Bone Marrow Regeneration Research Articles

Quantitative assessment of WT1 expression by real time quantitative PCR may be a useful tool for monitoring minimal residual disease in acute leukemia patients.

In order to verify if quantitative assessment of the WT1 transcript amount by the real time quantitative PCR (RQ-PCR) can be used as a marker for minimal residual disease detection, the WT1 transcript amount was determined in BM and PB samples of patients with myeloid and lymphoid acute leukemia, in normal controls, in regenerating bone marrow samples and in purified CD34-positive cells from normal subjects. In 10 patients bearing a fusion gene transcript suitable for minimal residual disease quantitative assessment, we performed a simultaneous analysis of the WT1 and of the fusion-gene transcript at sequential time intervals during follow-up. Sequential WT1 analysis was also performed in five AML patients lacking additional molecular markers. The data obtained show that normal and regenerating BM samples and purified CD34-positive cells consistently express minimal amounts of WT1 transcript and that this is extremely low and frequently undetectable in normal PB. By contrast, high levels of WT1 expression are present in the BM and PB samples of all acute leukemia (AL) cases at diagnosis. The WT1 levels during follow-up were found to follow the pattern of the other molecular markers (fusion gene transcripts) used for MRD monitoring and increased WT1expression in the BM and/or PB during follow-up of AL patients was always found to be predictive of an impending hematological relapse.

Allogeneic bone grafting of calvarial defects: an experimental study in the rabbit

Abstract. The purpose of this study was to investigate the incorporation of fresh frozen irradiated membranous allogeneic bone grafts into critical size calvarial defects in the rabbit. Fifteen rabbits had calvarial defects prepared. Twelve rabbits received allogeneic grafts and three received autogenous bone grafts. The rabbits were sacrificed at 9 and 12 months postoperatively, and the specimens were examined radiologically, histopathologically and with fluorescence microscopy. Neovascularization, bone marrow regeneration and new bone formation was evident throughout the grafts however revitalization of the entire graft was incomplete at 12 months. This study revealed that the FFI membranous grafts were well incorporated into rabbit calvarial defects.

Bone regeneration and Mesenchymal stem cells

Bone marrow mesenchymal stem cells have been considered to differentiate into cells in bone, cartilage, tendon, muscle, and fat. Further analyses revealed that these cell also give rise to myocardial cells, oval cells and nerve cells, indicating high plasticity of these cells. Recent researches have been focused to utilize these cells to regenerate not only bone but also the life-maintaining major organs. As a cell source for the future regeneration of multiple organs, regeneration of the bone marrow is critical and thus studies on the process of bone marrow regeneration will benefit not only bone physiology field alone but also that of many other organs.

Role of STAT3 for hematopoietic stem cells

Self-renewal of hematopoietic stem cells (HSCs) is a major factor determining the yield of ex-vivo manipulation of HSC or regeneration of bone marrow after transplantation. Despite the many interests, molecular mechanisms regulating the self-renewing process of HSC remains largely uncertain. Based upon previous finding that activating gp-130 signal enhance self-renewal of HSC, we speculated that STAT3, one of down stream molecule of gp-130, could be a effector molecule. In the expression analysis, higher level of STAT3 transcripts were detected in primitive human bone marrow cells (CD34+CD38−) cells compared to their in-vitro differentiated products or uncultured CD34+CD38+ cells. Similar quantitative difference was also observed in protein level of STAT3 in 5-FU treated murine bone marrow cells. To test the effect of STAT3 activity in various stages of hematopoiesis, murine fetal liver cells (TER119 depleted) were transduced with retrovirus (MSCV-IRES-GFP) encoding either wild type (wt) or dominant negative form (dn) of STAT3. Profound suppression of lympho-myeloid repopulation was observed in the cells transduced with do form of STAT3, but no changes in further down stage of hematopoietic activities as defined by spleen colony formation (CFU-S) or in-vitro colony formation (CFC). Similar results were observed murine bone marrow cells stimulated with 5-FU, suggesting that HSCs of more broad spectrum requires STAT3 activity for their repopulating activities. In contrast, overexpression of wild type STAT3 did not affect any of those hematopoietic activities. However, when similar study was performed using another mutant form of STAT3 (STAT3-C) that can dimerize without being phosphorylated, significant increases in the repopulating activities of transduced cells were observed. Our study suggests that the STAT3 plays a direct regulatory role for gp-130 mediated induction of self-renewal in HSCs.

