Effects of hypoxia on the small acetylcholinesterase-positive megakaryocyte precursor in bone marrow of mice

The number of small acetylcholinesterase-positive (SAChE+) cells in the marrow of hypoxic mice was measured. Mice were exposed to 6-7% O2 levels by enclosure in cages covered with dimethyl-silicone rubber membranes for 1-14 days. The mice showed a linear increase in packed cell volumes with time in the hypoxic atmosphere, but platelet counts showed a characteristic biphasic response, i.e., increased platelet counts were observed after 1-3 days of hypoxia, and significantly (P less than 0.05-P less than 0.0005) decreased platelet counts were observed thereafter (6-14 days). The total number of megakaryocytes in the marrow of hypoxic mice decreased significantly (P less than 0.005) with time. In agreement with the data on platelet counts, hypoxia caused the total number of SAChE+ cells in the marrow of mice to be biphasic. At Day 2 there was a significant increase (P less than 0.05) in the total number of SAChE+ cells/mm3 of bone marrow; however, by days 10-14 the numbers had decreased markedly (P less than 0.005). These data indicate that hypoxia decreases platelet production by action on a precursor cell to the SAChE+ cell. The hypoxia-induced thrombocytopenia is probably caused by stem-cell competition between the erythrocytic and megakaryocytic cell lines.     

Analysis of human dysplastic haematopoiesis in long-term bone marrow culture

In this paper we have analysed the behaviour of myelodysplastic marrow in a long-term bone marrow liquid culture system (LTBMC) from eleven patients with myelodysplastic syndromes with regard to cellularity, day-7 and day-14 CFU-GM growth, cluster formation, adherent cells and CFU-F formation. An altered CFU-GM pattern was found in 64% of cases at diagnosis, while normal growth was seen in the remaining cases, all of which were affected by refractory anaemia. The levels of CFU-GM, as well as cellularity, were reduced in myelodysplastic marrows compared to normal controls over the whole duration of LTBMCs. Cases with a normal CFU-GM level at diagnosis also showed pathological behaviour when examined in LTBMC. The duration of dysplastic haematopoiesis was significantly shorter than that of controls. The proliferative ability of CFU-F was reduced in 50% of cases as shown by replating experiments. In conclusion, myelodysplastic marrow shows an abnormal behaviour in LTBMC, even in those cases which present normal CFU-GM growth at diagnosis.     

Bone Marrow Hypoplasiaresponsive to Testosterone Therapyin a Patient with Panhypopituitarism:Need for Adherence to Androgen Replacement

ObjectiveTo describe the case of a young Saudi male patient with long-term panhypopituitarism and pancytopenia attributable to poor adherence to androgen replacement therapy, which resolved after institution of testosterone treatment and recurred after another interval of poor adherence to recommended therapy.MethodsWe present the clinical and laboratory data before and after treatment with testosterone. In addition, the corresponding histologic changes in the bone marrow are illustrated.ResultsAfter resection of a hypothalamic glioma, panhypopituitarism developed in a 14-year-old Saudi boy. At age 22 years, he had shunt-related meningitis. He was then noted to have pancytopenia, with a platelet count of 54 × 10³/μL, a hemoglobin concentration of 6.9 g/dL, and a leukocyte count of 2.7 × 10³/μL. After treatment of sepsis, the pancytopenia persisted. No underlying cause was detected. Bone marrow biopsy showed a hypocellular marrow with dysplastic megakaryocytes. The patient’s family indicated that he had not been taking his testosterone therapy. Testosterone decanoate (250 mg) was administered intramuscularly daily for 3 days. His platelet count increased to 74 × 10³/μL. Maintenance therapy with testosterone once weekly for 3 weeks and then once every 3 weeks resulted in improved hematologic findings. Repeated bone marrow biopsy after 6 weeks showed normocellular marrow, with disappearance of the megakaryocytic dysplasia. The patient again discontinued his testosterone treatment, and the hematologic abnormalities recurred but were again corrected after supervised testosterone therapy.ConclusionThis case emphasizes the importance of androgen replacement therapy in patients with hypopituitarism, not only for sexual potency, bone strength, and quality of life but also for normal bone marrow function. (Endocr Pract. 2008;14:229-232)     

Aberrant epigenetic and genetic marks are seen in myelodysplastic leukocytes and reveal Dock4 as a candidate pathogenic gene on chromosome 7q

