Biomedical subjects
D Green
Publications and source records attributed to D Green.
Constitutive production and thrombin-induced release of vascular endothelial growth factor by human megakaryocytes and platelets.
We have shown that coculture of bone marrow microvascular endothelial cells with hematopoietic progenitor cells results in proliferation and differentiation of megakaryocytes. In these long-term cultures, bone marrow microvascular endothelial cell monolayers maintain their cellular integrity in the absence of exogenous endothelial growth factors. Because this interaction may involve paracrine secretion of cytokines, we evaluated megakaryocytic cells for secretion of cytokines, we evaluated megakaryocytic cells for secretion of vascular endothelial growth factor (VEGF). Megakaryocytes (CD41a+) were generated by ex vivo expansion of hematopoietic progenitor cells with kit-ligand and thrombopoietin for 10 days and further purified with immunomagnetic microbeads. Using reverse transcription-PCR, we showed that megakaryocytic cell lines (Dami, HEL) and purified megakaryocytes expressed mRNA of the three VEGF isoforms (121, 165, and 189 amino acids). Large quantities of VEGF (> 1 ng/10(6) cells/3 days) were detected in the supernatant of Dami cells, ex vivo-generated megakaryocytes, and CD41a+ cells isolated from bone marrow. The constitutive secretion of VEGF by CD41a+ cells was stimulated by growth factors of the megakaryocytic lineage (interleukin 3, thrombopoietin). Western blotting of heparin-Sepharose-enriched supernatant mainly detected the isoform VEGF165. In addition, immunohistochemistry showed intracytoplasmic VEGF in polyploid megakaryocytes. Thrombin stimulation of megakaryocytes and platelets resulted in rapid release of VEGF within 30 min. We conclude that human megakaryocytes produce and secrete VEGF in an inducible manner. Within the bone marrow microenvironment, VEGF secreted by megakaryocytes may contribute to the proliferation of endothelial cells. VEGF delivered to sites of vascular injury by activated platelets may initiate angiogenesis.
An association between the flat back and postpolio syndromes: a report of three cases.
The cases of three patients with a history of paralytic poliomyelitis in childhood who developed the flat back syndrome before or after spinal fusion for degenerative disease as adults were reviewed. The flat back syndrome, a combination of an inability to stand erect because of forward flexion of the trunk and pain in the low back and/or legs, typically occurs in the setting of decreased lumbar lordosis as a result of distraction instrumentation of the spine for scoliosis, vertebral fracture, or degenerative disease. Focus was placed on determining the factors responsible for the development and/or persistence of the flat back syndrome in these patients despite maintenance of, or partial operative restoration of, lumbar lordosis. Considering the essential role that the trunk extensor musculature plays in maintaining upright posture, it may be that a new onset of weakness (postpolio syndrome) in this musculature represents a major contributing factor to the flat back syndrome in these patients. Spine surgeons considering operative procedures in patients with a remote history of paralytic poliomyelitis should be aware of the possible increased risk of the flat back syndrome in this population of patients.
Evaluation of prenatal care information on birth certificates.
Misclassification frequently leads to bias in epidemiological studies, and causes concern for perinatal epidemiologists interested in using birth certificates as a data source. We used a maximum likelihood method to estimate the classification probabilities (conditional probabilities that indicate the probability of classification in a particular category, given the person's true category) of two data sources for a three-category outcome of prenatal care. The probability that women receiving adequate or inadequate care were correctly classified was estimated to be greater than 90%. The probability was much lower (< 35%) that women receiving intermediate care were correctly classified. The misclassification women from the intermediate category resulted in poor predictive values (< 70%) of women classified as receiving either adequate or inadequate care. Because of these findings, we combined the adequate and intermediate categories to form a two-category classification system. This revision resulted in higher positive predictive values (> 90%) with only a slightly lower classification probability (> 85%) for the combined category. We conclude that the degree of accuracy for a two-category classification of prenatal care based upon birth certificate information is acceptable, but we question the accuracy of indices of prenatal care with more than two categories.
Clear cell adenocarcinoma affecting the bladder, kidney and ureter--an unusual distribution.
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Posttraumatic feeding disorders.
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Photothermal sclerosis of leg veins.
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Factor VII, cholesterol, and triglycerides. The CARDIA Study. Coronary Artery Risk Development in Young Adults Study.