Editorial

Our series of invited contributions on “Current Trends in Peptide Science” continues in this issue with a group of articles that encompass a spectrum of topics in peptide structure, pharmacology, and synthesis. In the first article, Mozsolits and Aguilar detail the application of surface plasmon resonance (SPR) spectroscopy to the detailed analysis of peptide affinity for a variety of membrane systems. The authors describe the technique of SPR; the preparation of immobilized lipid surfaces for SPR-based biophysical studies; and provide several examples of information that can be obtained, highlighting the membrane interactions of antimicrobial peptides. Lauer-Fields et al. then review the biological functions—and consequences—of the matrix metalloproteinase (MMP) family of enzymes, describing the capacity of MMPs to catabolize collagen (termed “collagenolysis”), and distinguishing this capacity from triple-helical peptidase activity. The authors point out that the ability to cleave a collagen triple helix is not the limiting factor for collagenolytic activity, which may reside in the enzyme's ability to properly orient and potentially destabilize collagen. The wide-ranging properties of osteogenic growth peptide (OGP) and its C-terminal pentapeptide Tyr–Gly–Phe–Gly–Gly [OGP(10–14)] as bone anabolic agents and hematopoietic stimulators are reviewed by Bab and Chorev. The authors describe, for example, the roles of OGP in the regulation matrix mineralization, in the expression of type I collagen, and as a stimulator of bone marrow regeneration after chemotherapy; and provide a detailed overview of OGP biosynthesis and activity, and of the structure–activity relationships germane to its functions. The final article of this issue features a review by Taylor on the use of side-chain lactam bridges as a valuable method for introducing conformational constraints into a peptide, notably for stabilization of α-helical structures. Through specifically chosen spacing, along with appropriately chosen selectively cleavable protecting groups, desired mono-, di-, and bicyclic lactam-bridged peptides can be prepared by solid-phase methods. The author reviews the key contributions of this methodology to protein biology, including studies of protein folding, ligand–receptor recognition, as well as development of peptide therapeutics with increased potency. The articles in this issue are particularly comprehensive and informative, and form a topical selection that should be of broad interest to peptide chemists and biologists.

Regeneration of Bone and Bone Marrow by Constructs Grafting of Human Cultured Bone and Porous Ceratite

Regeneration of Bone and Bone Marrow by Constructs Grafting of Human Cultured Bone and Porous Ceratite

Flow Cytometric Analysis of Reticulated Platelets

Flow Cytometric Analysis of Reticulated Platelets (G. Matic, G. Rothe, and G. Schmitz, University of Regensburg, Regensburg, Germany). In the last several years, flow cytometry has emerged as one of the tools of choice in evaluation of platelets. In particular, the distinction between reticulated platelets and mature platelets based on RNA fluorescence has proved to be a vital tool in the clinical hematology laboratory. Further, as is now well understood, it is important to evaluate platelets in whole blood rather than in isolated populations. Platelet quantification in a dual-color whole-blood method is of interest for the characterization of thrombocytopoiesis in (immune)-thrombocytopenia or in the regenerating bone marrow.

Population dynamics of clonogenic stromal precursor cells in hemopoietic and lymphoid organs during bone marrow regeneration

This paper describes our study on the regeneration of hemopoietic and stromal components of bone marrow after mechanically emptying the medullar cavity of the guinea pig tibia. The intensity of hemopoiesis was determined from the number of hemopoietic cells, while the concentration and total number of stromal precursor cells were used to estimate the ability of the bone marrow to produce stromal structures, including its ability to restore a specific microenvironment. We found that there was no direct correlation between the recovery characteristics of hemopoietic and stromal cells. An increase in the population size of stromal precursor cells takes place early after curettage, and stromal fibroblasts become phosphatase-positive according to Gomori, which is characteristic of osteogenic tissue. We have also demonstrated that curettage of 3-5 tubular bones results in the growth of this cell population in the bone marrow of nonoperated bones and even in the spleen, which in guinea pigs participates only in lymphopoiesis.

Regenerating normal B-cell precursors during and after treatment of acute lymphoblastic leukaemia: implications for monitoring of minimal residual disease.

