Biomedical subjects
M A Green
Publications and source records attributed to M A Green.
Subcellular distribution of tissue radiocopper following intravenous administration of 67Cu-labeled Cu-PTSM.
The subcellular distribution of radiocopper in the brain and liver of rats has been determined following i.v. administration of Cu-PTSM, pyruvaldehyde bis(N4-methylthiosemicarbazonato)copper(II), labeled with copper-67. Homogenized tissue samples were separated by differential centrifugation into four subcellular fractions: (I) cell membrane + nuclei; (II) mitochondria; (III) microsomes; and (IV) cell cytosol. Upon sacrifice at 10 min post-Cu-PTSM injection, brain fractions, I, II, III and IV contain 35 +/- 12, 11 +/- 3, 2.8 +/- 1.3 and 51 +/- 7% of brain activity, respectively (n = 4). In animals sacrificed 24 h post-injection the subcellular fractions of brain tissue show little change from the radiocopper distribution seen at 10 min post-injection, although the mitochondrial fraction may contain slightly more tracer and the cytosolic fraction slightly less (I, 40 +/- 10%; II, 18 +/- 5%; III, 3.4 +/- 1.5%; and IV, 38 +/- 5%; n = 5). Subcellular fractions I, II, III and IV of liver contain 25 +/- 5, 12 +/- 3, 17 +/- 4 and 46 +/- 6% of 67Cu tracer in animals sacrificed 10 min post-Cu-PTSM injection. An identical subcellular distribution of 67Cu, was found in the liver following i.v. administration of ionic radiocopper (as Cu-citrate). The liver and brain cytosolic fractions at 10 min post-injection were further separated by Sephadex column chromatography. In liver cytosol, three different radiocopper components with molecular weights of about 140,000, 41,000-46,000 and 10,000-16,000 Da were found. In the brain supernatant fraction, most of the radiocopper was bound to a single low molecular weight cytosolic component (14,000-16,000 Da).(ABSTRACT TRUNCATED AT 250 WORDS)
Increase in severity of graft versus host disease by cytomegalovirus.
An allogeneic transplant recipient developed severe graft versus host disease (GvHD) 48 days after transplantation that was concomitant with a cytomegalovirus (CMV) viraemia, from which she subsequently died. CMV infection was detected in blood by the polymerase chain reaction and later in tissue by immunohistochemical techniques. CMV should be considered in patients in whom GvHD does not respond to appropriate treatment, and this case suggests that herpes viruses may increase the severity of GvHD by synergistically enhancing the graft versus host reaction.
A study of cerebellar inositol 1,4,5-trisphosphate receptor following climbing and parallel fibre deafferentation.
We have examined the influence of climbing fibre and parallel fibre afferent inputs on the inositol 1,4,5-trisphosphate (InsP3) receptor in rat cerebellum. Lesions of the inferior olive-climbing fibre projections to Purkinje cells by 3-acetylpyridine (3-AP) significantly reduced the [3H]InsP3 binding density (-20%) with no apparent changes in the binding affinity 21 days after treatment. No further reduction in binding density was found in rats given a second dose 7 days after the initial injection. A significant reduction in the binding density was evident as early as 7 days post-lesion. However, 3-AP (0.5 mM) failed to inhibit [3H]InsP3 binding in vitro. Cerebellar granule cells were lesioned by two consecutive injections of methylazoxymethanol acetate at birth. In these 60-day-old granuloprival rats, the density and affinity of [3H]InsP3 binding sites in the cerebellum remained comparable to the controls. Since lesions of the climbing fibres increase Purkinje cell activity, we suggest that changes in InsP3 receptor density may reflect an adaptative response to the heightened Purkinje cell activity. In addition, the results also indicate that expression of the InsP3 receptor in the cerebellum is largely independent of the presence of granule cell-parallel fibre synaptic innervation onto the Purkinje cells.
A remote system for the synthesis of copper-62 labeled Cu(PTSM).
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Investigation of copper-PTSM as a PET tracer for tumor blood flow.
