Micropipette aspiration of guinea pig megakaryocytes: absence of fragmentation and dependence on maturation stage

Platelet release has been alternatively viewed as a fragmentation of platelet territories demarcated within the cytoplasm of mature megakaryocytes or as a later event involving segmentation of proplatelet pseudopodia extended from the cell. The mechanical constraints on platelet release were evaluated by measuring the resistance of guinea pig megakaryocytes to aspiration into micropipettes of similar diameter to the width of naturally forming proplatelet projections. Application of increasing negative pressure to the surface of the cells resulted in progressively longer extensions being drawn into the pipette until maximal extension lengths were reached. None of the passively aspirated cytoplasmic extensions fragmented off the cells even at the highest aspiration pressure under physiologic study conditions. The longest extensions were aspirated from megakaryocytes of the most advanced maturation stage, and a proportion of the mature cells yielded very long extensions over 50 mu and up to 150 mu in length. Surprisingly, the ease of aspiration did not correlate to cell size during any stage of maturation. The mechanical behavior of guinea pig megakaryocytes indicates a large availability of surface for extension in mature cells ideal for active proplatelet projection. The lack of mechanical fragility suggests that platelet release is a very late maturational event not yet initiated in the "mature" megakaryocytes available for study from marrow harvests.     

Splenic granulocytopoiesis and production of colony-stimulating activity in lymphoma and leukemia

Spleen cell production of granulocyte-macrophage colony stimulating activity (CSA) and colony forming capacity (CFU-GM) from 59 patients with Hodgkin's and non-Hodgkin's lymphoma, acute (AML) and chronic myeloid leukemia (CML), and control subjects was quantified to evaluate local cellular potential for modulating splenic granulocytopoiesis. Mononuclear spleen cell conditioned media stimulated myeloid CFU-GM by human nonadherent marrow target cells. In contrast to conditioned media produced by marrow and peripheral blood cells, the vast majority of spleen CSA was generated by nonadherent lymphoid cells rather than adherent monocytic cells. The nonadherent cells producing CSA were non- T cells (assessed by sheep erythrocyte rosetting), with 98% +/- 2% CSA produced by the nonrosetted fraction (B lymphocytes and null cells), and had a peak density heavier than that of the adherent spleen CSA- producing cells. Dose response curves demonstrated significantly increased cellular CSA production from patients with lymphomas and AML in remission. In a high proportion of patients, foci of immature granulocytic cells were found by specific cytochemical staining of histologic sections of spleens. A limited degree of splenic granulocytopoiesis was demonstrated morphologically and by CFU-GM incidence. CSA was not detectable in conditioned medium prepared from nonadherent spleen cells from 5 patients with CML, due to a nondialyzable substances(s) produced by the nonadherent cells which inhibited normal CFU-GM response to CSA. The high CFU-GM incidence and extensive leukemic granulocytopoiesis present in the CML spleens suggests diminished effect of this inhibitor on leukemic as opposed to normal granulocytic precursor cell proliferation.     

Isolated follicular lymphoma cells are resistant to apoptosis and can be grown in vitro in the CD40/stromal cell system

Low-grade follicular non-Hodgkin's lymphomas are characterized by the presence of a t(14;18) chromosomal translocation that results in deregulation of the B-cell lymphoma (Bcl-2) gene. Studies in cell lines and transgenic animal models have suggested that this results in the suppression of apoptotic cell death in germinal centers. B lymphocytes from normal germinal centers and lymph nodes infiltrated by follicular lymphoma were isolated by immunomagnetic depletion of cells bearing CD4, CD8, or slgD for study in vitro. Follicular lymphoma cells expressing Bcl-2 protein were shown to resist apoptosis after isolation, and could be induced to proliferate in a culture system previously described for the growth of normal B lymphocytes. By the use of a mouse fibroblast monolayer transfected with the CDw32 Fc receptor to present CD40 monoclonal antibody in the presence of interleukin-4, prolonged culture was possible. Karyotypic analysis of cultured lymphoma cells showed the t(14;18) translocation, with clonal identity confirmed by polymerase chain reaction amplification of the breakpoints and direct sequence analysis. These findings support the hypothesis that resistance to apoptosis is an influence on the initiation of follicular lymphoma, and provide a novel means of studying in vitro the intercellular reactions that may be important in progression of the disease.     

