Interferon affects nuclear proteins in cells of clinically sensitive chronic myelogenous leukemia patients.

Cytoplasmic protein extracts from chronic myelogenous leukemia (CML) cells contained an activity that altered the electrophoretic mobility of complexes formed between nuclear proteins and the transcriptional enhancers of interferon (IFN)-inducible genes. Exposure of CML cells to IFN-alpha diminished the effect of the CML cytoplasmic proteins on these nuclear protein-DNA complexes. The presence of clinical responsiveness to IFN-alpha correlated with the sensitivity to the IFN-induced change in the electrophoretic mobility of nuclear protein-DNA complexes. These data suggest that the action of IFN-alpha in CML may be linked to a pathway that can result in posttranslational modification of nuclear proteins.

Immunohistochemical observations on bone morphogenetic protein in normal and abnormal conditions.

Localization of bone morphogenetic protein (BMP) in human tissues and cells is important for investigating the mechanism of bone induction. A stable cell line secreting monoclonal antibody against bovine BMP (bBMP-McAb) was obtained by the hybridoma technique. The result of immunohistochemical staining (ABC method) showed that BMP is distributed along collagen fibers of normal bone, in periosteal cells, and in mesenchymal cells of marrow stroma. Little BMP can be found in bone cells of lamellar bone or in calcified bone matrix. BMP may be abundant in human tooth anlagen such as predentin, cells of the outer and inner enamel epithelium, and cells of dental sac generating bone. BMP is found in the cytoplasm of tumor cells of osteosarcoma and chondrosarcoma. Immunohistochemical staining showed that BMP plays a role in bone fracture healing. The ability of BMP-McAb to detect BMP and to inhibit the generation of new bone also makes it potentially useful in diagnosing, treating, and providing a prognosis for osteosarcoma and other bone diseases.

Tissue sources of murine megakaryocyte potentiator: biochemical and immunological studies.

The immunological and biochemical characteristics of murine megakaryocyte potentiator from lung and bone marrow were examined and compared with thrombopoietic stimulatory factor. Biological activity was not neutralized by anti-erythropoietin, but megakaryocyte potentiator activity from all three sources was abolished or reduced when the preparations were treated with anti-thrombopoietic stimulatory factor or anti-interleukin-6. Megakaryocyte potentiator levels in lung conditioned medium were not found to be enhanced from mice treated with lipopolysaccharide, in contrast to granulocyte-macrophage colony-stimulating factor (GM-CSF) levels. The biochemical properties of murine megakaryocyte potentiator from lung and bone marrow were compared and found to be similar in the elution profiles from anion exchange, gel filtration and reversed phase liquid chromatography. It is concluded that the activities in lung and bone marrow are very similar if not identical, to interleukin-6.

The long-term effects of MVPP chemotherapy for Hodgkin's disease on bone marrow function.

Using in vitro techniques, bone marrow (BM) function has been studied in 25 patients in complete remission and at least one year after the completion of MVPP chemotherapy for Hodgkin's disease. The numbers of granulocyte/macrophage (GM-CFC) and fibroblastoid (CFU-F) progenitors were significantly lower than controls and there was no evidence of any improvement with time (median months off treatment was 30 for GM-CFC and 34 for CFU-F). In long-term BM culture production of haemopoietic cells were strikingly lower in the post-MVPP group and the development of adherent stromal cell populations was also significantly less. In addition, the yield of GM-CFC in adherent layers after four weeks of culture was significantly lower than in controls. We conclude that following MVPP chemotherapy and in apparently disease free and haematologically normal individuals there is evidence of impaired BM function up to nine years after the completion of treatment. These abnormalities may be relevant to the known increased risk of acute non-lymphocytic leukaemias in this group of patients and are likely to render the BM less able to withstand subsequent insults such as further chemotherapy or infection. The eventual development of BM failure is also a possibility and long-term follow-up of these patients is essential.

A heparan sulfate-containing fraction of bone marrow stroma induces maturation of HL-60 cells in vitro.

