Evidence of calcium phosphate depositions in stenotic arteriovenous fistulas.

This study investigates vascular samples from patients with and without end-stage renal disease (ESRD) to determine the occurrence of calcium depositions. Findings in stenotic arteriovenous (AV)-fistula veins were compared with those of nonstenotic AV-fistula veins, non-AV-fistula veins, and atherosclerotic vessels. Calcium and phosphorus content was measured by means of scanning electron microscopy and its built-in method of energy-dispersive spectrometry (EDS) X-ray analysis. We found calcium and phosphorus in samples from AV fistulas with stenotic areas with a calcium/phosphorus molar ratio of 1. Based on EDS analysis and crystal shape comparison, we conclude that calcium phosphate precipitations in stenotic AV fistulas are brushites with the composition CaHPO(4)*2H(2)O. This specific calcium phosphate deposition was found solely in stenotic AV fistulas, not in nonstenotic AV-fistula veins or non-AV-fistula veins regardless of whether the patient had ESRD. Moreover, this calcium phosphate deposition was different from calcium compounds found in atherosclerotic samples. Whether the precipitation of brushite is primarily involved in the development of vascular-access stenosis or represents a secondary consequence cannot be determined from the present study.

Morphologic conversion of Helicobacter pylori from spiral to coccoid form. Scanning (SEM) and transmission electron microscopy (TEM) suggest viability.

Helicobacter pylori is a pathogen associated with type B gastritis, peptic ulcer disease, gastric atrophy, and stomach cancer. H. pylori exists in two morphological forms, spirals and coccoids. The latter has been described as viable but non-cultivable. The role of the coccoid form in the pathogenesis of gastric disease is disputed. Some authors consider the coccoid form to be a degenerative or dead form of H. pylori, while others consider it a resting but still metabolically active form. This study reports the conversion from spiral to coccoid form ultrastructurally. Dense material is accumulated in the periplasmic space, the spiral bacteria bend and the outer membrane is separated from the inner cell wall layer. Remodeling of inner structures takes place, ending in the coccoid form of the bacteria with preserved light polyphosphate areas. Reduction of surface takes place by production of surface membrane vesicles, which later are squeezed off. The finding of preserved subcellular structures and intact double membranes in combination with degenerative forms suggests that some of the coccoids are viable. Scanning electron microscopy (SEM) demonstrates coccoid form of bacteria with slightly ruffled surfaces but no spiral forms.

Multiple-system organ damage resulting from prolonged hepatic inflow interruption.

BACKGROUND: It has been reported that patients undergoing major hepatectomy tolerated 90 and 127 minutes of continuous hepatic inflow interruption with no evidence of permanent damage to the liver. We questioned the safety and feasibility of the interruption beyond 90 minutes in normothermic human beings. We also postulated that, besides injury to the liver per se, extended continuous hepatic inflow interruption would cause extrahepatic multiple-system organ damage in subjects exposed to continuous hepatic inflow interruption for 90 or 120 minutes. DESIGN: Fifty Sprague-Dawley rats were divided into three groups. Group 1 served as controls that had only laparotomy. Group 2 underwent continuous hepatic inflow interruption for 90 minutes, and group 3 was subjected to continuous hepatic inflow interruption for 120 minutes. Scanning electron microscopy and transmission electron microscopy were used to evaluate ultrastructural alterations in the liver, lung, heart, and intestine. SETTING: Lund (Sweden) University Hospital and Top Cancer Institute, Lund. INTERVENTIONS: Intraoperative and postoperative infusion and blood transfusion were given in all experimental animals. MAIN OUTCOME MEASURES: Animal survival and manifestations of multiple-system organ failure. RESULTS: In rats with continuous hepatic inflow interruption for 90 or 120 minutes, scanning electron microscopy showed a necrotic surface of the liver cells together with fibrin exudation. Hepatic sinusoids and intrahepatic nerves also had severe injury. Destruction of pulmonary structures and breakdown of microcirculation in the lung were characterized by thinned and ruptured walls of alveoli and a greatly decreased number of capillaries in the damaged alveolar wall. Transmission electron microscopy showed four types of ultrastructural changes, ie, necrosis of epithelial cells, extremely swollen mitochondria in intestinal cells, death of mucosal cells, and increased permeability of vessels in the injured intestine. The affected heart manifested highly enlarged mitochondria in myocardial cells, thickened vascular walls, and scattered necrotic lesions in myocardial tissue. CONCLUSIONS: Multiple-system organ failure resulting from ischemia-reperfusion injury and obstacle of portal hemodynamics in a subject subjected to an extended continuous hepatic inflow interruption is an unrecognized new disorder that may cause a high mortality rate. Our preliminary results indicated that animals subjected to continuous hepatic inflow interruption for 90 or 120 minutes developed various injuries to the liver, lung, heart, and gut. Therefore, we believe that continuous hepatic inflow interruption exceeding 90 minutes could also be hazardous in human beings.

