Dualism of mixed chimerism between hematopoiesis and stroma in chronic idiopathic myelofibrosis after allogeneic stem cell transplantation.

Scant knowledge exists concerning lineage-restricted mixed chimerism (mCh) after allogeneic peripheral blood stem cell transplantation (PSCT) in patients with chronic idiopathic myelofibrosis (CIMF). Following a sex-mismatched PSCT, a combined immunopheno- and genotyping by fluorescence in-situ hybridization (FISH) was performed on sequential bone marrow (BM) biopsies at standardized intervals. Results were compared with PCR analysis of corresponding peripheral blood samples in five patients. According to FISH, pretransplant specimens revealed a gender congruence of more than 99%, while in the first three months the total BM exhibited a persistent fraction of host cells (30% to 40%) with a tendency to decline after about one year. It is noteworthy that the majority of endothelial cells maintained a recipient origin, whereas CD34+ progenitors and especially CD61+ megakaryocytes exhibited only very few host-derived cells. In keeping with the prevalence of donor cells in the hematopoietic compartment, PCR analysis of peripheral blood cells displayed a non-significant degree of mCh. In conclusion, according to FISH and PCR analysis, successful PSCT in CIMF results in an almost complete chimeric (donor-derived) state of the hematopoietic cell population. The non-transplantable stromal compartment includes the vascular endothelium with a predominance of recipient cells. The minimal mCh of this population implies probably a donor-derived origin (endothelial progenitor cells).

Trisomy 8 in prefibrotic early stages of chronic idiopathic myelofibrosis: a fluorescence in situ study of bone marrow biopsies.

Repeatedly performed bone marrow biopsies were studied in 30 patients with chronic idiopathic myelofibrosis (CIMF) by fluorescence in situ hybridization to detect and quantify trisomy 8 anomaly during the evolution of disease. For the establishment of threshold values we used negative and positive control specimens. At least 500 cells were evaluated in each specimen and only nuclei with three distinctive signals were regarded as positive. According to the controls, 27 patients revealed false-positive signals ranging from 0 to 1.2% (0.88 +/- 1.12). On the other hand, 3 patients showed an incidence of more than 6.5% (up to 10.1%) in the initial prefibrotic as well as advanced fibro-osteosclerotic stages of CIMF. In conclusion, trisomy 8 has been demonstrated already in the prodromal stages of CIMF and therefore is not limited to classical fibro-osteosclerotic manifestations.

[Regression of the Philadelphia chromosome (bcr/abl)-positive myelo- and megakaryopoiesis after Imatinib (STI571) therapy in chronic myelogenous leukemia (CML)]. / Regression der Philadelphia-Chromosom (bcr/abl)-positiven Myelo- und Megakaryopoiese unter Imatinib(STI571)-Therapie bei chronischer myeloischer Leukämie (CML).

In chronic myeloid leukemia following therapy with Imatinib (STI571) hematologic and cytogenetic response is associated with conspicuous changes of bone marrow morphology. However, it is not known to which extent these alterations are accompanied by a loss of the bcr/abl translocation. To study regression of the leukemic cell population we recruited 14 patients lacking pretreatment. Therapy resulted in a reduction of CD61(+) megakaryopoiesis. Dwarf megakaryocytes characteristic for this disorder were replaced by large, normally appearing cells of this lineage. Morphometric analysis confirmed the significant decrease in the number of micromegakaryocytes and yielded planimetric parameters in keeping with normalization. Moreover, a fluorescence in-situ hybridization study in five patients of this cohort revealed that before therapy 70% of all myeloid cells exhibited the bcr/abl gene. Regarding megakaryopoiesis about 65% of the micromegakaryocytes displayed positive signals. Following treatment these bcr/abl(+) cell populations decreased significantly while the emerging large megakaryocytes lacked a proper labeling. Because cytogenetic response and reduction of atypical micromegakaryocytes are linked, this feature may be useful to monitor therapeutic efficacy.

Regeneration of heart muscle tissue: quantification of chimeric cardiomyocytes and endothelial cells following transplantation.