Constituents of the bone marrow microenvironment have the capacity to influence both normal and malignant hematopoietic cell behavior. For example, HL-60 human promyelocytic leukemia cells in vitro display a more mature phenotype when grown on a bone marrow stroma-derived matrix. To elucidate which component(s) of the stromal matrix is capable of modulating HL-60 cell phenotype, matrices were treated with a variety of chemicals and enzymes prior to being used in the differentiation assay. Treatment of matrices with collagenase, pronase, chondroitinase, or chloroform:methanol:ether could not abolish the differentiation-promoting activity of bone marrow stroma. In contrast, the activity was destroyed by alkali treatment (0.5 M NaOH for 18 h) or heparinase/heparitinase enzymes. Heparin added to cultures increased maturation of HL-60 cells as determined by esterase production, Fc rosette formation, and morphological appearance. Other stromal components such as laminin, fibronectin, collagen I, collagen IV, or chondroitin sulfate did not alter the HL-60 leukemia cell phenotype. Stroma-derived matrix material which labeled with [35S]sulfate and eluted on a DEAE ion-exchange column as a high ionic fraction in 1.5 M LiCl and 7.5% sodium dodecyl sulfate contained the active fraction. A heparan sulfate proteoglycan component isolated by polyacrylamide-agarose gel electrophoresis induced a more mature HL-60 phenotype, and digestion with heparinase/heparitinase in the presence of protease inhibitors abrogated the effects on HL-60 phenotype. We conclude that a heparan sulfate-associated fraction of the bone marrow matrix plays a key role in the regulation of leukemic cell maturation.

Epithelial tumor cells in bone marrow of patients with colorectal cancer: immunocytochemical detection, phenotypic characterization, and prognostic significance.

A monoclonal antibody (mAb) directed against the cytokeratin (CK) polypeptide no. 18 specifically expressed in cells derived from simple epithelia was used to detect epithelial tumor cells in bone marrow aspirates. Of 156 patients with colorectal carcinoma, 42 presented with cells at the time of primary surgery. The incidence of positive findings varied considerably with the size and the localization of the primary tumor, the involvement of regional lymph nodes, and the presence of clinically manifest metastases. Applying a sensitive double-staining procedure, we could demonstrate that epithelial cells in bone marrow showed a heterogeneic expression of receptors for epidermal growth factor (EGF-R) and transferrin (Tf-R) as well as of the proliferation-associated Ki67 antigen. Also human leukocyte antigen (HLA) class I antigens differed widely in their expression on the CK-positive cells. Clinical follow-up studies on 85 patients showed a significantly higher relapse rate in patients presenting with CK-positive cells in their bone marrow at the time of primary surgery. Twenty-three patients were monitored for the presence or absence of CK-positive cells in bone marrow over time. The majority of monitored patients (18 of 23) exhibited a constant pattern of immunocytochemical findings during the time of observation. Thus, the technique may be useful in identifying high-risk patients as well as in monitoring adjuvant therapeutic trials.

[Long-term cultures of marrow cells from the patients with aplastic anemia].

Long-term marrow cultures were established from 35 patients with aplastic anemia (AA) and the adherent stromal cell layers were assessed. Cultures from 23 of the 35 patients grew scanty stromal cell layers or did not produce any adherent cells. Long-term cultures from the remaining patients formed adherent cell layers that appeared to be morphologically normal. Cultures from 7 patients that grew apparently normal adherent cell layers were examined for expression of intermediate filament proteins using antibodies CGA-7 and HHF, which respectively recognize actin epitopes expressed in smooth muscle and normal marrow stromal cells. Cells from 4 of the 7 patients expressed vimentin (antibody 43 beta E8) but did not react with CGA-7 or HHF. It thus appears that most patients with AA have quantitative or qualitative abnormalities in the adherent cell layers from long-term marrow cultures, suggesting a defect in the hematopoietic microenvironment.

Correlation of the proliferative index of residual leukemia with outcome in patients treated with sequential high dose ara-C and asparaginase.

