Detection of the Epstein-Barr virus in blood and bone marrow mononuclear cells of patients with aggressive B-cell non-Hodgkin's lymphoma is not associated with prognosis

The Epstein-Barr virus (EBV) is associated with a large spectrum of lymphoproliferative diseases. Traditional methods of EBV detection include the immunohistochemical identification of viral proteins and DNA probes to the viral genome in tumoral tissue. The present study explored the detection of the EBV genome, using the BALF5 gene, in the bone marrow or blood mononuclear cells of patients with diffuse large B-cell lymphomas (DLBCL) and related its presence to the clinical variables and risk factors. The results show that EBV detection in 21.5% of patients is not associated with age, gender, staging, B symptoms, international prognostic index scores or any analytical parameters, including lactate dehydrogenase (LDH) or beta-2 microglobulin (B2M). The majority of patients were treated with R-CHOP-like (rituximab. cyclophosphamide, doxorubicin, vincristine and prednisolone or an equivalent combination) and some with CHOP-like chemotherapy. Response rates [complete response (CR) + partial response (PR)] were not significantly different between EBV-negative and -positive cases, with 93.2 and 88.9%, respectively. The survival rate was also similar in the two groups, with 5-year overall survival (OS) rates of 64.3 and 76.7%, respectively. However, when analyzing the treatment groups separately there was a trend in EBV-positive patients for a worse prognosis in patients treated with CHOP-like regimens that was not identified in patients treated with R-CHOP-like regimens. We conclude that EBV detection in the bone marrow and blood mononuclear cells of DLBC patients has the same frequency of EBV detection on tumoral lymphoma tissue but is not associated with the risk factors, response rate and survival in patients treated mainly with immunochemotherapy plus rituximab. These results also suggest that the addition of rituximab to chemotherapy improves the prognosis associated with EBV detection in DLBCL.

Protective effects of bone marrow mononuclear cell therapy on lung and heart in an elastase-induced emphysema model

We hypothesized that bone marrow-derived mononuclear cell (BMDMC) therapy protects the lung and consequently the heart in experimental elastase-induced emphysema. Twenty-four female C57BL/6 mice were intratracheally instilled with saline (C group) or porcine pancreatic elastase (E group) once a week during 4 weeks. C and E groups were randomized into subgroups receiving saline (SAL) or male BMDMCs (2 x 10(6), CELL) intravenously 3 h after the first saline or elastase instillation. Compared to E-SAL group, E-CELL mice showed, at 5 weeks: lower mean linear intercept, neutrophil infiltration, elastolysis, collagen fiber deposition in alveolar septa and pulmonary vessel wall, lung cell apoptosis, right ventricle wall thickness and area, higher endothelial growth factor and insulin-like growth factor mRNA expressions in lung tissue, and reduced platelet-derived growth factor, transforming growth factor-beta, and caspase-3 expressions. In conclusion, BMDMC therapy was effective at modulating the inflammatory and remodeling processes in the present model of elastase-induced emphysema. (c) 2012 Elsevier B.V. All rights reserved.

The effect of bone allografts combined with bone marrow stromal cells on the healing of segmental bone defects in a sheep model

Background: The repair of large bone defects is a major orthopedic challenge because autologous bone grafts are not available in large amounts and because harvesting is often associated with donor-site morbidity. Considering that bone marrow stromal cells (BMSC) are responsible for the maintenance of bone turnover throughout life, we investigated bone repair at a site of a critically sized segmental defect in sheep tibia treated with BMSCs loaded onto allografts. The defect was created in the mid-portion of the tibial diaphysis of eight adult sheep, and the sheep were treated with ex-vivo expanded autologous BMSCs isolated from marrow aspirates and loaded onto cortical allografts (n = 4). The treated sheep were compared with control sheep that had been treated with cell-free allografts (n = 4) obtained from donors of the same breed as the receptor sheep. Results: The healing response was monitored by radiographs monthly and by computed tomography and histology at six, ten, fourteen, and eighteen weeks after surgery. For the cell-loaded allografts, union was established more rapidly at the interface between the host bone and the allograft, and the healing process was more conspicuous. Remodeling of the allograft was complete at 18 weeks in the cell-treated animals. Histologically, the marrow cavity was reestablished, with intertrabecular spaces being filled with adipose marrow and with evidence of focal hematopoiesis. Conclusions: Allografts cellularized with AOCs (allografts of osteoprogenitor cells) can generate great clinical outcomes to noncellularized allografts to consolidate, reshape, structurally and morphologically reconstruct bone and bone marrow in a relatively short period of time. These features make this strategy very attractive for clinical use in orthopedic bioengineering

