The proliferative capacity of pure red cell aplasia bone marrow cells.

Pure red cell aplasia (PRCA) is a heterogeneous disorder. Immunologic abnormalities have recently been uncovered suggesting that both cell-mediated and humoral immune mechanisms may be of etiological importance in PRCA. Utilizing a technique for the cloning of bone marrow erythroid precursors, we determined the in vitro proliferative capacity of erythroid cells obtained from 21 patients with PRCA. Bone marrow cells from one group of patients produced normal or increased numbers of erythroid colonies while the in vitro proliferative capacity of bone marrow cells from a second group was characterized by subnormal erythroid colony formation. Sera obtained from the former group was frequently associated with demonstrable serum inhibitors of erythropoiesis, while PRCA in the latter group was probably the consequence of intrinsic erythroid stem cell defects or pathologic cellular interactions with nonerythroid regulatory cells. This survey of a relatively large population of patients with PRCA provides evidence for the multiple causative mechanisms that can be operative in the production of PRCA.     

An assay for serum cytotoxicity against erythroid precursor cells in pure red cell aplasia

Several reports have indicated that a circulating serum inhibitor (antibody) is involved in the pathogenesis of acquired pure red cell aplasia (PRCA). In the present study, the pathophysiologic significance of this inhibitor was assessed according to the status of erythroid progenitor cells in the bone marrow. So far, direct proof for the antibody acting against erythroid stemcells was lacking. Employing an "in vitro" assay, erythroid colony forming cell (CFU-e) numbers in PRCA marrow were quantified and the cytotoxic effect of PRCA serum on CFU-e was investigated. It was revealed that the CFU-e population size in the marrow of PRCA patients was severely reduced; at the same time the relative number of myeloid colony forming cells was normal. The serum was demonstrated to contain a factor cell which was cytotoxic to CFU-e, in the presence of complement. The results indicate that inhibition of erythropoiesis in PRCA is achieved by a complement dependent plasma factor which eliminates or inactivates CFU-e and which constitutes an effective block at the precursor cell level in the differentiation pathway of the erythroid line. The data present a practical assay for measuring cytotoxic factors affecting erythroid stem cells.     

Bone Marrow Graft in Man after Conditioning by Antilymphocytic Serum

Allogeneic bone marrow grafts carried out after previous administration of antilymphocytic serum alone were attempted in 16 patients. Of these, six had acute myeloblastic leukaemia, four acute lymphoblastic leukaemia, and one a blast cell crisis in polycythaemia vera. Ten of these patients were in an overt phase of the disease and resistant to chemotherapy, while nine had complete agranulocytosis. In five of these patients erythrocyte and leucocyte antigenic markers demonstrated the establishment of the graft. One patient had thalassaemia major, and four others had aplasia of the bone marrow, in one case due to chloramphenicol poisoning and in another to virus hepatitis. The grafts were successful in the last two patients and transformed their clinical condition.No signs of early acute secondary disease were noted in any of the patients, either when the donor had been given antilymphocytic serum or when he was untreated. The grafts had no adoptive immunotherapeutic effect on the acute leukaemia. These observations have clearly shown that antilymphocytic serum has an immunosuppressive effect in man when it is used alone.     

Progenitor white cells and serum inhibitors in patients with bone marrow aplasia.

In patients with various blood diseases, especially with bone marrow hypoplasia following parameters were determined: presence of antibodies against autologous bone marrow, number of granulocyte-progenitor cells in short term bone marrow cultures and stimulating or inhibiting factors in patient's serum influencing the proliferative activity of bone marrow colonies. The results as well as possible clinical aspects are discussed.     

DNA topoisomerase inhibitors block erythropoiesis and delay hemoglobinization in vitro

