Cytokine production by primary bone marrow megakaryocytes

Primary human bone marrow megakaryocytes were studied for their ability to express and release cytokines potentially relevant to their proliferation and/or differentiation. The purity of the bone marrow megakaryocytes was assessed by morphologic and immunocytochemical criteria. Unstimulated marrow megakaryocytes constitutively expressed genes for interleukin-1 beta (IL-1 beta), IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor-alpha (TNF-alpha), by the polymerase chain reaction (PCR) and Northern blot analysis. At the protein level, megakaryocytes secreted significant amounts of IL-1 beta (53.6 +/- 3.6 pg/mL), IL-6 (57.6 +/- 15.6 pg/mL), and GM-CSF (24 +/- 4 pg/mL) but not TNF-alpha. Exposure of human marrow megakaryocytes to IL-1 beta increased the levels of IL-6 (87.3 +/- 2.3 pg/mL) detected in the culture supernatants. Transforming growth factor- beta was also able to stimulate IL-6, IL-1 beta, and GM-CSF secretion, but was less potent than stimulation with phorbol-12-myristate-13- acetate (PMA). The secreted cytokines acted additively to maintain and increase the number of colony-forming unit-megakaryocytes colonies (approximately 35%). These studies demonstrate the production of multiple cytokines by isolated human bone marrow megakaryocytes constitutively or stimulated in vitro. The capacity of human megakaryocytes to synthesize several cytokines known to modulate hematopoietic cells supports the concept that there may be an autocrine mechanism operative in the regulation of megakaryocytopoiesis.     

Red cell abnormalities associated with sulphasalazine maintenance therapy for ulcerative colitis.

Of 52 patients receiving a mean dose of 2.5g sulphasalazine/day as maintenance therapy for ulcerative colitis, 35 were found to have one or more drug-induced red cell abnormalities, which were not found in 50 normal controls or in 10 colitics not receiving sulphasalazine. Twenty-three of the treated patients had contracted red cells, an abnormality that is thought to result in mild haemolysis. Red cell contraction was related to the dose of sulphasalazine (P smaller than 0.01), the serum total sulphapyridine level (P smaller than 0.001), and acetylator status. Eleven of the treated patients had a macrocytosis, 21 had elevated levels of methaemoglobin, and one had Heinz bodies. A dose of 1.5 g sulphasalazine/day was not associated with red cell contraction, and is suggested as a safer maintenance dose for the asymptomatic colitic.     

Administration of pegylated recombinant human megakaryocyte growth and development factor to humans stimulates the production of functional platelets that show no evidence of in vivo activation

This report describes the effect of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) on platelet production and platelet function in humans. Subjects with advanced solid tumors received PEG-rHuMGDF daily for up to 10 days. There was no increase in circulating platelet count at doses of 0.03 or 0.1 microgram/kg/d by day 12 of study. At doses of 0.3 and 1.0 microgram/kg/d there was a threefold median increase (maximum 10-fold) in platelet count by day 16. The platelets produced in vivo in response to PEG-rHuMGDF showed unchanged aggregation and adenosine triphosphate (ATP)-release responses in in vitro assays. Tests included aggregation and release of ATP in response to adenosine diphosphate (ADP) (10, 5, 2.5, and 1.25 mumol/L), collagen (2 micrograms/mL), thrombin-receptor agonist peptide (TRAP, 10 mumol/L) and ristocetin (1.5 mg/mL). Administration of aspirin to an individual with platelet count of 1,771 x 10(3)/L resulted in the typical aspirin-induced ablation of the normal aggregation and ATP-release response to stimulation with arachidonic acid (0.5 mg/mL), collagen, and ADP (2.5 and 1.25 mumol/L). There was no change in the expression of the platelet-surface activation marker CD62P (P-selectin) nor induction of the fibrinogen binding site on glycoprotein IIb/IIIa as reported by the monoclonal antibody, D3GP3. An elevation of reticulated platelets was evident after 3 days of treatment with PEG-rHuMGDF and preceded the increase in circulating platelet count by 5 to 8 days; this reflected the production of new platelets in response to PEG-rHuMGDF. At later time points, the mean platelet volume (MPV) decreased in a manner inversely proportional to the platelet count. Levels of plasma glycocalicin, a measure of platelet turnover, rose 3 days after the initial increase in the peripheral platelet count. The level of plasma glycocalicin was proportional to the total platelet mass, suggesting that platelets generated in response to PEG-rHuMGDF were not more actively destroyed. Thus, the administration of PEG-rHuMGDF, to humans, increased the circulating platelet count and resulted in fully functional platelets, which showed no detectable increase in reactivity nor alteration in activation status.     

