Prevention of bone marrow graft failure by an anti LFA-1 monoclonal antibody

Graft rejection is the major cause of failure of HLA mismatched bone marrow transplantation because of residual host immunity. we have proposed to use a monoclonal murine antibody specific for the LFA-1 molecule (25-3) to prevent graft failure in HLA mismatched bone marrow transplantation (BMT). The rationale for this approach is three fold: LFA-1 deficient patients (3/3) do not reject HLA mismatched BMT; anti LFA-1 blocka in vitro the induction of T cell responses and T/ non T cytotoxic functions; LFA-1 is not expressed by other cells than leucocytes. We have accordingly treated twenty two patients with inherited diseases and 8 with leikemia. The bone marrow was T cells depled by E rosetting of Campath antibody. The antibody was given at days -3, -1, +1, +3, +5 at dose of .1 mg/kg/d for the first 9 and then .2mg/kg/d from day -3 to +6. Engraftment occured in 23/30 patients as shown by at least HLA typing. Hematological recovery was rapid, GVH was limited. Side effects of antibody infusion included fever and possibly an increased incidence of early bacteral infection (sepsis, 1 death). Immunological reconstitution occured slowly leading in six cases to EBV-induced B cell poliferation (1 death and in two others to transient auto immune hemolytic anemia. There has been only one secondary graft rejection. Sisteen patients are alive 3 to 26 months post transplant with functional grafts. Although the number of patients treated is still low the absence of late rejection so far, gives hope for long term maintenance of the graft using anti LFA-1. Since the antibody is an IgG 1 unable to bind human complement, and since it is known to inhibit phagocytosis, there is a good suggestion that 25-3 act through functional blocking of host T and non T luymphocytes at both induction and effector levels.

Bone marrow contribution to eosinophilic inflammation

Allergen-induced bone marrow responses are observable in human allergic asthmatics, involving specific increases in eosinophil-basophil progenitors (Eo/B-CFU), measured either by hemopoietic assays or by flow cytometric analyses of CD34-positive, IL-3Ralpha-positive, and/or IL-5-responsive cell populations. The results are consistent with the upregulation of an IL-5-sensitive population of progenitors in allergen-induced late phase asthmatic responses. Studies in vitro on the phenotype of developing eosinophils and basophils suggest that the early acquisition of IL-5Ralpha, as well as the capacity to produce cytokines such as GM-CSF and IL-5, are features of the differentiation process. These observations are consistent with findings in animal models, indicating that allergen-induced increases in bone marrow progenitor formation depend on hemopoietic factor(s) released post-allergen. The possibility that there is constitutive marrow upregulation of eosinophilopoiesis in allergic airways disease is also an area for future investigation.

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.

Culture of equine bone marrow mononuclear fraction and adipose tissue-derived stromal vascular fraction cells in different media

The objective of this study was to evaluate the culture of equine bone marrow mononuclear fraction and adipose tissue - derived stromal vascular fraction cells in two different cell culture media. Five adult horses were submitted to bone marrow aspiration from the sternum, and then from the adipose tissue of the gluteal region near the base of the tail. Mononuclear fraction and stromal vascular fraction were isolated from the samples and cultivated in DMEM medium supplemented with 10% fetal bovine serum or in AIM-V medium. The cultures were observed once a week with an inverted microscope, to perform a qualitative analysis of the morphology of the cells as well as the general appearance of the cell culture. Colony-forming units (CFU) were counted on days 5, 15 and 25 of cell culture. During the first week of culture, differences were observed between the samples from the same source maintained in different culture media. The number of colonies was significantly higher in samples of bone marrow in relation to samples of adipose tissue.

