Preparation and investigation of 99m technetium-labeled low-density lipoproteins in rabbits with experimentally induced hypercholesterolemia.

Low-density lipoproteins (LDL) were radiolabeled in atherosclerosis studies. The aim was to investigate the biodistribution and uptake of 99mTc-labeled LDL by atherosclerotic plaques in experimentally induced hyperlipidemia. Rabbits were fed a diet containing 2% cholesterol for 60 days to develop hyperlipidemia and atheromatous aortic plaques. A combination of preparative and analytical ultracentrifugation was used to investigate human LDL aliquots, to prepare radioactive-labeled lipoproteins and in rabbits with induced hyperlipidemia. Preparative density gradient centrifugation was applied for the simultaneous isolation of the major lipoprotein density classes, which form discrete bands of lipoproteins in the preparative tubes. The cholesterol and protein levels in the lipoprotein fractions were determined. LDL was subsequently dialysed against physiological solution and sterilized and apolipoprotein fragments and aggregates were eliminated by passage through a 0.22-micron filter. LDL was radiolabeled with 99mTc by using sodium dithionite as a reducing agent. Radiochemical purity and in vitro stability were controlled by paper chromatography in acetone. The labelling efficiency was 85-90% for human LDL. Two months after the start of cholesterol feeding, the total cholesterol in the blood serum had increased approximately 33-fold in comparison with the basal cholesterol content of hypercholesterolemic rabbits. Investigation of LDL was performed by Schlieren analysis after adjustment of the density of serum and underlayering by salt solution in a spinning ultracentrifugation capillary band-forming cell. Quantitative results were obtained by measuring the Schlieren areas between the sample curves and the reference baseline curve by means of computerized numerical and graphic techniques. In this manner we measured the concentrations of human LDL and analyzed rabbit LDL levels in induced hyperlipidemia. Gamma scintillation camera scanning of the rabbits was performed. Overnight fasted rabbits were injected in the marginal ear vein with 99mTc-labeled human LDL (4-10 mCi, 0.5-1.5 mg protein). The initial scintigram showing a typical blood-pool scan, gradually changing with time to an image of specific organ uptake of radioactivity by the liver, kidneys and brain and in the bladder. Gamma camera in vivo scintigraphy on rabbits revealed visible signals corresponding to atherosclerotic plaques in the aorta and carotid arteries. Our results show that 99mTc-LDL can be used to assess the organ distribution pattern of LDL in the rabbit, and to detect and localize areas of arterial atherosclerotic lesions.

Plasma endotoxin level of healthy donors.

The plasma level of endotoxin was determined in 116 healthy blood donors. After a routine physical and laboratory investigations the endotoxin level was determined with Limulus amebocyte lysate assay (LAL-test) by the chromogenic kinetic method of Bio-Whittaker Co. (USA). Its sensitivity was 0.005-50 EU/ml. The plasma level of endotoxin in most of the healthy donors was less than 1 EU/ml (in the range of 0.01-1.0 EU/ml), but always measurable. The average +/- S.D. was 0.128 +/- 0.215 EU/ml. Because of the high standard deviation and high range of values, the data were distributed into two groups with the means of 0.05 +/- 0.022 EU/ml and 0.294 +/- 0.186 EU/ml. The difference between the groups was significant (p < 0.001). In conclusion, endotoxin can be measured in plasma of healthy individuals.

Radiodetoxified lipopolysaccharide fails to activate the hypophyseal- pituitary-adrenal axis in the rat.

Lipopolysaccharide (LPS) is known to raise the concentration of the circulating stress hormones such as ACTH, corticosterone and beta-endorphin. This effect of endotoxin is mediated by different immune system-released hormone-like factors (e.g. interleukins, tumor necrosis factor etc.). Gamma-ray irradiation of LPS alters its biological properties and results in a radiodetoxified LPS preparation with numerous beneficial effects and decreased toxicity. In this study we compared the neuroendocrine effects of a commercial LPS and our native and radiodetoxified LPS preparations in rats. Plasma ACTH, corticosterone and beta-endorphin levels were measured by specific radioimmunoassays 120 min after intraperitoneal LPS administration. Control animals were injected with saline. Results show a dramatic increase in all hormones after administration of commercial and our native LPS preparation. Hormone levels in saline-injected controls and radiodetoxified LPS-treated rats did not rise significantly. These results suggest that radio-detoxification disintegrated that part of the LPS molecule complex which is responsible for toxicity including an enhanced production of cytokines, which trigger the hypothalamo-pituitary-adrenal axis.