Copper-PTSM has been shown in previous studies to act as a fluid microsphere and to be useful in quantitating blood flow in brain, myocardium, and kidneys. In this study we have evaluated this agent as a PET tumor blood flow agent. 64Cu- or 67Cu-labeled Cu-PTSM was administered (i.v.) to Golden Syrian hamsters with colorectal carcinoma cell implants (GW39). One minute prior to sacrifice (10-60 min after Cu-PTSM was administered) 125I-iodoantipyrine (125I-IAP), an agent known to measure tumor blood flow, was administered intravenously by a 3-stage, 1 min ramp infusion. Following sacrifice, samples of tumor and brain were removed (within 40s) and the tumor and brain levels of Cu-PTSM and iodoantipyrine determined. Since the brain uptake of both Cu-PTSM and IAP is perfusion rate limited, the brain was used as a reference organ to normalize tumor levels of the two tracers. The plot of Cu-PTSM versus 125I-IAP tumor/brain ratios showed a good linear correlation (r value of 0.97), suggesting that Cu-PTSM could be used to quantify tumor blood flow. Since the mechanism of Cu-PTSM trapping is likely to be due to glutathione levels in the tissue, and because tumor tissue glutathione levels might vary, the temporal uptake of Cu-PTSM was investigated by PET imaging both the tumor-bearing hamsters and approximately 300 g Copenhagen rats bearing R3227 prostate tumors. The tumors were clearly visualized and the retained copper radioactivity in the tumor was constant over the 30 min imaging period.
The effects of glutathione depletion on the biodistribution of Cu(PTSM) in rats.
The tissue retention of radiocopper afforded by intravenous injection of the proposed blood flow imaging agent, 62Cu-labeled copper(II) pyruvaldehyde bis(N4-methylthiosemicarbazone) [Cu(PTSM)], is thought to result from reductive decomposition of the copper(II) complex by intracellular sulfhydryls (e.g. glutathione, GSH). To determine if the tissue uptake and retention of this tracer adequately measures perfusion in tissues containing altered GSH concentrations, the biodistribution of copper-67 labeled Cu(PTSM) was determined in GSH-depleted rats. Despite treatment to induce relatively large reductions in tissue GSH levels, it was found that only very small changes in the biodistribution of copper-labeled Cu(PTSM) occurred in the treated rats compared to untreated controls.
Immunopathology of early graft-versus-host disease--a prospective study of skin, rectum, and peripheral blood in allogeneic and autologous bone marrow transplant recipients.
The immunopathological appearances of skin and rectum in 64 autologous and allogeneic recipients were determined before and after bone marrow transplantation. Patients who developed acute graft-versus-host disease were biopsied as soon as a clinical diagnosis was made. At the same time peripheral blood samples were collected for comparative analysis. Immunohistological and morphometric techniques were employed using a panel of monoclonal antibodies to T lymphocytes and subsets, B lymphocytes, natural killer cells, macrophages, and Langerhans cells. A reduction in the CD4/CD8 ratio after BMT was seen in skin and rectal biopsies from both autologous and allogeneic recipients with or without GVHD. The same pattern was observed in blood samples taken at the same time. Langerhans cells were reduced in the skin in all patients after BMT, probably by the conditioning regimen. Only a few cells expressing activation or natural killer cell markers were present and there were no changes observed in the macrophage population. This study has provided no evidence to implicate either CD4- or CD8-positive T lymphocytes as the initiators of the cellular damage in acute GVHD. The distribution of lymphocyte subsets in the blood was similar to that in the tissues, suggesting that the tissue changes reflect the pattern of lymphocyte repopulation after BMT and may have little bearing on the pathogenesis of GVHD.
Subdural fat effusion complicating parenteral nutrition.
A preterm infant fed parenterally through a central venous catheter developed a subdural effusion containing fat emulsion. Subsequent postmortem examination failed to demonstrate any vascular abnormality that might have explained this rarely reported complication. Although retrograde flow of feeding solutions into cerebral veins seems a likely explanation, the exact mechanism remains uncertain.
In vivo comparison of copper blood-pool agents: potential radiopharmaceuticals for use with copper-62.
