Impact of different variables on the outcome of patients with clinically confined prostate carcinoma: prediction of pathologic stage and biochemical failure using an artificial neural network.

BACKGROUND: The advent of advanced computing techniques has provided the opportunity to analyze clinical data using artificial intelligence techniques. This study was designed to determine whether a neural network could be developed using preoperative prognostic indicators to predict the pathologic stage and time of biochemical failure for patients who undergo radical prostatectomy. METHODS: The preoperative information included TNM stage, prostate size, prostate specific antigen (PSA) level, biopsy results (Gleason score and percentage of positive biopsy), as well as patient age. All 309 patients underwent radical prostatectomy at the University of Colorado Health Sciences Center. The data from all patients were used to train a multilayer perceptron artificial neural network. The failure rate was defined as a rise in the PSA level > 0.2 ng/mL. The biochemical failure rate in the data base used was 14.2%. Univariate and multivariate analyses were performed to validate the results. RESULTS: The neural network statistics for the validation set showed a sensitivity and specificity of 79% and 81%, respectively, for the prediction of pathologic stage with an overall accuracy of 80% compared with an overall accuracy of 67% using the multivariate regression analysis. The sensitivity and specificity for the prediction of failure were 67% and 85%, respectively, demonstrating a high confidence in predicting failure. The overall accuracy rates for the artificial neural network and the multivariate analysis were similar. CONCLUSIONS: Neural networks can offer a convenient vehicle for clinicians to assess the preoperative risk of disease progression for patients who are about to undergo radical prostatectomy. Continued investigation of this approach with larger data sets seems warranted.

Mature micromegakaryocytes: an unusual developmental pattern in term infants.

We sought to gain perspective on platelet production in the fetus and the newborn by counting and characterizing megakaryocytes from available cord blood. Elutriation was used to isolate circulating megakaryocytes from umbilical arteries and veins obtained at scheduled caesarean sections of nine normal term fetuses. Megakaryocytes were identified by established criteria, their diameters measured, and maturation stages recorded. Large numbers of megakaryocytes, mostly mature, were found in both the umbilical arteries and veins, many times more than previously observed circulating in adult blood. In term infants more than a third of the mature megakaryocytes had unusually decreased nuclear lobation and were dwarf cells with diameters as small as 13 microns, which we considered to be micromegakaryocytes. The atypicality of these small but mature cells is seen as merely a leftward skewing in the development of megakaryocyte ploidies. We believe that in normal fetuses the extent of megakaryocyte ploidization and development is distinctive and probably regulated differently to the adult pattern.

The response of cord blood megakaryocyte progenitors to IL-3, IL-6 and aplastic canine serum varies with gestational age.

Fetal cord blood (CB) is rich in haemopoietic stem cells and progenitors. We studied the clonogenic, proliferative and maturational responses of megakaryocyte (MK) progenitors in CB, from different gestational ages, to various cytokines: IL-3, IL-6, IL-3 + IL-6, and aplastic canine serum (PICS-J), and compared their responses to those of progenitors in adult peripheral blood (PB) or bone marrow (BM). We found that 34-week gestation CB produced some spontaneous colonies 28 +/- 4.7 CFU-MK in the absence of exogenous cytokines, and produced more CFU-MK and BFU-MK in response to IL-3, IL-6 and IL-3 + IL-6 than the other samples tested. Proliferation of CFU-MK was maximal at 34 weeks and decreased gradually toward term. When compared to adult BM or PB, the CB-derived CFU-MK had increased cellularity and contained significantly more cells undergoing fragmentation into platelet-like particles after stimulation with IL-3 or IL-6. Post-irradiation aplastic canine serum (PICS-J) was a highly potent stimulator of MK progenitors at all developmental stages. Our results indicate that CB MK progenitors are exquisitely sensitive to exogenous cytokines and that the magnitude of their proliferative and maturational responses to cytokines is related to developmental age.

Circulating megakaryocytes: delivery of large numbers of intact, mature megakaryocytes to the lungs.

