The cultivation of bone marrow mesenchymal stem cells derived from patients with high altitude polycythemia.

The aim of this study was to isolate and culture bone marrow mesenchymal stem cells (MSCs) from patients with high altitude polycythemia (HAPC) in order to provide a foundation for further exploration of their biological characteristics. MSCs were isolated and cultured from 10 HAPC patients and 10 healthy controls by using a density gradient centrifugation and an adherent screening method. The morphous of MSCs were observed under an inverted microscope, and its surface antigens were determined using flow cytometry. The growth of the MSCs was also detected to evaluate its proliferation. Bone marrow mononuclear cells were isolated from the bone marrow using a density gradient centrifugation, and they were cultured in vitro. The bone marrow MSCs were successfully isolated and cultured, which presented as fusiform and adherent cells. The MSCs in both groups expressed CD90,CD44,CD29,CD105, CD106, CD146, CD166,Stro—1 and CD13, but they did not express CD45, CD4,CD8,CD19,CD20,CD80,CD14,CD3,CD34 or HLA—DR (P>0.05). The bone marrow MSCs from HAPC patients had a higher proliferation than the bone marrow MSCs from the healthy controls (P<0.01). The bone marrow MSCs from HAPC patients can be effectively cultured in vitro.

Iron deficiency modifies gene expression variation induced by augmented hypoxia sensing.

In congenital Chuvash polycythemia (CP), VHL(R200W) homozygosity leads to elevated hypoxia inducible factor (HIF) levels at normoxia. CP is often treated by phlebotomy resulting in iron deficiency, permitting us to examine the separate and synergistic effects of iron deficiency and HIF signaling on gene expression. We compared peripheral blood mononuclear cell gene expression profiles of eight VHL(R200W) homozygotes with 17 wildtype individuals with normal iron status and found 812 up-regulated and 2120 down-regulated genes at false discovery rate of 0.05. Among differential genes we identified three major gene regulation modules involving induction of innate immune responses, alteration of carbohydrate and lipid metabolism, and down-regulation of cell proliferation, stress-induced apoptosis and T-cell activation. These observations suggest molecular mechanisms for previous observations in CP of lower blood sugar without increased insulin and low oncogenic potential. Studies including 16 additional VHL(R200W) homozygotes with low ferritin indicated that iron deficiency enhanced the induction effect of VHL(R200W) for 50 genes including hemoglobin synthesis loci but suppressed the effect for 107 genes enriched for HIF-2 targets. This pattern is consistent with potentiation of HIF-1α protein stability by iron deficiency but a trend for down-regulation of HIF-2α translation by iron deficiency overriding an increase in HIF-2α protein stability.

Multisite evaluation of a monoclonal IMMULITE erythropoietin immunoassay.

BACKGROUND: Erythropoietin (EPO) measurements are useful in diagnosing anemias and polycythemias. We conducted a multisite evaluation of a monoclonal IMMULITE® EPO immunoassay.(1) DESIGN AND METHODS: The IMMULITE EPO assay is a solid-phase enzyme-labeled chemiluminescent immunometric assay. Method comparison to the Beckman ACCESS 2 assay using clinically characterized samples and reproducibility studies were conducted at three external independent laboratories. Internal evaluation conducted at Siemens included comparison of IMMULITE® 2000 and IMMULITE® 1000 assays to the ACCESS 2 assay; imprecision; linearity; limit of blank (LoB), limit of detection (LoD), and functional sensitivity; potential interference and cross-reactants; and reference interval determination. RESULTS: External method comparison gave Deming regression of (IMMULITE 2000)=0.96(ACCESS 2)+2.57IU/L, r=0.98 (n=217). Reproducibility ranged from 6.1% to 16.2%. Internal method comparisons gave Deming regressions of (IMMULITE 2000)=1.09(ACCESS 2)-3.51IU/L, r=0.98 and (IMMULITE 1000)=0.95(ACCESS 2)+0.52IU/L, r=0.95. Total imprecision ranged from 6.4% to 10.3% and linearity was confirmed from 3.5 to 562IU/L. LoB, LoD, and functional sensitivity were 0.5, 1.0, and 1.5IU/L, respectively. The assay was highly specific for EPO. Nonparametric reference interval was 4.3 to 29.0IU/L (n=170). CONCLUSIONS: The monoclonal IMMULITE EPO assay showed acceptable performance for EPO measurement.

Generation and characterization of a neutralizing monoclonal antibody against erythroid cell stimulating factor.