Cross-sectional studies have shown that factor VII coagulant activity (VIIc) is positively associated with plasma total cholesterol (TC), LDL cholesterol, and triglycerides (TG) as well as body mass index (BMI) and diastolic blood pressure. To determine whether changes in VIIc parallel changes in coronary risk factors over a period of 2 years, we examined data from 1514 participants in the Coronary Artery Risk Development in Young Adults Study (CARDIA), an ongoing investigation of lifestyles and evolution of cardiovascular risk factors. Subjects were 23 to 35 years old at the year 5 examination. Cross-sectional analyses at these examinations showed that VIIc was positively correlated (P < .001) with TC and TG in all race/sex groups except for TC in black women at the year 5 examination. Changes in VIIc over the 2-year period were correlated positively with changes in TC in all except black men and TG in all groups; the association of VIIc change with change in TC and TG was reduced only slightly with adjustment for age and BMI at year 5 and 2-year change in BMI. To determine whether the higher levels of VIIc in subjects with higher lipid values were due to activation of the factor or to an increase in the concentration of the factor VII clotting protein, we measured factor VII antigen (VIIag) in a randomly selected subsample of 223 subjects at the year 7 examination. In all sex/race groups, VIIag correlated with VIIc (r = .69 to 0.81). After adjustment for sex and race, the partial correlation coefficient between TG and VIIc was .28 (P = .0001); between TG and VIIag, .35 (P = .0001); between TC and VIIc, .39 (P = .0001); and between TC and VIIag, 0.43 (P = .0001). No associations were observed between lipid levels and the ratio of VIIc to VIIag. We conclude that the raised VIIc with higher lipid levels occurs in blacks as well as whites, in men and women, persists over time, and represents a true increase in the plasma concentration of this clotting factor.
Distribution, diversity, and host specificity of Bartonella in rodents from the Southeastern United States.
A number of Bartonella isolates were obtained from seven species of rodents sampled from 12 geographic sites representing the major biotic communities of the southeastern United States. Bartonella were isolated from the blood of 42.2% of 279 tested rodents. The highest prevalence of infection typically occurred among the most commonly captured species in the rodent community. Four phylogenetic groups, uniting 14 genotypic variants of Bartonella, were identified by sequence analysis of the citrate synthase gene. The level of sequence homology between genotypic groups varied from 88.8% to 96.4%, and the degree of homology among variants within groups was > or = 97%. Cotton rats (Sigmodon hispidus) harbored up to three phylogenetic groups of Bartonella at a single site, and Bartonella of two phylogenetic groups were isolated from a single rodent. All the Bartonella isolated from three species of Peromyscus clustered in a single distinct phylogenetic group, suggesting some host specificity may occur. Mouse ascitic fluids produced in BALB/c mice inoculated with Bartonella of three phylogenetic groups demonstrated high indirect fluorescent antibody (IFA) titers to homologous antigens. However, use of eight Bartonella antigens in an IFA test with sera from 394 wild-caught rodents resulted in either little or extremely low titers of antibody.
Unusual but memorable. Pancreatic arthropthy.
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Reduce the stress of calling the physician.
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Advance directives in Utah. Information from death certificates and informants.
BACKGROUND: Advance directives have been studied in different patient populations and institutions. Most reports have shown limited use and little medically observable effect. To our knowledge, no previous study has focused on the use of advance directives by individuals who have died or how their family members perceived the documents' effect. METHODS: We contacted informants listed on Utah Death Certificates from 1992 to estimate the prevalence and effect of advance directives. Eighty-two percent of 1398 informants we contacted agreed to our telephone interview. RESULTS: More than 50% of decedents reportedly completed an advance directive. Individuals older than 65 years (57.3%), women (58.1%), nursing home residents (63.4%), and hospice users (75.2%) were most likely to have had advance directives. Education, religion, religiosity, and location had no effect on prevalence. Most informants stated that advance directives had no effect on the decedent's care, but a minority felt they helped to limit treatment. Do-not-resuscitate orders were written more often for patients with advance directives. Feeding tubes were removed more often from decedents with living wills than from other decedents. Mechanical ventilatory support was not less frequent in patients with advance directives. CONCLUSIONS: Our study confirms others that found little evidence that advance directives affect life-sustaining treatments. In the infrequent situations when they apply, they may be more persuasive than family members in convincing physicians to limit treatment. We observed that survivors had 2 perceptions about advance directives, not emphasized in previous reports, that they seemed to limit treatment and to ease their burden of decision making.
Acid sphingomyelinase-deficient human lymphoblasts and mice are defective in radiation-induced apoptosis.