To examine the importance of topological constraints on DNA during erythroid development, we measured the effects of camptothecin and teniposide, two tumoricidal agents which are also specific inhibitors of type I and type II topoisomerases respectively, on the formation of hematopoietic colonies by cultured human bone marrow cells. When added to bone marrow culture, each inhibitor alone impairs the formation of early BFU-E-derived colonies, late CFU-E-derived colonies and mixed hematopoietic (CFU-GEMM-derived) colonies by up to 100%. Inhibition of colony formation is directly related to the time of inhibitor addition and the inhibitor concentration tested. Although either inhibitor alone reduces colony formation by 90%, when added together at a submaximal concentration, camptothecin and teniposide exert a synergistic suppressive effect. Furthermore, addition of topoisomerase inhibitors to culture impairs hemoglobinization of colony erythroblasts in a time-dependent fashion. In contrast to the effects of topoisomerase inhibitors, the antiproliferative agent aphidicolin reduces erythroid colony number and size without altering hemoglobinization of colony erythroblasts. Since neither topoisomerase inhibitor alters the morphology of cultured cells, the capacity of cells to exclude trypan blue or the potential to form erythroid colonies through the interval required for the first progenitor cell division, it is unlikely that camptothecin or teniposide are cytotoxic to hematopoietic cells. Human mononuclear cells enriched in bone marrow lymphocytes and nucleated erythroblasts from both human and mouse sources release DNA into the detergent soluble fraction. Release requires functional topoisomerases and is altered by acute exposure to topoisomerase inhibitors. Our results suggest that topoisomerases are critical not only to proliferation but also to differentiation of human marrow erythroid progenitor cells and stem cells in culture.     

Testosterone influences renal electrolyte excretion in SHR/y and WKY males

Background

Aldehyde dehydrogenase (ALDH) is a cytosolic enzyme highly expressed in hematopoietic precursors from cord blood and granulocyte-colony stimulating factor mobilized peripheral blood, as well as in bone marrow from patients with acute myeloblastic leukemia. As regards human normal bone marrow, detailed characterization of ALDH⁺ cells has been addressed by one single study (Gentry et al, 2007). The goal of our work was to provide new information about the dissection of normal bone marrow progenitor cells based upon the simultaneous detection by flow cytometry of ALDH and early hematopoietic antigens, with particular attention to the expression of ALDH on erythroid precursors. To this aim, we used three kinds of approach: i) multidimensional analytical flow cytometry, detecting ALDH and early hematopoietic antigens in normal bone marrow; ii) fluorescence activated cell sorting of distinct subpopulations of progenitor cells, followed by in vitro induction of erythroid differentiation; iii) detection of ALDH⁺ cellular subsets in bone marrow from pure red cell aplasia patients.

Results

In normal bone marrow, we identified three populations of cells, namely ALDH⁺CD34⁺, ALDH^-CD34⁺ and ALDH⁺CD34^- (median percentages were 0.52, 0.53 and 0.57, respectively). As compared to ALDH^-CD34⁺ cells, ALDH⁺CD34⁺ cells expressed the phenotypic profile of primitive hematopoietic progenitor cells, with brighter expression of CD117 and CD133, accompanied by lower display of CD38 and CD45RA. Of interest, ALDH⁺CD34^- population disclosed a straightforward erythroid commitment, on the basis of three orders of evidences. First of all, ALDH⁺CD34^- cells showed a CD71^bright, CD105⁺, CD45^- phenotype. Secondly, induction of differentiation experiments evidenced a clear-cut expression of glycophorin A (CD235a). Finally, ALDH⁺CD34^- precursors were not detectable in patients with pure red cell aplasia (PRCA).

Conclusion

Our study, comparing surface antigen expression of ALDH⁺/CD34⁺, ALDH^-/CD34⁺ and ALDH⁺/CD34^- progenitor cell subsets in human bone marrow, clearly indicated that ALDH⁺CD34^- cells are mainly committed towards erythropoiesis. To the best of our knowledge this finding is new and could be useful for basic studies about normal erythropoietic differentiation as well as for enabling the employment of ALDH as a red cell marker in polychromatic flow cytometry characterization of bone marrow from patients with aplastic anemia and myelodysplasia.     

Inhibitor of heme synthesis in polycythemic rabbit serum

Sera from hypertransfused polycythemic rabbits were found to significantly inhibit ⁵⁹Fe incorporation into heme in erythroid cells in normal rabbit bone marrow cultures when compared with that of normal serum controls suggesting a higher concentration of this inhibitor in polycythemic serum. This serum inhibitor delayed the time of peak cumulative heme synthesis $\text{in}$$\text{vitro}$ and the delay in peak cumulative heme synthesis was increase with increasing concentrations of polycythemic serum. It is suggested from these studies that this serum inhibitor may be involved in a negative feedback system in the control of erythropoiesis and may act specifically on differentiated nucleated erythroid cells to delay their entry into the cell cycle, consequently inhibiting heme synthesis.     

Tissue inhibitor of metalloproteinases from human bone marrow stromal cell line KM 102 has erythroid-potentiating activity, suggesting its possibly bifunctional role in the hematopoietic microenvironment

In this study, we demonstrated that tissue inhibitor of metalloproteinases (TIMP) produced by human bone marrow stromal cell line KM-102 had erythroid-potentiating activity (EPA) which stimulates the proliferation of erythroid progenitor cells. We, then, propose a scheme for the bifunctional role of TIMP/EPA in hematopoietic microenvironment, that is, the maintenance of the integrity of bone marrow matrix and the proliferation of erythroid progenitor cells proceeding on the matrix.     

