Biomedical subjects
Publications and source records attributed to U Pfeifer.
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4.5 months after successful kidney transplantation a Non-Hodgkin-lymphoma with polymorph centroblastic appearance of the tonsillar gland developed in a 21 years old male patient during immunosuppressive therapy with cyclosporine A and prednisone parallel to infection with Epstein Barr virus. Focal epileptic seizures occurred and were due to cerebral posttransplantation lymphomas as proven by brain biopsy. Reduction of immunosuppressive therapy led to complete remission as shown by CCT and MRI.
Cytoplasmic growth (hypertrophy) presupposes a positive metabolic balance brought about by increased anabolic and/or decreased catabolic processes. Degradation of cytoplasmic components takes place in autophagic vacuoles (AVs) whose volume fraction may be taken as a measure of the relative rate of degradation of cytoplasmic components. Male adult Sprague-Dawley rats (n = 80) were unilaterally nephrectomized (n = 40) or sham-operated (n = 40) and were killed 3.5-57.5 h p.o. The volume density of AVs in parenchymal cells of renal cortical convoluted tubules was determined morphometrically by systematic evaluation of large test fields in the electron microscope. During compensatory renal growth, the volume densities of autophagic vacuoles were reduced at day 0 (3.5-8 h p.o.), day 1 (20.5-33.5 h p.o.) and day 2 (44.5-57.5 h p.o.) by 49% (p less than 0.01), 43% (p less than 0.05), and 19% (n.s.), respectively, when compared with sham-operated controls. No decrease, and even an increase, in the AV-volume fraction was found in liver parenchymal cells of the unilaterally nephrectomized animals. This indicates that inhibition of autophagy is not a general response after unilateral nephrectomy, but is confined to the growing kidney, where it may represent a significant factor in the increase of cytoplasmic mass.
In an effort to investigate the functional relationship between cell-specific work and intracellular degradative processes, the effect of furosemide on cellular autophagy was investigated in two different portions of the nephron, namely, the thick ascending limb of Henle's loop (TAL), which is a main target of this drug, and the proximal convoluted tubule (PCT) as a reference structure. Eight male adult rats were treated with furosemide (60 mg/kg body weight, s.c.). Eight control animals received physiological saline. 1 to 4 h after the injections the animals were killed by perfusion fixation. Small specimens of kidney tissue from the inner stripe of the outer medulla and from the outer cortex were processed for electron microscopy; they were investigated morphometrically for volume fraction and numerical density of autophagic vacuoles (AVs). A significant increase of both parameters (volume fraction: 0.42 x 10(-4) to 1.09 x 10(-4); numerical density: 4.2 x 10(5)/mm3 to 15.5 x 10(5)/mm3) was seen under the influence of furosemide in TAL cells, whereas PCT cells did not show a significant increase in volume fraction or any increase in numerical density of AVs. These data suggest that the functional unloading of TAL, via blocking of the Na+-2Cl- -K+ co-transport by furosemide, results in adaptative "structural unloading", i.e., an increased sequestration of cytoplasmic components into AVs, within a short-time interval.
Endomyocardial biopsy in this study of 1250 biopsied patients (mean of five samples/patient) proved to be a remarkably safe technique with no lethal complications. It may help to detect the underlying cause of heart failure but is handicapped by sampling error in focal disease processes (such as myocarditis and sarcoid heart disease) when conventional light and electron microscopy are used. In this biopsy series 123 patients (9.8%) suffered from severe heart failure; lymphocytic infiltrates were found in only 10 (8%). Immunohistological data suggested a secondary humoral immunopathogenesis in all patients with myocarditis and perimyocarditis, in 75% of patients with postmyocarditic heart muscle disease and in 48% of patients with primary dilated cardiomyopathy. There may thus be a need for a new classification of heart muscle diseases that includes immunological parameters of humoral and cellular autoreactivity.