Morphology and morphometry of feline bone marrow-derived mesenchymal stem cells in culture

Mesenchymal stem cells (MSC) are increasingly being proposed as a therapeutic option for treatment of a variety of different diseases in human and veterinary medicine. Stem cells have been isolated from feline bone marrow, however, very few data exist about the morphology of these cells and no data were found about the morphometry of feline bone marrow-derived MSCs (BM-MSCs). The objectives of this study were the isolation, growth evaluation, differentiation potential and characterization of feline BM-MSCs by their morphological and morphometric characteristics. in vitro differentiation assays were conducted to confirm the multipotency of feline MSC, as assessed by their ability to differentiate into three cell lineages (osteoblasts, chondrocytes, and adipocytes). To evaluate morphological and morphometric characteristics the cells are maintained in culture. Cells were observed with light microscope, with association of dyes, and they were measured at 24, 48, 72 and 120h of culture (P1 and P3). The non-parametric ANOVA test for independent samples was performed and the means were compared by Tukey's test. On average, the number of mononuclear cells obtained was 12.29 (±6.05x10(6)) cells/mL of bone marrow. Morphologically, BM-MSCs were long and fusiforms, and squamous with abundant cytoplasm. In the morphometric study of the cells, it was observed a significant increase in average length of cells during the first passage. The cell lengths were 106.97±38.16µm and 177.91±71.61µm, respectively, at first and third passages (24 h). The cell widths were 30.79±16.75 µm and 40.18±20.46µm, respectively, at first and third passages (24 h).The nucleus length of the feline BM-MSCs at P1 increased from 16.28µm (24h) to 21.29µm (120h). However, at P3, the nucleus length was 26.35µm (24h) and 25.22µm (120h). This information could be important for future application and use of feline BM-MSCs.

Protocol for collection and separation of bone marrow mononuclear cells in Chlorocebus aethiops

Abstract: Chlorocebus aethiops is a species of non-human primate frequently used in biomedical research. Some research involves this species as an experimental model for various diseases and possible treatment with stem cells. The bone marrow is one of the main sources of these cells and provides easy access. The aim of this study was to standardize the protocol of collection and separation of bone marrow in C. aethiops. Ten animals were submitted to puncture of bone marrow with access to the iliac crest and cell separation by density gradient. The bone marrow of C. aethiops had an average of 97% viability. From the results achieved, we can conclude that C. aethiops is an excellent model to obtain and isolate mononuclear cells from bone marrow, fostering several studies in the field of cell therapy.

Genotoxic activity of the insecticide Nuvacron (Monocrotophos) detected by the micronucleus test in bone marrow erythrocytes of mice and in CHO cells

The organophosphorus insecticide Nuvacron (Monocrotophos) is a very toxic agent widely utilized in Brazilian agriculture. To evaluate the clastogenic potential of this insecticide, in vivo and in vitro micronucleus (MN) assay experiments were carried out on Swiss mice and on Chinese hamster ovary (CHO) cells, respectively. Nuvacron administered at doses of 2.5 and 5.0 mg/kg induced a statistically significant increase in the frequencies of MN detected in polychromatic bone marrow erythrocytes from animals (six/group) treated ip 24 h before. Exponentially growing CHAO cells were treated continuously (16h) with Nuvacron diluted in water to final concentrations of 1, 10, 100, 200, and 400 mug/ml. Three experiments were carried out using the cytokinesis-block method and a total of 6000 binucleated cells were scored to determine MN frequencies. A statistically significant increase in the frequencies of MN was observed for the cells treated with 1 and 10 mug/ ml Nuvacron. A marked decrease in cell proliferation rates was observed for CHO cultures treated with higher concentrations. These data demonstrate that Nuvacron has a genotoxic effect on both in vivo and in vitro mammalian test systems.

Alterations in proteins of bone marrow extracellular matrix in undernourished mice

The objective of the present study was to determine the effect of protein malnutrition on the glycoprotein content of bone marrow extracellular matrix (ECM). Two-month-old male Swiss mice were submitted to protein malnutrition with a low-protein diet containing 4% casein as compared to 20% casein in the control diet. When the experimental group had attained a 20% loss of their original body weight, we extracted the ECM proteins from bone marrow with PBS buffer, and analyzed ECM samples by SDS-PAGE (7.5%) and ECL Western blotting. Quantitative differences were observed between control and experimental groups. Bone marrow ECM from undernourished mice had greater amounts of extractable fibronectin (1.6-fold increase) and laminin (4.8-fold increase) when compared to the control group. These results suggest an association between fluctuations in the composition of the hematopoietic microenvironment and altered hematopoiesis observed in undernourished mice.