[Role of bile acids and endotoxins in the pathogenesis and therapy of psoriasis]. / Epesavak és endotoxinok szerepe a pikkelysömör kórfejlódésében és kezelésében.

The authors demonstrated significant curative effect of bile acids (Suprachol; Acidum dehydrocholicum) in 551 psoriatic patients. The clinical efficiancy was evaluated by means of PASI-score (Psoriasis Area Severity Index). During this treatment (1-8 weeks) 434 patients (78.8 per cent) became asymptomatic. However, the traditional therapy resulted in 62 patients (24.9 per cent) of 249 sick persons a recovery (p < 0.05). In acute form of psoriasis (184 patients) this curative effect of bile acids was elevated (95.1 per cent). Two years later 319 patients (57.9 per cent) of bile treated 551 people were asymptomatic in contrast with 15 people (6.0 per cent) of 249 traditional treated patients (p < 0.05). In same time among the patients which were treated in acute form of psoriasis 10 (7.2 per cent) of 139 controls and 147 (79.9 per cent) of 184 bile-treated individuals were asymptomatic (p < 0.01). On the basis of their clinical observations (digestive disorders, ultrasonical confirmed gallbladder complaints, etc.) authors supposed that the deficiency of bile acids and the consecutive endotoxin translocation might play a role in the pathogenesis of psoriasis. In normal conditions the bile acids as detergent (physico-chemical defense) can protect the body against enteral endotoxins while split them to atoxic fragments and so preventing consecutive cytokin liberation.

Natural immunity and neuroimmune host defense.

Innate resistance is mediated by non-immune defense and by natural immunity. Non-immune defense includes diverse mechanisms (e.g., physico-chemical defense by bile acids). Natural killer (NK) cells, gamma delta T lymphocytes and CD5+ B lymphocytes are key mediators of natural immunity. These cells utilize germ-line coded receptors that recognize highly conserved, homologous epitopes (homotopes). Typically, it is not the antigen, but cytokines and hormones that regulate the level of NK-mediated cytotoxicity. These include interleukin-2, interferons, prolactin and growth hormone. Less is known about gamma delta T lymphocytes. CD5+ B lymphocytes produce germ-line coded antibodies (predominantly IgM) that are polyspecific, and able to recognize a great variety of microorganisms, cancer-cells and self-components. Antigen is not an effective stimulus for natural antibody (NAb), but bacterial lipopolysaccharide (LPS) is. During the acute phase response (febrile illness) the T-cell-regulated adaptive immune response is switched off and natural immune mechanisms are amplified several hundred to a thousand times within 24-48 hours (immunoconversion). This immunoconversion is initiated by immune-derived cytokines, and involves profound neuroendocrine and metabolic changes, all in the interest of host defense. Immune recognition is assured by natural antibodies and by some liver-derived acute phase proteins, such as C-reactive protein or endotoxin-binding protein, the level of which is elevated in the serum. Thus, natural immunity is essential for a first and last line of defense and the neuroendocrine system is an important promoter of this activity.

[A new possibility for enhancing natural immunity: a radiation-detoxified endotoxin]. / A természetes ellenállóképesség fokozásának új lehetósége: a sugárzással méregtelenített endotoxin.

Using ionizing radiation the author and co-workers produced a detoxified endotoxin preparation (Tolerin) which seems to be a suitable product for the increase of natural immunity (nonspecific resistance)-including activation of bone marrow in immunosuppressions, immunodeficiencies-protection against various types of shocks-radiation injury, septic/endotoxic shock, etc.- and increase of immunogen effect of antigens (e.g. inactivated virus vaccines) as an immunoadjuvant in human beings and experimental animals.

[Bile acids and endotoxins: physico-chemical defense of the body]. / Epesavak és endotoxinok: a szervezet fiziko-kémiai védelme.