We studied 57 childhood acute lymphoblastic leukaemia (ALL) patients who remained in continuous complete remission after treatment according to the Dutch Childhood Leukaemia Study Group ALL-8 protocols. The patients were monitored at 18 time points during and after treatment [640 bone marrow (BM) and 600 blood samples] by use of cytomorphology and immunophenotyping for the expression of TdT, CD34, CD10 and CD19. Additionally, 60 BM follow-up samples from six patients were subjected to clonality assessment via heteroduplex polymerase chain reaction (PCR) analysis of immunoglobulin VH-JH gene rearrangements. We observed substantial expansions of normal precursor B cells in regenerating BM not only after maintenance therapy but also during treatment. At the end of the 2-week intervals after consolidation and reinduction treatment, B-cell-lineage regeneration was observed in BM with a large fraction of immature CD34+/TdT+ B cells. In contrast, in regenerating BM after cessation of maintenance treatment, the more mature CD19+/CD10+ B cells were significantly increased, but the fraction of immature CD34+/TdT+ B cells was essentially smaller. Blood samples showed a profound B-cell lymphopenia during treatment followed by a rapid normalization of blood B cells after treatment, with a substantial CD10+ fraction (10-30%). Heteroduplex PCR analysis confirmed the polyclonal origin of the expanded precursor B cells in regenerating BM. This information regarding the regeneration of BM is essential for the correct interpretation of minimal residual disease studies.

Transient expression of activin βA mRNA on osteoprogenitor cells in rat bone regeneration after drill-hole injury

We investigated the expression of activin βA on osteoprogenitor cells in the regenerating bone and bone marrow of the rat femur after drill-hole injury, by immunocytochemistry and in situ hybridization. The periosteum and endosteum adjacent to the wound region showed marked thickening at day 3 and abundant osteoprogenitor cells, which were immunoreactive for proliferating cell nuclear antigen and showed positive reactions for alkaline phosphatase activity, and existed in the inner layer of the periosteum as well as in the endosteum. During the same period, these osteoprogenitor cells began to exhibit activin βA immunoreactivity and mRNA expression. However, the latter expression gradually reduced the intensity as the cells started to express osteocalcin mRNA during their differentiation to osteoblasts participating in the periosteal and medullary bone formation from day 5. Immunoreactivity for activin type IB and II receptors was also found on activin βA-immunoreactive cells between days 3 and 7. The above findings suggest that proliferating osteoprogenitor cells, before their transformation to osteoblasts, transiently produce and release activin A, which may play crucial roles in bone and bone marrow regeneration in a receptor-mediated, autocrine and paracrine fashion.

Captopril inhibits in vitro and in vivo the proliferation of primitive haematopoietic cells induced into cell cycle by cytotoxic drug administration or irradiation but has no effect on myeloid leukaemia cell proliferation.

Angiotensin I-converting enzyme (ACE) has been shown to be involved in the catabolism of the tetrapeptide acetyl-Ser-Asp-Lys-Pro (AcSDKP). As AcSDKP is a physiological inhibitor of haematopoietic stem cell proliferation, we investigated the in vitro and in vivo effects of captopril, one of the specific inhibitors of ACE, on the proliferation of primitive haematopoietic cells. Regenerating bone marrow cells obtained from mice given one injection of cytosine arabinoside (100 mg/kg) as well as SA2 myeloid leukaemia cells were incubated in vitro for 24 h with 10-6 M captopril. Captopril significantly reduced the proportion of high proliferative potential colony-forming cells (HPP-CFC-1) in S-phase, whereas it had no effect on the proportion of SA2 leukaemic colony-forming cells in S-phase. When given in vivo to mice 1 h after 2 Gy gamma-irradiation or cytosine arabinoside (AraC) injection, captopril (100 mg/kg) was shown to prevent HPP-CFC-1 entry into S-phase induced by these cytotoxic treatments. The observed effects correlated with a reduction in ACE degradative activity and an increase in the level of endogenous AcSDKP both in the supernatants of captopril-treated bone marrow cells and in plasma of treated animals. The present findings suggest that AcSDKP might mediate the observed in vitro and in vivo inhibitory effects of captopril on primitive haematopoietic cell proliferation.

Bone marrow recovery after irradiation of the spine

Imaging of bone marrow by radio labeled antibodies against NCA-95 antigen of human granulocytes offers the possibility to visualize granulopoietic activity. After radiotherapy, a reduced uptake delineates the radiation field. Aim of our investigation was the study of strength and duration of this effect. We analyzed 80 cases of patients irradiated for different primary or metastatic malignancies including parts of the spine who received a bone marrow scan for re-staging or during follow-up. Activity uptake of vertebrae inside and outside the irradiation portal was evaluated by ROI and the ratio was taken as measure of bone marrow suppression. A depression of granulopoietic bone marrow activity was seen even after a few fractions of 1.8 or 2 Gy (Figure 1). Depending on time since irradiation (Figure 2), we found a bone marrow recovery. In patients without bone metastasis, regeneration could be complete, whereas in patients treated for metastases, it was incomplete in all cases (Figure 3). For a certain time after irradiation, radioimmunoimaging delineates the irradiation portal by showing depression of granulopoiesis. Later on, it also shows bone marrow regeneration after radiotherapy. This may be helpful in reconstruction of radiation portals or in toxicity estimation during multimodal cancer therapy.