Therapy of acute myelogenous leukemia (AML) with sequential high-dose ara-C and asparaginase (HiDAC----ASNase) on a day 1 and 8 schedule was designed to exploit potential recruitment of residual leukemia cells following initial cytoreduction from day 1 treatment. DNA flow cytometry was used to evaluate the proliferative index (%S + G2M) of bone marrow leukemia cells from pretreatment and day 8 marrow samples. The proliferative index on day 1, day 8, and incremental change (day 8 minus day 1) were analyzed for their correlation with bone marrow aplasia on day 15 and with the attainment of subsequent complete remission. Pretreatment (day 1) and the change in proliferative index did not correlate (p greater than 0.10) with day 15 marrow aplasia or with clinical outcome. However, the magnitude of the day 8 proliferative index did relate to the attainment of bone marrow aplasia on day 15 (p = 0.05) and the attainment of complete remission (p = 0.002). Recruitment of residual leukemia cells into the proliferative phases of the cell cycle may contribute to the unique efficacy of the day 1 and 8 schedule of HIDAC----ASNase. Additionally, the cytokinetics of residual leukemia after initial chemotherapy may be predictive of outcome and could be useful as a marker for the design of optimal therapeutic regimens.

Stromal hemopoietic microenvironment in aging.

The bone marrow content, proliferative potential and proliferative activity of precursor cells for stromal fibroblast colony forming cells (CFC-F) were investigated in young and old CBA mice. The relationship between CFC-F and the number of bone marrow nucleated cells and granulocytic-macrophagal precursors (GM-CFC) was studied as well. The results obtained showed increased CFC-F contents in old animals. The proliferative potential of old mice CFC-F did not appear to differ from that of young animals. The proliferative activity of bone marrow CFC-F and hemopoietic stem cells--spleen colony forming cells (CFC-S) was studied by determining the sensitivity to hydroxyurea administration. The responses were almost the same in young and old mice. A direct correlation between CFC-F and nucleated cells and GM-CFC precursors was found in young mice, but not in the old animals. The results of the present study have pointed to the reorganisation of the stromal tissue microenvironment in bone marrow in old age.

Anaemia of chronic disease: diagnostic significance of erythrocyte and serological parameters in iron deficient rheumatoid arthritis patients.

Erythrocyte and serological parameters were assessed in 44 anaemic rheumatoid arthritis (RA) patients to detect iron deficiency as assessed by stainable bone marrow iron. The anaemia was normochromic normocytic in 60% and hypochromic normocytic in 30% of those with anaemia of chronic disease (ACD). Iron deficiency was present in 55% and the anaemia was hypochromic microcytic in 54% and hypochromic normocytic or normochromic normocytic in 21%. Iron absorption was found to be higher in iron deficient patients. In ACD patients, iron absorption correlated inversely with ESR and CRP. For the detection of iron deficiency among RA patients with ACD, the MCV showed the highest specificity (90%) and predictive value (87%). Serum ferritin was the most sensitive (82%) and valid (86%) test. Combination of MCV, ferritin and transferrin resulted in 100% validity. It was concluded that iron deficiency can be detected accurately without bone marrow aspiration using combinations of blood parameters.

Use of polymerase chain reaction-detected sequence polymorphisms to document engraftment following allogeneic bone marrow transplantation.

Distinguishing between host and donor origin of cells after bone marrow transplantation is important in understanding the engraftment process. Restriction fragment-length polymorphism (RFLP) analysis, the most generally applicable approach for this purpose, is limited by a requirement for at least 10(6) cells per assay. The small number of cells available at early time points post-BMT has thus precluded studies of early engraftment kinetics. This report describes the application of the polymerase chain reaction (PCR) to engraftment analysis following allogeneic BMT. We describe a series of PCR polymorphisms (PCRFLP/s) that allows the distinction of most patient-donor pairs (excluding identical twins). Thirteen patient-donor pairs were evaluated using this approach, and engraftment data obtained at time points when leukocyte counts were often too low for conventional analysis. This approach is quantitative and significantly more rapid than conventional techniques. (Analysis can be completed in less than a day). Serial evaluation at early time points post-BMT in five patients demonstrated residual host cells early (days 7-14) followed by their subsequent rapid disappearance. In one patient an apparent resurgence of host elements occurred around days 28-35, followed by a sharp decline by day 42.

Bone marrow karyotypes in 94 children with acute leukemia.