Erythroid cell stimulating factor (ESF) is present in mouse serum and has been reported to function in concert with erythropoietin (EPO) in the formation of erythroid cells in in vitro culture systems. We report here the generation and characterization of a monoclonal antibody (MAb) directed against ESF, with potent anti-ESF-neutralizing activity. A hybridoma-producing MAb to ESF was selected following enzyme-linked immunosorbent assay (ELISA)-based screening of 270 colonies obtained from a fusion of immunized mouse splenocytes with NS1 myeloma cells. Western blot analyses of mouse serum using this antibody specifically detected a single protein (approximate molecular weight of 60 kDa and 120 kDa, under reducing and nonreducing conditions, respectively) corresponding to ESF, with no reactivity to EPO. Furthermore, this MAb demonstrated reactivity to a protein similar in molecular mass, across species, showing reactivity in sera obtained from human, horse, goat, guinea pig, rabbit, and rat. Immuno-chemical characterization demonstrated this antibody to be of IgG3 isotype, bearing kappa light chains. Injection of this monoclonal anti-ESF antibody to exhypoxic polycythemic mice at 6 and 24 h after EPO injection significantly reduced 59Fe incorporation into red blood cells, demonstrating its ability to neutralize in vivo erythropoiesis in our mouse model system. Thus, this novel erythroid cell-specific MAb will be an invaluable tool for further delineating the physiological role of ESF in in vivo erythropoiesis.

Combined in vitro effect of marijuana and retrovirus on the activity of mouse natural killer cells.

Both marijuana and retroviruses impair natural killer (NK) cell functions. No data on their simulataneous effects are available. Similarities to human AIDS induced early by Friend leukemia complex (FLC) and its replication competent helper Rowson-Parr virus (RPV) provides a mouse model to study drug-virus action. Leukemia susceptible BALB/c and resistant C57BL/6 mice were infected, then at time intervals their nylon wool-separated splenocytes were exposed to tetrahydrocannabinol (THC) for 3h. Natural killer (NK) cell activity against Yac-1 cells was assayed by 51Cr-release for 4 and 18h. Recovery of splenocytes was found to be suppressed by FLC, but in BALB/c only by RPV. After a transient enhancement in C57BL/6 by FLC, NK cell activity of both mice became suppressed early (2 to 4 days), normalized subsequently and enhanced late (11 to 14 days) postinfection. A moderate increase in BALB/c, no change in C57BL/6 were induced by low (1-2.5 microgram/ml) THC doses. NK cell activity of BALB/c became suppressed exponentially by higher (5-10 microgrtam/ ml) THC doses in 18h as compared to 4h assays, while its proportional and moderate impairment was seen in C57BL/6. The magnitude of NK cell activity of infected mice was determined by THC: enhancement or impairment followed those of untreated, infected counterparts, but on the level of THC-treated cells. Low doses hardly, high doses additively influenced NK cells of infected BALB/c. THC hardly affected very early and late enhancement in NK cell activiy of FLC infected C57BL/6, but augmented RPV induced suppression late in 18h assays. Genetic factors similar to endotoxin resistance, altered cytokine profile might determine these effects. Similar phenomena in humans might result in earlier manifestation of AIDS.

Activated phenotype in neutrophils and monocytes from patients with primary proliferative polycythaemia.

AIM: To investigate whether monocytes and neutrophils from patients with primary proliferative polycythaemia (PPP) exhibit increased expression of markers of cell activation and, if so, whether they are associated with the phagocytic activity of these cells and concentrations of circulating cytokines. METHODS: Expression of CD11b, CD14, CD18, and CD64 on monocytes and neutrophils was assessed by flow cytometry. Phagocytosis was analysed using immunoglobulin opsonised Escherichia coli. Serum concentrations of granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF) and macrophage CSF (M-CSF) were determined by bioassays, and interferon-gamma (IFN-gamma) by enzyme linked immunosorbent assay (ELISA). RESULTS: Patients with PPP (n = 18), when compared with normal subjects (n = 10), had increased percentages of CD64+ monocytes (52% v 36%) and neutrophils (42% v 11%) and of CD14+ neutrophils (36% v 18%). Monocytes from patients with PPP exhibited increased expression of CD64 (47 v 26) and of CD11b (65 v 36). These abnormalities were not found in patients with secondary (n = 8) or apparent (n = 13) polycythaemia. The percentage of neutrophils undergoing phagocytosis was higher in patients with PPP (mean 64%; n = 6) than in normal subjects (mean 42%; n = 5). G-CSF, GM-CSF and IFN-gamma concentrations in patients' serum samples were comparable with normal; M-CSF was not detected in any of the samples. There was no correlation between cytokine concentrations and the expression of CD11b, CD14, CD18, and CD64 on patients' phagocytes. CONCLUSIONS: Increased expression of CD11b and CD64 by monocytes, increased percentages of CD14+ and CD64+ neutrophils and the high phagocytic activity of neutrophils suggests that these cells are activated in vivo in patients with PPP. The phenotypic changes of PPP phagocytes were not associated with increased concentrations of circulating cytokines and probably reflect intrinsic abnormalities within the neoplastic PPP clone.

