Injection Of Bovine Serum Albumin Research Articles

Anaphylaxis in Mast Cell-Deficient Mice

Evidence of a functional deficit in mast cell-deficient mice was sought by testing for the development of systemic anaphylaxis. W/Wv mast cell-deficient mice and wild type control animals were sensitized by injection of bovine serum albumin and subsequently challenged. Nine of ten W/Wv mast cell-deficient mice and 10/10 control mice demonstrated signs of anaphylaxis, including death. Although histamine levels were higher in control animals, no differences were noted before and after antigen challenge. The numbers of peripheral blood basophils were the same in W/Wv and wild type mice. These studies suggest that caution is necessary in the use of these animals to study disease.

An acute ocular inflammatory reaction induced by intravitreal bovine serum albumin in presensitized rabbits: the effect of phentolamine.

An acute inflammatory reaction in the uvea was challenged by an intravitreal injection of bovine serum albumin (BSA) in presensitized rabbits. During the observation time of 3 h, intraocular pressure (IOP) increased, anterior uveal blood vessels were dilated, miosis was produced, and as a sign of the breakdown of the blood aqueous barrier, protein content in the aqueous humour was increased. In the contralateral eye, the reaction consisted of an increase in IOP and a disruption of the blood aqueous barrier. As an evidence of the immunological mechanism of this acute reaction, presensitized animals intravitreally injected with saline or unimmunized rabbits which were injected with BSA showed no significant changes in IOP, pupillary diameter or protein content in aqueous humour. Phentolamine significantly reduced protein leakage into the anterior chamber of the experimental eye. It also significantly inhibited the increase in IOP and protein leakage in the contralateral eye. The present findings demonstrate that a hypertensive inflammatory phase is present in the acute experimental uveitis. The mechanisms of the reaction and the mode of action of phentolamine are discussed.

Mouse paw anaphylaxis

Anaphylactic swelling passively transferred to the hind paws of mice with homologous antibodies was investigated. The ascites fluid containing homocytotropic antibodies of the IgG1 class in a high concentration was obtained by repeated injections of bovine serum albumin in complete Freund's adjuvant into the peritoneal cavity of female ICR mice. The standard procedure for mouse paw anaphylaxis defined was as follows: female ICR mice received subcutaneously 25 μl of a 1:20 diluted ascites in the hind footpad and 2 h later were given intravenously 1.0 mg of the antigen in saline, and subsequently at 5 min intervals paw thickness was assessed by means of a simple thickness gauge. Maximal paw edema occurred invariably 15 min after antigen challenge. Sex differene in response was not observed. Heating at 56°C for 4 h resulted in a reduction of the activity of mouse ascites by approximately 40%. Histological examinations revealed that paw anaphylaxis was accompanied by mast cell degranulation and release of biologically active constituents from mast cell granules. Promethazine, pyrilamine, diphenhydramine, methysergide, cyproheptadine, ketotifen and phenoxybenzamine provided significant protection from the anaphylactic reaction in mice, while bromocriptine, dexamethasone and indomethacin were inactive. Relative drug effectiveness in paw anaphylaxis, passive cutaneous anaphylaxis and active and passive systemic anaphylaxis in mice was compared and discussed. Consequently, it was shown that mouse paw anaphylaxis was not only convenient for quantitative assessment of homocytotropic antibodies, but also useful in he routine work for screeningof potential anti-allergic drugs.

The macrophage-phagocyte system in rabbits with experimentally induced serum sickness: functional studies.