Bone marrow cells of swine: collection and separation

Bone marrow is a source of stem cells for greater and easier access, which is widely studied as a provider of hematopoietic and mesenchymal cells for various purposes, mainly therapeutic by the advances in research involving cell therapy. The swine is an animal species commonly used in the pursuit of development of experimental models. Thus, this study aimed to standardize protocol for collection and separation of bone marrow in swines, since this species is widely used as experimental models for various diseases. Twelve animals were used, which underwent bone marrow puncture with access from the iliac crest and cell separation by density gradient followed by a viability test with an average of 98% of viable cells. Given our results, we can ensure the swine as an excellent model for obtaining and isolation of mononuclear cells from bone marrow, stimulating several studies addressing the field of cell therapy. Microsc. Res. Tech., 2012. (C) 2012 Wiley Periodicals, Inc.

Kidney injury and cell therapy: Preclinical study

The aim of this study is to show histological and immunofluorescence analysis of renal parenchyma of agoutis affected by gentamicin-induced renal disease after the infusion of bone marrow mononuclear cells (BMMC) stained with Hoechst (R). Nine agouti's males were divided into three groups: Test group (TG): renal disease by gentamicin induced (n = 3), cell therapy group (CTG): renal disease by gentamicin induced and BMMC infusion (n = 3), and control group (CG): nonrenal disease and BMMC infusion (n = 3). TG and CTG were submitted to the protocol of renal disease induction using weekly application of gentamicin sulfate for 4 months. CG and CTG received a 1 X 108 BMMC stained with Hoechst and were euthanized for kidney examination 21 days after BMMC injection and samples were collected for histology and immunofluorescence analysis. Histological analysis demonstrated typical interstitial lesions in kidney similarly to human disease, as tubular necrosis, glomerular destruction, atrophy tubular, fibrotic areas, and collagen deposition. We conclude that histological analysis suggest a positive application of agouti's as a model for a gentamicin inducing of kidney disease, beyond the immunofluorescence analysis suggest a significant migration of BMMC to sites of renal injury in CTG. Microsc. Res. Tech., 2012. (c) 2011 Wiley Periodicals, Inc.

The Aurora A and B kinases are up-regulated in bone marrow-derived chronic lymphocytic leukemia cells and represent potential therapeutic targets

Background The malignant B cells in chronic lymphocytic leukemia receive signals from the bone marrow and lymph node microenvironments which regulate their survival and proliferation. Characterization of these signals and the pathways that propagate them to the interior of the cell is important for the identification of novel potential targets for therapeutic intervention. Design and Methods We compared the gene expression profiles of chronic lymphocytic leukemia B cells purified from bone marrow and peripheral blood to identify genes that are induced by the bone marrow microenvironment. Two of the differentially expressed genes were further studied in cell culture experiments and in an animal model to determine whether they could represent appropriate therapeutic targets in chronic lymphocytic leukemia. Results Functional classification analysis revealed that the majority of differentially expressed genes belong to gene ontology categories related to cell cycle and mitosis. Significantly up-regulated genes in bone marrow-derived tumor cells included important cell cycle regulators, such as Aurora A and B, survivin and CDK6. Down-regulation of Aurora A and B by RNA interference inhibited proliferation of chronic lymphocytic leukemia-derived cell lines and induced low levels of apoptosis. A similar effect was observed with the Aurora kinase inhibitor VX-680 in primary chronic lymphocytic leukemia cells that were induced to proliferate by CpG-oligonucleotides and interleukin-2. Moreover, VX-680 significantly blocked leukemia growth in a mouse model of chronic lymphocytic leukemia. Conclusions Aurora A and B are up-regulated in proliferating chronic lymphocytic leukemia cells and represent potential therapeutic targets in this disease.