Hematopoietic growth factors for graft failure after bone marrow transplantation: a randomized trial of granulocyte-macrophage colony- stimulating factor (GM-CSF) versus sequential GM-CSF plus granulocyte- CSF

Delay in hematologic recovery after bone marrow transplantation (BMT) can extend and amplify the risks of infection and hemorrhage, compromise patients' survival, and increase the duration and cost of hospitalization. Because current studies suggest that granulocyte- macrophage (GM) colony-stimulating factor (CSF) may potentiate the sensitivity of hematopoietic progenitor cells to G-CSF, we performed a prospective, randomized trial comparing GM-CSF (250 micrograms/m2/d x 14 days) versus sequential GM-CSF x 7 days followed by G-CSF (5 micrograms/kg/d x 7 days) as treatment for primary or secondary graft failure after BMT. Eligibility criteria included failure to achieve a white blood cell (WBC) count > or = 100/microL by day +21 or > or = 300/microL by day +28, no absolute neutrophil count (ANC) > or = 200/microL by day +28, or secondary sustained neutropenia after initial engraftment. Forty-seven patients were enrolled: 23 received GM-CSF (10 unrelated, 8 related allogeneic, and 5 autologous), and 24 received GM- CSF followed by G-CSF (12 unrelated, 7 related allogeneic, and 5 autologous). For patients receiving GM-CSF alone, neutrophil recovery (ANC > or = 500/microL) occurred between 2 and 61 days (median, 8 days) after therapy, while those receiving GM-CSF+G-CSF recovered at a similar rate of 1 to 36 days (median, 6 days; P = .39). Recovery to red blood cell (RBC) transfusion independence was slow, occurring 6 to 250 days (median, 35 days) after enrollment with no significant difference between the two treatment groups (GM-CSF: median, 30 days; GM-CSF+G- CSF; median, 42 days; P = .24). Similarly, platelet transfusion independence was delayed until 4 to 249 days (median, 32 days) after enrollment, with no difference between the two treatment groups (GM- CSF: median, 28 days; GM-CSF+G-CSF: median, 42 days; P = .38). Recovery times were not different between patients with unrelated donors and those with related donors or autologous transplant recipients. Survival at 100 days after enrollment was superior after treatment with GM-CSF alone. Only 1 of 23 patients treated with GM-CSF died versus 7 of 24 treated with GM-CSF+G-CSF who died 16 to 84 days (median, 38 days) after enrollment, yielding Kaplan-Meier 100-day survival estimates of 96% +/- 8% for GM-CSF versus 71% +/- 18% for GM-CSF+G-CSF (P = .026). These data suggest that sequential growth factor therapy with GM-CSF followed by G-CSF offers no advantage over GM-CSF alone in accelerating trilineage hematopoiesis or preventing lethal complications in patients with poor graft function after BMT.(ABSTRACT TRUNCATED AT 400 WORDS)     

Humoral immune function in pediatric patients treated with autologous bone marrow transplantation for B cell non-Hodgkin's lymphoma. The influence of ex vivo marrow decontamination with anti-Y 29/55 monoclonal antibody and complement

Elimination of neoplastic B cell populations from autologous bone marrow grafts also removes normal B lymphocytes. This is potentially hazardous for the reconstitution of the immune system in patients undergoing high-dose chemotherapy and total body irradiation followed by autologous marrow rescue. Five pediatric patients with B cell non- Hodgkin's lymphoma in first remission undergoing such a regimen were studied. They received bone marrow pretreated with anti-Y 29/55 monoclonal antibody and complement. B and T lymphocyte subpopulations reached normal levels within 6 months after autologous bone marrow transplantation (ABMT), and serum immunoglobulin levels became normal within 4 to 9 months. Vaccination with diphtheria and tetanus toxoid, trivalent poliomyelitis vaccine of the Salk type, and pneumococcal capsular antigens (38 to 54 months after transplantation) gave rise to specific antibody production. ABO isoagglutinins could be demonstrated in all patients. The response pattern was similar to that of patients who received unmanipulated autologous bone marrow. It is concluded that ex vivo anti-Y 29/55 depletion of the marrow graft does not induce relevant disturbances of humoral immune functions.     