Disseminated Fusariosis Secondary to Neuroblastoma with Fatal Outcome

Disseminated fusariosis is an uncommon clinical condition in immunocompromised patients. We report a fatal case of disseminated fusariosis secondary to neuroblastoma in a male patient, 15 years old, who underwent a bone marrow transplant. The patient was admitted to the pediatric intensive care unit (PICU) of a public hospital in Recife-PE, Brazil, presenting bone marrow aplasia, severe leukopenia, and thrombocytopenia. After 15 days, the patient developed right knee effusion. Synovial fluid and blood samples were analyzed at the Medical Mycology Laboratory of the Federal University of Pernambuco. Mycological diagnosis was based on the presence of hyaline septate hyphae on direct examination and the isolation of Fusarium oxysporum in culture, confirming the case of disseminated fusariosis. In vitro, the isolate showed fluconazole resistance and sensitivity to amphotericin B, anidulafungin, and voriconazole. Therapy with voriconazole in combination with liposomal amphotericin B led to an improved clinical response; however, due to underlying disease complications, the patient progressed to death.     

Oct-4, Rex-1, and Gata-4 expression in human MSC increase the differentiation efficiency but not hTERT expression

Micro-environment seems to exert an important influence on human mesenchymal stem cell (MSC) differentiation and proliferative capacity in bone marrow as well as in culture ex vivo. Oct-4, Rex-1, and TERT genes are well-known for the maintenance of pluripotentiality differentiation and the proliferative capacity of embryonic stem cells. Some previous data report expression of these embryonic factors in selected clones from bone marrow adult stem cells. Our goal was to study expression of Oct-4, Rex-1, and TERT in primary cultured human MSC according to the serum concentration. In addition, we have studied the expression of Gata-4 since this factor plays a key role in organogenesis. We hypothesized that low serum concentration with appropriate growth factors may induce an undifferentiated status with a re-expression of embryonic factors and extend differentiation capacity. Thus, using a defined culture medium, we report on the increased expression of Oct-4, Rex-1, and Gata-4 in human MSC. We have correlated this expression to an increase in differentiation efficiency towards osteogenic and adipogenic phenotypes. Our data suggest that the culture medium used permits the emergence of adult stem cells with a high differentiation capacity and expression of embryonic factors. These cells may have important implications for cell therapy.     

Hematopoietic precursor cells in radiation chimeras restored by bone marrow from thymectomized mice]

Radioprotective capacity of bone marrow CFUs of adult thymectomized mice was studied. Lethally irradiated mice were inoculated with bone marrow of mice thymectomized 8-11 months before. The colony forming capacity and proliferative rate of CFUs were studied 1-7.5 months after obtaining the radiation chimeras. It has been shown that proliferative capacity of bone marrow of adult thymectomized mice was reduced in comparison with that of normal animals. It is related to the decrease (4-fold) of the proliferative rate of bone marrow of thymectomized mice which was inoculated into lethally irradiated recipients 1 month before. We also found that the content of CFUs in bone of those chimeras was reduced later--after 7.5 months. In this period (1-7.5 months) the cellularity of bone marrow did not change.     

Granulopoiesis in long-term culture by marrow from mice with busulfan-induced chronic latent aplasia

Mice given high-dose busulfan therapy develop a chronic latent marrow aplasia characterized by normal peripheral blood neutrophil numbers, hematocrits and marrow cellularity but reduced numbers of pluripotent hemopoietic stem cells (CFU-s) and granulocyte-monocyte progenitor cells (CFU-gm). To study the pathogenesis of this lesion, bone marrow was propagated in long-term marrow cultures (LTMC). Small amounts of normal marrow readily established and sustained long-term granulopoiesis in vitro. In contrast, inocula of marrow from busulfan-treated animals containing three to five times as many stem and progenitor cells failed to establish long-term granulopoiesis in vitro. These results suggest that high-dose busulfan therapy produces a qualitative defect in either the hemopoietic stem cells, the stromal-forming elements, or both, rendering them incapable of establishing long-term granulopoiesis in vitro. Furthermore, mixing experiments employing normal and busulfan-damaged marrow demonstrate that this qualitative defect is not due to the emergence of a suppressor cell population. LTMC can show types of marrow damage not detectable by other techniques currently available and represent a powerful tool for studying latent bone marrow failure.     

