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/æL and with the ProCount™ method we found 36.6 ± 23.2 CD34+ cells/æL. With the ProCount™ method, CD34+ bright cell counts were 9.3 ± 8.2 cells/æL. CD34+ bright and regular cells were individually analyzed by the ISHAGE protocol. Only about 1.8 percent of the bright CD34+ cells are alive, whereas a small part (19.0 percent) is undergoing apoptosis and most of them (79.2 percent) 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.

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 percent CD34+ cells, 2.6 ± 2.1 percent 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 percent. On the other hand, the fraction of CD34+CD38- cells became 52.0 ± 29 percent 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.

The hematopoietic stroma

All blood cells are derived from a small common pool of totipotent cells, called hematopoietic stem cells. The process is strictly regulated by the hematopoietic microenvironment, which includes stromal cells, extracellular matrix molecules and soluble regulatory factors. Several experimental in vitro assays have been developed for the study of hematopoietic differentiation, and have provided valuable information on the stroma, which includes, among other cell types, macrophages, fibroblasts, adipocytes, and endothelial cells. The composition, ontogeny, and function in physiological as well as pathological conditions of stroma are discussed

Produçäo e caracterizaçäo de anticorpos monoclonais contra o vírus da rinotraqueíte infecciosa bovina (BHV-1) / Production and characterization of monoclonal antibodies to infectious bovine rhinotracheitis virus (BHV-1)

Um painel de anticorpos monoclonais (AcM) foi produzido contra antígenos da amostra "Oxford" do Vírus de Rinotraqueíte Infecciosa Bovina ou Herpesvírus Bovino tipo 1 (BHV-1). Foram selecionados cinco AcM pertencentes à classe IgG que, embora näo-neutralizantes, reconheceram todas as amostras de BHV-1.1 e BHV-1.2 quando testados por imunoperoxidase sobre cultivos de células infectadas e ELISA. Os AcM näo foram capazes de distinguir entre os subtipos BHV-1.1 e BHV-1.2

Frequency of B cells in normal mice which recognize self proteins

The mechanism whereby the immune system avoids self-aggression is one of the central issues of Immunology. The discovery of natural autoantibodies, mainly of IgM isotype, and of idiotypic interactions between antibodies indicates that elements of the immune system interact with self constituents and with themselves. Results of studies with soluble antibodies have indicated that the pool of circulating IgM represents the end result of a highly selective process of B cell activation and differentiation by self proteins resulting in the formation of a network. The objective of the present work was to determine the frequency of self-reacting B cells in normal mice. We were able to detect B cells that recognize self proteins present in extracts of different organs in normal adult, 2-3-month old, BALB/c and C57BL/6 mice with an ELISA spot assay. About 1 per cent of total IgM-secreting cells among small, LPS-stimulated spleen cells reacted with organ extracts, whereas among large spleen cells the frequency was 5- to 10- fold lower. Immunization induced an increase in the frequency of IgM-secreting cells. The present results provide cellular evidence for the results of studies done at the serological level. The physiological role of these self-recognizing cells, as well as their participation in autoimmune processes, remain to be established.

Effect of IL-3 and E. coli LPS on the proliferation of mouse bone marrow cells in vitro

Bone marrow cells from adult BALB/c mice were cultured at 37-C, with 5% CO2 in air, in RPMI 1640 medium complemented with fetal serum. The addition of IL-3 (5% of WEHI-3-conditioned medium) or E. coli lipopolysaccharides (LPS, 50 µg/ml) to the cultures stimulated cell proliferation (1.29 and 1.22-fold, respectively, relative to control culture), whereas the simultaneous addition of the two factors reduced the number of cells recovered by 38% relative those from control cultures (which were around 2.83 x 10***5 cells for each 10***6 plated cells). The frequency of blasts and cells with surface Ig presented the same pattern of variation (o.07 and 0.02%, respectively, in control cultures). The inhibitory effect of IL-3+LPS on cell proliferation was evident from the first day of culture, but more apparent on day 3. Macrophage-colony stimulating factor (M-CSF, L929-conditioned medium) and LPS each given alone stimulated proliferation but reduced it when given together. In contrast, fetal liver cells were not affected by the simultaneous addition of IL-3 and LPS or by M-CSF and LPS. The mechanism of action of the cumulative effect of these two factors in unknown. Since crude cell-conditioned medium was used as the source of IL-3, it is possible that another factor present in this medium interacts with LPS to cause the inhibitory effect on cell proliferation

In vitro differentiation of Wuchereria bancrofti (Filariidae)

Wuchereria bancrofti microfilariae were isolated from blood of infected individuals and cultured in vitro under several conditions. RPMI 1640 and TC-199 media supplemented with fetal or human serum were able to support the microfilariae for periods up to 35 days at 37-C (viability > 85%). In contrast, in minimal essential medium the microfilariae did not survive for more than 48h. In culture kept at 28-C, where viability was much lower (approximately 10% on day 15), microfilariae differentiated into type IV larvae ("sausage form). The in vitro maintainance of microfilarial larval forms is particularly important in the case of W. bancrofti due to the absence of an experimental model for the disease

Antimitotic action of integerrimine in rats

Alguns alcalóides pirrolizidínicos hepatóxicos inibem a divisäo celular no fígado de ratos, após hepatectomía parcial. A atividade antimitótica da integerrimina, um alcalóide extraido de Senecio brasiliensis Less.var.tripartitus foi testada em ratos machos de uma linhagem endocruzada. Como estímulo `a divisäo celular, foi utilizada a hepatectomia parcial. Injeçöes intraperitoneais em doses de 0,025, 0,05, 0,2 e 0,4 da DL50 de integerrimina, 40 dias antes da hepatectomia, resultaram em 72,85, 82,02, 92,25 e 94,22% de inibiçäo da mitose, respectivamente. Nos animais que receberam as doses mais altas do alcalóide, foram observadas alteraçöes megalocíticas iniciais