The objective of this study was to measure the phagocytic functions of the spleen and the liver and, if possible, to correlate them with the morphology and the immunohistology of these organs in rabbits with serum sickness. Serum sickness was induced by repeated intravenous injections of bovine serum albumin (BSA). Rabbits with acute serum sickness (stage A of serum sickness), with chronic serum sickness (stage C), rabbits in a stage of immunization between stage A and stage C (stage B), and rabbits that did not respond to the antigenic challenge [nonresponders (NR)], as well as nonimmunized (NI) rabbits were used. The function of the spleen and of the liver was measured by the clearances of 51Cr-labeled heat damaged (HE), periodate-treated (PE), or immunoglobulin G (IgG)-coated (IgGE) autologous erythrocytes. In NR rabbits the functions of the spleen and liver were normal. In rabbits in stage A of serum sickness the clearance of HE, the only one measured in this stage, was decreased. In rabbits in stage B the clearances of HE and IgGE were determined; both clearances were decreased. In rabbits in stage C, the clearances of HE and IgGE were decreased, while the time of clearance of 50% (T 1/2) of PE was not significantly prolonged. In addition, in rabbits in stages A, B, and C the ratio of whole spleen vs liver radioactivity was increased. In rabbits in stage B the clearance of HE returned to normal 3 days after discontinuation of BSA injections. While all animals with detectable morphologic and immunohistologic changes of the spleen and the liver had prolonged clearances of treated erythrocytes, some animals with similarly prolonged clearances lacked evidence of pathology of these organs. The results obtained indicate that: (1) In NI and in NR rabbits, treated erythrocytes are rapidly removed by the macrophage-phagocyte system (MPS) and preferentially by the liver. (2) In rabbits in stages A, B, and C of serum sickness the clearances of treated erythrocytes are significantly prolonged. Moreover, treated erythrocytes appear to be preferentially removed by the spleen, indicating that the ability of the spleen to clear treated erythrocytes is less impaired than that of the liver. (3) In stages A, B, and C of serum sickness the nonimmunologic clearance of autologous erythrocytes is as altered as the clearance that occurs via Fc receptors. (4) In rabbits in stage B of serum sickness recovery of MPS function occurs after the interruption of BSA injections, suggesting reversible damage.(ABSTRACT TRUNCATED AT 400 WORDS)

Intravenously Administered Lecithin Liposomes: A Synthetic Antiatherogenic Lipid Particle