The toxic effects of endotoxin--the cell wall component of Gram negative intestinal bacteria--under experimental conditions can be induced only when they are administered parenterally. However, in naturally occurring enteroendotoxemic diseases (e.g. septic and various shocks, etc.), the endotoxin absorbs from the intestinal tract. The cause and mode of translocation was unknown. The generally used experimental shock models differ from natural diseases only in the mode by which endotoxin enters the blood circulation. If the common bile ducts of rats were chronically cannulated (bile deprived animals) perorally administered endotoxin was absorbed from the intestinal canal into blood circulation and provoked endotoxin shock. The translocation of endotoxins and consequent shock can be prevented by sodium deoxycholate or natural biles. The bile acids can split the endotoxin macromolecule (atoxic fragments). A similar destructive detergent action might will be a significant factor against potential infectious agents with lipoprotein outer structure (e.g. so-called "big" viruses). This defense mechanism of macrooganisms based on the detergent activity of bile acids is called as physico-chemical defense system. On the basis of this knowledge the bile acids might be used in the prevention and therapy of some clinical processes (e.g. hepatorenal syndrome; psoriasis).

[Human herpesvirus 6 A enhances HIV-1 replication in vitro by soluble mediators, this effect is diminished by endotoxin]. / Az emberi 6-os herpeszvírus A változata szolubilis mediátorok által fokozza a HIV-1 szaporodását in vitro, mely hatást az endotoxin csökkenti.

Simultaneous HHV-6A infection can activate HIV-1 latency and promote AIDS progression, but in this process the effects of HHV-6A induced soluble mediators on HIV-1 have not been studied yet. Recently, supernatants of HSB-2 cultures infected with HHV-6A and/or treated with endotoxin have been filtered virus free at time intervals until the cytopathic effect developed. Biological activity of some cytokines which might participate in HIV-1 activation was quantitated. Filtered supernatants were mixed into CEM-ss cultures, which had been HIV-1 infected at 1:1 cell:virus ratio, subsequently HIV-1 replication was quantitated and compared to controls. Supernatants filtered during the first 96 hours of HHV-6A replication without visible cytopathic effect augmented HIV-1 syncytium formation by tenfold, reverse transcriptase activity by threefold, p24 antigen production by 6-fold. Filtered supernatants obtained at onset of HHV-6A cytopathic effect did not modify HIV-1 replication. HSB-2 cultures produced no IL-2, and IFN-gamma induced by endotoxin diminished HIV-1 replication. HHV-6A delayed IFN-gamma release. An increase in the tumour necrosis factor activity upon the effect of HHV-6A and endotoxin was not parallel to HIV-1 activation. The putative mediator, different from those above which characterisation is in progress, might transmit similar transactivating effects between immune cells of lymph nodes and circulation.

[Plasma endotoxin level in healthy blood donors]. / Egészséges véradók plazma endotoxin szintje.

The plasma level of endotoxin was determined in 116 healthy blood donors in this laboratory. After a routine physical and laboratory investigations the endotoxin level was determined with Limulus amebocyte lysate assay (LAL test) by the chromogenic kinetic method of Bio-Whittaker Co. (USA). Its sensitivity was 0.005-50 EU/ml. The plasma level of endotoxin in most of healthy donors was less than 1 EU/ml (in the range of 0.01-1.0 EU/ml), but always measurable. The average +/- S. D. was 0.128 +/- 0.215 EU/ml. Because of the high standard deviation and high range of values, the data were distributed to two groups with the means of 0.05 +/- 0.022 EU/ml and 0.294 +/- 0.186 EU/ml. The difference between the groups was significant found (p < 0.001). In conclusion, endotoxin can be measured in plasma of healthy individuals.

Endotoxins and endocrine system.

