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Inside Experimental Hematology: January and February 2015

Posted By Connections Editor, Friday, February 27, 2015
Updated: Monday, February 23, 2015


An ALL xenograft model that simulates the process of clonal selection of chemoresistant clones
Nowak et al., pages 32-93.

The combination of high throughput molecular techniques such as next generation sequencing and copy number micro arrays in combination with xenotransplantation models have revealed that the leukemic cell pool of patients suffering from acute lymphoblastic leukemia (ALL) is not only composed of a sequentially developed dominant clone, but also of a disturbingly large collection of additional highly variable subclonal populations with a complex branching architecture of ancestry. This subclonal diversity, which is formed by a high genetic plasticity of ALL cells, is thought to constitute a reservoir for disease relapse and for cell populations resistant to therapy. This study by Nowak et al tested the feasibility of simulating the process of clonal selection of chemoresistant ALL clones in an in vivo xenograft model. Exposing ALL cells to the selective pressure of chemotherapy led to a rapid outgrowth of clones harboring genomic lesions conferring resistance. Even by next-generation deep sequencing, an emerging chemoresistant clone could not be detected in the samples before drug selection. This finding shows a need to further investigate the genetic plasticity of ALL cells under treatment with chemotherapy to elucidate the dynamics by which resistant subclones emerge. The presented model for induction of chemoresistance provides a platform for performing such studies.

Mixed chimerism in C3a-deficient mice
Baskiewicz-Ha1asa et al., pages 14-22

Transplantation tolerance is an important goal in the effort to reduce long-term morbidity and mortality in organ transplant recipients. Mixed chimerism promises transplant tolerance because
donor-derived antigen-presenting cells migrate to the thymus, present donor antigens to developing recipient thymocytes, and promote the elimination of donor reactive cells through negative selection. Following up on recent studies showing that the C3a-complement component plays a major role in hematopoietic cell circulation and attraction to hematopoietic niches after
transplantation, Ba_skiewicz-Ha1asa et al. investigated in this study the role of C3a in immune tolerance induction in a chimeric mice model. The analysis of mixed chimerism rate in peripheral blood leukocytes during the 20-week experiment revealed that stable multilineage mixed chimerism was not effectively established in C3a_/_ mice, in contrast to wild-type animals. The tolerance to donor antigens (Balb/c) related to mixed chimerism was also not achieved in C3a_/_ mice, as it was observed employing lymphocytes expressing Vb5 and Vb11 TCRs measurement and skin graft acceptance analysis. The present study demonstrates that C3a is essential for achieving stable mixed chimerism and resulting transplant tolerance. Although the levels of hematopoietic stem and progenitor cells were comparable in C3a_/_ and wild-type mice, the hematopoietic environment in C3a-deficient animals was defective for engraftment of transplanted cells. Because blocking C3a anaphylatoxin has been considered as a potential technique to avoid graft rejection, the authors suggest that blocking C3a might not be applicable in transplantation procedures that are aimed at mixed chimerism.

Donor chimerism in CD25D-activated leukocytes as a predictor of GvHD after stem cell transplantation
Martínez -Laperche et al., pages 4-13

Development of severe graftvs-host disease (GvHD) remains the main complication of hematopoietic stem cell transplantation.CD3þ T lymphocytes (TL) chimerism is currently the cornerstone of leukocyte lineage chimerism analysis for the evaluation of events related to the development of GvHD. However, the TL subset may include both alloreactive and naive cells, what underscores the need to further dissect the TL subset, targeting activated leukocytes (AL) for chimerism studies, since only alloreactive cells would be considered in the analysis. In this study, Martínez -Laperche et al. evaluated the impact of the status of ALs (CD25þ) chimerism on the incidence and clinical course of GvHD in allogeneic transplant recipients after myeloablative conditioning. This retrospective study shows that the analysis of chimerism in AL at day 30 and day 90 after stem cell tranplantation adds to the study of TL and could be useful for the improved anticipation of acute GvHD and chronic GvHD, respectively. The association between
chimerism in AL and GvHD would aid in clinical decision-making by allowing early post-transplant modification of immunomodulatory therapies.


Increase level of intact CXCL12 in the spleen of patients with myelofibrosis: a mechanism underlying the development of extramedullary hematopoiesis
Wang et al., pages 100-109