INTRAVENOUSLY ADMINISTERED LECITHIN UPOSOMES: A SYNTHETIC ANTIATHEROGENIC UPID PARTICLE KEVINJON WILUAMS* VICTORIA P. WERTHJ andJON A. WOLFFt 1. Introduction Almost 3 decades ago, intravenous administration of lecithin was shown to induce regression of experimental atherosclerosis in rabbits [1—6]. This was confirmed by several groups using different animal models [7-9]. No satisfactory explanation for this phenomenon has been offered [4, 5, 8, 10-12]. We propose that lecithin induces regression of experimental atherosclerosis by forming a synthetic antiadierogenic lipid particle, the lecithin liposome. We are proposing the following. Intravenously injected lecithin forms circulating liposomes, which take up cholesterol from many sources, including the arterial wall. In this way, lecithin liposomes become carriers ofendogenous cholesterol. These liposomes are gradually removed from the circulation by the liver, which catabolizes liposomes and excretes their cholesterol. Mobilization and excretion of endogenous cholesterol are further enhanced by the interactions of circulating liposomes with native lipoproteins and red blood cells. For example, circulating liposomes pick up apoproteins from native lipoproteins. Apoproteins directly augment the ability of liposomes to extract tissue cholesterol and may facilitate their catabolism by the liver. In addition, liposomes remove cholesterol from, and donate phospholipid to, native lipoproteins. These modifications The authors thank Simeon Margolis and Henry Seidel of The Johns Hopkins University , Ferd E. Williams of the University of Delaware, and Anne L. Williams for their generous guidance and support. ?Department of Internal Medicine, Division of Endocrinology, Yale University, 333 Cedar Street, New Haven, Connecticut 06510. (Work done while at the University of Chicago.) tDepartment of Medicine, Northwestern University, Chicago, Illinois 60611.¿Department of Pediatrics, University of California, San Diego, California 92103.©1984 by The University of Chicago. AU rights reserved. 0031-5982/84/2703-0396$01.00 Perspectives in Biology and Medicine, 27, 3 ¦ Spring 1984 \ 417 may enhance the ability of native lipoproteins to extract cellular cholesterol and may reduce any further lipoprotein-mediated transport of cholesterol into the arterial wall. Finally, liposomes remove cholesterol from the membranes of red blood cells. Cholesterol-depleted red blood cells may act as additional intermediaries in the extraction of arterial cholesterol deposits. In die following sections, we first review die studies demonstrating regression of experimental atherosclerosis with intravenous administration of lecithin. We then examine the evidence diat lecidiin mobilizes tissue cholesterol by the formation of liposomes composed of lecithin and cholesterol, that these liposomes are disposed of by die liver, and Uiat interactions of liposomes with lipoproteins and red cells enhance these processes. Finally, we review literature from which we infer that die cholesterol of human atheromata may be susceptible to mobilization and excretion by intravenously administered liposomes. 2. Regression ofExpérimental Atherosclerosis with I.V. Lecithin The cholesterol-fed rabbit is widely used as an experimental model of atfierosclerosis. In diis model, rabbits fed on a diet containing 1-5 percent added cholesterol develop witfiin 4—12 weeks deposits of cholesterol in many tissues, including die intima of large arteries. These intimai deposits remain die same size or even progress when the rabbits are returned to their usual cholesterol-free diet [13]. In contrast, intravenous administration of lecidiin preparations of varying purity (0.53 .5 g two to three times a week) resulted in rapid, substantial regression of the intimai lesions, with resolution in 1-4 weeks or after a total phospholipid dose of about 2-12 g [1-4, 6]. Lecithin-induced regression occurred even while the rabbits were maintained on die cholesterol-rich, adierogenic diet [6]. Similar results were observed in other models. Experimental atherosclerosis produced in rabbits by a semisyndietic cholesterol-free diet rich in saturated fat regressed with infusions of lecithin [5]. In baboons, the incidence and severity of experimental atherosclerosis, induced over 6 months by combining cholesterol feeding widi injections of bovine serum albumin (to produce vasculitis and intimai injury), were significandy reduced by concurrent thrice-weekly infusions of polyunsaturated lecidiin [7]. Quail fed on a high-cholesterol diet for 3 mondis developed intimai deposits diat resolved during 3 subsequent months of intravenous lecidiin administration, despite continuation of the adierogenic diet [8]. Extravascular cholesterol deposits in the subcutaneous tissues of rats regressed witfi topical lecidiin [9]. In several of diese experimental models, polyunsaturated lecidiin from soybeans...

Cardiac cAMP and cGMP dynamics in rats with transient coronary insufficiency

Experiments on rabbits have shown hyperlipidemia to develop within the first 48 h after a single intravenous injection of bovine serum albumin (BSA, fraction V). The mean concentration of blood plasma triglycerides (TG) was considerably higher than normal (by 262% after 24 and by 625% after 48 h). The cholesterol content was also elevated (by 80 and 270%, respectively). Following 7 and 14 days the lipid concentration returned to normal. The plasma post-heparin lipoprotein lipase activity (PHP-LPL) was lower 24 h and 7 days after BSA injection and the hypotriglyceridemic effect of heparin was less pronounced. The data obtained support the hypothesis that hyperlipidemia provoked by a single intravenous injection of BSA to rabbits results from low PHP-LPL activity and possible changes in TG-rich lipoprotein substrate affinity for the enzyme.

Participation of auto-anti-idiotypes in immune complex glomerulonephritis in rabbits.

We report the presence of auto-anti-Id antibodies in the glomerular immune deposits of a rabbit with chronic serum sickness induced by daily i.v. injections of bovine serum albumin. Auto-anti-Id antibodies were also documented in the serum of the same animal during the phase of chronic serum sickness in which immune complex glomerulonephritis develops. These results suggest that auto-anti-Id may normally participate in pathologic reactions and may be one of the mechanisms of potentiation and/or progression of immunopathologic lesions.