Effects of increasing cyclic AMP or calcium on feline erythroid progenitors in vitro: normal cells are stimulated while cells from retrovirus-infected cats are suppressed

Pharmacologic modulators of cyclic 3',5'-adenosine monophosphate (cAMP) and calcium were added to cultures of bone marrow cells from normal cats and cats with retrovirus-induced erythroid aplasia (EA). Treatment with the following reagents increased the number of erythroid progenitors (CFU-e and BFU-e) in cells from normal cats: isoproterenol, dibutyryl cAMP, forskolin, RO-20-1724 and A23187. However, treatment of cells collected from viremic cats not only failed to enhance CFU-e and BFU-e but inhibited their growth. These studies suggest that EA is related to a non-reversible block of primitive erythroid progenitors or to direct inhibition of BFU-e and CFU-e growth.     

CARDIAC SURGERY

The occurrence of red cell aplasia of the bone marrow and benign thymoma is a rare but well-defined syndrome. Data presented suggest the presence in the serum of a patient who had thymoma, of a humoral factor which may inhibit erythropoiesis. Further attempts should be made to clarify the association of thymoma and anerythroid anemia.     

Attempt to restore lymphocytes with defective T-cell function by mixing with normal cells in vitro

Blood leukocytes from a patient with T-cell immunodeficiency failed to respond to stimulation with phytohemagglutinin (PHA) in vitro although ample lymphocytes were present. Leukocytes from this patient were mixed in various proportions with normal leukocytes, and the mixtures were stimulated with PHA and cultured for three days. There was no restoration of PHA responsiveness of the patient''s cells in the presence of normal cells. This would indicate that the defect in the patient''s cell population was not due to the absence of any facilitating factor or cooperative function that could be provided by normal T cells or other blood leukocytes. Since in thymic aplasia the patient''s own cells are rapidly restored to normal PHA responsiveness by an unknown facilitating factor after thymic transplantation, we suggest that this mixed-cell culture method might differentiate between patients who need a bone marrow transplant and those who will respond to a thymus transplant.     

Selective inhibition of erythropoiesis by sera from patients with chronic renal failure

The pathogenesis of anemia in patients with chronic renal failure was studied by analyzing the effect of uremic sera on the in vitro colony growth of erythroid (CFU-E) and granulocyte-macrophage (CFU-GM) progenitor cells. Uremic sera from 20 of 30 patients inhibited erythroid colony growth below 70% of control even when cultured with normal human bone marrow of the same blood type. On the other hand, only one of the sera inhibited colony growth of CFU-GM as compared with normal sera. On Sephadex G-15 gel filtration, the CFU-E-inhibiting activity appeared in two different fractions: the void volume peak and the delayed eluant before the second peak. The inhibiting activity in the former fraction was noted only in uremic sera. The results of this study suggest the existence of a serum inhibitor(s) of erythropoiesis with a relative molecular mass of more than 1500 Da which are virtually impossible to dialyze by conventional membranes.     

Partial myeloperoxidase deficiency in preleukemia

In seven subjects with partial and apparently acquired form of myeloperoxidase (MPO) deficiency, some functional properties of neutrophils (PMNs) were studied. Five patients suffered from preleukemia, one from diabetes mellitus and one from carcinoma of the breast with bone marrow metastases. Intracellular bactericidal activity, oxygen consumption and superoxide radical production were within normal limits. In three patients with preleukemia, the serum opsonic activity was markedly reduced (less than m-3SD) in an autologous system, but normal in the presence of pooled normal serum. Decreased opsonic activity was also found when these patient's sera were assayed in the presence of normal PMNs. Since the levels of IgG and C3 were comparable in the patients' sera and the pooled serum, a deficiency of another unknown opsonin or the presence of an opsonization inhibitor has to be postulated. The partial MPO defect apparently doesn't decrease the intracellular killing of Staphylococcus aureus by PMNs. The known susceptibility to bacterial infections in preleukemia may be explained by the reduction of serum opsonization conducing to a secondary decrease of the ingestion and killing of bacteria by the PMNs.     

