Analysis of acylcarnitine profiles in umbilical cord blood and during the early neonatal period by electrospray ionization tandem mass spectrometry

Acylcarnitine profiling by electrospray ionization tandem mass spectrometry (ESI-MS/MS) is a potent tool for the diagnosis and screening of fatty acid oxidation and organic acid disorders. Few studies have analyzed free carnitine and acylcarnitines in dried blood spots (DBS) of umbilical cord blood (CB) and the postnatal changes in the concentrations of these analytes. We have investigated these metabolites in healthy exclusively breastfed neonates and examined possible effects of birth weight and gestational age. DBS of CB were collected from 162 adequate for gestational age neonates. Paired DBS of heel-prick blood were collected 4-8 days after birth from 106 of these neonates, the majority exclusively breastfed. Methanol extracts of DBS with deuterium-labeled internal standards were derivatized before analysis by ESI-MS/MS. Most of the analytes were measured using a full-scan method. The levels of the major long-chain acylcarnitines, palmitoylcarnitine, stearoylcarnitine, and oleoylcarnitine, increased by 27, 12, and 109%, respectively, in the first week of life. Free carnitine and acetylcarnitine had a modest increase: 8 and 11%, respectively. Propionylcarnitine presented a different behavior, decreasing 9% during the period. The correlations between birth weight or gestational age and the concentrations of the analytes in DBS were weak (r £ 0.20) or nonsignificant. Adaptation to breast milk as the sole source of nutrients can explain the increase of these metabolites along the early neonatal period. Acylcarnitine profiling in CB should have a role in the early detection of metabolic disorders in high-risk neonates.

Salvaged single-unit cord blood transplantation for 26 patients with hematologic malignancies not in remission

Treatments for patients with hematologic malignancies not in remission are limited, but a few clinical studies have investigated the effects of salvaged unrelated cord blood transplantation (CBT). We retrospectively studied 19 patients with acute leukemia, 5 with myelodysplastic syndrome (MDS with refractory anemia with excess blasts [RAEB]), and 2 with non-Hodgkin's lymphoma who received 1 CBT unit ≤2 loci human leukocyte antigen (HLA)-mismatched after undergoing myeloablative conditioning regimens between July 2005 and July 2014. All of them were in non-remission before transplantation. The infused total nucleated cell (TNC) dose was 4.07 (range 2.76-6.02)×107/kg and that of CD34+ stem cells was 2.08 (range 0.99-8.65)×105/kg. All patients were engrafted with neutrophils that exceeded 0.5×109/L on median day +17 (range 14-37 days) and had platelet counts of >20×109/L on median day +35 (range 17-70 days). Sixteen patients (61.5%) experienced pre-engraftment syndrome (PES), and six (23.1%) patients progressed to acute graft-versus-host disease (GVHD). The cumulative incidence rates of II-IV acute GVHD and chronic GVHD were 50% and 26.9%, respectively. After a median follow-up of 27 months (range 5-74), 14 patients survived and 3 relapsed. The estimated 2-year overall survival (OS), disease-free survival (DFS), and non-relapse mortality (NRM) rates were 50.5%, 40.3%, and 35.2%, respectively. Salvaged CBT might be a promising modality for treating hematologic malignancies, even in patients with a high leukemia burden.

Ultrastructural characterization of bovine umbilical cord blood cells

The umbilical cord blood (UCB) is an important source of pluripotent stem cells, which motivated researches on ontogeny and transplantation. The morphological characterization of umbilical cord cells is the first step to establish subsequent experiments on these areas. Although some information on humans can be found, no data on UCB is available for bovines. Therefore, this work is the first attempt to conduct an ultrastructural characterization of bovine umbilical cord blood. Blood was collected from the umbilical cord of twenty fetuses by punction of the umbilical vein. Samples were processed for whole leucocytes observation by centrifugation and the buffy coat was collected. Cells were washed and pelleted and prepared according to the standard protocol of the transmission electron microscopy. The presence of cells with morphologic characteristics compatible with the precursors from the erythrocytic, neutrophilic, eosinophilic, basophilic, and lymphocytic lineages was observed. Atypical cells with peculiar morphological features, strongly similar to apoptotic cells, were seen. Bovine neutrophils with three types of cytoplasmic granules were also found in the blood. The ultrastructural characteristics of observed bovine UCB cells where similar to those found in other species, suggesting that bovines could possibly constitute an experimental model for approaches on UCB cells research.