New insight into the evolution of the vertebrate respiratory system and the discovery of unidirectional airflow in iguana lungs

The generally accepted framework for the evolution of a key feature of the avian respiratory system, unidirectional airflow, is that it is an adaptation for efficiency of gas exchange and expanded aerobic capacities, and therefore it has historically been viewed as important to the ability of birds to fly and to maintain an endothermic metabolism. This pattern of flow has been presumed to arise from specific features of the respiratory system, such as an enclosed intrapulmonary bronchus and parabronchi. Here we show unidirectional airflow in the green iguana, a lizard with a strikingly different natural history from that of birds and lacking these anatomical features. This discovery indicates a paradigm shift is needed. The selective drivers of the trait, its date of origin, and the fundamental aerodynamic mechanisms by which unidirectional flow arises must be reassessed to be congruent with the natural history of this lineage. Unidirectional flow may serve functions other than expanded aerobic capacity; it may have been present in the ancestral diapsid; and it can occur in structurally simple lungs.Energetically demanding forms of locomotion, such as powered flight, require a great capacity for gas exchange and selection for aerobic stamina may underlie many unique features of the avian respiratory system (1, 2). The avian respiratory system consists of highly vascularized lungs and avascular air sacs, which are membranous structures that effect ventilation and, in some species, extend between the muscles and even enter the bones (3). The topography of the conducting airways is complex; they form a circular system of tubes, analogous to the loop formed by the blood circulatory system in which arteries connect to veins through numerous small diameter vessels, the capillaries. Likewise, the avian conducting airways connect to each other through numerous tubules, the parabronchi, to form a circular path for respiratory gases (3). Gases flow through most of the parabronchi in the same direction during both inhalation and exhalation (unidirectional flow). This is due to the presence of aerodynamic valves (4–10). In contrast, the mammalian conducting airways arborize with the branch tips ending in blind sacs, there are no valves, and gases travel in the opposite direction along the conducting airways during expiration from the direction followed during inspiration (tidal flow). The presence of aerodynamic valves and unidirectional flow has generally been thought to be a highly derived feature found, among extant animals, only in birds and having evolved either in the crown group with flight or somewhere along the saurischian lineage leading to birds (11), perhaps as a mechanism to meet the high energetic demands of endothermy.The discovery of unidirectional flow in the lungs of alligators (12, 13) and the savannah monitor lizard (14) indicates that we do not understand the distribution of this phenomenon among different lineages of vertebrates and raises questions about its underlying value. It is possible that unidirectional flow evolved convergently in crocodilians and monitor lizards and serves to expand aerobic capacity. Although monitor lizards are ectotherms, their lifestyles are largely convergent with small predatory mammals (15) and they have high aerobic capacities compared with other lizards (16). In contrast, extant alligators have limited aerobic stamina (17) but their common ancestor with birds may have had a great aerobic capacity (18) or may have been endothermic (19, 20). Crocodilians and monitor lizards also share a suite of features of their pulmonary and cardiac anatomy that have been purported to give rise to, or coevolve with, birdlike patterns of flow. These features are: (i) a bronchus that has grown deep into the lung as a mesobronchium, (ii) partitioning of the respiratory system into a mechanical part that functions in ventilation and a gas-exchanging region, (iii) intercameral perforations, and (iv) separation of the heart into right and left sides (1, 21). Crocodilians and monitors are also derived in having evolved mechanisms to supplement costal ventilation while exercising (18, 22, 23). Thus, unidirectional flow in these lineages may be one of many derived traits underpinning exceptionally high rates of oxygen consumption during activity.It is also possible, however, that this pattern of flow evolved before the split of Diapsida into the Lepidosauromorpha (tuatara, lizards, snakes) and Archosauromorpha (crocodilians and birds) in an ectothermic ancestor lacking expanded aerobic capacities and living as long ago as the Permian Period. Unidirectional flow has been purported to serve ectotherms by harnessing the heart as a pump for air during periods of breath-holding (apnea) (12). Light can be shed on this pattern of evolution with observations of more squamates (snakes, lizards), which are the most diverse and largest (∼9,000 species) group of living reptiles (24).To test the hypothesis that unidirectional flow is present in squamates other than varanid lizards; to better understand anatomical features that give rise to these patterns of flow; and to gain insight into the underlying value of this pattern of flow, green iguanas (Iguana iguana) were studied. Green iguanas differ from monitors because they are herbivores and because they have structurally simple lungs that lack an enclosed intrapulmonary bronchus. Iguanas lack septation of the cardiac ventricle and have poor locomotor stamina. The poor stamina is due in part from an impairment during running in their blood and air circulatory systems (19, 25, 26).     