Myelofibrosis (MF) is characterized by the constitutive mobilization of hematopoietic stem and progenitor cells (HSCs/HPCs) and the establishment of extramedullary hematopoiesis (EMH). The mechanisms underlying this abnormal HSC/HPC trafficking pattern remain poorly understood. In this study, Wang and coworkers identified a possible mechanism by which MF CD34+ cells lodge in the spleens of MF patients, eventually leading to the development of EMH. The authors observed a dramatically higher number of splenic MF CD34+ cells that migrated in response to splenic homogenate supernatant (plasma) of MF patients, as compared with peripheral blood (PB) MF plasma. They documented that the concentration of the intact HSC/HPC chemoattractant CXCL12 was greater in splenic MF plasma than PB MF plasma, as quantified using mass spectrometry. By contrast, comparable concentrations of the four functionally inactive truncated products of CXCL12, which are due to proteolytic degradation by serine proteases, were detected in both splenic and PB MF plasmas. The authors further showed that the treatment with an anti-CXCL12 neutralizing antibody resulted in a reduction in the degree of migration of splenic MF CD34+ cells toward both PB and splenic MF plasma, validating the role of CXCL12 as a functional chemoattractant. Their data indicate that the MF splenic microenvironment is characterized by increased levels of intact, functional CXCL12, which contributes to the homing of MF CD34+ cells to the spleens, rather than the marrows, of MF patients, ultimately leading to EMH in the spleen. Therapeutic strategies that would be capable of reversing such pathological chemokine gradients might represent a possible means of correcting the abnormal cellular trafficking characteristic of MF-HSC/HPC and delaying disease progression. 


A novel anti-HLA monoclonal antibody with anti-GVHD activity
Nakauchi et al., pages 79-88

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) using cord blood or haploidentical donor cells is a promising alternative option for patients who cannot find a human leukocyte antigen (HLA)-matched donor. However, HLA-mismatched allo-HSCT may be complicated by graft-versus-host disease (GVHD), a major cause of nonrelapse mortality mediated by alloreactive T cells. Although several agents have been developed for effective control of GVHD, serious side effects are the major issues to be addressed. In this paper, Nakauchi et al. established a novel therapeutic approach to GVHD using allele-specific anti-HLA monoclonal antibodies (ASHmAbs) that specifically recognize donor-derived cells and cause their death. One such ASHmAb against HLA-A*02:01 (A2-kASHmAb) was examined for its effectiveness in preventing disease in a xenogeneic GVHD mouse model. To induce fatal GVHD, nonirradiated immunodeficient mice were injected with healthy-donor human peripheral blood mononuclear cells with or without expression of HLA-A*02:01. Besides the induction of complement-dependent cell death on their target cells in vitro, the administration of the allele-specific anti-HLA antibody effectively ameliorated the GVHD symptoms of the xenogeneic nonirradiated immunodeficient mice and markedly improved their survival. Furthermore, the authors observed the recovery of human blood-cell chimerism after the antibody treatment, indicating that human hematopoietic stem cells were preserved. This study is the first reported instance of the effectiveness of a cytotoxic anti-HLA antibody against GVHD. This novel therapeutic approach may offer hope for effective treatment of GVHD, favorably influencing the outcome of allo-HSCT.


Do osteoclasts have a role in HSC trafficking?
Rao et al., pages 110-114

The role of osteoclasts in regulating hematopoietic stem/progenitor cell (HSPC) trafficking in the bone marrow is controversial. Initial reports suggested that osteoclasts promote HSPC egress from the bone marrow. On the other hand, genetic or pharmacologic inhibition of osteoclast activity is associated with accentuated granulocyte colony–stimulating factor (G-CSF)-induced HSPC mobilization. This question has potential clinical relevance, since pharmacologic agents that inhibit osteoclasts are widely used in the clinic to treat osteoporosis. Definitive studies addressing this issue have been limited by the osteopetrosis and extramedullary hematopoiesis associated with transgenic mouse models of osteoclast deficiency. To overcome this limitation, Rao et al. developed in this study two complementary nonosteopetrotic mouse models to assess the impact of osteoclast deficiency on HSPC trafficking. In the first model, they generated Rank−/− fetal liver chimeras in Csf3r−/− recipient mice. These mice have normal osteoclast numbers but the osteoclasts lack G-CSF receptor expression. In the second model, they acutely depleted osteoclasts by treating wild-type mice with osteoprotegrin-Fc (OPG-Fc, Figure). In both models, HPSC mobilization at baseline and after G-CSF treatment was comparable to control mice. These data show that osteoclasts are not required for the efficient retention of HSPCs in the murine bone marrow and are dispensable for HSPC mobilization by G-CSF. According to these data, pharmacologic strategies to augment HSPC mobilization by disrupting osteoclast function are unlikely to be effective.


Mature adipocytes as potential cell source for therapeutic angiogenesis
Poloni et al., pages 137-146.

Mature adipocytes are generally considered terminally differentiated because they have lost their proliferative abilities. However, this group has previously reported that adipocytes can dedifferentiate to mesenchymal stem cells. In this study, Poloni et al. show that human adipocytes can also differentiate toward the endothelial lineage without change in karyotype. The cells formed cord- or tube-like structures when cultured in specific medium and expressed several endothelial marker genes and proteins, such as vascular endothelial growth factor receptor 2, vascular endothelial cadherin, von Willebrand factor, and CD133. The characteristics displayed by the cells may reflect an intrinsic plasticity of mature adipocytes to change into different cell types and, moreover, to revert into an immature phase without any chromosomal alterations. These results also highlight the concept that adipose lineage cells may represent a suitable new cell source for therapeutic angiogenesis in ischemic disease.

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