Umbilical cord blood and neonatal endothelin-1 levels in preterm newborns with and without respiratory distress syndrome

Increased pulmonary vascular resistance in preterm newborn infants with respiratory distress syndrome is suggested, and endothelin-1 plays an important role in pulmonary vascular reactivity in newborns. We determined umbilical cord blood and neonatal (second sample) levels of endothelin-1 in 18 preterm newborns with respiratory distress syndrome who had no clinical or echocardiographic diagnosis of pulmonary hypertension and 22 without respiratory distress syndrome (gestational ages: 31.4 ± 1.6 and 29.3 ± 2.3 weeks, respectively). Umbilical cord blood and a second blood sample taken 18 to 40 h after birth were used for endothelin-1 determination by enzyme immunoassay. Median umbilical cord blood endothelin-1 levels were similar in both groups (control: 10.9 and respiratory distress syndrome: 11.4 pg/mL) and were significantly higher than in the second sample (control: 1.7 pg/mL and respiratory distress syndrome: 3.5 pg/mL, P < 0.001 for both groups). Median endothelin-1 levels in the second sample were significantly higher in children with respiratory distress syndrome than in control infants (P < 0.001). There were significant positive correlations between second sample endothelin-1 and Score for Neonatal Acute Physiology and Perinatal Extension II (r = 0.36, P = 0.02), and duration of mechanical ventilation (r = 0.64, P = 0.02). A slower decline of endothelin-1 from birth to 40 h of life was observed in newborns with respiratory distress syndrome when compared to controls. A significant correlation between neonatal endothelin-1 levels and some illness-severity signs suggests that endothelin-1 plays a role in the natural course of respiratory distress syndrome in preterm newborns.

Immunophenotype of hematopoietic stem cells from placental/umbilical cord blood after culture

Identification and enumeration of human hematopoietic stem cells remain problematic, since in vitro and in vivo stem cell assays have different outcomes. We determined if the altered expression of adhesion molecules during stem cell expansion could be a reason for the discrepancy. CD34+CD38- and CD34+CD38+ cells from umbilical cord blood were analyzed before and after culture with thrombopoietin (TPO), FLT-3 ligand (FL) and kit ligand (KL; or stem cell factor) in different combinations: TPO + FL + KL, TPO + FL and TPO, at concentrations of 50 ng/mL each. Cells were immunophenotyped by four-color fluorescence using antibodies against CD11c, CD31, CD49e, CD61, CD62L, CD117, and HLA-DR. Low-density cord blood contained 1.4 ± 0.9% CD34+ cells, 2.6 ± 2.1% of which were CD38-negative. CD34+ cells were isolated using immuno-magnetic beads and cultured for up to 7 days. The TPO + FL + KL combination presented the best condition for maintenance of stem cells. The total cell number increased 4.3 ± 1.8-fold, but the number of viable CD34+ cells decreased by 46 ± 25%. On the other hand, the fraction of CD34+CD38- cells became 52.0 ± 29% of all CD34+ cells. The absolute number of CD34+CD38- cells was expanded on average 15 ± 12-fold when CD34+ cells were cultured with TPO + FL + KL for 7 days. The expression of CD62L, HLA-DR and CD117 was modulated after culture, particularly with TPO + FL + KL, explaining differences between the adhesion and engraftment of primary and cultured candidate stem cells. We conclude that culture of CD34+ cells with TPO + FL + KL results in a significant increase in the number of candidate stem cells with the CD34+CD38- phenotype.