Stress is believed to activate sphingomyelinase to generate ceramide, which serves as a second messenger in initiating the apoptotic response. Conclusive evidence for this paradigm, however, is lacking. In the present study, we used a genetic approach to address this issue directly. We show that lymphoblasts from Niemann-Pick patients, which have an inherited deficiency of acid sphingomyelinase activity, fail to respond to ionizing radiation with ceramide generation and apoptosis. These abnormalities are reversible up on restoration of acid sphingomyelinase activity by retroviral transfer of human acid sphingomyelinase cDNA. Acid sphingomyelinase knockout mice also expressed defects in radiation-induced ceramide generation and apoptosis in vivo. Comparison with p53 knockout mice revealed that acid sphingomyelinase-mediated apoptosis and p53-mediated apoptosis are likely distinct and independent. These genetic models provide definitive evidence for the involvement of acid sphingomyelinase in one form of stress-induced apoptosis.
A prospective study on the reliability and cost effectiveness of preoperative ultrasound screening of the "marginal" liver donor.
With the growing shortage of available liver donors, many donors with risk factors that would have traditionally precluded liver procurement are now being considered. In this prospective study, we evaluated 50 "marginal" liver donors with pre-procurement abdominal ultrasounds and correlated results with findings at procurement and with subsequent allograft function. The results show that the ultrasounds have a specificity of 96% and a sensitivity of 68% in predicting abnormalities in donor livers that precluded transplantation. In addition, using ultrasound to screen marginal donors would result in significant savings in manpower and hospital resource utilization without "missing" any normal liver organs. Our results also show that, when properly selected, livers from donors with one or more high-risk factors function well with acceptable primary nonfunction rates.
Wild type and mutant human heart (R)-3-hydroxybutyrate dehydrogenase expressed in insect cells.
(R)-3-Hydroxybutyrate dehydrogenase (BDH) is a lipid-requiring mitochondrial enzyme with a specific requirement of phosphatidylcholine (PC) for function. PC is an allosteric activator that enhances NAD(H) binding to BDH. The enzyme serves as a paradigm to study specific lipid-protein interactions in membranes. Analysis of the primary sequence of BDH, as determined by molecular cloning, predicts that lipid binding and substrate specificity are contributed by the C-terminal third of the protein [Marks, A. R., McIntyre, J. O., Duncan, T. M., Erdjument-Bromage, H., Tempst, P., & Fleischer, S. (1992) J. Biol. Chem. 267, 15459-15463]. The mature form of human heart BDH has now been expressed in catalytically active form in insect cells (Sf9, Spodoptera frugiperda) transfected with BDH-cDNA in baculovirus. Endogenous PC in the insect cells fulfills the lipid requirement for the expressed BDH since enzymatic activity is lost upon digestion with phospholipase A2 and restored selectively by reconstitution with PC vesicles. The K(m)s for NAD+ and (R)-3-hydroxybutyrate (R-HOB) of expressed BDH are similar to those for bovine heart or rat liver BDH in mitochondria. Replacing Cys242 (the only cysteine in the C-terminal domain) with serine by site-directed mutagenesis resulted in a 10-fold increase in K(m) for R-HOB with no change in the K(m) for NAD+, indicating a role for Cys242 in substrate binding. Carboxypeptidase cleavage studies had indicated a requirement of the C-terminal for catalysis and a role in lipid binding [Adami, P., Duncan, T. M., McIntyre, J. O., Carter, C. E., Fu, C., Melin, M., Latruffe, N., & Fleischer, S. (1993) Biochem J. 292, 863-872]. We now show that deletion of twelve C-terminal amino acids to form a truncated BDH mutant results in loss of enzymic function. The expression in Sf 9 cells of the constitutively active full-length mature form of human heart BDH and the first expression and characterization of BDH mutants validate this system for structure-function studies of BDH.
Thrombopoietin-stimulated ex vivo expansion of human bone marrow megakaryocytes.