Proliferating cell nuclear antigen expression by erythroid precursors in normal bone marrow, in reactive lesions and in polycythaemia rubra vera.

Using a sequential double-immunostaining technique, a morphometric analysis was performed on routinely processed bone marrow trephines from 20 patients with secondary (reactive) polycythaemia and 28 patients with polycythaemia rubra vera to determine the proliferation capacity of erythropoiesis. Monoclonal antibodies PC10--anti-proliferating cell nuclear antigen (PCNA)--and Ret40f--anti-glycophorin C--were employed. For comparison with the PCNA-labelling index, in a pilot study, Ki-67 was additionally used on frozen-section material. In comparison with normal bone marrow (15 patients) morphometric and statistical evaluation revealed a numerical increase in erythroid precursors (pro-, erythro- and normoblasts) in secondary polycythaemia and to a pronounced degree in polycythaemia rubra vera. In comparison with secondary polycythaemia and the control group, in polycythaemia rubra vera there was a significant enhancement of proliferation according to PCNA-staining reactivity in all haematopoietic cell elements and particularly in the erythroid series. Evaluation of PCNA v. Ki-67 immunostaining disclosed only a slight difference, which could be mainly attributed to various modalities of antigen expression during the cell cycle. Our findings are in keeping with in vitro studies on cultured erythroid progenitor cells and, in problematic cases, may present a valuable aid in differentiation between reactive lesions and polycythaemia rubra vera.

The association of erythrocytosis and chronic lymphocytic leukemia.

The authors report the clinical course of three patients with well-documented chronic lymphocytic leukemia (CLL) and concomitant erythrocytosis. Associated disorders included immune cytopenias, Hashimoto struma and Richter syndrome. Durable complete remissions of CLL have occurred in two patients. Inasmuch as a chance association of these two relatively rare hematologic disorders is unlikely, the available information suggests that a pluripotent stem cell with the capacity to differentiate into lymphoid and erythroid pathways is the most attractive hypothesis.

Synergistic effect of human lactoferrin and recombinant murine interferon-gamma on disease progression in mice infected with the polycythemia-inducing strain of the Friend virus complex.

Mice infected with the polycythemia-inducing strain of the Friend virus complex (FVC-P) have been used as a leukemic mouse model. In the present study, purified iron-saturated human lactoferrin (LF) and recombinant murine (rmu) interferon-gamma (IFN-gamma), alone or in combination, were used to influence disease progression in virally infected mice. DBA/2 mice were injected i.v. with FVC-P, and were treated s.c. with 100 micrograms LF at day 7, and/or rmuIFN-gamma at 5 x 10(4) units/day for 3 days beginning at day 6 after viral infection. Mice were assessed for survival, and also 14 days after virus inoculation, the mice were killed and spleen extracts were assessed for spleen focus forming virus (SFFV) titers by spleen focus forming unit (SFFU) assay, SFFV mRNA and genomic DNA expression, and natural killer (NK) cell activity. Treatment with LF or rmuIFN-gamma alone had little or no effect on SFFU numbers or SFFV mRNA or genomic DNA expression. However, dramatically decreased SFFV titers and levels of SFFV mRNA and genomic DNA were observed in mice treated with the combination of LF and rmuIFN-gamma. NK cell activity decreased by FVC-P was returned to normal levels by LF and rmuIFN-gamma. The combined treatment also enhanced the survival rates of FVC-P-infected mice. The results suggest synergistic suppressive effects of LF with rmuIFN-gamma on disease progression in FVC-P-infected mice. This information might be of significance as a potential therapy for patients with leukemia and those infected with retroviruses.

Abnormal membrane physical properties of red cells in McLeod syndrome.

