Red blood cell survival and morphology during and after intraoperative autotransfusion.

In an animal experimental model the survival of untreated red blood cells (RBC) tagged with 51Cr was compared with cells processed by an autotransfusion device separating and washing the RBC tagged with 111In. Intraoperative autotransfusion (IAT) was done by the Haemonetics Cell Saver. Additionally the morphology of human RBC was investigated in different stages of IAT by the aid of scanner electron micrographs. The survival of RBC after processing was slightly decreased in comparison to untreated cells. The morphology of autologous RBC is little affected compared with older homologous RBC in erythrocytic concentrates. Cell detritus due to haemolysis is reliably eliminated by microfiltration. Especially regarding survival and morphologic alterations of RBC IAT with a system separating and washing the cells seems to be superior to the transfusion of homologous blood.

Characterization of the cytolytic activity of a cloned antigen-specific T suppressor cell derived from a tolerant CBA/J mouse.

The antigen-specific T suppressor cell clone HF1 isolated from a CBA/J mouse made tolerant by low doses of bovine serum albumin has suppressive and cytolytic activity. The analysis of the latter gave the following results. Natural killer (NK)-sensitive YAC-1 (H-2a) and RBL-5 (H-2b) target cells are lysed whereas other NK targets, like EL4 (H-2b) or the human K562 cell line are resistant. Cytolytic activity is not antibody mediated. Its inhibition by sugar phosphate or monoclonal antibodies against LFA-1 antigens is such that HF1 can neither by typed as T killer nor as NK cells. It seems to represent a distinct T lymphocyte type.

T cell lines and variant subclones with differing susceptibility to tumor promoters.

The effect of tumor-promoting phorbol esters on cloned cytotoxic T cell (CTL) lines, H-2 restricted and specific for minor histocompatibility antigens, was studied. We found that two of the three CTL lines tested can be induced by 12-O-tetradecanoylphorbol 13 acetate (TPA or PMA) to synthesize DNA. One of the lines did not respond. TPA concentrations in the range of 1 nmol were active, and induction of DNA synthesis was independent from cell densities. Testing of other phorbol esters showed correlations with their tumor-promoting activity, because TPA and phorbol 12,13-didecanoate were active, whereas phorbol and 4 alpha phorbol 12,13-didecanoate had no effect. Interestingly, two subclones isolated from one of the lines responding to TPA behaved differently: one of the subclones synthesized DNA when incubated with TPA, whereas the other remained completely negative. This difference was reflected also in the dissimilarity of morphologic alterations (observed by scanning electron microscopy) occurring in the responding cells. The nonresponsiveness, in terms of DNA synthesis, by some of the CTL lines is not due to a lack of interaction between TPA and the cells. When incubated simultaneously with TPA and T cell growth factor (IL 2), all lines responded with greater DNA synthesis than when incubated with IL 2 or TPA alone, thus demonstrating a synergistic action between TPA and IL 2. We have also done experiments to test whether TPA induces the synthesis and secretion of a factor leading to proliferation of the cells. From bystander experiments it appears that the action of TPA is not mediated by a secreted product but is a direct one.

Variability of the topography of low-density lipoprotein (LDL) receptors in the plasma membrane of cultured human skin fibroblasts as revealed by gold-LDL conjugates in conjunction with the surface replication technique.

In this investigation the membrane-perturbing effect of filipin, a polyene antibiotic which reacts specifically with cholesterol or cholesterol-like compounds in cell membranes, has been exploited to study the distribution of coated pits in cultured human skin fibroblasts. The coated pits, showing no filipin-cholesterol complexes, occurred singly or in clusters without apparent localization of either type to a particular region of the fibroblast membrane. Colloidal gold, conjugated to low-density lipoprotein, has proven to be an excellent marker, allowing the localization of low-density lipoprotein receptors on the surface of cultured cells. A pattern similar to that for the coated pits in the plasma membrane fracture faces was observed in the distribution of gold-low-density lipoprotein conjugates in surface replicas, indicating that the low-density lipoprotein receptors are associated with these coated pits. It was shown that there is an apparent heterogeneity in the distribution of low-density lipoprotein receptors, from cell to cell and even among different areas of the same cell membrane. The binding capacity for gold-low-density lipoprotein complexes, as represented by the extent of surface labeling, was directly related to the cell variety within the culture or to the particular experimental procedure. The observation of differences in the distribution of gold-low-density lipoprotein conjugates, even among adjacent coated pits, provides evidence for various stages of activity of the low-density lipoprotein receptors corresponding to incorporation, mobility, and internalization.