To determine the locus of platelet production, we sought to determine if sufficient megakaryocytes reach the lungs in a state that could produce platelets. Elutriation was used to isolate megakaryocytes from blood reaching and leaving the lungs of 20 patients undergoing routine cardiac catheterizations. A mean of 5.0 intact megakaryocytes/ml were found in pulmonary artery blood, compared to only 0.5 megakaryocytes/ml, with partial cytoplasmic content, in aortic samples. The megakaryocytes in central venous and aortic samples were all mature. The identity of these cells as megakaryocytes, their maturity and normal morphology were confirmed by standard and immunoelectron microscopy. Cardiac outputs were obtained for each patient at the time of blood sampling, allowing an extrapolation that 40 x 10(6) intact, mature megakaryocytes were being delivered to the lungs every day in the average patient, compared to only 4.0 x 10(6) partially spent megakaryocytes exiting the lungs daily. About 98% of megakaryocyte cytoplasm reaching the lungs did not exit as recognizable megakaryocytes or fragments. The number and state of the megakaryocytes apparently filtered in the lungs is consistent with the hypothesis that megakaryocytes may shed platelets within the pulmonary microvasculature, which may be the primary site of platelet production.

Effect of bone marrow T lymphocytes treated with CAMPATH 1G on megakaryocyte colony formation.

Specific populations of lymphocytes play a significant role in the regulation of megakaryocyte (MK) development. CAMPATH 1G (C1G) and CAMPATH 1M (C1M) are T lymphocyte (TL)-specific monoclonal antibodies (MABs) that are routinely employed to reduce graft-vs.-host disease (GVHD) in allogeneic bone marrow transplantation (BMT). Following BMT, engraftment of the erythroid and myeloid lineages is prompt, but prolonged thrombocytopenia often prevails. We therefore studied the effect on MK colony formation of treating donor bone marrow (BM) with the CAMPATH MABs. MK colonies were grown in plasma clots using postirradiation aplastic canine serum (PICS-J) as MK colony-stimulating factor (MK-CSF). C1M, which causes TL destruction by complement-dependent lysis, had no effect on MK cloning efficiency. C1G is not lytic but causes the elimination of TL in the BMT recipients via antibody-dependent cell cytotoxicity (ADCC). Treatment of donor BM with C1G significantly enhanced the number of early burst-forming units (BFU-MK) and late colony-forming units (CFU-MK) and had no effect on granulocyte-macrophage (CFU-GM) or erythroid (BFU-E) colonies. Enhancement of MK cloning efficiency was concentration-dependent between 0.03 and 3 micrograms MAB/10(6) BM mononuclear cells (MNC). Similar results were observed when C1G-treated TL or purified CD4+ TL were co-cultured with untreated autologous BMMNC or peripheral blood (PB) MNC. Conditioned medium (CM) from C1G-treated TL and CD4+ TL contained soluble factors that, when combined with suboptimal doses of PICS-J, potentiated MK growth. C1G in combination with PICS-J also stimulated TL proliferation in a dose-dependent manner. The T cell CM did not contain elevated levels of interleukin-3 (IL-3), IL-6, IL-1 beta, or GM-CSF. Our data provide additional evidence for the involvement of activated TL, and perhaps novel soluble T cell products, in the immunomodulation of megakaryocytopoiesis.

Megakaryocytes and megakaryocyte progenitors in human cord blood.

Thrombocytopenia contributes significantly to morbidity in the sick term or preterm infant. However, few data exist on newborn's megakaryocytes and megakaryocyte progenitor cells (CFU-MK). We therefore studied CFU-MK in term and preterm infant cord blood and compared the results with data on CFU-MK from adult bone marrow and adult peripheral blood in a plasma clot culture with postirradiated aplastic canine serum (PIACS) as a source of megakaryocyte colony-stimulating activity. The number of CFU-MK and the number of cells per CFU-MK were counted with an immunofluorescent method at day 12. The effect of T-lymphocyte depletion on cord blood cultures for CFU-MK was studied with PIACS and a partially purified product of PIACS. We also studied individual megakaryocytes from newborns. The number and sizes of circulating megakaryocytes, isolated from adult peripheral blood and term venous cord blood by elutriation, were compared. Term and preterm cord blood contained more CFU-MK than adult peripheral blood. The numbers of CFU-MK in preterm cord blood were comparable to those in adult bone marrow. When the number of cells per colony were compared, cord blood contained significantly more cells than adult marrow CFU-MK. The depletion of T lymphocytes did not significantly change the growth of CFU-MK compared to nondepleted cultures. A substantial number of circulating megakaryocytes were obtained from venous cord blood, though they were significantly smaller than adult peripheral blood megakaryocytes. Since cord blood is easily obtained and contains large numbers of megakaryocytes and CFU-MK, it may provide a convenient model for studying the regulation of fetal megakaryocytopoiesis.