Persuasive evidence has been recently provided that adult bone marrow (BM) cells exert greater plasticity than previously assumed. This review is focused on the quantification of mixed chimerism (mCh) in the hearts (cardiomyocytes and endothelial cells) of patients after orthotopic heart to heart transplantation (HHT) in comparison to full (unmanipulated) allogeneic BM and peripheral blood stem cell (PBSC) transplants. Following a sex-mismatched transplantation constellation heart muscle tissue obtained at autopsy was examined. Evaluation of mCh was most often performed by immunophenotyping combined with fluorescence in-situ hybridization (FISH) applying x- and y-chromosome-specific DNA probes. When comparing our data with the results of former studies that were regularly based on the detection of the y-chromosome alone, the quantity of chimeric cardiomyocytes after HHT ranged from 0% to 9%. On the other hand, after full BM transplantats (chimeric) cardiomyocytes of donor-type origin appeared at an incidence between 0.23% to 6.4%. These disturbing inconsistencies were assumed to be related to methodology: the restriction to the y-chromosome, disregard of the plane of section (detection sensitivity ranging between 35% and 67%) and state of tissue preservation (cadaver hearts). Therefore, when strictly applying dual color FISH and limiting the recognition of chimeric cardiomyocytes and endothelial cells to the presence of two distinctive signals detection sensitivity was significantly enhanced. Contrasting a total congruence with the genotyping in control specimens of normal cadaver hearts, a striking disparity in the extent of mCh was found depending on the different modes of transplantation. After allografting with PBSC a considerably low incidence (1.6%) of chimeric cardiomyocytes was determined contrasting with 5.3% of donor-derived cells after full BM transplants. Following HHT host-type endothelial cells (16.2%) of the intramural and subepicardial vessel walls were more often encountered than following BM and PBSC allografting. These findings are in keeping with the assumption of a sprouting and migration of vascular structures into the donor heart from the site of surgical aligment and injury between retained host and donor atrial walls. When considering the other methods of transplantation (BM, PBSC) the data on chimeric endothelial cells support the hypothesis of a common hemangioblast. Concerning the cardiomyocytes it seems most reasonable to assume that primitive mesenchymal stem cells of the BM play a pivotal role in the development of mCh. This phenomenon is more extensively expressed than previously expected and may be related to an enforced repair of the damaged myocardium during the post-transplant period as the sequel of myeloablative (cardiotoxic) conditioning.

Mixed chimerism of erythro- and megakaryopoiesis following allogeneic bone marrow transplantation.

Until now, studies on mixed chimerism (MCh) after allogeneic bone marrow transplantation (BMT) have predominantly focused on the B- and T-lymphocyte population, but not on distinct myeloid cell lineages like nucleated erythroid precursors and megakaryocytes. To evaluate the lineage-restricted MCh more explicitly in 10 patients with chronic myelogenous leukemia (CML), a quantitative analysis was performed on bone marrow biopsies following a sex-mismatched host/donor constellation. Techniques included immunophenotyping (antiglycophorin C, CD61) for the identification of erythro- and megakaryopoiesis and a simultaneously conducted genotyping with x- and y-chromosome-specific DNA probes. Normal bone marrow and specimens taken before BMT served as controls. Contrasting a total gender-dependent sex-typing in the latter samples in the early and late posttransplant period (up to 586 days), 3-9% erythroid precursors and about 16% megakaryocytes revealed a host-type origin. This significantly higher number of host megakaryocytes is explained by their polyploidy generating an increased probability to detect positive signals at a certain section level of the corresponding biopsies. A striking conversion of MCh to a recipient cell type was found in leukemic relapse with a more than 90% host-derived erythroid and megakaryocytic cell population in 4 patients approximately 643 days after BMT.

Dynamics of lineage-restricted mixed chimerism following sex-mismatched allogeneic bone marrow transplantation.

Scant knowledge is available about the dynamics of lineage-specific mixed chimerism (Ch) following bone marrow transplantation (BMT). This review is focused on findings derived from bone marrow (BM) biopsies in patients with chronic myeloid leukemia (CML) including a sex-mismatched host/donor constellation. Appropriate techniques involved immunophenotyping by monoclonal antibodies to identify the various cell lineages, dual color fluorescence in situ hybridization (FISH) with x- and y-chromosome-specific DNA-probes and a proper detection system for a simultaneous labeling of the bcr/abl locus. A significant degree of Ch with more than 20% host CD34+ progenitors was found in the early and late (up to 200 days after BMT) posttransplant period. However, only 10% of these cells harbored the bcr/abl translocation gene. This result fits well with corresponding molecular biological findings of so-called minimal residual disease. Conversion of Ch evolved during leukemic relapse with 90% host progenitors of which 50% revealed the bcr/abl locus. A Ch of nucleated erythroid percursors (5%) and CD68+ macrophages (8%) was expressed to a significantly lower degree. The slightly increased frequency found in CD61+ megakaryocytes (16%) was probably due to the polyploid state of these cells. Similar to the CD34+ progenitor cells abrupt changes from donor to host type was associated with an insidious transformation into recurrent leukemia. The CD34+ endothelial cells showed a minor degree of Ch, because donor-derived elements ranged from 18% to 25%. Leukemic relapse was characterized by an almost complete conversion of the endothelial cells to a host type. These findings point towards a CD34+ progenitor cell origin of the (leukemic) endothelial cell layer and suggests that their dysfunction may contribute to an expansion of the neoplastic clone.

[Chimerism of cardiomyocytes and endothelial cells after allogeneic bone marrow transplantation in chronic myeloid leukemia. An autopsy study]. / Der Chimärismus von Kardiomyozyten und Endothelzellen nach allogener Knochenmarkstransplantation bei chronischer myeloischer Leukämie. Eine Autopsiestudie.