Forced Expression of Nanog in Human Bone Marrow-Derived Endothelial Cells Activates Other Six Pluripotent Genes

Human endothelial cells (ECs) have the ability to make up the lining of blood vessels. These cells are also capable of neovascularization and revascularization and have been applied in various clinical situations. With the aim of understanding the effect of NANOG superexpression on ECs, we transduced the Nanog gene into the ECs. Nanog is highly expressed in embryonic stem cells (ESCs) and is essential for pluripotency and self-renewal. However, Nanog can also be expressed in somatic stem cells, and this gene is related to great expansion capacity in vitro. We found that ECs expressing Nanog showed expression of other stemness genes, such as Sox2, FoxD3, Oct4, Klf4, c-myc, and beta-catenin, that are not normally expressed or are expressed at very low levels in ECs. Nanog is one of the stemness genes that can activate other stemness genes, and the upregulation of the Nanog gene seems to be critical for reprogramming cells. In this study, the introduction of Nanog was sufficient to alter the expression of key genes of the pluripotent pathway. The functional importance of Nanog for altering the cell expression profile and morphology was clearly demonstrated by our results.

A quantitative proteomic and transcriptomic comparison of human mesenchymal stem cells from bone marrow and umbilical cord vein

Human mesenchymal stem cells (hMSCs) are adult multipotent cells that have high therapeutic potential due to their immunological properties. They can be isolated from several different tissues with bone marrow (BM) being the most common source. Because the isolation procedure is invasive, other tissues such as human umbilical cord vein (UCV) have been considered. However, their interchangeability remains unclear. In the present study, total protein extracts of BM-hMSCs and UCV-hMSCs were quantitatively compared using gel-LC-MS/MS. Previous SAGE analysis of the same cells was re-annotated to enable comparison and combination of these two data sets. We observed a more than 63% correlation between proteomic and transcriptomic data. In silico analysis of highly expressed genes in cells of both origins suggests that they can be modulated by microRNA, which can change protein abundance. Our results showed that MSCs from both tissues shared high similarity in metabolic and functional processes relevant to their therapeutic potential, especially in the immune system process, response to stimuli, and processes related to the delivery of the hMSCs to a given tissue, such as migration and adhesion. Hence, our results support the idea that the more accessible UCV could be a potentially less invasive source of MSCs.

Annexin A1 regulates neutrophil clearance by macrophages in the mouse bone marrow

Under homeostatic conditions, a proportion of senescent CXCR4(hi) neutrophils home from the circulation back to the bone marrow, where they are phagocytosed by bone marrow macrophages. In this study, we have identified an unexpected role for the anti-inflammatory molecule annexin A1 (AnxA1) as a critical regulator of this process. We first observed that AnxA1(-/-) mice have significantly increased neutrophil numbers in their bone marrow while having normal levels of GM and G colony-forming units, monocytes, and macrophages. Although AnxA1(-/-) mice have more neutrophils in the bone marrow, a greater proportion of these cells are senescent, as determined by their higher levels of CXCR4 expression and annexin V binding. Consequently, bone marrow neutrophils from AnxA1(-/-) mice exhibit a reduced migratory capacity in vitro. Studies conducted in vitro also show that expression of AnxA1 is required for bone marrow macrophages, but not peritoneal macrophages, to phagocytose apoptotic neutrophils. Moreover, in vivo experiments indicate a defect in clearance of wild-type neutrophils in the bone marrow of AnxA1(-/-) mice. Thus, we conclude that expression of AnxA1 by resident macrophages is a critical determinant for neutrophil clearance in the bone marrow.-Dalli, J., Jones, C. P., Cavalcanti, D. M., Farsky, S. H., Perretti, M., Rankin, S. M. Annexin A1 regulates neutrophil clearance by macrophages in the mouse bone marrow. FASEB J. 26, 387-396 (2012). www.fasebj.org

Morphological characterization of the progenitor blood cells in canine and feline umbilical cord

The umbilical cord blood (UCB) is an important source of hematopoietic stem cells with great deal of interest in regenerative medicine. The UCB cells have been extensively studied as an alternative to the bone marrow transplants. The challenge is to define specific methods to purify and characterize these cells in different animal species. This study is aimed at morphological characterization of progenitor cells derived from UCB highlighting relevant differences with peripheral blood of adult in dog and cats. Therefore, blood was collected from 18 dogs and 5 cats' umbilical cords from fetus in various developmental stages. The mononuclear cells were separated using the gradient of density Histopaque-1077. Characterization of CD34+ cells was performed by flow cytometric analysis and transmission electron microscopy. Granulocytes (ancestry of the basophiles, eosinophiles, and neutrophiles) and agranulocytes (represented by immature lymphocytes) were identified. We showed for the first time the ultrastructural features of cat UCB cells. Microsc. Res. Tech. 75:766770, 2012. (C) 2011 Wiley Periodicals, Inc.