A mechanism of transient immune deposition in the skin.

ABSTRACTTo gain further insight into the mechanism of immune deposition in the skin, antigens and antibodies were administered intravenously at different times into normal guinea pigs, and the presence of immune complexes in frozen sections of skin was determined immunohistochemically, using light microscopy. Cutaneous immune deposits were found after primary injection of anti‐horseradish peroxidase antibody (IgG) followed by secondary administration of horseradish peroxidase, while no immune complexes were detected after the same injections in the reverse order. This disparity may possibly be explained by the length of time the antigen or antibody remains in the skin. Using a primary injection of bovine serum albumin (BSA), followed by secondary injection of anti‐BSA antibody, immune deposits were demonstrated in the skin. In these cases, if the initially injected antigen or antibody remained for a long time at the site of exudation, they would react with and form immune complexes with the subsequently injected antibody or antigen at the same site.

Colloidal gold, a useful marker for antigen localization on follicular dendritic cells

Bovine serum albumin (BSA) bound to colloid gold particles (BSA-gold; 20 nm diameter) and injected into preimmunized mice was found at ultrastructural level in different locations of the lymph nodes. It was detected particularly in the secondary lysosomes of macrophages and between the cytoplasmic processes of the follicular dendritic cells. Between these processes the gold particles were isolated or grouped in clusters; they were in close contact with cell membranes or embedded in dense material. Colloidal gold injected alone was not retained on these cells. The presence of anti-BSA antibodies in the serum was necessary for trapping of BSA-gold particles on follicular dendritic cells. Injections of BSA alone after BSA-gold had been administered to preimmunized mice eliminated most of the BSA-gold from the dendritic processes. BSA-gold is thus trapped in the form of immune complexes which behave characteristically. BSA-gold is thus a suitable marker for antigen localization. Being small and electron dense it permits more precise location than radioactive markers.

Resistance of T suppressor cells to cyclophosphamide administration in total-lymphoid-irradiated mice.

The effect of administration of cyclophosphamide (Cy) on the susceptibility of mice to tolerance induction and its influence on the rate of induced T suppressor cells in antigen-specific and nonspecific systems was assessed using mice that had undergone total lymphoid irradiation (TLI). Tolerance to bovine serum albumin (BSA) was induced in adult mice conditioned by a short course of fractionated total lymphoid irradiation with an injection of nondeaggregated BSA; Cy was administered upon termination of TLI, two days prior to administration of the tolerizing antigen. Susceptibility to tolerance induction and rate of induced T suppressor cells were assessed one month later. Cy-treated TLI mice and mice treated with TLI alone did not respond to a challenge with DNP-BSA in complete Freund's adjuvant, whereas a good response was obtained to an unrelated protein antigen. The induced T suppressor cell rate was measured by inhibition of a secondary anti DNP-BSA response in an adoptive transfer system. The rate of non-antigen-specific T suppressor cells was measured by inhibition of the mixed leukocyte reaction with Concanavalin A-induced T suppressor cells obtained from spleen cells of TLI-treated, or Cy-treated mice, or those given both treatments. The results reveal that Cy treatment of TLI-conditioned mice does not reduce the rate of induced of T suppressor cells in antigen-specific and antigen-nonspecific systems.

Antibody production suppressed in the domestic fowl(gallus domesticus)by zinc deficiency

Fowls maintained on a zinc-deficient diet from hatching and immunised either by intraperitoneal injections of bovine serum albumin (BSA) or by a combination of ocular drops and intraperitoneal injections showed no antibody response as judged by immunoelectrophoresis and immuno-fluorescence, whereas birds on a zinc-sufficient diet had serum antibody and anti-BSA cells in the Harderian gland, Peyer's patches, spleen and thymus. The localisation of immunoglobulins did npt differ between control and deficient birds except in the bursa of Fabricius where follicle-associated epithelium of deficient fowls stained for immunoglobulins, especially IgA, whereas in sections from control birds these cells remained unstained.