The results of a number of animal experimental studies are in keeping with the finding that hematopoietic progenitors can generate cardiomyocytes and endothelial cells. As a consequence innovative therapeutic strategies have been suggested to possibly ameliorate the outcome of coronary artery disease. However, there is no information available at present whether this pathomechanism is also effective in humans, in particular without prior ischemic lesion of the myocardium. Therefore an autopsy study was performed on cadaver hearts derived from five male patients with chronic myeloid leukemia who received full unmanipulated bone marrow grafts from female donors 21-631 days before death. The purpose of this investigation was to detect and quantify a putative chimerism of cardiomyocytes and endothelial cells (intramural and subepicardial vessels). Genotyping was carried out by applying X- or Y-chromosome-specific DNA probes (fluorescence in-situ hybridisation) on routinely formalin-fixed specimens of the myocardium. To test the sensitivity of our method, cadaver hearts from two males and two females without a history of transplantation served as controls. In contrast to a totally corresponding sex-matched genotyping in 780 cardiomyocytes and 155 endothelial cells of the control group, the five male patients with a previous transplantation revealed significantly different results. A mixed chimerism was identifiable in 57 out of 890 counted cardiomyocytes (6.4%) and in 19 out of 322 endothelial cells (5.8%). These findings support the assumption that in addition to endothelial cells there is also a cardiomyogenic potential of bone marrow stem cells which exists without prior (ischemic) damage to the heart. However, further investigations are necessary to identify, isolate and enrich the cardiomyocytic stem cells more specifically for future curative therapeutic options in patients with severe ischemic cardiomyopathy.

Distribution of lectin binding sites in human bone marrow. Identification by use of an ultrastructural postembedding technique.

The purpose of this ultrastructural study was to detect various carbohydrate residues on mature elements of the major human haematopoietic cell lines (granulopoiesis, erythropoiesis and megakaryopoiesis), sinus endothelium and plasma cells under comparable experimental conditions. Marrow specimens were processed according to a modified postembedding technique with Unicryl as embedding resin. A broad panel of 10 digoxigenin (dig)-conjugated lectins was applied for staining and specificity was evaluated by incubation with their corresponding inhibitory sugars. Lectins under study were derived from Canavalia ensiformis (Con A), Triticum vulgare (WGA), Ulex europaeus-I (UEA-I), Baubinia purpurea (BPA), Erythrina cristagalli (ECA), Glycine max (SBA), Helix pomatia (HPA), Arachis hypogaea (PNA), Griffonia simplicifolia-I (GS-I) and its isotype GS-I-B4. As a common feature WGA was shown to be a prominent marker of cytoplasmic membranes, except for plasma cells. On the other hand, Con A turned out to be reactive with the nuclear envelopes in all haematopoietic cells and, additionally, exhibited a strong labelling of the rough endoplasmic reticulum in plasma cells. Granules of eosinophilic granulocytes revealed staining of varying intensity with all lectins; however, inhibition was mostly incomplete. Several lectins (WGA, Con A, UEA, BPA, ECA, SBA and PNA) disclosed a clear cut differentiation of at least two subpopulations of granules in polymorphonuclear leukocytes. UEA-I (H type 2 specific) exhibited a high affinity to cytoplasmic membranes of erythropoietic precursor cells. In keeping with the blood group of our patient (O Rh+) membranes of red blood cells were completely negative with those lectins that are known to exhibit a group A or B specificity (HPA, GS-I-B4). As a remarkable finding the luminal and abluminal surfaces of sinusoidal endothelium revealed a specific reaction with UEA-I. Carbohydrate binding sites on the surface of endothelial cells may play a pivotal role in several functional processes such as cellular adhesion, traffic of mature cell elements across the marrow blood barrier and "homing' of haematopoietic stem cells.

An improved postembedding technique for ultrastructural studies of lectin binding sites in bone marrow: a critical evaluation.

Little is known about the ultrastructural localization of lectin binding sites in human bone marrow tissue. This is probably due to the lack of suitable methods yielding both satisfactory tissue preservation and optimal labelling results. For this reason, we developed a modified postembedding technique for electron-microscopic studies of the glycosylation pattern of haematopoietic cells. Fixation with a 2.5% glutardialdehyde solution was shown to be an important prerequisite and could be even improved by postfixation with tannic acid and uranyl acetate. In particular, embedding with the acrylic resin UnicrylR (Bioacryl) resulted in an optimal ultrastructural preservation of bone marrow tissue. Employment of this hydrophilic resin in combination with a two-step labelling method which included digoxigenin-conjugated Concanavalin A (Con A) followed by ultrasmall anti-digoxigenin-gold and silver amplification, delivered a highly specific staining pattern. Our results with this lectin in different bone marrow cells revealed nuclear and cytoplasmic membranes, rough endoplasmic reticulum as well as granules to display reactivity of varying intensity. These findings underline the validity of our method, for they confirm and extend formerly reported histochemical and biochemical evaluations on the cellular binding sites of Con A.