Comparative quantification of umbilical cord blood CD34+ and CD34+ bright cells using the ProCount™-BD and ISHAGE protocols

The total number of CD34+ cells is the most relevant clinical parameter when selecting human umbilical cord blood (HUCB) for transplantation. The objective of the present study was to compare the two most commonly used CD34+ cell quantification methods (ISHAGE protocol and ProCount™ - BD) and analyze the CD34+ bright cells whose 7-amino actinomycin D (7AAD) analysis suggests are apoptotic or dead cells. Twenty-six HUCB samples obtained at the Placental Blood Program of New York Blood Center were evaluated. The absolute numbers of CD34+ cells evaluated by the ISHAGE (with exclusion of 7AAD+ cells) and ProCount™ (with exclusion of CD34+ bright cells) were determined. Using the ISHAGE protocol we found 35.6 ± 19.4 CD34+ cells/µL and with the ProCount™ method we found 36.6 ± 23.2 CD34+ cells/µL. With the ProCount™ method, CD34+ bright cell counts were 9.3 ± 8.2 cells/µL. CD34+ bright and regular cells were individually analyzed by the ISHAGE protocol. Only about 1.8% of the bright CD34+ cells are alive, whereas a small part (19.0%) is undergoing apoptosis and most of them (79.2%) are dead cells. Our study showed that the two methods produced similar results and that 7AAD is important to exclude CD34 bright cells. These results will be of value to assist in the correct counting of CD34+ cells and to choose the best HUCB unit for transplantation, i.e., the unit with the greatest number of potentially viable stem cells for the reconstitution of bone marrow. This increases the likelihood of success of the transplant and, therefore, the survival of the patient.

Human umbilical cord blood cells in infarcted rats

Therapy with bone marrow-derived cells has been used in ischemic patients with reported success. The aim of this study was to determine the therapeutic efficacy of fresh and frozen human umbilical cord blood cells (hUCB) in Wistar rats submitted to permanent occlusion of the left coronary artery. Three hours after myocardial infarction, 2 x 10(7) hUCB cells or vehicle were administered by intramyocardial injection. The animals were divided into five groups: control (N = 10), sham operated (N = 10), infarcted that received vehicle (N = 9), infarcted treated with cryopreserved hUCB (N = 7), and infarcted treated with fresh hUCB (N = 5). Cardiac function was evaluated by electrocardiogram (ECG) and echocardiogram (ECHO) before cell therapy, and by ECG, ECHO, cardiopulmonary test, and left ventricular pressure measurements 3 weeks later. After 3 weeks, both groups treated with hUCB still had Q wave present in L1, âQRS >90° and reduced shortening fraction (less than 50%). In addition, cardiac indexes of left ventricular contractility and relaxation were 5484 ± 875 and -4032 ± 643 mmHg (cryopreserved hUCB) and 4585 ± 955 and -2862 ± 590 mmHg (fresh hUCB), respectively. These values were not statistically different from those of saline-treated animals. Cardiopulmonary exercise test profile was typical of infarcted hearts; exercise time was about 14 min and maximal VO2 was 24.77 ± 5.00 mL·kg-1·min-1. These data show that hUCB therapy did not improve the cardiac function of infarcted animals or prevent cardiac remodeling.

Transplantation of mononuclear cells from human umbilical cord blood promotes functional recovery after traumatic spinal cord injury in Wistar rats

Cell transplantation is a promising experimental treatment for spinal cord injury. The aim of the present study was to evaluate the efficacy of mononuclear cells from human umbilical cord blood in promoting functional recovery when transplanted after a contusion spinal cord injury. Female Wistar rats (12 weeks old) were submitted to spinal injury with a MASCIS impactor and divided into 4 groups: control, surgical control, spinal cord injury, and one cell-treated lesion group. Mononuclear cells from umbilical cord blood of human male neonates were transplanted in two experiments: a) 1 h after surgery, into the injury site at a concentration of 5 x 10(6) cells diluted in 10 µL 0.9% NaCl (N = 8-10 per group); b) into the cisterna magna, 9 days after lesion at a concentration of 5 x 10(6) cells diluted in 150 µL 0.9% NaCl (N = 12-14 per group). The transplanted animals were immunosuppressed with cyclosporin-A (10 mg/kg per day). The BBB scale was used to evaluate motor behavior and the injury site was analyzed with immunofluorescent markers to label human transplanted cells, oligodendrocytes, neurons, and astrocytes. Spinal cord injury rats had 25% loss of cord tissue and cell treatment did not affect lesion extension. Transplanted cells survived in the injured area for 6 weeks after the procedure and both transplanted groups showed better motor recovery than the untreated ones (P < 0.05). The transplantation of mononuclear cells from human umbilical cord blood promoted functional recovery with no evidence of cell differentiation.