Effects of exogenous glucocorticoids on colony-forming activity of the bone marrow under cytotoxic exposure

Experiments on CBA/CaLac mice receiving a half-maximum tolerated dose of 5-fluorouracil demonstrated an inhibitory effect of physiological concentrations of dexamethasone (127×10−9M) on colony-forming activity of erythroid and granulomonocytic precursors in intact and regenerating bone marrow. Dexamethasone exhibited protective effects on granulocyte, macrophage, and fibroblast precursors during maximum myelosuppression. Various responses of hemopoietic progenitor cells to exogenous corticosteroidin vitro are probably determined by their functional state.

Leukaemia‐associated immunophenotypes (LAIP) are observed in 90% of adult and childhood acute lymphoblastic leukaemia: detection in remission marrow predicts outcome

Analysis of differentiation antigens on leukaemic blasts is routinely done for diagnostic purposes, i.e. determination of stage of differentiation and lineage assignment. Acute lymphoblastic leukaemias are also frequently characterized by a leukaemia-associated immunophenotype (LAIP), either the coexpression of differentiation antigens physiologically restricted to other stages of differentiation (asynchronous LAIP) or cell lineages (aberrant LAIP). We defined LAIP in 241 consecutive unselected B-lineage (n = 193) and T-lineage (n = 48) ALL by three-colour flow cytometry using directly conjugated monoclonal antibodies. The incidence of LAIP was found to be 91.7%. In 63% of patients two to six leukaemia-associated expression patterns were detected. In order to study the specificity of LAIP in a therapy-relevant setting, remission bone marrow samples from patients with B-lineage ALL were analysed for the expression of T-lineage-associated phenotypes on the normal bone marrow cells and vice versa. The frequency of all T-lineage LAIP+ cells and all aberrant B-lineage LAIP+ cells was <1% in regenerating bone marrow samples at different timepoints. The incidence and clinical significance of LAIP+ cells was studied in 196 remission marrows of 70 ALL patients (55 remaining in CCR, 14 with bone marrow relapse, one with isolated CNS relapse). The presence of >1% LAIP+ at two consecutive timepoints predicted 5/8 bone marrow relapses in B-lineage ALL. The occurrence of LAIP+ cells >1% in T-lineage ALL after induction therapy predicted relapse in 7/7 cases. In conclusion, flow cytometric detection of LAIP+ cells appears to be a powerful tool for the prediction of outcome in ALL.

Leukaemia-associated immunophenotypes (LAIP) are observed in 90% of adult and childhood acute lymphoblastic leukaemia: detection in remission marrow predicts outcome

Analysis of differentiation antigens on leukaemic blasts is routinely done for diagnostic purposes, i.e. determination of stage of differentiation and lineage assignment. Acute lymphoblastic leukaemias are also frequently characterized by a leukaemia-associated immunophenotype (LAIP), either the coexpression of differentiation antigens physiologically restricted to other stages of differentiation (asynchronous LAIP) or cell lineages (aberrant LAIP). We defined LAIP in 241 consecutive unselected B-lineage (n = 193) and T-lineage (n = 48) ALL by three-colour flow cytometry using directly conjugated monoclonal antibodies. The incidence of LAIP was found to be 91.7%. In 63% of patients two to six leukaemia-associated expression patterns were detected. In order to study the specificity of LAIP in a therapy-relevant setting, remission bone marrow samples from patients with B-lineage ALL were analysed for the expression of T-lineage-associated phenotypes on the normal bone marrow cells and vice versa. The frequency of all T-lineage LAIP+ cells and all aberrant B-lineage LAIP+ cells was <1% in regenerating bone marrow samples at different timepoints. The incidence and clinical significance of LAIP+ cells was studied in 196 remission marrows of 70 ALL patients (55 remaining in CCR, 14 with bone marrow relapse, one with isolated CNS relapse). The presence of >1% LAIP+ at two consecutive timepoints predicted 5/8 bone marrow relapses in B-lineage ALL. The occurrence of LAIP+ cells >1% in T-lineage ALL after induction therapy predicted relapse in 7/7 cases. In conclusion, flow cytometric detection of LAIP+ cells appears to be a powerful tool for the prediction of outcome in ALL.