Yoshida AH-130 ascites hepatoma cells were grown in rats and were examined by quantitative electron microscopy 4 days (exponential growth phase) and 10 days (stationary phase) after intraperitoneal inoculation. No significant differences between growing and growth-inhibited tumors were found in the composition of the cytoplasm, except for a slight increase in the cytoplasm, except for a slight increase in the volume fraction of mitochondria (from 9.6 to 12.1%) and, in particular, a prominent (4.2-fold) increase in the volume fraction of early stage of autophagic vacuoles (from 0.31 x 10(-4) at day 4 to 1.37 x 10(-4) at day 10; P less than 0.001). At the same time, the rate of cell protein degradation was increased twofold, namely from 0.67%/h at day 4 to 1.37%/h at day 10, as measured in vitro after labeling cells with 3H-leucine in vivo. Such elevated proteolytic activity was entirely suppressed by ammonia, which inhibits the lysosomal pathway for protein degradation. The data show that: (i) the regulation of autophagic degradation of cytoplasmic constituents depending on the growth state was maintained in these tumor cells, and (ii) the increase in autophagy measured by morphometric analysis contributed to, yet did not quantitatively explain, the acceleration of protein degradation characterizing the transition from the logarithmic growth phase to the growth-inhibited state.
Autophagic vacuoles (AVs) in adrenal parenchymal cells of the outer part of the zona fasciculata were investigated by means of electron microscopic morphometry in 40 adult male Sprague Dawley rats. Four test groups and four control groups received twice daily subcutaneous injections of ACTH (12.5 U/kg body weight) or physiological saline, respectively, for 3, 6, 9 and 12 days. At 12 days the total dry weight of the adrenals was increased in ACTH-treated animals from 17.2 to 71.0 mg (about fourfold) and the average volume of zona fasciculata cells from 1,500 to 5,100 microns3 (about threefold). The relatively larger increase in total mass reflects mitotic activity in the growing adrenal tissue. The calculated growth rate of total tissue expressed as the percentage increase in dry weight per day was 12% at day 3 and decreased to 4.6% at day 12 of ACTH stimulation. During the time interval investigated the cytoplasmic volume fraction of autophagic vacuoles was significantly reduced from 10 X 10(-5) in controls to 1 X 10(-5) in ACTH-treated animals (p less than 0.001). Assuming a half-life for AVs of 7.9 min (Papadopoulos and Pfeifer 1986), autophagic degradation of cytoplasm can be calculated as 1.3%/day in controls, but only 0.15%/day in ACTH-treated animals. Thus growth of 1.15%/day can be attributed to inhibition of autophagic degradation. Although this is only part of total growth, it is evident from the present results that anticatabolic growth induction is utilized nearly to its maximum in adrenocortical fasciculata cells under the influence of ACTH.
The present study examines the short-term (i.e. 10 min after injection) influence of isoproterenol on cellular autophagy in the rat submandibular gland. The volume fraction of autophagic vacuoles was significantly reduced, suggesting that an anticatabolic reaction, namely inhibition of cellular autophagy, is an early and significant event in the growth of the submandibular gland which is known to occur with long-term isoproterenol treatment.
The relationship between growth, protein degradation, and cellular autophagy was tested in growing and in growth-inhibited 3T3 cell monolayers. For the biochemical evaluation of DNA and protein metabolism, growth-inhibited 3T3 cell monolayers with high cell density and growing 3T3 cell monolayers with low cell density were labeled simultaneously with [14C]thymidine and [3H]leucine. The evaluation of the DNA turnover and additional [3H]thymidine autoradiography showed that 24 to 5% of 3T3 cells continue to replicate even in the growth-inhibited state, where no accumulation of protein and DNA can be observed. Cell loss, therefore, has to be assumed to compensate for the ongoing cell proliferation. When the data of protein turnover were corrected for cell loss, it was found that the rate constant of protein synthesis in nongrowing monolayers was reduced to half the value found in growing monolayers. Simultaneously, the rate constant of protein degradation in nongrowing monolayers was increased to about 1.5-fold the value of growing monolayers. In parallel to the increased rate constant of protein degradation, the cytoplasmic volume fraction of early autophagic vacuoles (AVs) as determined by electron microscopic morphometry was found to be increased twofold in nongrowing 3T3 cell monolayers when compared with the volume fraction of early AVs in growing 3T3 cell monolayers. These data are in agreement with the assumption that cellular autophagy represents a major pathway of regulating protein degradation in 3T3 cells and that the regulation of autophagic protein degradation is of relevance for the transition from a growing to a nongrowing state.