The effect of thrombopoietin (TPO) on megakaryocytopoiesis (MKP) has been mainly studied using clonogenic assays in murine systems. In this study, we evaluated MKP in liquid culture using human bone marrow cells. While interleukin 3 (IL-3) and stem cell factor (SCF) are potent activators of TPO-stimulated MKP in the murine system, only IL-3 exhibited synergistic activity with TPO in cultures of human bone marrow. The IL-3 effect on TPO-stimulated megakaryocyte (MK) proliferation, expressed as the absolute number of MKs per seeded CD34+ cell, was more pronounced with purified CD34+ cell (8 +/- 1.6 SE versus 2.8 +/- 0.7 SE in the presence and absence of IL-3, respectively) than with mononuclear cells (MNC) (16 +/- 2.8 SE versus 11 +/- 2.0 SE). This effect of IL-3 on TPO-stimulated MK proliferation was due to a general proliferation of all cell types since the relative frequency of MKs (32.1 +/- 3 SE and 55.8 +/- 3 SE in MNC and CD34+ cells, respectively) was not affected by IL-3. The effect of TPO alone, TPO + IL-3, TPO + SCF, and TPO + IL-3 + SCF on MK proliferation was examined in MNC and CD34+ cultures. Greater numbers of MK per seeded CD34+ were observed in MNC compared to CD34+ cultures under all conditions except when TPO was added with both IL-3 and SCF. The enhancing effect of MNC was also observed on MK ploidy in the presence of TPO and IL-3. While proliferation and ploidy increase with TPO concentration in the murine system, they are inversely related in the human system. A significant 2.5-fold enhancement of TPO-induced MK proliferation was observed when purified CD34+ cells were cultured in inserts separated from human bone marrow stroma, indicating that soluble stimulatory factors are released from the stroma. These observations will be useful for ex vivo expansion of MKs to treat post-transplant or chemotherapy-associated thrombocytopenia.
Comparison of bone densitometry of the phalanges, distal forearm and axial skeleton in early postmenopausal women participating in the EPIC Study.
We present baseline bone densitometry from the Early Postmenopausal Interventional Cohort study (EPIC, sponsored by Merck, Sharp & Dohme) for the first time, in which 1609 women from England, Oregon, Hawaii and Denmark are participating to investigate the efficacy of daily oral alendronate to prevent early postmenopausal bone loss. We compared radiographic absorptiometry (RA) of the phalanges for bone mineral density (BMD) measurement with single-energy X-ray absorptiometry (SXA) of the distal forearm, and dual-energy X-ray absorptiometry (DXA) of the lumbar spine, proximal femur and distal forearm. In a random subgroup of 308 women, aged 45-60 years, on average 6 years since menopause (YSM), bone densitometry was measured once at baseline by RA of the phalanges besides the mandatory measurements by DXA. Bone densitometry was furthermore measured by SXA at the Danish site (89 women). Sixty-eight of the women had duplicate measurements performed within 1-3 weeks to evaluate the short-term precision error (CV%). One hundred and one healthy premenopausal women, aged 25-48 years, were recruited at the Danish and Hawaiian sites to establish a reference group. The precision error was 1.5% for RA of the phalanges and in the range 1.0-2.2% for SXA and DXA. BMD by RA correlated with BMD measured by SXA and DXA in the range 0.45 < r < 0.72 (p < 0.001). In conclusion, bone densitometry by RA of the phalanges is highly correlated with bone densitometry by SXA and DXA. RA of the phalanges has a short-term precision error comparable to that of SXA and DXA.
Humidification reduces coughing and breath-holding during inhalation induction with isoflurane in children.
PURPOSE: Inhalation induction using isoflurane is associated with airway irritability, coughing, breath-holding and laryngospasm. These complications are more common in children. This study was designed to determine if humidification of isoflurane in oxygen/nitrous oxide would reduce respiratory complications and hypoxic episodes at induction. METHODS: Fifty-nine unpremedicated children, aged three months to 12 yr, were enrolled in the study and randomised to receive either humidified isoflurane (n = 27, Group A), or non-humidified isoflurane (n = 32, Group B). All inductions of anaesthesia were with isoflurane 4% in 50% oxygen/nitrous oxide. Subjects were observed for the occurrence of breath-holding, coughing, laryngospasm, bronchospasm, secretions, and hiccoughs. The severity of each complication was graded on a scale of 0-3. The need to administer 100% oxygen and/or succinylcholine was also identified. RESULTS: Coughing (33% vs 53%) was more frequent in Group B (P < 0.05). Coughing severity scores (13 vs 36) and breath-holding severity scores (8 vs 19) were also greater in Group B (P < 0.05). A change in FIO2 was required more frequently in Group B (4% vs 16%). Although there was a high incidence of laryngospasm in both groups (52% vs 59%), no other differences were identified, breath-holding (26% vs 31%), secretions (30% vs 31%), hiccough (11% vs 12.5%) (P > 0.05). CONCLUSION: Humidification of inspired isoflurane reduces the frequency and severity of coughing, the severity of breath-holding, and the need to increase supplemental inspired oxygen concentration, when isoflurane is used for inhalation induction of anaesthesia in children. Humidification has no effect, however, on the frequency and severity of laryngospasm, or on the frequency of occurrence of arterial oxygen desaturation.