Two techniques for labeling of albumin with copper-67 (67Cu) and 62Cu were investigated; one using the native Cu(II) binding site of the protein and the other employing a bifunctional chelate, 6-bromoacetamidobenzyl-1,4,8,11-tetraazacyclotetradecane- N,N'N",N"'-tetraacetic acid (Br-benzyl-TETA or BAT), conjugated to the protein. Rat biodistribution experiments with 67Cu demonstrated retention of i.v. 67Cu-benzyl-TETA-albumin in the blood pool identical to co-injected 125I-albumin. By contrast, i.v. administration of either [67Cu]-Cu-acetate or [67Cu]-Cu-acetate pre-mixed with albumin results in relatively rapid clearance of blood-pool radioactivity as the tracer is excreted into the urine. The 62Cu-benzyl-TETA-albumin radiopharmaceutical was obtained in ca. 17% radiochemical yield (end of synthesis, without decay correction) following a procedure that can be completed in 15-18 min. In PET experiments with a baboon, myocardial blood volume images with 62Cu-benzyl-TETA-albumin were identical to those obtained with C15O. Use of the 62Cu-benzyl-TETA-albumin image for blood-pool subtraction of a 62Cu-PTSM myocardial perfusion image is illustrated. Copper-62-benzyl-TETA-HSA should be a useful, generator-produced radiotracer for the detection of the vascular pool at PET facilities without cyclotrons.
Brain cancer mortality at a manufacturer of aerospace electromechanical systems.
Standardized proportional mortality ratios and mortality odds ratios were calculated for 583 deaths between 1950 and 1986 among employees who had worked for at least 10 years at a facility manufacturing missile and aircraft guidance systems. There was a statistically significant excess of brain cancer proportional mortality (PMR = 16/3.8 = 4.2, p = .0001). Among hourly employees, 12 brain cancer deaths occurred for 2.7 expected (PMR = 4.4, p = .00005). The PMR for brain cancer increased from 1.8 (p = .45) among hourly workers with less than 20 years to 8.7 (p = .000003) in those with more than 20 years employment. Work in "clean rooms," where gyroscopes were assembled, was associated with the brain cancer excess but did not fully account for it. Among 105 deceased hourly women, all three brain cancer deaths occurred among gyro assemblers working in clean rooms, and the risk increased with duration in clean rooms. Although the proportion of brain cancer deaths among hourly men with clean-room experience was similar to that for women, only three of the seven male brain cancer deaths occurred in this group. The suspect agents include gyro fluids and chlorofluorocarbon solvents.
Acute toxicity and mutagenicity of the copper complex of pyruvaldehyde-bis (N-4-methylthiosemicarbazone), Cu-PTSM.
Cu-PTSM is a potential imaging agent for the heart and brain when labeled with either 64Cu or 62Cu. Unlabeled Cu-PTSM was evaluated for its acute toxicity and mutagenicity. Cu-PTSM had an i.v. LD50 of 26 mg kg-1 in the rat and 2 mg kg-1 in the rabbit. At necropsy, rats exhibited severely hemorrhagic lungs, histological findings of acute pulmonary congestion, hemorrhage and edema, and mild congestion in kidney, liver and brain. The rabbit displayed marked polymorphonuclear infiltration in alveoli, peribronchial and periarterial areas with marked macrophage hyperplasia, congestion and mild hemorrhage into alveolar spaces. No effects were found in kidney, liver, testes or brain. Administration of 2.16 micrograms kg-1 day-1 for 5 days per week for 2 weeks resulted in no changes in histopathology, hematology or clinical chemistry parameters. This daily dose is at least 300 times the diagnostic dose intended for use in man. Cu-PTSM was not mutagenic when tested in the absence of S9 supernatant, but elicited a weakly mutagenic response in the presence of S9. Since acute effects in the lung occur at doses approaching 300,000 times the diagnostic dose, it is highly unlikely that the clinical use of Cu-PTSM would result in any acute adverse effects.
Structure-activity relationships for metal-labeled blood flow tracers: comparison of keto aldehyde bis(thiosemicarbazonato)copper(II) derivatives.