There is good evidence for the interaction of neuroendocrine and immune systems. Endotoxin (LPS)-induced mediators (e.g., cytokines, prostaglandins etc) set on endocrine organs (e.g., the hypothalamo-pituitry-adrenal axis; thyroid glands etc). Endotoxin-treated, intestinal ischemic, or irradiated rats show decreased T4 levels of blood. These animals cannot respond to TSH because the TSH-receptors of follicular membranes are disturbed by LPS in the thyroid glands. Radiodetoxified endotoxin is an effective immunstimulator and does not disturb the follicular membrane of thyroid gland. Thus, the T4 production remains normal. The bile acids--as the end-product of cholesterol metabolism--play an important role in the physiological defense of macroorganisms against endotoxin and other lipid-like agents (Physico-chemical defense) and in the regulation of endocrine system, including the reproduction.

Effect of trace element combination on the immune response of rats treated with cytostatic drug.

The effect of trace element combination, Béres Drop Plus (BDP) on the immune response of rats following single treatment with cytostatic drug (5-fluorouracil, 5-FU) was tested. Animals were treated with 5-FU and SRBC simultaneously, but separately. Rats were pretreated with BDP for 21 days. The body and spleen weight, furthermore the number of spleen cells and antibody producing cells were determined. The antibody titres in blood serum were also measured. The immune system of rats, 5 days following the 5-FU treatment, showed considerable regeneration. Pretreatment of rats with BDP had a beneficial effect on all parameters investigated.

[Bacterial endotoxins and their effects]. / Bakteriális endotoxinok és hatásaik.

The lipopolysaccharide endotoxin macromolecules are cell wall's components of the Gram negative bacteria. The endotoxins are produced by Gram negative bacteria of intestinal flora. If the endotoxins are translocated from the intestinal tract to the circulation or injected into bloodstream, they elicit (depending from the quantity of endotoxin), slight or serious effects (e.g. endotoxin shock). In the effects of endotoxin certain cell populations (e.g. thrombocytes, macrophages, leukocytes, etc.), certain organs and organ-systems (e.g. liver, spleen, bone marrow, endocrine and lymphoreticular systems etc.) are involved. Effects of endotoxin are produced by mediators (e.g. endotoxin binding proteins, cytokines, prostaglandins, prostacyclins, NO etc.). The endotoxin sensitivity of vertebrate organisms is dependent from the phylogenetical status of the species. Most sensitive species is the human. Generally accepted that endotoxin has an important role in the pathogenesis of septic shock. In other pathological processes (e.g. intestinal syndrome of radiation disease, Gram negative infections, various shock forms etc.) are supposed or proved the role of endotoxins. Lead acetate induced endotoxin hypersensitivity or LAL methods are good tools for demonstration of the role of endotoxin in the pathogenesis of various processes. For this reason, the experimental endotoxin shock is used a model of septic and other shocks.

Role of endotoxins and bile acids in the pathogenesis of septic circulatory shock.

It has long been known that the toxic effects of endotoxins under experimental conditions can be induced only when they are administered parenterally. However, in naturally occurring enteroendotoxemic diseases (e. g. septic and intestinal ischemic shocks) the endotoxins--which are produced by gram negative members of intestinal flora-, absorb from the intestinal tract to the blood circulation and can elicit pathological processes. It is an important distinction between natural and experimental endotoxin shock. If the common bile duct of rats were chronically cannulated a significant amount of perorally administered endotoxin was absorbed into the blood. This endotoxin shock can be prevented by bile acids. The physiological surfactants, the bile acids, are important facts in the defense of macroorganisms against endotoxins (physico-chemical defense). The production and passage of bile acids depend from the function of liver and the cholecystokinine (CCK) synthesis of small intestine wall. If the bile (bile acid) content of the intestinal canal decreases the endotoxin can translocate to the body and elicits toxic symptoms. So most important parts of defense against endotoxins in natural conditions are the CCK and bile acids. The consequence of damage of liver (place of bile acid synthesis) or small intestine (place of CCK synthesis) is the absorption of endotoxins.

Insoluble glycogen, a metabolizable internal adsorbent, decreases the lethality of endotoxin shock in rats.