McLeod red cells (RBCs) lack Kx antigens and have weak expression of the Kell antigens. Individuals who carry the McLeod phenotype have acanthocytic RBCs and a compensated hemolytic state. To elucidate the role of the protein on which the Kx antigens reside in maintaining membrane deformability, the rheologic properties of McLeod RBCs were determined by ektacytometry. RBCs were obtained from normal individuals and from four patients with McLeod syndrome. Osmotic gradient deformability profiles of McLeod RBCs showed decreased whole cell deformability. Resealed ghosts from McLeod RBCs also showed decreased deformability, partly because of the decreased cell surface area and partly because of an intrinsic membrane stiffness in this syndrome. For the measurement of membrane mechanical stability, resealed ghosts were subjected to constant high shear stress in the ektacytomer, and deformability was recorded continuously as the deformable ghosts fragmented into rigid spherical vesicles. Membranes from McLeod RBCs showed a noticeable increase in mechanical stability. Acquired causes of acanthocytosis, such as liver disease, did not cause the rheologic abnormalities observed in McLeod cells. Other abnormalities noted in McLeod RBCs were decreased RBC potassium content and an increased number of dense RBCs, as determined by centrifugation on a discontinuous density gradient. The data indicate that McLeod RBCs are rigid and have decreased surface area and that their membranes are intrinsically rigid with increased mechanical stability. These abnormalities may account for the reduced RBC survival observed in McLeod syndrome. The protein that carries the Kx surface antigen seems to be required for the maintenance of the normal physical function of RBC skeletal proteins.

Delayed immune response in chorea-amyotrophy with spherocytosis.

Progressive CSF lymphocytic pleocytosis and intrathecal IgG-synthesis occurred late in familial amyotrophy, neuropathy, chorea, and dementia with spherocytosis. Immunoblotting showed a serum and CSF antibody apparently directed against glial fibrillary acidic protein. A secondary autoimmune response was probably triggered during the evolution of the neurodegenerative process.

Serum immunoreactive erythropoietin in hypoxic lung disease with and without polycythaemia.

We studied 20 patients with chronic airflow obstruction, 10 patients without polycythaemia and 10 patients with compensatory polycythaemia having respectively mean red cell mass 24.7 (SD 4.2) and 47.8 (SD 7.5) ml/kg, mean daytime PaO2 7.6 and 6.9 kPa, mean FEV1 0.85 and 0.821. Groups were matched for severity of daytime arterial hypoxaemia but nocturnal arterial oxygen desaturation was more severe in the patients with polycythaemia than in those without. We also studied six additional patients with chronic airflow obstruction and polycythaemia and 19 normal controls. Estimates of serum immunoreactive erythropoietin (siEp) in those without polycythaemia were 19 m-i.u./ml (geometric mean) with 95% confidence range 11-35 m-i.u./ml and stable during 3 months. In those with polycythaemia they were similar and consistent in five and, in the other five, higher on at least one occasion. There was no significant difference between siEp in daytime (12.00 hours to 16.00 hours) and morning (07.00 hours) samples but geometric mean estimates of erythropoietin in paired daytime and morning samples were higher and more variable in patients with polycythaemia than in those without. The geometric mean estimate of siEp in all patients with chronic airflow obstruction and polycythaemia was greater than in normal subjects but, despite secondary polycythaemia, siEp could be in the range for normal subjects. In the patients with polycythaemia we were unable to predict the finding of normal or elevated siEp. Changes in siEp after erythrapheresis (10-26% reduction in packed cell volume) were observed in the 10 patients with polycythaemia and in one without. One month after erythrapheresis, packed cell volume remained below and siEp was above initial pretreatment levels, implying an erythropoietin secretory response and that the development of secondary polycythaemia had induced a fall in siEp.

Pre-B cells in bone marrow: peanut agglutinin binding and separation of cytoplasmic mu chain-bearing cell populations in normal, post-irradiation and polycythemic mice using fluorescence-activated cell sorting.

Mouse bone marrow cells exposed to fluorescein-conjugated peanut agglutinin (PNA) showed subsets of highly labeled cells when analyzed in a fluorescence-activated cell sorter. After separating three cell fractions of large and small PNA-binding cells and PNA-nonbinding cells, respectively, the B lymphocyte precursor (pre-B) cells, having cytoplasmic mu chains (c mu) without surface mu chains (s mu), were recovered solely in the PNA-binding fractions. Only a minority of s mu+ small lymphocytes having the lowest densities of s mu bound PNA. Small and large c mu+ s mu- pre-B cell populations were separated in high degrees of purity in the PNA-binding fractions, especially when obtained from bone marrow undergoing lymphoid regeneration after sublethal X-irradiation and during stimulation of lymphocyte production in post-polycythemic erythroid suppression. Characteristic shifts in the size distribution profile of PNA-binding cells reflected changes in the maturation stage of the pre-B cells. The results demonstrate that surface membrane components with strong PNA-binding capacities characterize c mu+ s mu- pre-B cells in the bone marrow during both normal and perturbed primary B lymphocyte genesis. The PNA-binding sites become undetectable soon after the first expression of s mu. This property permits the isolation from the bone marrow of high concentrations of subsets of large and small c mu+ s mu- cells in a viable state suitable for use in further functional studies.