Delivery of the Sox9 gene promotes chondrogenic differentiation of human umbilical cord blood-derived mesenchymal stem cells in an in vitro model

SRY-related high-mobility-group box 9 (Sox9) gene is a cartilage-specific transcription factor that plays essential roles in chondrocyte differentiation and cartilage formation. The aim of this study was to investigate the feasibility of genetic delivery of Sox9 to enhance chondrogenic differentiation of human umbilical cord blood-derived mesenchymal stem cells (hUC-MSCs). After they were isolated from human umbilical cord blood within 24 h after delivery of neonates, hUC-MSCs were untreated or transfected with a human Sox9-expressing plasmid or an empty vector. The cells were assessed for morphology and chondrogenic differentiation. The isolated cells with a fibroblast-like morphology in monolayer culture were positive for the MSC markers CD44, CD105, CD73, and CD90, but negative for the differentiation markers CD34, CD45, CD19, CD14, or major histocompatibility complex class II. Sox9 overexpression induced accumulation of sulfated proteoglycans, without altering the cellular morphology. Immunocytochemistry demonstrated that genetic delivery of Sox9 markedly enhanced the expression of aggrecan and type II collagen in hUC-MSCs compared with empty vector-transfected counterparts. Reverse transcription-polymerase chain reaction analysis further confirmed the elevation of aggrecan and type II collagen at the mRNA level in Sox9-transfected cells. Taken together, short-term Sox9 overexpression facilitates chondrogenesis of hUC-MSCs and may thus have potential implications in cartilage tissue engineering.

Red blood cells derived from peripheral blood and bone marrow CD34+ human haematopoietic stem cells are permissive to Plasmodium parasites infection

The production of fully functional human red cells in vitro from haematopoietic stem cells (hHSCs) has been successfully achieved. Recently, the use of hHSCs from cord blood represented a major improvement to develop the continuous culture system for Plasmodium vivax. Here, we demonstrated that CD34+hHSCs from peripheral blood and bone marrow can be expanded and differentiated to reticulocytes using a novel stromal cell. Moreover, these reticulocytes and mature red blood cells express surface markers for entrance of malaria parasites contain adult haemoglobin and are also permissive to invasion by P. vivax and Plasmodium falciparum parasites.

Age-related changes of the multidrug resistance P-glycoprotein function in normal human peripheral blood T lymphocytes

The multidrug resistance P-glycoprotein is a transmembrane efflux pump expressed by lymphocytes and is involved in their cytolytic activity. In the present study, we investigated the age-related changes of P-glycoprotein function in normal peripheral blood lymphocytes. Blood samples from 90 normal volunteers (age range, 0 to 86 years) were analyzed. P-glycoprotein function was assessed by the flow cytometric rhodamine 123 assay. P-glycoprotein function was highest in cord blood and progressively declined with age in peripheral blood T CD4+ and CD8+ cells. In contrast, P-glycoprotein function did not vary with age in CD19+ B or CD16+CD56+ natural killer cells. These data suggest that the decline in P-glycoprotein function in T CD4+ and CD8+ lymphocytes as a function of age may contribute to the decrease in T cell cytolytic activity with aging.

Incidence of congenital toxoplasmosis in southern Brazil: a prospective study

The study aimed to determine the incidence of congenital infection by Toxoplasma gondii and to describe neonatal and maternal characteristics regarding newborn infants treated at a teaching hospital in the town of Passo Fundo, State of Rio Grande do Sul, Brazil. Cord blood samples collected from 1,250 live newborns were analyzed. The laboratory diagnosis was established by the detection of Toxoplasma gondii IgM using an enzyme linked fluorescent assay. Gestational age, intrauterine growth, anthropometric measures, and prenatal characteristics were assessed. The incidence of congenital toxoplasmosis at birth was 8/10,000 (95%CI 0.2-44.5). Mean birthweight was 3,080 ± 215.56 grams and mean gestational age was 38.43 ± 1.88 weeks. With regard to prenatal care, 58% of the pregnant patients visited their doctors five times or more and 38.9% were serologically tested for toxoplasmosis in the first trimester of pregnancy. The incidence of congenital toxoplasmosis was similar to that found in most studies conducted in our country and abroad. Our study sample is representative of the town of Passo Fundo and therefore it is possible to consider the frequency observed as the prevalence of the disease in this town during the study period.