Familial thrombocytopenia with micromegakaryocytes.

Chronic thrombocytopenia was noted in two siblings and a first cousin. The initial impression was of immune thrombocytopenic purpura (ITP) with decreased megakaryocytes. One patient had splenectomy for presumed chronic ITP but showed no improvement. Bone marrow buffy coat slides were examined in the three children with thrombocytopenia, four normal controls, and five children with "classic" acute ITP. Megakaryocyte size, maturation, and ploidy were determined with Wright-Giemsa and Feulgen-stained material. Mean megakaryocyte diameters were 23.1 microns in the three related patients, 30.8 microns in normal controls, and 63.1 microns in children with "classic" acute ITP. Many "micromegakaryocytes" were noted in the three related children with chronic thrombocytopenia. An exhaustive family history was obtained, which showed multiple points of consanguinity. These patients represent an apparently new autosomal recessive disorder of megakaryocytopoiesis, characterized by disturbed megakaryocyte ploidization and maturation. More sensitive recognition of micromegakaryocytes should be attempted in children with atypical chronic thrombocytopenia, familial history of thrombocytopenia, or patients who have ITP and who have not responded to initial therapy.

The effect of flow on the interaction of isolated megakaryocytes with subendothelial extracellular matrix.

We have previously shown that human, guinea pig, or rat megakaryocytes, incubated under static conditions on an extracellular matrix (ECM) produced by endothelial cells, readily adhered to the matrix and underwent platelet-like shape change and thromboxane A2 secretion. We have now exposed megakaryocytes to ECM in a perfusion system similar to that used to study platelets circulated over aortic subendothelium. We used a continuous flow circuit incorporating a parallel plate perfusion chamber. Megakaryocytes were isolated to high purity from guinea pig marrow by centrifugal elutriation and velocity sedimentation. The cells were introduced into the flowing medium while the surface of an ECM-coated coverslip mounted in the chamber was observed continuously by phase-contrast video microscopy for up to 18 hours. Megakaryocytes from the flowing suspension started to adhere to the ECM within seconds. Significant adhesion occurred over a range of shear rates, from 10 to 190 seconds-1, did not appear above 300 seconds-1 and was greatest at a shear rate of 60 seconds-1. Adhesion to the ECM was specific, since there was no adherence to glass coverslips, glutaraldehyde-fixed ECM-coated coverslips, or to endothelial cells cultured on ECM-coated coverslips. At low shear rates large aggregates of megakaryocytes formed on the ECM surface; these could be detached and washed away by higher shear forces. Megakaryocytes thus acquire, even before platelet formation, an adhesive capacity similar to that of platelets. In addition, a significant fraction of the adherent megakaryocytes underwent elongation and pseudopod formation similar to that seen in marrow sinusoids.

Thrombocytopenia and absent radii syndrome: defective megakaryocytopoiesis-thrombocytopoiesis.

Thrombocytopenia and absent radii (TAR) syndrome is a congenital defect with osseous abnormalities and thrombocytopenia. It is inherited as an autosomal recessive trait, but the mechanism of thrombocytopenia in this disorder is not clear. We have had the opportunity to study the mechanism of thrombocytopenia in an infant with TAR syndrome. The infant had normal levels of thrombopoietin and megakaryocyte colony-stimulating activity in spite of marked thrombocytopenia. However, the megakaryocyte progenitor cells in the bone marrow produced abnormal colonies with increased numbers of megakaryocytes per colony and small megakaryocytes similar to the small megakaryocyte seen in vivo. These findings suggest that the TAR syndrome in this infant is due to a failure in the production of thrombopoietin or to an abnormal progenitor cell with a maturational defect.