Immunophenotype of normal and leukemic bone marrow B-precursors in a Brazilian population. A comparative analysis by quantitative fluorescence cytometry

The distinction between normal and leukemic bone marrow (BM) B-precursors is essential for the diagnosis and treatment monitoring of acute lymphoblastic leukemia (ALL). In order to evaluate the potential use of quantitative fluorescence cytometry (QFC) for this distinction, we studied 21 normal individuals and 40 patients with CD10+ ALL. We characterized the age-related changes of the CD10, CD19, TdT, CD34 and CD79a densities in normal and leukemic BM. Compared to normal adults, the B-precursors from normal children expressed significantly lower values of CD34-specific antibody binding capacity (SABC) (median value of 86.6 vs 160.2 arbitrary units (a.u.) in children and adults, respectively). No significant age-related difference was observed in the expression of the other markers in the normal BM, or in any of the markers in the leukemic BM. Based on the literature, we set the cut-off value for the normal CD10 expression at 45 x 10³ a.u. for both age groups. For the remaining markers we established the cut-off values based on the minimum-maximum values in the normal BM in each age group. The expression of CD10 was higher than the cut-off in 30 ALL cases and in 18 of them there was a concomitant aberrant expression of other markers. In 9 of the 10 CD10+ ALL with normal CD10 SABC values, the expression of at least one other marker was aberrant. In conclusion, the distinction between normal and leukemic cells by QFC was possible in 38/40 CD10+ ALL cases.

Bone marrow fibroblasts in patients with advanced lung cancer

In a previous study we demonstrated that the incidence of fibroblast colony-forming units (CFU-F) was very low in bone marrow primary cultures from the majority of untreated advanced non-small lung cancer patients (LCP) compared to normal controls (NC). For this reason, we studied the ability of bone marrow stromal cells to achieve confluence in primary cultures and their proliferative capacity following four continuous subcultures in consecutive untreated LCP and NC. We also evaluated the production of interleukin-1ß (IL-1ß) and prostaglandin E2 (PGE2) by pure fibroblasts. Bone marrow was obtained from 20 LCP and 20 NC. A CFU-F assay was used to investigate the proliferative and confluence capacity. Levels of IL-1ß and PGE2 in conditioned medium (CM) of pure fibroblast cultures were measured with an ELISA kit and RIA kit, respectively. Only fibroblasts from 6/13 (46%) LCP confluent primary cultures had the capacity to proliferate following four subcultures (NC = 100%). Levels of spontaneously released IL-1ß were below 10 pg/ml in the CM of LCP, while NC had a mean value of 1,217 ± 74 pg/ml. In contrast, levels of PGE2 in these CM of LCP were higher (77.5 ± 23.6 pg/ml) compared to NC (18.5 ± 0.9 pg/ml). In conclusion, bone marrow fibroblasts from LCP presented a defective proliferative and confluence capacity, and this deficiency may be associated with the alteration of IL-1ß and PGE2 production.

Reimmunization after bone marrow transplantation: current recommendations and perspectives

Autologous and allogeneic bone marrow transplantation (BMT) recipients lose immune memory of exposure to infectious agents and vaccines accumulated through a lifetime and therefore need to be revaccinated. Diphtheria toxoid, tetanus toxoid, pertussis vaccine (children <7 years old), Haemophilus influenzae type b conjugate, 23-valent pneumococcal polysaccharide, inactivated influenza vaccine, inactivated polio vaccine and live-attenuated measles-mumps-rubella vaccine are the currently recommended vaccines to be included in a vaccination program after BMT. For most of them, the best time to vaccinate, the number of vaccine doses and/or the duration of immunity after vaccination have not been established. Vaccination protocols vary greatly among BMT centers, suggesting that the lack of sufficient data has not permitted the formulation of reliable recommendations. The use of other vaccines and the perspectives for different vaccination protocols are analyzed in this review.