The influence of propranolol and verapamil, i.e. two cardiodepressant drugs differing in their pharmacological actions, on cellular autophagy in the left ventricular myocardium of the rat was investigated. In the first experimental series 10 animals were given propranolol subcutaneously (3 mg/kg body weight). Ten controls received physiological saline. In the second series 8 animals were treated with verapamil subcutaneously (9 mg/kg body weight) and 8 controls with physiological saline. Two to 4 h after the injections in the first series and 1 3/4 to 3 h in the second series retrograde perfusion fixation was carried out via the abdominal aorta with a paraformaldehyde-glutaraldehyde mixture. Myocardial tissue from the left anterior wall was processed for electron microscopy and was morphometrically evaluated for volume fraction and numerical density of early stages of autophagic vacuoles (AVs). Propranolol and verapamil significantly increased the AV volume fraction 4.3- and 2.7-fold, respectively. The numerical density was increased by the two drugs, although to a lesser degree (2.3-fold, and 1.7-fold, respectively). As verapamil affects neither the beta-adrenoreceptors nor the intracellular levels of the second messenger cAMP, the only common denominator for the stimulation of cellular autophagy seems to be the cardiodepressive effect of the two drugs. The data suggest that the rise in cellular autophagy is an early regulatory step in the adaptation of heart muscle mass to reduced work load.
It is shown in the present study by means of quantitative electron microscopy that the volume fraction of autophagic vacuoles is significantly reduced to about half the control value within a short time interval (about 10 min) after low dose administration of isoproterenol. The data suggest that stimulation of cardiac work is accompanied by an immediate anticatabolic reaction which can be assumed to represent an early contribution to the adaptative increase in myocardial mass known to result from long-term stimulation of cardiac work.
Ultrastructural evidence is presented that intracellular autophagic degradation of cytoplasmic constituents is reduced during pressure induced hypertrophy of left ventricular myocardium after supravalvular aortic constriction in rats. This anti-catabolic reaction has to be considered as an important mechanism for shifting the balance between synthesis and degradation to the positive side. Short term studies after administration of isoproterenol suggest a close functional relationship between work load on the one hand and the anti-catabolic reaction on the other.
Defined areas of the intact liver surface of male Sprague-Dawley rats were exposed by means of small plastic rings to cycloheximide (1.8 X 10(-6) M to 1.8 X 10(-2) M). The incorporation of systemically applied [3H]leucine in a subcapsular tissue layer of 40 in micron thickness, was completely abolished at 1.8 X 10(-2) M and was reduced to 20 to 30% by concentrations between 1.8 X 10(-4) M and 1.8 X 10(-6) M cycloheximide as assessed by autoradiography. After fixation in situ with OsO4 this tissue layer was used for electron microscopy. Early autophagic vacuoles (AVs) whose contents could clearly be identified as being derived from cytoplasm were evaluated morphometrically. Their volume fraction in hepatocyte cytoplasm was reduced by cycloheximide within 10 minutes to about 50% of control tissue exposed to Ringer solution only. Maximal reduction was already observed with concentrations as low as 1.8 X 10(-5) M. When exposing the test areas to 1.8 X 10(-4) M cycloheximide for time intervals of 10, 20, 30, and 60 minutes, an exponential decrease in the AV volume fraction was seen in each individual animal over 20 minutes without further decrease between 30 and 60 minutes. The half-life of AV contents which could be calculated from this exponential decrease, was different for the different AV types, as distinguished according to their contents. Glycogen sequestered within AVs showed the largest morphological half-life of about 45 minutes, whereas, the half-life of the remaining components of AV contents, i.e., mitochondria, microbodies and endoplasmic reticulum together with cytoplasmic ground substance, was 10.1, 5.7, and 5.2 minutes, respectively. The average half-life of total AVs excluding those containing glycogen was 7.9 minutes. (first order rate constant: 0.088 min-1). The coefficient of variation (s/x), as calculated from single animal values, was lower for AV half-life (0.21) than for control AV volume fraction (0.67).