In vivo modifications of AcSDKP metabolism and haematopoiesis in mice treated with 5-fluorouracil and Goralatide.

The tetrapeptide acetyl-Ser-Asp-Lys-Pro (AcSDKP), a physiological inhibitor of the proliferation of haematopoietic stem cells, is degraded by the angiotensin-I-converting enzyme (ACE). Whereas synthetic AcSDKP (Goralatide) protects normal mice from the haematological toxicity of chemotherapy, it has a lower beneficial effect in humans. This discrepancy could be dependent on Goralatide administration schedules, as well as on the endogenous concentrations of AcSDKP and ACE, which vary during chemotherapy. We investigated the effect of one myelotoxic dose of 5-fluorouracil (5-FU, 200 mg kg-1) administered without or with Goralatide on blood, bone marrow (BM) and spleen AcSDKP concentrations, ACE activity, nucleated cell counts and survival of the primitive haematopoietic progenitors high proliferative potential colony-forming cells (HPP-CFCs). The 5-FU treatment dramatically decreased the BM concentrations of AcSDKP by 73% and increased the ACE activity in plasma by 50% during the period of active BM regeneration. Repeated injections of Goralatide from 24 h before to 36 h after the i.p. injection of 5-FU spared BM HPP-CFCs. As an injection of 10 mg of Goralatide induced a short peak of plasma AcSDKP without modifying its BM concentrations, we suggest that its protective effect on HPP-CFCs could be mediated by its interference with other plasma molecules targeting to the BM. By improving our knowledge of the biology of AcSDKP in vivo during chemotherapy, our results could help to better define the therapeutic use of Goralatide.

Role of Thy 1.2+ cells in hemopoietic regulation in cytostatic-induced hemosuppression

Effect of Thy 1.2+ cells (direct and mediated by stromal elements) on the growth of granulomonocyte and erythroid colonies in the bone marrow is studied on CBA mice with cytostatic disease induced by single injection of adriamycin, cyclophosphamide, and 5-fluorouracil in maximum permissible doses. It is shown that Thy 1.2+ cells stimulate colony formation in regenerating bone marrow, the effect depending on functional activity of hemopoiesis-inducing microenvironment.

422 - Lymphocyte abnormalities during post-chemotherapy myelosuppression and bone marrow regeneration in patients with solid tumors

422 - Lymphocyte abnormalities during post-chemotherapy myelosuppression and bone marrow regeneration in patients with solid tumors

1223 - Relationship between rate of “in-field” bone marrow regeneration and radiation dose in patients with Hodgkin's disease

1223 - Relationship between rate of “in-field” bone marrow regeneration and radiation dose in patients with Hodgkin's disease

Isolation of osteogenic growth peptide from osteoblastic MC3T3 E1 cell cultures and demonstration of osteogenic growth peptide binding proteins

The osteogenic growth peptide (OGP) was recently characterized in regenerating bone marrow. In experimental animals in increases osteogenesis and hemopoiesis. In stromal cell cultures OGP stimulates proliferation, alkaline phosphatase activity, and matrix mineralization. OGP in high abundance is present in normal human and animal serum mainly complexed to OGP binding protein (OGPBP) or proteins. Here we show the presence of two OGPBPs, OGPBP-1, and OGPBP-2, in cultures of osteoblastic MC3T3 E1 cells. Immunoreactive OGP (irOGP) also accumulates in the medium of these cultures and in cultures of NIH 3T3 fibroblasts. A large amount of irOGP was released by heat inactivation of OGPBP-2 and purified by ultrafiltration and hydrophobic HPLC. The purified irOGP was identical to OGP obtained previously from rat regenerating bone marrow and human serum in terms of its amino acid sequence, immunoreactivity, and mitogenicity. Osteoblastic and fibroblastic cell proliferation can be arrested by anti-OGP antibodies and rescued by exogenous OGP, indicating that in the absence of serum or other exogenous growth stimulators the endogenously produced OGP is both necessary and sufficient for baseline proliferation. The OGP production is up- and down-regulated, respectively, by low and high doses of exogenous OGP in a manner consistent with an autoregulated feedback mechanism. The most effective OGP dose in MC3T3 E1 cells is at least two orders of magnitude lower than that in non-osteoblastic cell systems. This differential sensitivity of the osteoblastic cells could result in a preferential anabolic effect of OGP in bone.