Insoluble glycogen is an enzymatically modified form of naturally occurring soluble glycogen with a great adsorbing capacity. It can be metabolized by phagocytes to glucose. In this study we used insoluble glycogen intravenously in the experimental endotoxin shock of rats. Wistar male rats were sensitized to endotoxin by Pb acetate. The survival of rats were compared in groups of animals endotoxin shock treated and non-treated with insoluble glycogen. Furthermore, we have determined in vitro the binding capacity of insoluble glycogen for endotoxin, tumour necrosis factor alpha, interleukin-1 and secretable phospholipase A2. Use of 10 mg/kg dose of insoluble glycogen could completely prevent the lethality of shock induced by LD50 quantity of endotoxin in rats. All animals treated survived. Insoluble glycogen is a form of 'metabolizable internal adsorbents'. It can potentially be used for treatment of septic shock.

Bacterial endotoxins and nonspecific resistance.

The stress situations, including medical intervention (e.g. operations, antitumor drugs, irradiation, etc.) decrease the nonspecific resistance of the body. In these situations patients people have greater chance to get an opportunistic infection than healthy ones. The restoration or elevation of the activity of immune system in injured patients is a very important task of medicine. Minute amounts of bacterial endotoxin (LPS)--given parenterally--can elevate the nonspecific resistance. Unfortunately this beneficial influence is associated with noxious properties. Irradiation (60 Co-gamma; 150 kGy) is a good technique for the detoxification of LPS. The radiodetoxified endotoxin (RD-LPS) preparation (so-called TOLERIN) is less toxic but its beneficial properties is preserved. On the basis of animal experiments and clinical trials TOLERIN could be a suitable preparation for regeneration of the lymphoreticular-immune system and elevation of nonspecific resistance.

The effect of trace elements on the immune response of rats with intact or injured immune system.

We studied the effect of a trace element combination (Béres Drop Plus; BDP) on the immune response of rats, with intact and with injured immune system. Rats were treated with different doses of BDP for 26 days. The immune system was injured by 7.0 Gy whole body gamma irradiation on the first day of BDP treatment. Rats were immunized on the 31st day of the BDP treatment with sheep red blood cells. In the spleen the cell count and the number of antibody producing cells were determined. In the serum the titre of hemolysin was tested. In rats with intact immune system all used doses of BDP induced elevation of the immune response. The moderate high doses were especially effective. In rats with injured immune system the effect was less. Only the 250 microliters/kg body weight dose could elevate the immune response, and the highest (500 microliters/kg) dose reduced the response.

[In vitro synergic lymphocyte stimulation in HIV-1 infected patients using inosine derivatives and radio-detoxified endotoxin]. / HIV-1 fertözöttek lymphocytáinak in vitro együttes serkenthetösége inozin származékokkal és sugárdetoxikált endotoxinnal.

Restoration of immune functions through promoting cell cycle might delay acquired immunodeficiency syndrome development. Therefore, stimulation of peripheral lymphocytes of human immunodeficiency virus-1 infected patients in successive clinical stages was studied by phytohaemagglutinin and other stimulants. In vitro blastogenesis was quantitated by 3H-thymidine uptake. Stimulation by phytohaemagglutinin decreased in patients with AIDS related complex to 63.1%, with AIDS to 13.6% of control values. Small amount of recombinant interleukin-2 or indomethacin solely not promoting lymphocytes, increased response to phytohaemagglutinin minimally. Alone ineffective methyl-ester and methyl-phosphonate inosine derivatives augmented phytohaemagglutinin-response of controls and patients with AIDS related complex by approx. 1.5-fold, but the effect in the case of AIDS patients was minimal. Radio-detoxified endotoxin alone or in combination with phytohaemagglutinin stimulated lymphocytes of both controls and patients with AIDS related complex slightly. Lymphocyte stimulation of patients with AIDS related complex was augmented in concentration-dependent manner, and by synergic effect it approached phytohaemagglutinin-stimulated blastogenesis of controls. Anergy due to human immunodeficiency virus-1 infection damages synchronisation of secondary messenger systems induced on cell surface receptors, therefore their selective influence by recombinant interleukin-2 or indomethacin is less efficient. Inosine derivatives promote cell cycle by inhibiting cyclic adenosine 3',5'-monophosphate production. In the early stage of virus infection, radio-detoxified endotoxin might bind to receptors of immature T cells and facilitate cell cycle through cyclic guanosine 3',5'-monophosphate stimulation. The clinical trials of radio-detoxified endotoxin (Tolerin) have already been launched.