Myelodysplastic syndromes (MDS) are characterized by abnormal and dysplastic maturation of all blood lineages. Even though epigenetic alterations have been seen in MDS marrow progenitors, very little is known about the molecular alterations in dysplastic peripheral blood cells. We analyzed the methylome of MDS leukocytes by the HELP assay and determined that it was globally distinct from age-matched controls and was characterized by numerous novel, aberrant hypermethylated marks that were located mainly outside of CpG islands and preferentially affected GTPase regulators and other cancer-related pathways. Additionally, array comparative genomic hybridization revealed that novel as well as previously characterized deletions and amplifications could also be visualized in peripheral blood leukocytes, thus potentially reducing the need for bone marrow samples for future studies. Using integrative analysis, potentially pathogenic genes silenced by genetic deletions and aberrant hypermethylation in different patients were identified. DOCK4, a GTPase regulator located in the commonly deleted 7q31 region, was identified by this unbiased approach. Significant hypermethylation and reduced expression of DOCK4 in MDS bone marrow stem cells was observed in two large independent datasets, providing further validation of our findings. Finally, DOCK4 knockdown in primary marrow CD34(+) stem cells led to decreased erythroid colony formation and increased apoptosis, thus recapitulating the bone marrow failure seen in MDS. These findings reveal widespread novel epigenetic alterations in myelodysplastic leukocytes and implicate DOCK4 as a pathogenic gene located on the 7q chromosomal region.     

Bleeding time in patients with hepatic cirrhosis.

OBJECTIVE--To determine the frequency of an abnormal bleeding time in patients with cirrhosis and to relate this to known factors that affect primary haemostasis and to the severity of liver disease. DESIGN--Prospective clinical and laboratory study in patients admitted for complications or investigations of liver disease. SETTING--Royal Free Hospital hepatobiliary and liver transplantation unit. SUBJECTS--100 Consecutive inpatients aged 17-74 with various forms of cirrhosis, including alcoholic, biliary, autoimmune, viral, and cryptogenic. At least 10 days had elapsed since any episodes of bleeding, resolution of sepsis, or alcohol intake. No patient was taking any drug known to affect primary haemostasis. MAIN OUTCOME MEASURES--Bleeding time as measured with the Simplate double blade template device. A bleeding time longer than 10 minutes was considered abnormal. Other measures were platelet count, prothrombin time, partial thromboplastin time, packed cell volume, and blood urea, serum bilirubin, and serum albumin concentrations, all measured on each subject at the same time by standard laboratory methods. RESULTS--A weak but significant correlation existed between the bleeding time and the platelet count (rs = 0.483; p less than 0.001). There were significantly lower platelet counts, longer prothrombin times, and higher blood urea and serum bilirubin concentrations in the 42 patients with bleeding times of 10 minutes or more compared with the 58 patients with bleeding times less than 10 minutes. Multiple linear regression analysis showed that the bilirubin concentration as well as the platelet count was independently correlated with the bleeding time. The combination of a platelet count greater than 80 x 10(9)/l and a prothrombin time less than 17 seconds (usually taken as safe limits for performing routine liver biopsy) did not predict a normal bleeding time. Ten of 39 patients fulfilling these criteria had a prolonged bleeding time. CONCLUSIONS--Prolonged bleeding time is common in patients with cirrhosis, even in those with prothrombin times and platelet counts within "safe limits" for invasive procedures. The severity of liver disease as assessed by the bilirubin concentration plays an important part in determining the bleeding time in cirrhosis. The bleeding time should be measured when assessing patients for invasive procedures who have a raised bilirubin concentration or poor hepatic function, even if the platelet count and prothrombin time are considered adequate.     