During the last 10 years, we have cytogenetically analyzed at diagnosis bone marrow cells from a total of 94 children with acute leukemia. Of the 78 children with acute lymphatic leukemia (ALL), 53 (68%) had clonal acquired chromosome abnormalities; in the group with acute nonlymphatic leukemia (ANLL), the corresponding proportion was 13 out of 16 (81%). Among the cytogenetically abnormal ALL patients, the most numerous subset was the hyperdiploid cases with stemlines containing 51 or more chromosomes (26 of 53 abnormal cases; 49%). This is a clearly higher proportion than has been reported in large series from other centers. Deletions of 6q were present in 8 cases and rearrangements of 12p in 5. Of the 7 T-cell ALLs, 3 had translocations of the distal part of 7q, i.e., of the region where the beta T-cell receptor is encoded. Only 2 of 26 (8%) patients with leukemic stemlines with more than 50 chromosomes have relapsed; the remainder are still in first remission (mean observation time 42 months). This may be contrasted with 6 of 25 (24%) relapses among the cytogenetically normal (observation time 41 months), and 8 of 27 (30%) relapses among ALL patients with aberrations but with less than 51 chromosomes (observation time 26 months). Our results support the conclusion that the finding of a markedly hyperdiploid leukemia karyotype is indicative of good prognosis in ALL.

Bone marrow fat content in relation to bone remodeling and serum chemistry in intact and ovariectomized dogs.

It was previously shown that 11 months after ovariectomy the volume fraction of trabecular bone in the spine and 11th rib medullary canal of Beagle dogs (6 control, 9 ovariectomized) was significantly reduced. In this paper it is shown that these changes are accompanied by increased marrow fat volume in the 11th rib (59.0 +/- 9.5% vs. 44.3 +/- 10.0%). Conversely, the volume fraction of functional (hematopoietic) cells in the marrow was reduced by ovariectomy. Additionally, variations in marrow fat volume were tested for correlation with 22 other variables pertinent to bone physiology. Marrow fat volume was significantly positively correlated with serum osteocalcin, rib trabecular bone porosity, rib cross-sectional area, and gains in body weight. It was negatively correlated with serum estrogen concentrations and the extent of rib trabecular surfaces labeled with tetracycline.

Diagnosis of iron-deficiency anemia in the elderly.

PURPOSE: To determine the value of serum ferritin, mean cell volume, transferrin saturation, and free erythrocyte protoporphyrin in the diagnosis of iron-deficiency anemia in the elderly. PATIENTS AND METHODS: We prospectively studied consecutive eligible and consenting anemic patients over the age of 65 years, who underwent blood tests and bone marrow aspiration. The study consisted of 259 inpatients and outpatients at two community hospitals in whom a complete blood count processed by the hospital laboratory demonstrated previously undiagnosed anemia (men: hemoglobin level less than 12 g/dL; women: hemoglobin level less than 11.0 g/dL). RESULTS: Thirty-six percent of our patients had no demonstrable marrow iron and were classified as being iron-deficient. The serum ferritin was the best test for distinguishing those with iron deficiency from those who were not iron-deficient. No other test added clinically important information. The likelihood ratios associated with the serum ferritin level were as follows: greater than 100 micrograms/L, 0.13; greater than 45 micrograms/L but less than or equal to 100 micrograms/L, 0.46; greater than 18 micrograms/L but less than or equal to 45 micrograms/L, 3.12; and less than or equal to 18 micrograms/L, 41.47. These results indicate that values up to 45 micrograms/L increase the likelihood of iron deficiency, whereas values over 45 micrograms/L decrease the likelihood of iron deficiency. Seventy-two percent of those who were not iron-deficient had serum ferritin values greater than 100 micrograms/L, and in populations with a prevalence of iron deficiency of less than 40%, values of greater than 100 micrograms/L reduce the probability of iron deficiency to under 10%. Fifty-five percent of the iron-deficient patients had serum ferritin values of less than 18 micrograms/L, and in populations with a prevalence of iron deficiency of greater than 20%, values of less than 18 micrograms/L increase the probability of iron deficiency to over 95%.

Identification of monoclonal antibodies that recognize novel epitopes in native chondroitin/dermatan sulfate glycosaminoglycan chains: their use in mapping functionally distinct domains of human skin.