Radiodetoxified endotoxin-induced tolerance. Effect on endotoxin lethality and macrophage arachidonic acid metabolism.

Endotoxin (LPS) tolerance produces changes in macrophage mediator production which is thought to be responsible for the acquired LPS resistance. Detoxification of LPS by gamma irradiation has been reported to diminish certain noxious properties while retaining its tolerance inducing actions. We compared the efficacy of LPS and radiodetoxified (RD)-LPS from Escherichia coli O101 on stimulating rat peritoneal macrophage arachidonic acid (AA) metabolism, measured by thromboxane (TXB2). Changes in macrophage production of these mediators were also assessed after tolerance induction. LPS tolerance was induced by i.p. injection of LPS, RD-LPS or vehicle on day 1 (100 micrograms/kg, i.p.) and day 2 (500 micrograms/kg, i.p.). On day 5 or 4 weeks after pretreatment, peritoneal macrophages were harvested for in vitro studies, or rats were tested for lethality resistance. Macrophages were incubated +/- LPS (0.1 ng to 50 micrograms/ml), lipid A (1 or 10 micrograms/ml) or Ca+2 ionophore A23187 (10 microM) for determination of TXB2 production. Minimum effective concentrations of LPS and RD-LPS for stimulation (P < 0.05) of TXB2 were 100 ng/ml and 1 microgram/ml, respectively. Maximal stimulation of TXB2 occurred at 10 micrograms/ml of LPS or RD-LPS. Macrophages from LPS or RD-LPS tolerized rats were refractory to stimulated TXB2 with LPS or RD-LPS (0.1 ng to 50 micrograms/ml). The suppressed in vitro macrophage TXB2 production was apparent 4 weeks after rats were tolerized with LPS or RD-LPS. In subsequent mortality studies, LPS challenge of control or tolerance rats at day 5 in vivo with Salmonella enteritidis LPS (15 mg/kg, i.v.) produced a 90% mortality in control rats (N = 22), versus 13% mortality in the LPS pretreated group (N = 23) and a 20% in the RD-LPS pretreated group (N = 10) (P < 0.05 vs control). However, this lethality resistance was not apparent at 4 weeks after LPS or RD-LPS pretreatment. Both LPS and RD-LPS appear to be equipotent in inducing macrophage alterations, and in lethality resistance during LPS tolerance induction. However, these observations suggest that during LPS tolerance suppression of LPS-stimulated AA in peritoneal macrophage metabolism persists longer than acquired lethality resistance.

Effect of irradiation on the GTP binding kinetics of chicken embryo brain plasma membranes.

The membrane bound form of the catalytic subunit of adenylate cyclase of chicken embryo brain has been found earlier to be rather radioresistant [28]. The radiation induced changes of G proteins of membrane preparations of 19 day old chicken embryo brains were investigated in this work. The activation of catalytic subunit of adenylate cyclase by G protein dependent activators (Gpp/NH/p and NaF) was found elevated at lower radiation doses (0-400 Gy), while both basal enzyme activity (measured without any activator) and the activity measured in the presence of activators decreased at higher doses (above 800 Gy). Heterogeneity of GTP binding sites measured with 3-H-Gpp/NH/p a GTP-ase resistant GTP analogue was observed in the case of control (with Kd1 = 0.0663 +/- 0.034 mumol/l, Bmax = 0.0079 +/- 0.0022 nmol/ml and Kd2 = 2.038 +/- 0.4779 mumol/l, Bmax2 = 0.0291 +/- 0.0017 nmol/ml). The lower affinity high capacity binding sites seemed to be more radiosensitive, than the higher affinity sites. A marked decrease was observed in the number of low affinity binding sites above 200 Gy and these low affinity binding sites practically disappeared after irradiation with 400 Gy. At high doses (above 1600 Gy) the catalytic subunit was damaged, too. On the basis of the decrease of low affinity binding sites together with an increase in activation of the catalytic subunit via G proteins one can conclude that it is caused by radiation induced damage of Gi protein that can be more radiosensitive, than Gs protein.