Relationship between chromosome fragility,aneuploidy and severity of the haematological disease in Fanconi anaemia

Fanconi anemia (FA) is a chromosome instability syndrome, characterized by progressive pancytopenia and cancer susceptibility. Other cellular features of FA cells are hypersensitivity to DNA cross-linking agents and accelerated telomere shortening. We have quantified overall genome chromosome fragility and euploidy as well as chromosomes 7 and 8 aneuploidy in peripheral blood lymphocytes from a group of FA patients and age-matched controls that were previously measured for telomere length. The haematology of FA samples were also characterized in terms of whole blood cell, neuthrophil and platelet counts, transfusion dependency, requirement of androgens, cortico-steroids or bone marrow transplantation, and the development of bone marrow clonal cytogenetic abnormalities, myelodysplastic syndrome or acute myeloid leukemia. As expected, a high frequency of spontaneous chromosome breaks was observed in FA patients, especially of chromatid-type. No differences in chromosomes 7 and 8 monosomy, polysomy and non-disjunction were detected between FA patients and controls. The same was true for overall genome haploidy or polyploidy. Interestingly, the spontaneous levels of chromosome fragility but not of numerical abnormalities were correlated to the severity of the haematological disease in FA. None of the variables included in the present investigation (chromosome fragility, chromosome numerical abnormalities and haematological status) were correlated to telomere length.     

Oral Ezatiostat HCl (TLK199) and Myelodysplastic syndrome: A case report of sustained hematologic response following an abbreviated exposure

Treatment options for patients with lower risk non-del(5q) myelodysplastic syndromes (MDS) who fail erythroid stimulating agents are restricted to one of the hypomethylating drugs with an expected response rate of ~50%. Ezatiostat HCl, an agent with the potential for producing multi-lineage responses in this population is currently in clinical investigation phase. This case report describes a 77 year old male who received less than two cycles of therapy with ezatiostat HCl which had to be aborted due to intolerable side effects, but which produced a sustained normalization of all three blood counts. This trilineage response has now lasted for more than a year. Interestingly, the patient began with a del(5q) abnormality and responded briefly to lenalidomide. Upon relapse of the anemia, a bone marrow showed the disappearance of the del(5q) but the appearance of a new clonal abnormality t(2;3). Given that the patient had a complete cytogenetic response to a truncated exposure to lenalidomide followed by a trilineage response to an even briefer course of ezatiostat HCl suggests a potential role for ezatiostat HCl in del(5q) patients who relapse following lenalidomide.     

Gray platelet syndrome: selective alpha-granule deficiency and thrombocytopenia due to increased platelet turnover

Clinical and laboratory studies of two siblings, both suffering from gray platelet syndrome (GPS) are described. The patients had a mild bleeding disorder, their platelets were blue-gray in panoptic stains, and alpha-granules were markedly reduced, as shown by electron microscopy. The platelet content of platelet factor 4 and that of beta-thromboglobulin were significantly reduced (3%-7% of normal). Platelet count was decreased (33-150 X 10(9)/1) and small platelets were increased in platelet volume distribution. Bleeding time was prolonged on most occasions. Bone marrow aspiration was performed in one patient and revealed increased reticulin fibers, however, megakaryocyte count was normal. The mean platelet survival was 4.8 days using 111indium-labelled platelets. In this patient, platelet-associated IgG was within the normal range. Prednisone therapy failed to increase platelet count. Dental surgery was performed under cover of desmopressin and no bleeding complication occurred; however, no improvement of bleeding time was observed. The patient delivered a healthy male infant without hemorrhaging while under concurrent platelet transfusion therapy.     

Therapeutic evaluation of interleukin-1 for stimulation of hematopoiesis in primates after autologous bone marrow transplantation

A multiple dose IL-1 therapy was evaluated for its capability to stimulate hematopoiesis in normal primates and to restore hematopoiesis after autologous bone marrow transplantation. The administration of IL-1 to normal animals over a dose range of 0.5 to 10 ug/kg/d led to a 7-12 fold increase in peripheral blood neutrophil and monocyte counts after 24 hours. This increase in the mature peripheral blood myeloid cells was followed by changes in the myeloid composition of the bone marrow, where the percentage of myeloid elements increased along with a transient increase in myeloid progenitor cell activity. IL-1 treatment also led to an initial decrease in platelet counts of 10-30% during the first 3 days of treatment. However, a striking finding was a significant and long lasting stimulation of increased platelet production with platelet counts increasing to 77% of baseline 3 days after cessation of treatment and remaining elevated for the next 10 days. The therapeutic potential of the IL-1 regimen to restore hematopoiesis was further evaluated in an established autologous bone marrow transplantation model. In monkeys receiving IL-1 doses, 1.0 and 5.0 ug/kg/d, neutrophil counts recovered to >0.5 x 10e9/1 on day 16, one day earlier than control, but the recovery to baseline neutrophil counts occurred 5 days sooner than control. IL-1 therapy had its greatest effect on the restoration of platelet counts after transplantation, reaching >100 x 10e9/l by day 21, two weeks earlier than control. This work demonstrates that IL-1 therapy stimulates myelopoiesis but its most promising clinical application is the stimulation of platelet production.Views presented in this paper are those of the authors: no endorsement by the Department of the Navy or the Defense Nuclear Agency has been given or should be inferred. This work was supported by the Naval Medical Research and Development Command, Research Task No. 63706N MM095.003.1007 and the Defense Nuclear Agency Work Unit No. 132082.     