Radiocopper-labeled pyruvaldehyde bis(N4-methylthiosemicarbazonato)copper(II), Cu[PTSM], is under investigation as a radiopharmaceutical for evaluation of regional blood flow in the brain, heart, and kidneys because it affords relatively high levels of radioactivity in these organs upon intravenous injection, followed by prolonged tissue retention of the radiolabel. To probe and differentiate the physicochemical properties that are critical for blood-brain barrier (BBB) penetration and tissue retention in complexes of this type, 17 67Cu-labeled copper(II) bis(thiosemicarbazone) derivatives of Cu[PTSM] have been prepared and characterized, focusing on the bis(thiosemicarbazone), bis(N4-methylthiosemicarbazone), bis(N4-dimethylthiosemicarbazone), and bis(N4-ethylthiosemicarbazone) derivatives of several alkylglyoxals (R(1) = Me, Et, n-Pr, i-Pr, n-Bu, or Me(EtO)CH) and phenylglyoxal. The compounds studied varied in lipophilicity from log P = 0.75 to log P = 3.5 (where P is the octanol/water partition coefficient). In rat biodistribution studies the N4-methylthiosemicarbazone (R(1)TSM) and N4-dimethylthiosemicarbazone (R(1)TSM2) complexes always show comparable cerebral uptake at 1 min postinjection (iv) for any given R(1) group, while the thiosemicarbazone (R(1)TS) complex always penetrates the BBB less efficiently. Comparison of the various Cu[R(1)TS] derivatives shows that their brain uptake does tend to increase with increasing lipophilicity over the range 0.75 less than log P less than 2.4, although it never reaches that of the N4-alkylated derivatives. The Cu[R(1)TS] and Cu[R(1)TSM] complexes are found to exhibit prolonged cerebral retention of activity, consistent with their known susceptibility to reductive decomposition by intracellular sulfhydryl groups, while the more inert Cu[R(1)TSM2] complexes clear from the brain relatively rapidly. Tracer clearance kinetics in the heart and kidney are similar to those observed for the brain with each of the tracers examined.
Effect of N-methyl-d,l-aspartate on luteinizing hormone secretion in ovariectomized ewes in the absence and presence of estradiol.
N-methyl-d,l-aspartate (NMA), a potent agonist of the neuroexcitatory amino acids aspartate and glutamate, stimulates release of luteinizing hormone (LH) in rats and nonhuman primates. The objective of the experiments described here was to determine the effect of NMA on LH secretion in ovariectomized ewes, in both the absence and presence of estradiol. In Experiment 1, blood samples were collected from 16 ewes every 12 min for 4 h. At Hour 2, ewes received i.v. injections of either 0, 6, 12, or 24 mg NMA/kg body weight dissolved in 0.9% saline (n = 4 per treatment). Mean LH concentrations were unaltered by any dose of NMA (p greater than 0.3). Immediately after completion of Experiment 1, each ewe received an s.c. Silastic implant designed to maintain circulating concentrations of estradiol of approximately 1 pg/ml. Three weeks later, Experiment 2 was conducted, using the same blood sampling regimen and doses of NMA as Experiment 1. The estradiol implants decreased serum LH concentrations in all animals. Treatment with saline failed to alter mean LH concentrations (p greater than 0.3). In contrast, 6, 12, and 24 mg NMA/kg body weight increased mean LH concentrations by 326% (p less than 0.03), 1125% (p less than 0.02), and 441% (p less than 0.0001), respectively. These results demonstrate that exogenous estradiol suppresses LH release in sheep in a manner antagonized by NMA.
Diphtheria with a difference--a rare Corynebacterium fatality with associated apoptotic cell death.
A case of infection with Corynebacterium ulcerans (C. ulcerans), resulted in the sudden death of a previously healthy 73-year-old woman. Death from Corynebacterium diphtheriae (C. diphtheriae) infection is well-documented. Fatalities following infection with C. ulcerans are unreported; this would appear to be the first documented death due to such infection.
Assessment of regional myocardial and renal blood flow with copper-PTSM and positron emission tomography.