Kidney tubule cell injury of male rats after estrogen treatment.

Ultrastructural changes in the tubular epithelium of the rat kidney following a large dose of estrogen (300 micrograms per week for 20 weeks) were studied by means of electron microscopy. The ultrastructural changes in estrogen-treated rats were confined to the proximal tubule. The changes consisted of intracytoplasmic vacuoles and dilatation of the intracellular space between adjacent tubule cells. The cisternae of the endoplasmic reticulum exhibited some degree of vesiculation characterized by club-like formations. In addition, large masses of collagen could be observed within the peritubular capillaries. In contrast to the controls the tubular cells of estrogen-treated rats exhibited large areas or granules with dense inclusions and membranous, filamentous material in a process of being transformed into an autolysosome and residual body. The results are discussed in the light of those of other authors.

Ultrastructural study of cholestasis induced by longterm treatment with estradiol valerate. I. Tight junctional analysis and tracer experiments.

Over a period of 20 weeks estradiol valerate (1.5 mg/kg body weight/week) was administered subcutaneously to male Wistar rats from which the livers were examined at four week intervals employing a freeze-fracture technique and colloidal lanthanum tracer studies. In connection with intrahepatic cholestasis, distinct alterations in the tight junctions were observed, consisting of disorganization, rarification and proliferation. Disruption of the tight junctions was not seen and colloidal lanthanum did not penetrate into the bile canalicular lumen. Holding the view that the term "leakiness" of tight junctions should be defined with reference to the tracer employed, we conclude that in the liver one tight junctional strand is sufficient to prevent the escape of larger bile constituents such as bile acids and that a back diffusion of bile acids over the tight junctional barrier does not play a role in the pathogenesis of the estrogen-induced cholestasis. Interruptions of tight junctions, as described by other authors, are interpreted as a secondary mechanical effect. On the other hand, we consider an increased permeability of the tight junctions to water and small solute molecules as probable; possibly this increased permeability is brought about by alterations in the microfilaments. A model for the pathogenesis of the estrogen-induced intrahepatic cholestasis is proposed.

Cytochalasin B affects the gap and tight junctions of mouse hepatocytes in vivo.

This study investigates the effect of cytochalasin B at a dosage of 0.2 mg per mouse per day for a period of 7 days in an in vivo experiment on mouse liver. Using thin-sectioning and freeze-fracture technique both quantitative and qualitative analysis was made of membranes and cell contacts (gap and tight junctions). Significant alterations of both membranes and junctions were observed. The intercellular space showed vacuolar dilatation in some cases and there were vacuoles observed within the cytoplasm. The microvillar bile canaliculi were dilated. However, no colloidal tracer was observed within the lumen following lanthanum perfusion. With the aid of the freeze-fracture method it was possible to demonstrate that the strands of the tight junctions were highly disorganized. In some cases reduction and in other cases proliferation of tight junctions was observed. Large, proliferative plaques of tight junctions were found both in contact with the tight junctions of the bile canaliculus and ending freely on the plasmalemma. The gap junctions appeared enlarged as well. Their average size increased from 0.42 micron 2 to 0.90 micron 2 (p less than 0.005). The enlargement was also accompanied by an increase in the proportion of the plasma membrane occupied by the junctions: 3.42% in control animals, 10.25% in the livers of mice treated with cytochalasin B. Frequently evaginated and internalized gap junctions were seen in the experimental group. In view of the fact that cytochalasin B, in addition to other effects, also has an effect on the microfilament system of the cell, it may be supposed that microfilaments play a role in maintenance of the orderly structure or in the formation of tight and gap junctions. This remains hypothetical, however, and additional studies are necessary in order to conclusively clarify this issue.