Immunological detection of residual leukaemic disease in the bone marrow of children with acute lymphoblastic leukaemia

Peripheral blood lymphocytes incubated with tumour cells or extracts may undergo blastogenesis. This is the basis of a technique studied in children with acute lymphoblastic leukaemia (ALL) in childhood in an attempt to predict relapse. Samples of peripheral blood and bone marrow from 82 children with varying degrees of ALL were analysed. Cultures were prepared by incubating a lymphocyte suspension with an autologous bone-marrow suspension. Final ratios of lymphocytes to bone-marrow cells (L: BM) were 1: 1 and 2: 1. Control wells received bone-marrow or lymphocyte suspension only. Cultures were incubated for 72, 96, and 120 hours. All were pulse-labelled with ³H-TdR and radioactivity was measured by scintillation counting. Results were expressed as the stimulation index, calculated by dividing the mean counts per minute (cpm) of wells containing both lymphocytes and bone-marrow cells by the sum of the mean cpm for control wells. If the stimulation index exceeded 1 at 72, 96, or 120 hours at either L: BM ratio a positive response was recorded.Seventy-six children were in clinical remission at the time of testing (group A) and six were in clinical relapse (group B). In group A 24 patients showed stimulation and relapsed later at a mean time of 3·8 months (21 with marrow disease, two with testicular infiltration, and one with lung infiltration). Sixteen patients showed stimulation and had up to 4% blasts in their bone marrow but remained in remission. Nineteen other patients showed a positive response and several factors may have contributed to this: two underwent a “rebound” lymphocytosis after stopping treatment, nine had current or intercurrent infections, two had persistent unexplained bone-marrow lymphocytosis, but six had no causative symptoms and thus their responses were “true false-positives.” Seventeen patients from group A showed no response and remained in remission for a mean of 22·9 months after testing. None of the six children in group B responded, and at testing had 17-85% blasts in their bone marrow.During the study no patient relapsed who had not shown a positive response. The technique merits further study as a guide to the presence of leukaemic cells.     

The effect of food and water deprivation on the peripheral blood parameters of the mouse

Various peripheral blood and bone marrow parameters were determined during food and water deprivation and during food deprivation alone in order to obtain base lines that may be used to make comparisons with similar data from irradiated mice. The peripheral blood parameters following food and water deprivation were similar to those following food deprivation alone. The mean survival time was about 5 days and the weight loss 40% of the control weight. There was an absolute decrease in the total circulating lymphocyte and platelet counts, while the total granulocyte count remained unchanged or increased. The blood volume decreased, while the hematocrit and specific gravity of the blood increased. The bone marrow parameters following food and water deprivation showed that erythropoiesis was more markedly depressed than myelopoiesis. The tritiated thymidine labeling index for granulopoietic cells and megakaryocytes decreased progressively during starvation. The variations in the white blood count and the bone marrow parameters are not comparable with those found in irradiated mice having the G.I. syndrome; the changes in mean survival time, weight loss, hematocrit, and blood volume are similar.     

Chronic neutrophilic leukemia with marked myelodysplasia terminating in blast crisis

A case of chronic neutrophilic leukemia (CNL) is reported. The patient had a history of bleeding, and showed sustained mature neutrophilic leukocytosis, hepatosplenomegaly, a high leukocyte-alkaline phosphatase score, elevated serum vitamin B12 and uric acid, and the presence of D?hle bodies in the neutrophils. In addition to the above typical features, marked myelodysplastic changes in the erythroid and megakaryocytic series were observed in the bone marrow. Three months after the diagnosis of CNL had been established, the hematological picture evolved into a blast crisis of monocytic type. Such findings give support to the statement of CNL as a distinct entity belonging to the spectrum of the myeloproliferative disorders.     