Megakaryocyte function and dysfunction.

More than a hundred years have passed since platelets were recognized as cells and their haemostatic functions discovered. However, the process of platelet production is still not understood. The location, the mechanism and the regulation of thrombopoiesis remain elusive. Megakaryocytes are known to be the source of platelets. Investigations of megakaryocytes have revealed their normal functions and some of the abnormalities present in various diseases which affect platelets. In recent years, new techniques of cell isolation and tissue culture have been developed and have made possible advances in characterizing megakaryocyte precursors and differentiation. The primary function of megakaryocytes is to synthesize and assemble platelet components and organelles. Although debated for a long time, new data seems to indicate that the lung may be a central locus of platelet production. The new techniques for megakaryocyte investigations have barely begun to be of use in the study of abnormal platelet production in disease.

Elutriation for isolation of megakaryocytes.

Successful isolation of guinea pig megakaryocytes in large numbers was first achieved with a combination of techniques, taking sequential advantage of the low relative densities and large diameters of most megakaryocytes. Several laboratories have made minor improvements, but this approach retains the disadvantage of losing a significant fraction of the megakaryocyte population, the small immature ones. Counterflow centrifugal elutriation has been shown to eject cells from a chamber progressively, according to their sizes. Because almost all the megakaryocytes are bigger than the other marrow cells, the megakaryocytes can be retained while rejecting the contaminants. With this technology, yields of 1.4-2.0 x 10(6) megakaryocytes from one guinea pig are routine, recoveries have been 93%-94% of the input number of megakaryocytes, and final purities now average 72%. A split-specimen comparison with our previous method found elutriation to provide much greater yield and recovery with at least as great a purification as the density-velocity combination. This new technique was easily adapted to isolation of megakaryocytes in single aspirates from normal human marrow. Fifty-fold purification with near total recovery and a yield of 27,000 megakaryocytes per donor allows easy and reliable cytologic studies. Elutriation appears to be the current method of choice for isolation of megakaryocytes.

Genetics of resistance to the African trypanosomes. VI. Heredity of resistance and variable surface glycoprotein-specific immune responses.

The question of genetic linkage of parasite-specific immune responses to resistance to infection in experimental African trypanosomiasis was addressed. For this purpose, major histocompatibility complex-compatible resistant and susceptible inbred mouse strains and their F1 hybrid, F2 hybrid, and backcross offspring were infected with Trypanosoma brucei rhodesiense LouTat 1. Immunologic control of the first peak of parasitemia and survival times were the parameters measured. As we have reported previously (R. F. Levine and J. M. Mansfield, J. Immunol. 133:1564, 1984), B10.BR/SgSnJ mice are relatively resistant and controlled the growth of the infecting variant antigenic type (VAT) by mounting an antibody response to exposed epitopes of the variable surface glycoprotein (VSG). Fluctuating parasitemias resulting from sequential growth of different variable antigenic types occurred subsequently, and these mice died with a median survival time of 48 days. C3HeB/FeJ mice, relatively susceptible, did not control the infecting VAT and did not exhibit VSG-specific antibodies. These mice died with a median survival time of 22 days. The (B10.BR X C3H)F1 hybrids derived from crosses between resistant and susceptible mice all exhibited VSG-specific antibody responses and controlled the infecting VAT population. However, the median survival time of the F1 hybrids (24 days) was not significantly different from the survival time of the susceptible C3H parent. These findings demonstrate for the first time that antibody-mediated control of parasitemia is inherited as a dominant trait; that overall resistance, as measured by survival time, is inherited as a recessive trait (e.g., susceptibility is dominant); and that the two events segregate independently of one another. Further analyses of the inheritance of immunity and resistance (survival time) were made in which the F2 hybrid and backcross studies revealed that there are multiple genes controlling the VSG-specific antibody response as well as determining susceptibility. An extension of the present studies to a similar but non-major histocompatibility complex-mouse model system of resistance and susceptibility (C57BL/6J and C3H/HeJ mice, F1 hybrids, and 11 recombinant inbred B X H strains derived from them) was made in order to link the strain distribution patterns of known genetic markers with control of VSG-specific antibody responses or with control of susceptibility. Results of this study showed that resistance varied independently of the ability to control parasitemia with VSG-specific B cell responses.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of ethanol on thrombopoiesis.