Five monoclonal antibodies (MAb), 7D4, 4C3, 6C3, 4D3, and 3C5, were produced in mice immunized with high buoyant density embryonic chick bone marrow proteoglycans (PGs) as antigen. All of these MAb recognized epitopes in native chick bone marrow and cartilage PGs which could be selectively removed by chondroitinase ABC and chondroitinase AC II, indicating that their epitopes were present in chondroitin sulfate glycosaminoglycans (GAGs). These MAb recognized epitopes present in purified cartilage PGs obtained from a wide variety of different vertebrate species. However, none of the new MAb detected epitopes in Swarm rat chondrosarcoma PG. On the basis of these results, we propose that these MAb recognize novel epitopes located in chondroitin sulfate/dermatan sulfate glycosaminoglycan (CS/DS GAG) chains, representing at least four and possibly five different structures. Immunocytochemical studies have shown that the epitopes identified by these new MAb are differentially distributed in tissues. All of these MAb immunocytochemically detected epitopes in embryonic chick cartilage and bone marrow. Three of them (4C3, 7D4, and 6C3) recognized epitopes in adult human skin. All three detected epitopes in the epidermis, one (6C3) strongly detected epitopes in the papillary dermis, and two (4C3, 7D4) detected epitopes in the reticular dermis. Immunostaining patterns in skin using the new MAb directed against native CS/DS structures were distinctly different from those obtained using MAb against the common CS isomers. The distribution of these CS epitopes in functionally distinct domains of different tissues implies that these structures have functional and biological significance.

Anaemia in rheumatoid arthritis: the role of iron, vitamin B12, and folic acid deficiency, and erythropoietin responsiveness.

Thirty six patients with rheumatoid arthritis (RA) (25 with anaemia) were studied to establish the role of iron, vitamin B12, and folic acid deficiency, erythropoietin responsiveness, and iron absorption in the diagnosis and pathogenesis of anaemia in RA. Iron deficiency, assessed by stainable bone marrow iron content, occurred in 13/25 (52%), vitamin B12 deficiency in 7/24 (29%), and folic acid deficiency in 5/24 (21%) of the anaemic patients. Only 8/25 (32%) had just one type of anaemia. The iron deficiency of anaemia of chronic disease (ACD) was distinguished by ferritin concentration, which was higher in that group. Mean cell volume (MCV) and mean cell haemoglobin (MCH) were lower in both anaemic groups, but most pronounced in iron deficient patients. Folic acid, and especially vitamin B12 deficiency, masked iron deficiency by increasing the MCV and MCH. Iron absorption tended to be highest in iron deficiency and lowest in ACD, suggesting that decreased iron absorption is not a cause of ACD in RA. No specific causes were found for vitamin B12 or folic acid deficiency. Haemoglobin concentration was negatively correlated with erythrocyte sedimentation rate in the group with ACD. Erythropoietin response was lower in ACD than in iron deficient patients. It was concluded that generally more than one type of anaemia is present simultaneously in anaemic patients with RA. The diagnosis of each type may be masked by another. Studies on pathogenesis of the anaemia are difficult as deficiencies generally coexist with ACD. Disease activity and, possibly, erythropoietin responsiveness are major factors in ACD pathogenesis.

Bursin localization in mammalian bone marrow and epithelial cells of intrahepatic bile ducts.

Bursin is a tripeptide (lysyl-histidyl-glycyl-amide) found in follicular and dendritic reticular epithelial cells of the avian bursa of Fabricius that selectively induces the differentiation of committed B-lymphocyte precursor cells but not of committed T-lymphocyte precursor cells. We now show, in immunoassays with tissue extracts, that bursin is also present in avian and bovine bone marrow. There was, however, a categorical difference between avian liver (bursin-negative) and bovine liver (bursin-positive). Bursin was therefore isolated from bovine liver and bone marrow and the structure of mammalian bursin was determined; it was identical to avian bursin. Immunohistochemical examination of bovine liver showed the presence of bursin within epithelial cells of the intrahepatic bile ducts. These cells have previously been suspected of having an endocrine function because of the rich periductal capillary plexus, which coalesces to form a portal system draining into the liver sinusoids. These findings suggest that bone marrow is a site of bursin production and associated B-cell differentiation in both birds and mammals. The bursin-containing cells of the intrahepatic bile ducts are not associated with developing B cells and it would appear that mammals have evolved a local hepatic function for bursin.