Cyclosporine A treatment in four cases of aplastic anemia

Cyclosporine A (CyA) treatment of 4 patients with severe aplastic anemia, who were ineligible for bone marrow transplantation, was carried out for periods of between 12 weeks to 20 months. A normalization of Hb and bone marrow, together with a marked improvement in WBC and platelet counts, were observed in only one of these four patients. The remission was maintained for 20 months under continuous treatment. A relapse occurred only when the patient himself interrupted treatment. No serious side effects were observed with CyA doses of 4-10 mg/kg/daily and blood concentrations of 200-400 ng/ml. No significant changes in T helper/T suppressor ratios were noted during the course of CyA treatment.     

Amplification of RNA and DNA specific for erb B in unbalanced 1;7 chromosomal translocation associated with myelodysplastic syndrome

Previous work has established the presence of an unbalanced chromosome abnormality [+der(1),t(1;7)(p11;p11)] in some therapy-associated myelodysplastic disorders. Recently the EGF receptor has been found to reside at 7p11. Using a probe specific for erb B oncogene, which encodes a truncated form of the EGF receptor, we examined RNA and DNA derived from bone marrow and peripheral blood mononuclear cells from three patients with myelodysplastic syndromes (MDS) and one with acute lymphocytic leukemia (ALL), all bearing an abnormal clone in their bone marrow with a similar unbalanced 1;7 translocation. DNA-excess slot blot hybridization to 5'-32p-labeled cellular RNA revealed from ten- to thirtyfold enhancement in accumulation of mRNA specific for erb B in both peripheral blood and bone marrow cells of the three MDS patients when compared to normal controls. In addition, enhancement of H-ras mRNA accumulation was detected in some, though expression of other genes such as actin, N-ras, myc, src, B-lym, and 20 other genes was not found to be enhanced. Increased erb B expression was not apparent in mononuclear cells from patients with other hematologic disorders such as chronic lymphocytic leukemia, Hodgkin's disease, or lymphoma. Southern blot analysis of restriction-enzyme-cleaved DNA from three MDS patients with an unbalanced 1;7 translocation revealed that erb B gene was amplified at least twentyfold in peripheral blood white blood cells, while levels of actin hybridization were comparable to those of the controls. No such amplification was evident in the ALL patient. Our data suggest that +der(1),t(1;7)(p11;p11) chromosomal anomalies can be specifically associated with amplification of erb B DNA and RNA sequences.     

Reactivation of a hematopoietic endocrine program of pregnancy contributes to recovery from thrombocytopenia

Regulation of blood platelet levels involves an array of cytokines, including the placental hormone PRL-like protein E (PLP-E). The PLP-E receptor is present on megakaryocytes in pregnant mice, nonpregnant female mice, and male mice. Other known megakaryocytic cytokines do not share the PLP-E receptor, and thus the presence of this receptor in nonpregnant animals suggests that PLP-E may be expressed in tissues other than the placenta. Consistent with this prediction, PLP-E is produced in thrombocytopenic mouse bone marrow, primarily in granulocytes, but not in normal mouse bone marrow. Serum from thrombocytopenic mice, purified thrombopoietin or IL-6, or pregnancy can induce bone marrow cell expression of PLP-E. The induction of PLP-E gene expression in response to thrombocytopenia is physiologically significant, as injection of PLP-E into thrombocytopenic mice restores normal platelet levels with no effect on granulocytes, erythrocytes, and total white blood cell counts. We conclude that inducible expression of PLP-E in bone marrow is part of the mechanism of recovery from thrombocytopenia. These results also suggest a more general concept: that the endocrine program of pregnancy, which in mammals has evolved to support the intrauterine growth and development of the fetus, can also be harnessed to respond to pathophysiology.     

Two Populations of Rh Groups together with Chromosomally Abnormal Cell Lines in the Bone Marrow

At the onset of his disease a man with polycythaemia vera had chromosomally normal cells in the bone marrow and Rh blood group CDe/cDE. Five years later he developed pancytopenia with erythroid hyperplasia of the bone marrow. This was associated with the presence of a major abnormal clone, 45,XY,B-,C-,16+, a minor clone, 45,XY,2+,3-,C-, and a few apparently normal cells. At the same time Rh blood grouping showed two populations of red cells, one CDe/cDE and one giving the reactions of CDe/CDe which can be interpreted as CDe. If monosomic CDe be the correct interpretation the case provides a strong hint that the Rh complex locus is sited either on the long arm of a B-group chromosome or, less probably, on an autosome of the C group.     