Serial bone marrow transplantation reveals in vivo expression of the pCLPG retroviral vector

Abstract Background Gene therapy in the hematopoietic system remains promising, though certain aspects of vector design, such as transcriptional control elements, continue to be studied. Our group has developed a retroviral vector where transgene expression is controlled by p53 with the intention of harnessing the dynamic and inducible nature of this tumor suppressor and transcription factor. We present here a test of in vivo expression provided by the p53-responsive vector, pCLPG. For this, we used a model of serial transplantation of transduced bone marrow cells. Results We observed, by flow cytometry, that the eGFP transgene was expressed at higher levels when the pCLPG vector was used as compared to the parental pCL retrovirus, where expression is directed by the native MoMLV LTR. Expression from the pCLPG vector was longer lasting, but did decay along with each sequential transplant. The detection of eGFP-positive cells containing either vector was successful only in the bone marrow compartment and was not observed in peripheral blood, spleen or thymus. Conclusions These findings indicate that the p53-responsive pCLPG retrovirus did offer expression in vivo and at a level that surpassed the non-modified, parental pCL vector. Our results indicate that the pCLPG platform may provide some advantages when applied in the hematopoietic system.

IGFBP7 participates in the reciprocal interaction between acute lymphoblastic leukemia and BM stromal cells and in leukemia resistance to asparaginase

The interaction of acute lymphoblastic leukemia (ALL) blasts with bone marrow (BM) stromal cells (BMSCs) has a positive impact on ALL resistance to chemotherapy. We investigated the modulation of a series of putative asparaginase-resistance/sensitivity genes in B-precursor ALL cells upon coculture with BMSCs. Coculture with stromal cells resulted in increased insulin-like growth factor (IGF)-binding protein 7 (IGFBP7) expression by ALL cells. Assays with IGFBP7 knockdown ALL and stromal cell lines, or with addition of recombinant rIGFBP7 (rIGFBP7) to the culture medium, showed that IGFBP7 acts as a positive regulator of ALL and stromal cells growth, and significantly enhances in-vitro resistance of ALL to asparaginase. In these assays, IGFBP7 function occurred mainly in an insulin-and stromal-dependent manner. ALL cells were found to contribute substantially to extracellular IGFBP7 levels in the conditioned coculture medium. Diagnostic BM plasma from children with ALL had higher levels of IGFBP7 than controls. IGFBP7, in an insulin/IGF-dependent manner, enhanced asparagine synthetase expression and asparagine secretion by BMSCs, thus providing a stromal-dependent mechanism by which IGFBP7 protects ALL cells against asparaginase in this coculture system. Importantly, higher IGFBP7 mRNA levels were associated with lower leukemia-free survival (Cox regression model, P = 0.003) in precursor B-cell Ph(-) ALL patients (n = 147) treated with a contemporary polychemotherapy protocol.

Gene expression profiles displayed by peripheral blood mononuclear cells from patients with type 2 diabetes mellitus focusing on biological processes implicated on the pathogenesis of the disease