Energy dispersive X-ray microanalysis of the dentin in rat molars after corticosteroid treatment.

The aim of this study was to investigate whether the calcium (Ca) and phosphorus (P) composition of corticosteroid induced dentin was the same as in normally developed dentin. Seven rats were given corticosteroids intravenously and three rats served as controls. Energy dispersive X-ray microanalysis (EDX) was carried out on the axially sawn roots of the molars. Measurements were made at 20 sites, equally distributed in the buccal, mesial, lingual and distal direction. The results showed that the Ca/P ratio (weight %) was slightly above 2.0 in both the experimental and the control group, indicating that the corticosteroid induced dentin had a normal Ca/P ratio. However, different degrees of mineralization were found in different directions of the roots.

Uptake of radiolabeled and colloidal gold-labeled chyle chylomicrons and chylomicron remnants by rat platelets in vitro.

This study examined the uptake of chyle chylomicrons (CMs) and chylomicron remnants (CMRs) by rat platelets in vitro. CMs and CMRs were doubly labeled with [3H]arachidonate ([3H]-20:4) and [14C]cholesterol and were incubated with platelets for up to 4 hours. A significant uptake (binding and/or internalization) of CMs by the platelets occurred, as indicated by the parallel increase of [3H]20:4 and [14C]cholesterol in platelets with incubation time. Addition of unlabeled CMs, VLDLs, LDLs, and HDLs decreased the uptake of labeled CMs. The competition experiments suggested that there is both a saturable binding and a nonspecific uptake of CMs. During incubation with CMs, the proportion of [3H]20:4 in phospholipids decreased and that in 1,2-x-diacylglycerol increased. The data indicated that a phospholipase C-mediated degradation of phosphatidylcholine and phosphatidylethanolamine occurred, whereas [3H]20:4 in triglyceride and 14C in cholesterol ester did not change. Electron microscopic studies after incubation with colloidal gold-labeled CMs (CM-Au's) demonstrated an accumulation of CM-Au particles in the open canalicular system of the platelets. Some CM-Au particles were localized in cytoplasmic vacuoles that were not stained by ruthenium red. Some CM-Au's or free gold particles were in vacuoles that showed acid phosphatase activity, indicating that some true endocytosis of CM occurred. The uptake of [3H]-20:4- and [14C]cholesterol-labeled CMRs was low compared with the uptake of CMs. After incubation with colloidal gold-labeled CMRs (CMR-Au's), only a few platelets contained CMR-Au in their open canalicular systems, and no CMR-Au particles were seen in the cytoplasm or in acid phosphatase-positive vacuoles. Rat platelets can thus interact with CMs by a process that leads to a sequestration in the open canalicular system and endocytosis and a net degradation of CM phospholipids. The conversion of CMs to CMRs counteracts this interaction.

Pro- and antifibrinolytic effects of ultrasound on streptokinase-induced thrombolysis.