A correlative study on the topographical distribution of the receptors for low density lipoprotein (LDL) conjugated to colloid gold in cultured human skin fibroblasts employing thin section, freeze-fracture, deep-etching, and surface replication techniques.

The topographical distribution of the receptors for low-density lipoprotein (LDL) conjugated to colloidal gold on cultured human skin fibroblasts was studied using a surface replication technique following critical point drying. In this correlative study, also employing thin sectioning, freeze-fracture and deep-etching techniques in conjunction with the polyene antibiotic filipin, it could be shown that LDL preferentially binds to certain microdomains of the plasma membrane, the so-called coated pits. The coated pits can be recognized after filipin treatment both on the P face and E face of the membrane, due to the fact that they are free of filipin-sterol complexes. Filipin was also of value in clearly delimiting the structure of nascent coated pits from their surroundings in surface replicas. Using filipin has the effect of increasing the contrast between the smooth surface of a coated pit and the rough surface of the remaining membrane caused by formation of filipin-sterol complexes. This study has shown that there are differences in the topographical distribution and number of gold particles from fibroblast to fibroblast and even between different areas of the same cell. The surface replication technique using goldlabelled LDL provides s suitable method for improving the interpretation of the spatial arrangement of the LDL receptors. The advantages of this technique are discussed in comparison with the methods previously employed to visualize LDL receptors.

Changes in mouse hepatocytes caused by vincamin. A thin-sectioning and freeze-fracture study.

Mouse liver was examined following a single intraperitoneal application of 25 mg/kg body weight vincamine. The studies were made using the technique of thin-sectioning for electron microscopy as well as the freeze-fracture method. The thin-sectioning technique was useful for observing discrete changes such as an increase in the mitochondria and alterations in the bile canaliculi, e.g. a loss of microvilli and dilatation of the lumen. No other cytoplasmic changes could be observed. It was only with the aid of the freeze-fracture method that alterations in the cell contacts became visible. In contrast to the control animals the tight junctions in the liver tissue of mice treated with vincamin were disorganized and irregularly arranged. The gap junctions showed a very irregular contour as well. The question arises as to the extent to which the freeze-fracture method should be applied in the testing of pharmaceuticals, so as to exclude the possibility of damage to membranes.

[The effect of (+)-cyanidanol-3 on toxic fatty infiltration of liver parenchyma (author's transl)]. / Der Einfluss von (+)-Cyanidanol-3 auf die toxische Verfettung des Leberparenchyms. Eine tierexperimentell feinstrukturell-morphometrische untersudhung.

Polychlorinated biphenyls, pentachlorophenol, d-galactosamine or alcohol are well known substances which produce toxic liver damage in animal experiments. Of special significance is the toxic fatty infiltration in the live parenchyma of these models. The effects following (+)-Cyanidanol-3 (Catergen)--application after liver damage was studied by qualitative and quantitative electron microscopy. (+)-Cyanidanol-3 reduces the volume density of intracytoplasmatic fat following intoxication significantly to normal values.

Freeze-fracture elucidation of hepatocyte membrane alterations following beta-pyridylcarbinol application.

The effect of beta-pyridylcarbinol on mice was investigated in long-term studies (21 days, daily dose of 0.1 mg) making use of thin-sectioning and freeze-fracture techniques. Thin sectioning merely revealed only subtle pathological changes including dilatation of the intercellular space and foundation of hepatocytic vacuoles. Only upon investigation using freeze-fracture was it possible to demonstrate more profound alterations, primarily involving the cell contacts, i.e. the gap and tight junctions. The organized structure of the tight junctions appeared dissipated. The meshwork was disorganized in some cases and reduced in size. Large, proliferative tight junctional maculae were frequently observed on the plasmalemma. In some cases the gap junctions had lost their round to oval shape and had increased in size to form large plaques protruding at numerous points. Moreover, even the bile canaliculi, which presented no pathological alterations on ultrathin section, exhibited damage as seen in freeze-fracture preparations. The lumen was uneven in contour and processes were visible extending into the adjacent cytoplasm. The present investigation shows that hepatocytic damage primarily affecting the membranes was first made evident using freeze-fracture technique. The issue is addressed as to the extent to which freeze-fracture technique should be employed in routine investigations of pharmaceuticals.