Patients with type 2 diabetes mellitus (T2DM) exhibit insulin resistance associated with obesity and inflammatory response, besides an increased level of oxidative DNA damage as a consequence of the hyperglycemic condition and the generation of reactive oxygen species (ROS). In order to provide information on the mechanisms involved in the pathophysiology of T2DM, we analyzed the transcriptional expression patterns exhibited by peripheral blood mononuclear cells (PBMCs) from patients with T2DM compared to non-diabetic subjects, by investigating several biological processes: inflammatory and immune responses, responses to oxidative stress and hypoxia, fatty acid processing, and DNA repair. PBMCs were obtained from 20 T2DM patients and eight non-diabetic subjects. Total RNA was hybridized to Agilent whole human genome 4x44K one-color oligo-microarray. Microarray data were analyzed using the GeneSpring GX 11.0 software (Agilent). We used BRB-ArrayTools software (gene set analysis - GSA) to investigate significant gene sets and the Genomica tool to study a possible influence of clinical features on gene expression profiles. We showed that PBMCs from T2DM patients presented significant changes in gene expression, exhibiting 1320 differentially expressed genes compared to the control group. A great number of genes were involved in biological processes implicated in the pathogenesis of T2DM. Among the genes with high fold-change values, the up-regulated ones were associated with fatty acid metabolism and protection against lipid-induced oxidative stress, while the down-regulated ones were implicated in the suppression of pro-inflammatory cytokines production and DNA repair. Moreover, we identified two significant signaling pathways: adipocytokine, related to insulin resistance; and ceramide, related to oxidative stress and induction of apoptosis. In addition, expression profiles were not influenced by patient features, such as age, gender, obesity, pre/post-menopause age, neuropathy, glycemia, and HbA(1c) percentage. Hence, by studying expression profiles of PBMCs, we provided quantitative and qualitative differences and similarities between T2DM patients and non-diabetic individuals, contributing with new perspectives for a better understanding of the disease. (C) 2012 Elsevier B.V. All rights reserved.

Interferon-gamma and interleukin-10 production by mononuclear cells from patients with advanced head and neck cancer

OBJECTIVE: This study aims to evaluate the production of interferon-gamma and interleukin-10 by stimulated peripheral blood mononuclear cells isolated from patients with supraglottic laryngeal cancer before and after surgical treatment. METHODS: Fourteen patients with advanced supraglottic laryngeal cancer were studied. Cultures of peripheral blood mononuclear cells isolated during the preoperative and late postoperative periods were stimulated with concanavalin A and Bacille Calmette-Guerin, and the supernatant concentrations of interferon-gamma and interleukin-10 were measured. RESULTS: For non-stimulated cultures, the interferon-gamma levels produced by the preoperative period and the late postoperative period cultures were lower than the levels produced by the control group cultures. The interferon-gamma levels after stimulation with concanavalin A were higher in the late postoperative period cultures than in the preoperative evaluation cultures. Stimulation with Bacille Calmette-Guerin led to the production of similar levels of interferon-gamma and interleukin-10 by all cultures; thus, stimulation increased the levels of interferon-gamma produced by both the preoperative and postoperative cultures relative to the levels produced by the corresponding unstimulated cultures. CONCLUSION: Patients with advanced supraglottic laryngeal cancer exhibit an in vitro deficiency in interferongamma secretion by mononuclear cells. Stimulated cells seem to recover this function during the postoperative period.

GM-CSF Priming Drives Bone Marrow-Derived Macrophages to a Pro-Inflammatory Pattern and Downmodulates PGE(2) in Response to TLR2 Ligands

In response to pathogen recognition by Toll-like receptors (TLRs) on their cell surface, macrophages release lipid mediators and cytokines that are widely distributed throughout the body and play essential roles in host responses. Granulocyte macrophage colony-stimulating factor (GM-CSF) is important for the immune response during infections to improve the clearance of microorganisms. In this study, we examined the release of mediators in response to TLR2 ligands by bone marrow-derived macrophages (BMDMs) primed with GM-CSF. We demonstrated that when stimulated with TLR2 ligands, non-primed BMDMs preferentially produced PGE(2) in greater amounts than LTB4. However, GM-CSF priming shifted the release of lipid mediators by BMDMs, resulting in a significant decrease of PGE(2) production in response to the same stimuli. The decrease of PGE(2) production from primed BMDMs was accompanied by a decrease in PGE-synthase mRNA expression and an increase in TNF-alpha and nitric oxide (NO) production. Moreover, some GM-CSF effects were potentiated by the addition of IFN-gamma. Using a variety of TLR2 ligands, we established that PGE(2) release by GM-CSF-primed BMDMs was dependent on TLR2 co-receptors (TLR1, TLR6), CD14, MyD88 and the nuclear translocation of NF kappa B but was not dependent on peroxisome proliferator-activated receptor-gamma (PPAR-gamma) activation. Indeed, GM-CSF priming enhanced TLR2, TLR4 and MyD88 mRNA expression and phospho-I kappa B alpha formation. These findings demonstrate that GM-CSF drives BMDMs to present a profile relevant to the host during infections.