We have earlier documented that ultrasound can enhance the fibrinolytic effect of streptokinase and shorten the reperfusion time under experimental conditions. The present study concerns the magnitude of the effect in relation to ultrasound frequency, intensity and exposure time. The effect of ultrasound on the structural properties of the fibrin network was evaluated by scanning electron microscopy. Whole blood clots were created and exposed to streptokinase (5000 U/mL). In paired experiments, one clot was also exposed to pulsed ultrasound. Lysis is expressed as the percentage decrease in clot weight. Clot lysis was equally affected by ultrasound in the range of 0.5 to 2.3 MHz. Below 0.5 W/cm2 no enhancing effect was observed, while intensities between 0.5 and 1.5 W/cm2 resulted in a significant enhancement of clot lysis. Intensities above 4 W/cm2 inhibited the fibrinolytic effect of streptokinase but not of rt-PA. From scanning electron microscopy there was no evidence that ultrasound disrupted the clot surface or damaged the fibrin structure.

Energy-dispersive X-ray microanalysis of the bone mineral content in human trabecular bone: a comparison with ICPES and neutron activation analysis.

To evaluate the accuracy of bone mineral composition determination by electron microprobe analysis (EDX) the measurements have been compared to instrumental neutron activation analysis (INAA) and chemical analysis (ICPES). Bone specimens from five femoral heads were used. The trabecular content of calcium (Ca) and phosphorus (P) was analyzed by the three different methods. The EDX method allows for a microstructural analysis of intact, methylmetacrylate-embedded, undecalcified bone and the measuring points can thus be distinctly identified centrally in each trabecula. The analysis yielded 25.8 +/- 0.7 wt % Ca and 10.5 +/- 0.1 wt % P, compared with 22.2 +/- 0.5 and 23.0 +/- 1.0 wt % Ca, and 9.83 +/- 0.21 and 10.02 +/- 0.44 wt % P for INAA and ICPES, respectively. The EDX analysis was calibrated by consecutive measurements of a hard, pressed tablet of hydroxyapatit of known content. The mean Ca content deviated with -0.38 wt % from the given content and P with -0.89 wt %. We could not verify any particular interference from the embedding procedure, however, it is possible that the relatively lower P content still may reflect this. The magnesium (Mg) concentration was 0.31 +/- 0.02 wt % by EDX and 0.26 +/- 0.02 wt % by INAA. The EDX analytical method provides a useful tool for simultaneous elemental quantification in bone. It has the advantage of permitting the use of regular bone biopsy material and thus allowing for a unique microstructural evaluation of the degree of mineralization.(ABSTRACT TRUNCATED AT 250 WORDS)

Phospholipid impregnation of abdominal rubber drains: resistance to bacterial adherence but no effect on drain-induced bacterial translocation.

In order to evaluate the effect of surface modification of biomaterials on bacterial adherence and bacterial translocation after intraperitoneal biomaterial implantation, phosphatidylcholine- or phosphatidylinositol-impregnated rubber drain pieces, which had been intraperitoneally implanted in the rat for 2 and 7 days, or unimplanted, were incubated in vitro with 3H-labelled Escherichia coli and Enterobacter cloacae. As compared with unimpregnated pieces, the adherence of bacteria significantly decreased to phosphatidylcholine- and phosphatidylinositol-impregnated rubber drain pieces that were either unimplanted or implanted for 2 days, but not for 7 days. The supplementation of albumin in the medium reduced the adherence of bacteria to the unimplanted, unimpregnated drain pieces, but did not further decrease adherence of bacteria to the unimplanted, phospholipid-impregnated brain pieces. Bacterial growth was inhibited after incubation in nutrient broth supplemented with phospholipids. The incidence of enteric bacterial translocation induced by intraperitoneal drain implantation did not differ between phospholipid-impregnated and unimpregnated drain pieces. Scanning electron microscopy revealed a large amount of biofilm and fibrous deposition on the surface of the implanted, phospholipid-impregnated rubber drain pieces. Thus, phospholipid impregnation of rubber drains reduces bacterial adherence and inhibits bacterial growth, without influencing the incidence of bacterial translocation.

Adherence of blood cells to dialyzer membranes as a measure of biocompatibility.

