasia. A potential risk of GF administration is a stimulatory signal on the leukemic population. In the present study we investigated the proliferative and programmed cell death (PCD) effect of two cytokines that have recently entered clinical use, stem cell factor (SCF) and the granulocyte colony stimulating factor/IL-3 fusion molecule (PIXY-321), on 14 ALL samples. The activity of IL-7, a cytokine involved in the regulation of ALL cell proliferation, was also tested alone and in combination with these two cytokines. Using the acridine orange flow cytometric technique and the clonogenic assay, we showed that none of these cytokines was capable of significantly increasing the mean percentage of S-phase cells and CFU-L number. A mean decrease of G0 cells from 60.6% to 52.6% (p = 0.02), coupled by a significant increase of G1 cells from 28.2% to 37.9% (p = 0.003) was demonstrated in the presence of PIXY-321. IL-7 alone and in combination with either PIXY-321 or SCF induced similar changes in the percentage of cells in G0 and G1. SCF showed no activity on G0 depletion. When each individual samples was analyzed separately, some heterogeneity was observed. An increase of S phase was recorded in a proportion of cases after SCF and PIXY-321 exposure. However, none of the cytokines evaluated by a clonogenic assay following liquid culture was capable of maintaining or promoting self-renewal of leukemic precursors, as determined by plating fresh cells at time 0. Detection of cytokine effects of apoptosis showed that SCF and PIXY-321 did not significantly reduce the mean percentage of cells in PCD, whereas a significant protective effect was observed in the presence of IL-7 (p = 0.02). We conclude that PIXY-321 and, to a further extent, SCF fail to induce leukemic lymphoid cell proliferation, and do not protect cells from entering apoptosis. These in vitro findings may be useful for ALL clinical trial design.