Hemolytic disease of the newborn due to anti-U

Anti-U is a rare red blood cell alloantibody that has been found exclusively in blacks. It can cause hemolytic disease of the newborn and hemolytic transfusion reactions. We describe the case of a female newborn presenting a strongly positive direct antiglobulin test due to an IgG antibody in cord blood. Anti-U was recovered from cord blood using acid eluate technique. Her mother presented positive screening of antibodies with anti-U identified at delivery. It was of IgG1 and IgG3 subclasses and showed a titer of 32. Monocyte monolayer assay showed moderate interaction of Fc receptors with maternal serum with a positive result (3.1%). The newborn was treated only with 48 hours of phototherapy for mild hemolytic disease. She recovered well and was discharged on the 4th day of life. We conclude that whenever an antibody against a high frequency erythrocyte antigen is identified in brown and black pregnant women, anti-U must be investigated.

Glycemia in newborns of hypertensive mothers according to maternal treatment

PURPOSE: To evaluate the evolution of glycemic levels in newborns of hypertensive mothers according to maternal treatment. METHODS: Prospective randomized study, including 93 newborns of mothers treated with isradipine (n = 39), atenolol (n = 40), or low sodium diet (control group - n=14). Glycemia was determined at birth (mother and newborn by the oxidase glucose method) and in the 1st, 3rd, 6th, 12th, and 24th hours after birth (newborn by a test strip method). The evolution of glycemia was analyzed in each group (Friedman test). The groups were compared regarding glycemia (Kruskall-Wallis test), and linear regression models were constructed for the analyses (independent variable = maternal glycemia; dependent variables = umbilical cord, 3rd, and 6th hour glycemia). RESULTS: There were no statistically significant differences among the mean blood glucose levels of the 3 groups in any of the assessments. There was a correlation between maternal and umbilical cord blood glucose in the isradipine (r = 0.61; P <.05) and control (r = 0.84; P <.05) groups. Regarding glycemia levels of the mothers and newborns in the third and sixthhours postpartum, this correlation was present only in the control group (maternal x third hour: r = 0.65; P <.05; maternal x sixth hour: r = 0.68; P <.05). There were no correlations in the atenolol group. Hypoglycemia was detected in 51.3% of the isradipine group, 60% of the atenolol group, and 35.7% of the control group, and it was more frequent in the first hour postpartum in all groups. CONCLUSIONS: The results suggest a similar effect of the 3 types of treatment upon newborn glycemia. The correlation analysis suggests that isradipine could have effects upon newborn glycemia only after birth (correlation only in umbilical cord blood), whereas atenolol could act earlier (there was no correlation at any moment). The results also point to the need for glycemic control from the first hour postpartum of newborns of hypertensive mothers whether they have or have not undergone treatment with antihypertensive drugs.

A comparative study of congenital toxoplasmosis between public and private hospitals from Uberlândia, MG, Brazil

The main purpose of the present study was to examine if there is difference in terms of incidence rates of congenital toxoplasmosis among populations assisted in public and private hospitals from Uberlândia, state of Minas Gerais, Brazil. A total of 805 serum samples from cord blood were collected, being 500 from public hospital and 305 from private hospital, and all patients answered a questionnaire about pregnancy and newborns. An indirect enzyme linked immunosorbent assay (ELISA) was performed to detect IgG antibodies to Toxoplasma gondii and the positive samples were retested to verify the presence of specific IgM and IgA antibodies in a capture ELISA. We found significant differences among data from both hospitals with respect to maternal age, origin city, gestational age, number of visits to physicians during pregnancy, type of delivery, and birth weight. Seroprevalence of IgG antibodies against T. gondii for patients from public and private hospitals was 57.6% and 41.9% respectively, and this difference was statistically significant (P < 0.0001). In addition, the frequency of congenital toxoplasmosis measured by the presence of IgM and/or IgA antibodies toward T. gondii was exclusively located in samples from public hospital (0.8%), and no positive sample was seen in private hospital (0%). Considering that almost all babies suffering from congenital toxoplasmosis, if undiagnosed and untreated, will develop visual or neurological impairments by adulthood, the results presented herein emphasized the importance to accomplish screening programs for toxoplasmosis during pregnancy, particularly in the public hospitals, due to the expressive rate of congenital disease showed in the patients attended at these centers.