Alterations of tight and gap junctions in mouse hepatocytes following administration of colchicine.

Following the administration of colchicine at a dosage (1 mg/mouse) known to cause an antimicrotubular effect, membranes as well as tight and gap junctions of hepatocytes were studied using the thin-sectioning and freeze-fracturing technique. As early as 1 h after administration of colchicine the intercellular spaces were dilated and vacuoles were visible within the cytoplasm. The bile canaliculi became enlarged, and after lanthanum perfusion the tracer was found in the canalicular lumen, i.e., the tight junctions became permeable to the tracer. These findings correlated with a disorganized arrangement of the tight junctional strands of the zonula occludens. In some regions the strands showed interruptions and frequently ended freely in a diffuse pattern on the plasma membrane. Proliferation of tight junctions could be observed at various locations on the plasma membrane. The gap junctions also exhibited alterations. They showed an irregular outline with outpouchings, in addition to an enlargement in their total area from approximately 0.5 microns 2 in controls up to approximately 2 microns 2 in treated mice. The surface area occupied by these junctions increased from 4% (controls) to 20% (treated) of the hepatic plasma membrane. In the cytoplasm of hepatocytes from colchicine-treated mice gap-junctional vesicles were frequently observed. In view of the antimicrotubular effect of colchicine it is tentatively suggested that the intact microtubular system of the cell may play a decisive role in the regular formation of gap and tight junctions, either directly or indirectly via microfilaments.

Ultrastructural study of cholestasis induced by longterm treatment with estradiol valerate. II. Gap junctional analysis.

Treatment of male Wistar rats with estradiol valerate induced alterations in hepatic gap junctions as visualized by the freeze-fracture technique. The alterations involved the spacing, and regularity of packing of the membrane particles of the P face (PF) and complementary pits on the E face (EF), as well as internalization and changes in the number, size and shape of the junctional domains. In approximately 20% of the PF's of the lateral membrane of treated animals the nonjunctional IMPs were aggregated, while the bile canalicular membrane was never involved, maintaining its random distribution of particles. It is proposed that the changes in junctional area and the more general arrangement of the junctional particles may indicate a decrease in coupling between hepatocytes. The invaginations of gap junctions may represent a means for removing gap junctional membrane from the surface or may be an expression of a higher turnover of gap junctions. We assume that the alterations observed here are due to the specific effects of estrogen. This study addresses in detail a number of possible sites of activity and modes of action for estrogen.

Structural relationship between desmosomes and mitochondria in human livers exhibiting a wide range of diseases.

Liver biopsies from ten patients (five women and five men, aged 25-63) with a number of different diseases were studied with a transmission electron microscope. In addition to many different pathologic changes in the hepatocytes, all clinical diagnoses showed 5-70% of mitochondria with paracrystalline inclusions. A peculiar finding was that the desmosomes that join two cells had mitochondria associated with the intracytoplasmic component in both cells in 5-15% of desmosomes observed. In addition, the association involved only one side in one cell in 7-23% of desmosomes observed. Only speculation can be made regarding the function of these mitochondrial-desmosomal associations.

Ultrastructural alterations in the mouse liver following vincristine administration.