Decreased nucleotide and serotonin storage associated with defective function in Chediak-Higashi syndrome cattle and human platelets

Prolonged mean template bleeding time of 14 min observed in seven cattle with the Chediak-Higashi syndrome (CHS) prompted the examination of platelet function in these animals. There was no distinguishable difference in concentration of circulating platelets between CHS and control cattle. CHS bovine platelets failed to aggregate in vitro in response to concentrations of acid-soluble collagen which induced aggregation in all normal samples. The primary platelet response to 5 muM ADP was normal in CHS cattle. A markedly decreased amount of serotonin (1.2% of normal) was detected in CHS bovine platelets. Bovine CHS platelet ATP and ADP contents were significantly less than normal, and the ATP/ADP ratios were 5.04 in normal and 29.38 in CHS platelets. Results of these animal investigations prompted a similar study of two patients with CHS. In humans, an increased bleeding time greater than 15 min and an in vitro impaired aggregation response to acid-soluble collagen and 5 muM adrenaline were discovered. Both ATP and ADP were reduced in CHS human platelets, and the ATP/ADP ratio was 3.96, compared to a ratio of 1.52 for platelets of two normal subjects. These findings suggested the presence of a "storage pool disease" of CHS platelets.     

Adult hemoglobins are synthesized in murine fetal hepatic erythropoietic cells

The hemoglobins present in murine fetal hepatic erythroblasts on days 12-15 of gestation were studied by biochemical and immunocytologic techniques. In addition, fetal hepatic hemopoietic progenitor cells obtained from normal and mutant f/f mouse fetuses on days 11-13 of gestation were cultured in vitro with added erythropoietin and adult spleen cell conditioned medium to form large erythroid colonies. In all instances, adult hemoglobin synthesis was detected in the fetal hepatic erythroblasts and in the erythroid cell cultures in vitro. The tumor promoter, 12-O-tetradecanoylphorbol 13-acetate, enhanced the fetal hepatic erythroid colony growth in vitro, but did not alter the hemoglobin phenotypic expression.     

Excessive bronchoconstriction induced by histamine and effects of volume history in patients with bronchial asthma

Abstract The aim of this study was to examine the inter-relationships between the different effects of deep breaths and histamine provocation on airway function in patients with bronchial asthma. Group 1 consisted of 38 consecutive out-patients with newly diagnosed mild asthma, group 2 consisted of 20 patients with bronchial asthma of varying severity who were studied during clinical remission. We measured bronchial responsiveness (BR) to histamine inhalation as the dose of histamine which provoked a 20% fall in FEV₁ (PD₂₀). The fall in forced vital capacity (FVC) after inhaling the highest dose of histamine during each BR test was calculated and expressed as percentage of the value measured at baseline (δFVC in percentage). We studied the effects of deep breaths on airway caliber in group 2 patients by comparing isovolumic flow rates on partial (P) and maximal (M) forced expiratory flow volumes curves expressed as the M/P ratio. The changes in residual volume (RV) after deep breaths (δRV) were expressed as a percentage of the largest VC measured on the composite M and P curves. The patients in group 1 had significantly higher PD₂₀ and lower δFVC than patients in group 2. There was, however, no significant correlation between PD₂₀ and δFVC measurements in individual patients (r<0.1, P>0.05). The M/P ratio was significantly related to δFVC (r=?0.6, P<0.006). There was also a significant positive relation between the magnitude of increase in residual volume following deep breaths (δRV) and the degree of fall in FVC following histamine inhalation (δFVC) (r= 0.65, P= 0.001). This significant relationship between the degree of airway closure after a deep breath and airway closure after histamine challenge is a new finding. In patients with bronchial asthma, the effects of a deep breath on airway function may be indicative of the tendency for airway closure during BR testing.     

Pharmacokinetics and immunomodulatory properties of intravenously administered recombinant human interleukin-10 in healthy volunteers

Normal volunteers received single doses of recombinant human interleukin-10 (rhIL-10; n = 6 per group) or placebo (n = 3 per group) by intravenous injection to characterize pharmacokinetics, tolerability, and immunomodulatory effects. Dosages were 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 25.0, 50.0, and 100.0 micrograms/kg. Dose-related adverse effects consisted of a mild-to-moderate flu-like syndrome characterized by fever with chills, headache, and myalgias at the highest dose. The mean terminal phase t1/2 ranged from 2.3 +/- 0.5 to 3.7 +/- 0.8 hours. Dose-related effects of rhIL-10 included transient increases of circulating neutrophils and monocytes and decreases of lymphocytes. rhIL-10 markedly suppressed, in a time- and dose-dependent manner, the synthesis of the inflammatory cytokines IL-1 beta and tumor necrosis factor alpha by whole blood stimulated ex vivo with bacterial lipopolysaccharide. Circulating numbers of CD14+/HLA-DR+ cells at 24 hours after the dose were increased in a dose-dependent manner. Effects on expression of HLA-DR by CD14+ cells were variable. There was no apparent effect on HLA-DR expression by CD20+ cells. The immunomodulatory effects of rhIL-10 merit further clinical investigation.