We recently demonstrated in isolated, perfused hearts that radiolabeled pyruvaldehyde bis(N4-methylthiosemicarbazonato)copper(II) (Cu-PTSM) is well extracted throughout a range of conditions including ischemia, hypoxia, and hyperemia. Once extracted, binding of radioactivity by the isolated heart was essentially irreversible, giving this tracer microspherelike qualities. Because Cu-PTSM can be readily prepared with the generator-produced positron-emitting copper 62 and other gamma- or positron-emitting copper radionuclides, we evaluated its usefulness for measuring regional myocardial and renal blood flow in vivo in intact dogs at rest, after ischemia, or after coronary hyperemia was induced by intravenous administration of dipyridamole. After intravenous administration of radiolabeled Cu-PTSM, the tracer cleared rapidly from the blood. Myocardial uptake of single photon-emitting 67Cu-labeled Cu-PTSM was measured directly in myocardial samples 15 minutes after tracer administration, and it increased proportionally with blood flow throughout the flow range (estimated concomitantly with radiolabeled microspheres) of 0.0-6.0 ml/g/min (n = 340 samples from 17 dogs, r = 0.99, Ycopper radioactivity = 85Xmicrosphere flow -7 chi 2 + 17). Renal uptake of radiolabeled Cu-PTSM was also proportional to blood flow. Positron emission tomography was performed in four intact dogs after intravenous administration of 64Cu-labeled Cu-PTSM (19% positron decay, t1/2 = 12.8 hours). High-quality images of heart and kidney were obtained. Accordingly, radiolabeled Cu-PTSM should be a useful, generator-produced tracer for estimating regional myocardial and renal blood flow with positron emission tomography.
Pathological aspects of ricin toxicity in mammalian lymph node and spleen.
In an earlier study (Griffiths et al., 1987) we demonstrated that two toxic plant proteins, ricin and abrin bring about apoptotic rather than necrotic cell death in mammalian lymphoid tissues and intestine. Here we expand upon the previous study, and report further observations relating purely to ricin and its pathology in lymph node and spleen. Rats were injected with ricin and killed at time intervals, tissue being excised and examined by light or electron microscopy. As well as previously reported apoptotic changes in the lymph node, we observed sinusoidal haemorrhage accompanied by erythrophagocytosis and loss of normal structure. With the aid of lymphocyte typing, we noticed a complete relocation of T and B lymphocyte populations, with destruction of B cells. Follicular centres of the spleen exhibited similar pathology to that seen in lymph nodes, along with loss of lymphocytes in areas adjacent to the central artery of the peri-arteriolar lymphoid sheath (PALS).
Effects of free fatty acids on luteinizing hormone and growth hormone secretion in ovariectomized lambs.
The effects of FFA on circulating LH and GH concentrations in ovariectomized ewe lambs were investigated. Lambs (n = 14) were weaned at 2.5 months, ovariectomized at 6.5 months, and used at 8.5 months of age. From weaning until day 0 of the experiment, lambs were fed to maintain body weights (23 kg). On day 0, serum FFA concentrations and mean serum LH concentrations and number and amplitude of LH pulses, as assessed in blood samples collected every 12 min for 4 h, were 6.4 +/- 0.6 mg/100 ml, 0.57 +/- 0.08 ng/ml, 0.45 +/- 0.09 pulses/h, and 0.73 +/- 0.11 ng/ml, respectively. Double the maintenance feeding, beginning day 1, increased (P less than 0.01) body weights by 16% and LH pulse frequency by 82%, but had no effect (P greater than 0.1) on FFA concentrations, mean LH concentrations, or LH pulse amplitude by day 14. On day 14, lambs were infused with lipid (n = 9; 95.8 mg/min) or 0.9% saline solution (n = 5) for 8 h. Blood samples were collected at 12-min intervals for 12 h, beginning 4 h before infusions. FFA levels increased (P less than 0.01) in lipid-infused animals to 27.6 +/- 2.9 mg/100 ml by 4 h of infusion. Mean LH concentrations and LH pulse frequency and amplitude were unaffected (P greater than 0.1) by treatment. In contrast, mean GH concentrations and GH pulse frequency, which were similar (P greater than 0.1) between groups before infusion (14.0 +/- 0.8 ng/ml and 0.36 +/- 0.07 pulses/h, respectively) were decreased by FFA treatment by 51% (P less than 0.01) and 81% (P less than 0.006), respectively. GH pulse amplitude was highly variable and unaffected (P greater than 0.1) by treatment. In summary, elevated FFA levels appear to inhibit the release of GH, but not LH, in the ovariectomized ewe lamb.