Chronic ethanol abuse causes thrombocytopenia but the underlying mechanism is unknown. To determine the target cells involved, we examined the effects of the drug in vitro on both megakaryocyte progenitor cells (CFU-Meg) and isolated, maturing megakaryocytes. In the presence of ethanol concentrations of 0.05-2.0 g/dl, megakaryocyte colony formation by mouse CFU-Meg in soft agar was normal. At 5 g/dl ethanol, colony formation was reduced by 50%; with 7 g/dl ethanol, no megakaryocyte colonies were formed. Acetaldehyde did not inhibit colony formation unless very high concentrations (100 mg/dl) were employed. Isolated guinea-pig megakaryocytes can maintain their viability and incorporate 3H-leucine into TCA-precipitable protein for at least 24 h. Incubation of these maturing megakaryocytes with ethanol did not affect their viability, but at concentrations greater than 120 mg/dl ethanol progressively inhibited protein synthesis. At 0.5 g/dl ethanol, protein synthesis was decreased by 23% while viability was still 93% of control. Like CFU-Meg, maturing megakaryocytes were resistant to the toxic effects of acetaldehyde. To determine the in vivo correlates of these results, guinea-pigs were fed 5 g/dl ethanol in a liquid diet. By 11 d, when blood ethanol levels were 20-150 mg/dl, platelet counts in the animals were reduced by 17-29%, while the number of marrow megakaryocytes was unaltered. These data indicate that the site of action of ethanol in suppressing thrombopoiesis is at the level of the maturing megakaryocyte.

Adhesion, spreading and fragmentation of human megakaryocytes exposed to subendothelial extracellular matrix: a scanning electron microscopy study.

Platelet agonists and subendothelial extracellular matrix (ECM) induce morphological and biochemical changes in animal megakaryocytes, reminiscent of the response of platelets to the same substances. We have examined the behavior of human megakaryocytes exposed for up to 36 hours to the ECM produced by cultured bovine corneal endothelial cells. By phase contrast and scanning electron microscopy these megakaryocytes demonstrated non-reversible adherence and flattening with formation of long filopodia, thus confirming that human megakaryocytes acquire platelet functional capacities. In addition, megakaryocyte fragmentation into prospective platelets was apparently induced by the ECM. Up to 50% of the adherent megakaryocytes underwent spontaneous fragmentation into small particles which individually reacted like platelets on the ECM. The interaction of the megakaryocytes with the ECM was specific since no adherence, flattening or fragmentation occurred upon incubation of the megakaryocytes on regular tissue culture plastic or glutaraldehyde fixed ECM. Thus we have demonstrated platelet like behaviour of human megakaryocytes in response to this physiological basement membrane and a possible role of the subendothelium in platelet production which may occur in vivo as megakaryocytes cross the sinusoid walls and enter the blood stream.

A non-programmatic approach to hemopoiesis.

The currently prevailing concept of hemopoietic differentiation is that the pathway starts at the multipotential stem cell and proceeds in a programmatic, relatively fixed fashion to the development of circulating blood cells. We have presented considerations supporting a more dynamic regulation. Self-renewal capacity within a compartment is seen as a measure of the cell's resistance to differentiation pressures. Mutable environmental factors, including feedback interactions, are proposed to regulate in each compartment the relative probabilities of renewal and maturation. We question the rigid distinction between multipotential and committed cells, generally believed to be separated by a discrete "determination" event. We prefer to see commitment as a manifestation of a gradually increasing bias for a given developmental fate. Such commitment might be modulated under different environmental conditions. Multipotency and stemness are two aspects of cellular resistance to maturation pressures.