Ring chromosome 18 in a child with febrile seizures

Ring chromosomes are uncommon cytogenetic findings but have meanwhile been reported for nearly all human chromosomes. Among the rare observations of ring chromosomes in man, the diagnosis of ring chromosome 18 represents a prominent group. We here describe on the cytogenetic analysis results obtained for a 9 years old male patient of non-consanguineous parents. He had growth and developmental delay, mental and motor retardation, microcephaly, microphtalmia, triangle face, small dysplastic ears, strabismus, epicanthal folds on the left, short stature, cryptorchidism, spasticity, pes equinovarus, pes planus, hypothroidism, stereotypic movements and febrile seizures. Also he had hypomyelinization and multiple hyperintense focuses within the white matter on the MRI. The generalized epileptiform abnormality originated from bilateral Centroparietal region. The metabolic investigations including blood and urine amino acids and lysosomal screening tests were normal. The chromosome analysis identified [46,XY,r(18)/46,XY] in 35% of cells a ring 18 and in 65% of cells normal karyotype in peripheral blood cells examined by standard G-bands by Trypsin using Giemsa (GTG) analysis. The dysmorphic features of the presented patient are discussed to the identification of the genotype-phenotype correlation related to his karyotype.     

Characterization of mice lacking the tetraspanin superfamily member CD151

The tetraspanin membrane protein CD151 is a broadly expressed molecule noted for its strong molecular associations with integrins, especially alpha3beta1, alpha6beta1, alpha7beta1, and alpha6beta4. In vitro functional studies have pointed to a role for CD151 in cell-cell adhesion, cell migration, platelet aggregation, and angiogenesis. It has also been implicated in epithelial tumor progression and metastasis. Here we describe the generation and initial characterization of CD151-null mice. The mice are viable, healthy, and fertile and show normal Mendelian inheritance. They have essentially normal blood and bone marrow cell counts and grossly normal tissue morphology, including hemidesmosomes in skin, and expression of alpha3 and alpha6 integrins. However, the CD151-null mice do show phenotypes in several different tissue types. An absence of CD151 leads to a minor abnormality in hemostasis, with CD151-null mice showing longer average bleeding times, greater average blood loss, and an increased incidence of rebleeding occurrences. CD151-null keratinocytes migrate poorly in skin explant cultures. Finally, CD151-null T lymphocytes are hyperproliferative in response to in vitro mitogenic stimulation.     

大鼠异基因骨髓移植急性移植物抗宿主病模型的建立

目的建立较稳定的异基因骨髓移植急性移植物抗宿主病动物模型,为异基因骨髓移植后的急性移植物抗宿主病（aGVHD）的相关研究提供实验参照。方法以雄性SD大鼠为供鼠,雌性Wistar大鼠为受鼠,受体大鼠随机分成A、B、C、D、E 5组,移植当天所有受鼠均接受8.5 GY的全身照射（TBI）,于照射后4～6 h内,A组回输等量培养液,B组经尾静脉输注供鼠骨髓细胞（2×10^8个/kg）,C、D、E组分别回输供鼠骨髓细胞（2×10^8个/kg）＋不同比例的脾细胞。观察各组大鼠生存期、外周白细胞计数、及有无aGVHD的临床及病理表现。结果A组大鼠于15d内全部死亡,外周血白细胞计数明显减低,骨髓病理示造血组织减少,提示死于造血衰竭。B、C、D、E组大鼠外周血白细胞计数均有明显恢复,B组大鼠8只存活超过50 d,C、D、E组大鼠均于50 d观察期内死亡,并有aGVHD的临床表现及病理表现,但C组大鼠aGVHD的程度较轻且时间不集中,其中D、E组大鼠可于相对集中的时间内观察到典型aGVHD临床及病理。结论TBI预处理的方式是可行的,单纯输入异基因骨髓细胞不能引起明显的aGVHD,骨髓细胞与脾细胞1∶1及1∶1.5混合组均可作为异基因骨髓移植后理想的aGVHD动物模型。