Cell adherence to plate dialyzer membranes was analyzed at the end of 4 hours of dialysis. Three types of membranes were examined: Cuprophan Hemophan and Gambrane, (a polycarbonate membrane). The membranes were mounted in dialyzers that contained 23 layers of one membrane type and one layer of each of the two other. Less leukocytes adhered to the Pc than to the Cu and He membranes. Transient initial complement activation during dialysis, which was considerably lower with dialysers containing mainly Pc membrane, was not correlated to adherence of cells to the membranes. Instead flow geometry is proposed as the main factor determining the adherence. Contrary to what has been earlier suggested, we think that leukocyte adherence is not a very suitable measure of membrane biocompatibility. The reason is that the influence of membrane surface-chemical factors can not be separated from mechanical factors due to the design of the device.

Histopathological lesions in the pancreas of a rat model of diabetes induced with complete Freund's adjuvant and low-dose streptozotocin.

Neither injection of complete Freund's adjuvant (CFA) alone nor the administration of low doses of streptozotocin (STZ) to rats produced remarkable histopathological changes in the endocrine pancreas, but treatment with the combination of both resulted in necrosis of beta cells. When the combination of CFA/STZ was given two times, necrosis progressed, and the beta cell reserve was depleted to such an extend that persistent hyperglycemia ensued. These changes were associated with a significant reduction in the apparent islet size. A single injection of CFA induced pancreatitis and inflammatory lesions in the exocrine parenchyma with no insular involvement. Three injections caused extensive destruction of pancreatic acinar tissue but only moderate beta cell injury in the minority of islets. Apart from mild degranulation of beta cells, treatment with STZ did not produce histopathological changes in the pancreas. These results suggest that the acute inflammatory process induced by CFA may initially damage the beta cells, increasing thereby their susceptibility to the action of STZ.

Development of cytotoxic islet cell antibodies in rats following damage of the pancreas by complete Freund's adjuvant combined with a nondiabetogenic dose of streptozotocin.

The possible relationship between destruction of pancreatic beta cells and islet cell surface antibodies (ICSA) was examined in a rat model using complete Freund's adjuvant (CFA), a lymphocyte activator, in combination with the beta cell toxin, streptozotocin (STZ). In addition to this treatment, the rat insulinoma cell line, RIN5AH, as a readily accessible source of insulin-producing cells, was utilized to potentiate the production of ICSA. Intraperitoneal injections of CFA to male Lewis rats, followed 24 h later by a single nondiabetogenic dose of STZ, produced a 47% (p less than 0.01) reduction in pancreatic insulin content associated with degranulation and necrosis of insulin-immunoreactive cells. Eight weeks after treatment, ICSA were detectable that mediated the complement-dependent lysis of neonatal rat islet cells. Injections of RIN5AH cells, following treatment with CFA/STZ, did neither increase the severity of histopathological changes in the exocrine pancreas nor the extent of beta cell necrosis, but gave rise to higher levels of cytotoxic ICSA. Immunization with RIN cells alone, although increasing ICSA levels above those of the other experimental groups, produced no major histopathological changes. These results indicate that ICSA are the consequence of beta cell damage, and they are not capable of promoting or initiating beta cell necrosis in this model.

Production of pro-insulin, C-peptide, and insulin in nesidioblastosis, focal islet-cell adenomatosis, and genuine insulomas. A correlated radioimmunochemical, immunohistochemical, and ultrastructural investigation with particular regard to the occurrence of argyrophil and pro-insulin immunoreactive cells.

Subtotal pancreatectomy specimens from one case of nesidioblastosis, one case of focal adenomatosis, and two cases of insulin-producing islet-cell tumours were studied with special reference to their production of pro-insulin, C-peptide and insulin, and their contents of argyrophil parenchymal cells. Specific immunostaining revealed the presence of abundant cells reacting with pro-insulin, C-peptide, and insulin antiserum; at least the great majority of them were obviously non-argyrophil cells. The content of extractable immunoreactive insulin (IRI) was higher in the cases of nesidioblastosis and focal adenomatosis than in the two insulomas. Molar ratios of IRI to C-peptide immunoreactivity (CPR) varied between 7 and 100. Gel filtration analysis of the extracts revealed two peaks of CPR, corresponding to 3,000 and 10,000 daltons, respectively. Ultrastructurally, the insulin cells in cases of nesidioblastosis and focal adenomatosis contained numerous typical beta granules. In the islet-cell neoplasms some "polycrine" islet cells were also found, containing typical as well as atypical granules with electron dense or pale cores. Some cells even showed a mixture of apparent beta and alpha granules. Despite structural differences and variable contents of IRI and CPR, the predominance of cells reactive with antibodies to pro-insulin, C-peptide, and insulin, and the absence of argyrophil pro-insulin cells in adenomatosis and insulomas indicates that the hormonal products of these parenchymal cells are not any chemically modified insulin or any other member of the insulin family.