Dual phase hepatic CT: influence of scanning direction on liver attenuation

OBJECTIVE: We measured changes in hepatic attenuation during arterial and portal phase acquisition of hepatic CT in the craniocaudal and caudocranial directions. SUBJECTS AND METHODS: In 10 of 20 patients undergoing dual phase helical CT during staging for colorectal cancer, images in both phases were obtained in the craniocaudal direction. Ten patients underwent imaging in the caudocranial direction. Attenuation values in the aorta and in the peripheral and central liver regions of interest were measured on each slice. Central and peripheral liver attenuation was also measured in 10 additional patients undergoing unenhanced CT. RESULTS: Both peripheral and central regions of interest revealed progressively increasing attenuation during the arterial phase, irrespective of scanning direction. During the portal phase, hepatic attenuation was stable in the craniocaudal direction but decreased in the caudocranial direction (p < 0.05, Wilcoxon's signed rank sum test). Central hepatic attenuation was lower than peripheral attenuation in unenhanced livers and in enhanced livers during both phases of caudocranial acquisition. We determined no significant difference during the arterial phase of enhancement in the craniocaudal direction. CONCLUSION: The direction of acquisition does not influence sequential liver enhancement during the arterial phase. Craniocaudal acquisition produces more stable enhancement during the portal phase. Differences in attenuation between the central and peripheral areas of the liver are probably unrelated to contrast administration.     

Mesenteric artery disease in the elderly

PURPOSE: The purpose of this study was to estimate the population-based prevalence of mesenteric artery stenosis (MAS) and occlusion among independent elderly Americans. METHOD: As part of an ancillary investigation to the Cardiovascular Health Study (CHS), participants in the Forsyth County, NC cohort had visceral duplex sonography of the celiac arteries and superior mesenteric arteries (SMAs). Critical MAS was defined by celiac peak systolic velocity >or=2.0 m/s and/or SMA peak systolic velocity >or=2.7 m/s. Occlusion of either vessel was defined by lack of a Doppler-shifted signal within the imaged artery. Demographic data, blood pressures, and blood lipid levels were collected as part of the baseline CHS examination. Participants' weights were measured at baseline and before the duplex exam. Univariate tests of association were performed with two-way contingency tables, Student t tests, and Fisher exact tests. Multivariate associations were examined with logistic regression analysis. RESULTS: A total of 553 CHS participants had visceral duplex sonography technically adequate to define the presence or absence of MAS. The study group had a mean age of 77.2 +/- 4.9 years and comprised 63% women and 37% men. Participant race was 76% white and 23% African-American. Ninety-seven participants (17.5%) had MAS. There was no significant difference in age, race, gender, body mass index, blood pressure, cholesterol, or low-density lipoproteins for participants with or without MAS. Forward stepwise variable selection found renal artery stenosis (P =.008; odds ratio [OR], 2.85; 95% confidence interval [CI], 1.31, 6.21) and high-density lipoprotein >40 (P =.02; OR, 3.03; 95% CI, 1.17, 7.81) significantly associated with MAS in a multivariate logistic regression model. Eighty-three of the 97 participants with MAS (15.0% of the cohort) had isolated celiac stenosis. Seven participants (1.3% of the cohort) had combined celiac and SMA stenosis. Five participants (0.9% of the cohort) had isolated SMA stenosis. Two participants (0.4% of the cohort) had celiac occlusion. Considering all participants with MAS, there was no association with weight change. However, SMA stenosis and celiac occlusion demonstrated an independent association with annualized weight loss (P =.028; OR, 1.54; 95% CI, 1.05, 2.26) and with renal artery stenosis (P =.001; OR, 9.48; 95% CI, 2.62, 34.47). CONCLUSION: This investigation provides the first population-based estimate of the prevalence of MAS among independent elderly Americans. MAS existed in 17.5% of the study cohort. The majority had isolated celiac disease. SMA stenosis and celiac artery occlusion demonstrated a significant and independent association with weight loss and concurrent renal artery disease.