Multicellular spheroids of bone marrow stromal cells: a three-dimensional in vitro culture system for the study of hematopoietic cell migration

Cell fate decisions are governed by a complex interplay between cell-autonomous signals and stimuli from the surrounding tissue. In vivo cells are connected to their neighbors and to the extracellular matrix forming a complex three-dimensional (3-D) microenvironment that is not reproduced in conventional in vitro systems. A large body of evidence indicates that mechanical tension applied to the cytoskeleton controls cell proliferation, differentiation and migration, suggesting that 3-D in vitro culture systems that mimic the in vivo situation would reveal biological subtleties. In hematopoietic tissues, the microenvironment plays a crucial role in stem and progenitor cell survival, differentiation, proliferation, and migration. In adults, hematopoiesis takes place inside the bone marrow cavity where hematopoietic cells are intimately associated with a specialized three 3-D scaffold of stromal cell surfaces and extracellular matrix that comprise specific niches. The relationship between hematopoietic cells and their niches is highly dynamic. Under steady-state conditions, hematopoietic cells migrate within the marrow cavity and circulate in the bloodstream. The mechanisms underlying hematopoietic stem/progenitor cell homing and mobilization have been studied in animal models, since conventional two-dimensional (2-D) bone marrow cell cultures do not reproduce the complex 3-D environment. In this review, we will highlight some of the mechanisms controlling hematopoietic cell migration and 3-D culture systems.

Linking immunity and hematopoiesis by bone marrow T cell activity

Two different levels of control for bone marrow hematopoiesis are believed to exist. On the one hand, normal blood cell distribution is believed to be maintained in healthy subjects by an "innate" hematopoietic activity, i.e., a basal intrinsic bone marrow activity. On the other hand, an "adaptive" hematopoietic state develops in response to stress-induced stimulation. This adaptive hematopoiesis targets specific lineage amplification depending on the nature of the stimuli. Unexpectedly, recent data have shown that what we call "normal hematopoiesis" is a stress-induced state maintained by activated bone marrow CD4+ T cells. This T cell population includes a large number of recently stimulated cells in normal mice whose priming requires the presence of the cognate antigens. In the absence of CD4+ T cells or their cognate antigens, hematopoiesis is maintained at low levels. In this review, we summarize current knowledge on T cell biology, which could explain how CD4+ T cells can help hematopoiesis, how they are primed in mice that were not intentionally immunized, and what maintains them activated in the bone marrow.

Pharmacokinetic and parasitological evaluation of the bone marrow of dogs with visceral leishmaniasis submitted to multiple dose treatment with liposome-encapsulated meglumine antimoniate

The aim of the present study was to evaluate the impact of a multiple dose regimen of a liposomal formulation of meglumine antimoniate (LMA) on the pharmacokinetics of antimony in the bone marrow of dogs with visceral leishmaniasis and on the ability of LMA to eliminate parasites from this tissue. Dogs naturally infected with Leishmania chagasi received 4 intravenous doses of either LMA (6.5 mg antimony/kg body weight, N = 9), or empty liposomes (at the same lipid dose as LMA, N = 9) at 4-day intervals. A third group of animals was untreated (N = 8). Before each administration and at different times after treatment, bone marrow was obtained and analyzed for antimony level (LMA group) by electrothermal atomic absorption spectrometry, and for the presence of Leishmania parasites (all groups). There was a significant increase of antimony concentration from 0.76 µg/kg wet organ (4 days after the first dose) to 2.07 µg/kg (4 days after the fourth dose) and a half-life of 4 days for antimony elimination from the bone marrow. Treatment with LMA significantly reduced the number of dogs positive for parasites (with at least one amastigote per 1000 host cells) compared to controls (positive dogs 30 days after treatment: 0 of 9 in the LMA group, 3 of 9 in the group treated with empty liposomes and 3 of 8 in the untreated group). However, complete elimination of parasites was not achieved. In conclusion, the present study showed that multiple dose treatment with LMA was effective in improving antimony levels in the bone marrow of dogs with visceral leishmaniasis and in reducing the number of positive animals, even though it was not sufficient to achieve complete elimination of parasites.