Biochemical, immunohistochemical and ultrastructural studies of the alteration of pancreatic beta cells resulting from the combined effects of complete Freund's adjuvant and non-diabetogenic doses of streptozotocin.

Injection of complete Freund's adjuvant (CFA) 24 h prior to treatment of rats with non-diabetogenic doses of streptozotocin (STZ) produced a reduction in pancreatic insulin content associated with ultrastructural changes and a decrease of the volume density of insulin-immunoreactive cells without affecting other islet cells. After one injection of CFA and STZ, pancreatic insulin content was decreased by 69% (p less than 0.01) within 48-96 h, while plasma glucose concentrations were not changed. The volume density of beta cells in pancreata of these rats was reduced by 40% (p less than 0.01) compared with citrate-saline treated control rats. No significant lymphocytic infiltration was detectable in islets, but the exocrine pancreas exhibited inflammatory lesions. Degranulation and vacuolation was evident by ultrastructural analysis. Following administration of CFA or STZ alone, pancreatic insulin content was insignificantly reduced, and major histopathological changes in pancreatic islets were apparently absent. These results support the hypothesis that activation of the immune system by CFA allows an anti-beta cell reaction to develop following exposure to the beta cell toxic agent STZ.

Destruction of pancreatic beta cells in rats by complete Freund's adjuvant combined with non-diabetogenic doses of streptozotocin.

Non-specific activation of the immune system by complete Freund's adjuvant (CFA) in combination with non-diabetogenic doses of streptozotocin (STZ) was used to study autoimmune reactions against pancreatic islets. Male Lewis RT1a rats received either CFA (0.5 ml/kg) 24 hr prior to the injection of STZ (25 mg/kg), or CFA or STZ alone. The injections of CFA followed by STZ, but not CFA or STZ alone, produced a 69% (p less than 0.01) reduction in pancreatic insulin content associated with necrosis and a decrease of the relative volume density of insulin-immunoreactive cells without affecting other islet cells. Two injections of CFA and STZ induced hyperglycemia. This was associated with a depletion of pancreatic insulin and a significant reduction in the relative volume density of insulin-immunoreactive cells (p less than 0.01) and in the mean islet area (p less than 0.01). On day 20, after treatment with 3 injections of CFA and STZ, the animals developed persistent hyperglycemia. The pancreata in these rats contained less than 12% B-cells, and the insulin content was reduced by 96% (p less than 0.01). The main components of the remaining atrophic islets were glucagon- and somatostatin-immunoreactive cells. No significant lymphocytic infiltration into the islets was detectable, but the exocrine parenchyma exhibited severe inflammatory lesions. Degranulation and vacuolation of B-cells was evident by ultra-structural analysis. The volume densities of insulin containing cells and islet area were not changed after 3 injections of either CFA or STZ alone, although the pancreatic insulin content decreased by 61% (p less than 0.01) and 39% (p less than 0.05), respectively. These treatments did not produce an increase in plasma glucose. The present results demonstrate that CFA in combination with non-diabetogenic doses of the beta cytotoxic agent STZ induces B-cell destruction without significant insulitis. Our observations support the hypothesis that activation of the immune system by CFA allows an anti-B-cell immune reaction to develop following exposure to STZ.

A simple method for processing cytologic samples obtained from body cavity fluids and by fine needle aspiration biopsy for ultrastructural studies.

A simple method for processing routine cytologic samples for electron microscopy is described. The method has been successfully applied to body cavity fluids and fine needle aspiration biopsies. The technique neither requires additional time and personnel nor interferes with the normal activity of the laboratory.

The concentration of calcium and phosphorus in trabecular bone from the iliac crest.