The damage pattern in mouse liver was investigated following a single administration of 0.15 mg vincristine sulfate using the conventional thin-section technique and freeze-fracture. Numerous cytoplasmic changes were observed. Foremost of these was the increase in autophagosomes and vacuoles with very-low-density-lipoprotein (VLDL)-like vesicles. The rough endoplasmic reticulum exhibited a vesicular structure. The mitochondria also exhibited alterations such as enlargement and polymorphism. Both the interstitium and bile canaliculi were dilated. Lanthanum penetrated into the lumen of the canaliculi, i.e. the sinusoid/canaliculus compartmentalization did not remain intact, thus permitting bile to pass into the blood. Of particular significance are the alterations in the intercellular junctions. In contrast to the controls, the tight junctions were reduced and irregularly arranged. The gap junctions exhibited hypertrophy as well as a very irregular contour. The alterations may either be attributed to non-specific toxic effects of vincristine and/or the destructive properties of the agent on microtubules.

Ultrastructural changes in mouse hepatocytes exposed to vinblastine sulfate with special reference to the intercellular junctions.

The effects of a single dose (0.5 mg) of the antimicrotubular drug vinblastine sulfate on the ultrastructure of mouse hepatocytes was studied by thin sectioning and freeze-fracturing after intravenous injection of the drug. The following cytoplasmic modifications occurred in the hepatocytes: storage of lipid droplets, heavy accumulation of autophagosomes and vacuoles with very low density lipoprotein (VLDL)-like vesicles, pathological changes in the mitochondria, and dilatation of the Golgi complexes. From 30 minutes onwards the bile canaliculi appeared altered. The tight junctions surrounding the bile canaliculi became permeable to lanthanum. This increased permeability was correlated with a disorganized arrangement of tight junctional strands and localized interruptions within the zonulae occludentes. In contrast to controls the gap junctions appeared more numerous and larger in size, exhibiting a high degree of pleomorphism. In the cytoplasm gap junctional vesicles could be observed indicating the removal of gap junctions from the surface by a process of internalization. We cannot establish whether the described changes are a direct or indirect effect of vinblastine sulfate treatment. In view of the antimicrotubular effect of vinblastine sulfate we hypothesize that normal formation of gap- and tight junctions is dependent directly or indirectly on intact microtubules.

Paracrystalline inclusions in lysosomes of mouse kidney tubule cells induced by vinblastine and vincristine.

Following the simultaneous administration of vinblastine and vincristine, crystalline and paracrystalline structures were observed in the lysosomes of kidney tubule cells of mice. These structures appeared as hexagonal elements with an average diameter of 22 nm, with filaments about 5,5 nm thick located at each angle of the hexagons. They were formed following the aggregation and fusion of several mitochondria, probably from the mitochondrial cristae. In addition, highly osmiophilic, membrane-bound lysosomes were observed, which in cross-section exhibited regular arrays of straight membrane plates with a periodicity of about 10 nm. The extent to which, or even whether, these are associated with the above-mentioned paracrystalline inclusions could not be clearly established.

Combined effects of vinblastine and vincristine on mouse hepatocytes with respect to ultrastructural elements.

The results of a single dose of the microtubule-destroying agents vinblastine/vincristine (0.5 mg/0.05 mg) on the ultrastructural elements of mouse hepatocytes was studied using the techniques of thin-sectioning and freeze-fracture following intravenous injection of the drugs. Several cytoplasmic modifications were observed in the hepatocytes. These included the storage of lipid droplets, a heavy accumulation of autophagosomes and vacuoles with very low density lipoprotein (VLDL)-like vesicles, large glycogen fields and dilated intercellular spaces with intrahepatocytic vacuoles. The rough endoplasmic reticulum exhibited pathological changes with loss of ribosomes and the bile canaliculi exhibited in some cases the loss of microvilli as well as dilatation of the lumen. The tight junctions surrounding the bile canaliculi exhibited alterations as well. The strands were reduced in number and showed a less organized arrangement. The gap junction showed an increase in size as well as an irregular outline in contrast to controls. These findings are interpreted as non-specific toxic phenomena. However, the possibility cannot be precluded that certain phenomena, such as alterations in the cell junctions, may be attributable to specific microtubule-destroying properties of the drugs.