Hematopoietic differentiation cell surface antigen switching in the bone marrow of different aged chickens

Abstract. Immune cytolysis and immunofluorescence were used to examine chicken fetal antigen CFA) and chicken adult antigen (CAA) expression on the differentiation/maturation series of definitive erythroid cells obtained from the bone marrow of different aged chickens. We found that erythroid cells undergo changes in CFA/CAA antigenic expression dependent on their differentiation/maturation stage as well as the developmental age of the chicken. All differentiation/maturation stages of erythroid cells in the bone marrow of 12 and 18-day-old embryos express CFA only. Erythroblasts obtained from 7-day post-hatched chickens express either CFA or CAA. All three CFA/CAA phenotypes (i.e., CFA, CAA, and CFA + CAA) are observed in subsequent maturation stages, but only the CFA + CAA phenotype is observed in mature erythroid cells in the bone marrow of 7day post-hatched chickens. Erythroblasts from 62 day post-hatched chickens exhibit all three CFA/CAA phenotypes. Cells in the subsequent maturation stages express various CFA, CAA, or CFA + CAA phenotypes resulting in a majority of the mature erythrocytes expressing both CFA and CAA, and a small population of mature erythrocytes expressing CAA only. Erythroblasts from adult chickens express both CFA and CAA; however, CFA is lost during erythroid maturation resulting in mature erythrocytes which express CAA only. These studies indicate that both the erythroid differentiation/maturation stage and the developmental age of the chicken influence CFA and CAA antigenic expression on erythroid cells undergoing cellular differentiation/maturation in the bone marrow.     

Retrovirus-induced feline pure red cell aplasia: the kinetics of erythroid marrow failure

Cats viremic with feline leukemia virus subgroup C (FeLV-C) develop pure red cell aplasia (PRCA) characterized by the loss of detectable late erythroid progenitors (CFU-E) in marrow culture. Normal numbers of early erythroid progenitors (BFU-E) and granulocyte-macrophage progenitors (CFU-GM) remain, suggesting that the maturation of BFU-E to CFU-E is impaired in vivo. We have examined the cell cycle kinetics of BFU-E and their response to hematopoietic growth factor(s) to better characterize erythropoiesis as anemia develops. Within 3 weeks of FeLV-C infection, yet 6-42 weeks before anemia, the traction of BFU-E in DNA synthesis as determined by tritiated thymidine suicide increased to 43 +/- 4% (normal 23 +/- 2%) while there was no change in the cell cycle kinetics of CFU-GM. In additional studies, we evaluated the response of marrow to the hematopoietic growth factor(s) present in medium conditioned by FeLV-infected feline embryonic fibroblasts (FEA/FeLV CM). With cells from normal cats or cats viremic with FeLV-C but not anemic, a 4-fold increase in erythroid bursts was seen in cultures with 5% FEA/FeLV CM when compared to cultures without CM. However, just prior to the onset of anemia, when the numbers of detectable CFU-E decreased, BFU-E no longer responded to FEA/FeLV CM in vitro. BFU-E from anemic cats also required 10% cat or human serum for optimal in vitro growth. These altered kinetics and in vitro growth characteristics may relate to the in vivo block of BFU-E differentiation and PRCA. Finally, when marrow from cats with PRCA was placed in suspension culture for 2 to 4 days in the presence of cat serum and CM, the numbers of BFU-E increased 2- to 4-fold although no CFU-E were generated. By 4 to 7 days, CFU-E were detected, suggesting that conditions contributing to the block of erythroid maturation did not persist. The suspension culture technique provides an approach to study further the defect in erythroid differentiation characteristic of feline PRCA.     

Acute Myeloid Leukemia (AML) with Erythroid Predominance Exhibits Clinical and Molecular Characteristics that Differ from Other Types of AML

The clinical importance of erythroid predominance in bone marrow of patients with acute myeloid leukemia (AML) is controversial. These cases represent a heterogeneous group of diseases that historically have been classified into different categories. We studied 313 AML patients and specifically compared the clinical, cytogenetic, and molecular features of cases of AML with erythroid predominance, arbitrarily defined as ≥50% erythroid precursors, to AML cases without erythroid predominance. We also assessed 51 patients with a high-grade myelodysplastic syndrome (MDS), refractory anemia with excess blasts (RAEB). All neoplasms were classified according to the World Health Organization classification. With the exception of therapy-related AML/MDS, the presence of erythroid predominance in variously classified categories of AML was associated with a survival advantage. In addition, AML with erythroid predominance had a lower frequency of cytogenetic abnormalities as well as a lower frequency of mutations involving NPM1, NRAS and FLT3 as compared with AML without erythroid predominance. We conclude that the clinical, cytogenetic, and molecular features of AML with erythroid predominance in the non-therapy-related setting are much closer to those of a high-grade myelodysplastic syndrome than they are to other types of AML.