The concentrations of calcium and phosphorus were determined in central parts of bone trabeculae in the human iliac crest by means of an energy dispersive microanalysis technique. Two age groups, each consisting of six suddenly diseased male subjects, were investigated. It was found that the concentrations of calcium and phosphorus did not differ between young and old men. The obtained mean values were fairly alike and determined to 40 and 17 weight %, respectively.

Alteration in density, morphology and parathyroid hormone release of dispersed parathyroid cells from patients with hyperparathyroidism.

Dispersed parathyroid cells from normal human and bovine glands and from 10 patients with primary (7 adenomas, 3 hyperplasias) and 4 patients with uraemic hyperparathyroidism (HPT) have been investigated with respect to density, morphology and parathyroid hormone (PTH) release. Percoll density gradients enabled an efficient isolation of viable parathyroid cells which generally banded between 1.035-1.090 g/ml. The average density was significantly higher in cells from the normal than the abnormal glands. The pathological glands contained large chief cells, oxyphil and transitional oxyphil cells and, in one case, water-clear cells which were enriched in fractions with densities below 1.055 g/ml. Measurements of cell diameters revealed an increased proportion of enlarged cells in the preparation of abnormal glands. Nuclear diameters were similar in the normal human glands, adenomas and hyperplasias, but the variability was greater among the adenomas. In comparison to normal bovine parathyroid cells, PTH release of cells from the pathological human glands was reduced and abnormally insensitive to extracellular calcium. The oxyphil and water-clear cells secreted similar amounts of PTH as the chief cells of the abnormal glands. The disturbed PTH release in secondary HPT seemed to be confined mainly to cells within nodules of the hyperplastic glands. The results show that the disturbed hormone regulation in HPT is related to morphological changes of the cells and that buoyant density gradients can be used to accumulate the abnormal cells.

The use of a new dextran gradient medium for rapid isolation of functionally intact neonatal rat pancreatic islets.

The use of a new density gradient medium, Dextran M 70, is described for isolation of islets of Langerhans from collagenase digested pancreases of neonatal Wistar rats. Centrifugation in continuous as well as in discontinuous gradients of a less expensive dextran with a mol. wt. of 70,000 was performed and the results were compared with those obtained with Ficoll gradients. About 100 islets free from exocrine acinar parenchyma were obtained from each neonatal rat pancreas. There were no differences in glucose-stimulated insulin release and in potentiation of glucose-stimulated insulin release by the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX) between the islets isolated by centrifugation in Dextran M 70 or Ficoll 400 and those directly harvested from the pancreatic digest without density media. These data demonstrate that high numbers of pancreatic islets can be rapidly isolated in dextran gradients without any impairment of their functional integrity. Dextran M 70 gradients can be readily formed as continuous or 4-step discontinuous gradients without special osmotic compensators and its maximum density of 1.097 g/ml at a concentration of 23% (w/w) seems to be sufficient for the purification of other cell types too.

Electron microprobe investigation of calcium and phosphorus concentration in human bone trabeculae--both normal and in posttraumatic osteopenia.

With electron probe microanalysis the concentration of calcium and phosphorus was determined in central parts of tibia epiphyseal bone. Trabeculae from five men who had had a fracture of the ipsilateral tibia diaphysis were analyzed. The results were compared with those of five men who had suffered sudden death. There was an even distribution of calcium in the control cases: the mean value was 45 +/- 1 wt%. In the posttraumatic group of patients there was a lower mean and a greater scatter: 29 +/- 10 wt%. Also, the concentration of phosphorus was lower in the posttraumatic cases as compared with the cadavers.

Energy dispersive X-ray microanalysis of zinc and calcium in organelles of insulin-producing cells of the mouse, rat, and a fish.

By means of energy dispersive X-ray microanalysis in the scanning-transmission electron microscope, spectra were obtained from quick-frozen, cryo-ultramicrotome cut, freeze-dried sections of insulin cells from a fish and a mouse. It was shown that both zinc and calcium are present in significant quantities in native islet-cell beta-granules. In the beta-granules of the rat RINm5F insuloma cells calcium, but not zinc, seemed to accumulate; the zinc contents in the secretion granules of these neoplastic beta-cells were probably below the detection limit.