Left ventricular biopsies from 376 patients (including 78 patients undergoing bypass surgery) were analyzed by light microscopy (necrosis, infiltration with or without fibrosis) and by immunohistology (bound antibodies). Circulating antisarcolemmal antibodies (ASA) were determined at the time of biopsy using a double-sandwich technique. Circulating antimyolemmal antibodies were assessed in intact rat and human cardiocytes. Histologic findings, heart catheterization, and echocardiography together with the patient's history established the diagnosis of perimyocarditis, myocarditis, postmyocarditic dilated cardiomyopathy, healed myocarditis, and healed perimyocarditis. Both bound and circulating ASA were found in up to 100% of cases in acute inflammatory heart disease and postmyocarditic cardiomyopathy, indicating a secondary immunopathogenesis of the myocardial disease. Analysis of immunoglobulin subclasses revealed: IgG-binding does not discriminate between acute/healing/healed carditis and postmyocarditic dilated heart disease (61.1%-91.7% positive); IgM binding is diagnostic for acute or healing perimyocarditis but has a relatively low incidence (33.3%); IgA binding occurs in acute or healing myocarditis (45.5%), perimyocarditis (33.3%), and in postmyocarditic heart disease (39.4%), but not in controls; complement fixation was never seen in controls, but was seen in acute myocarditis (45.4%), perimyocarditis (25%), and postmyocarditic heart disease (46%). Pretreatment of cryostat sections with collagenase to avoid "nonspecific" binding of antibodies to collagen considerably reduced the sensitivity but increased the specificity. Thus, endomyocardial biopsy proved a safe and valuable method for the further analysis of patients with carditis and myocardial disease of unknown origin.
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Three adult dialysis patients developed ascites after having received repeatedly the plasma substitute hydroxyethyl starch (HES 40/0.5). In two cases (total dose 180, and 330 g HES, respectively) the ascites was reversible after discontinuation of the HES administration. In the third case (total dose 915 g HES) the ascites could be controlled only by implantation of a Denver shunt. In this latter case it was shown by histological, electron microscopical, and biochemical findings that the ascites was caused by hepatic sinusoidal obstruction due to an extreme storage of HES in the sinusoidal lining cells. Additional storage was detected in hepatocytes, bile duct epithelia, endothelial cells, and fibroblasts in the portal tracts. Biochemically HES was found in liver tissue at a concentration of 4% (w/w). Although in renal impairment plasma clearance of HES is not significantly different from normal individuals, long-term administration of HES must be regarded inadvisable because of tissue storage which apparently is especially significant in this condition.
In six cases of medullary carcinoma of the thyroid rather characteristic findings were seen in cytological specimens obtained by aspiration or imprint. The roundish-polygonal or spindle-shaped cells were predominantly disseminated, but occasionally arranged in small groups. The nuclei were frequently found in an excentric position; binuclear cells were sometimes encountered. In addition, five of the six cases showed intranuclear cytoplasmic inclusions which were also investigated by electron microscopy in specimens of two cases. Such inclusions in aspiration biopsies of the thyroid have so far been regarded, as characteristic for papillary carcinoma. In smears as well as in histological sections amyloid and calcitonin could be demonstrated by congo red staining and by immunocytochemistry, respectively. The cytomorphology of the tumor cells, their pattern of arrangement, and the peculiar nuclear inclusions make up a combination of findings that may justify the diagnosis of medullary carcinoma in aspiration biopsies. In smears the diagnosis can already be confirmed when (immuno-) cytochemical methods are applied to smear preparations.