Induction of membranous nephropathy in rabbits by administration of an exogenous cationic antigen.

We examined the role of antigenic electrical charge as a determinant of glomerular immune complex localization in the rabbit. Serum sickness nephritis was induced in groups of New Zealand white rabbits by daily 25-mg intravenous injections of bovine serum albumin (BSA) chemically modified to be cationic (pI > 9.5) or more anionic (pI, 3.5-4.6); an additional group received unmodified native BSA (pI, 4.5-5.1). Factors known to influence immune complex localization, e.g., molecular size of the administered antigen and resulting circulating immune complexes, immunogenicity, and disappearance time from the circulation were examined and found to be similar for both anionic and cationic BSA. Charge modification did increase the nonimmune clearance of cationic and anionic BSA compared with native BSA. Injected cationic BSA was shown in paired label experiments to bind directly to glomeruli compared with native BSA. The renal lesion produced by cationic BSA was markedly different from that found in rabbits given anionic or native BSA. Animals receiving cationic BSA uniformly developed generalized diffuse granular capillary wall deposits of IgG, C3, and BSA detected after 2 wk of injections and increasing until death at 6 wk. Qualitatively similar deposits were produced by the administration of low doses of cationic BSA of only 1 or 10 mg/d. In contrast, the injection of both anionic and native BSA resulted in mesangial deposits at 2 and 4 wk with capillary wall deposits appearing by 6 wk. Ultrastructural examination of animals receiving cationic BSA revealed pure, extensive formation of dense deposits along the lamina rara externa of the glomerular basement membrane whereas such deposits were absent or rare in animals injected with the anionic or native BSA. Albuminuria was significantly greater at 6 wk in the groups receiving cationic BSA with a mean of 280 mg/24 h compared with 53 mg/24 h in the combined groups injected with anionic or native BSA. Blood urea nitrogen values were similar in all groups at 2 and 4 wk but higher in the animals receiving cationic BSA at 6 wk. These experiments describe the reproducible induction of epimembranous immune deposits by administration of an exogenous cationic antigen. They suggest that antigenic charge can play an important role in the pathogenesis of membranous nephropathy by permitting direct glomerular binding of an antigen and predisposing to in situ immune complex formation.

Involvement of the macrophage in experimental chronic immune complex glomerulonephritis.

Serial renal biopsies for glomerular culture, histochemical staining for beta-glucuronidase, electron microscopy (EM) and light microscopy, were used to study macrophage involvement in experimental chronic immune complex (IC) glomerulonephritis (GN) induced in rabbits by daily intravenous injections of bovine serum albumin (BSA). In the 26 animals studied, proliferative GN of variable severity was induced, with mild disease in 5 animals, moderate proliferation in 15 and crescentic GN in 6. Macrophages first appeared in glomerular culture outgrowths during the 2nd and 3rd weeks, coincident with the onset of proteinuria and rising serum creatinine concentration. Large numbers of macrophages (in excess of 20 per glomerulus) were seen by the 5th weeks and persisted to the 9th week. The number of macrophages in outgrowths was not significantly greater in animals with crescentic disease. EM demonstrated macrophages in capillary loops, and in glomeruli with crescents, macrophages could be seen in the urinary space. Histochemical staining for beta-glucuronidase also demonstrated macrophages in the glomerular tuft and in crescents when present. These results indicate that macrophages constitute a considerable proportion of the glomerular hypercellularity seen in chronic IC glomerulonephritis.

Effect of immune complexes on plasma HDL-cholesterol in rabbits

The effect of immune complexes on plasma lipids, especially on high density lipoprotein (HDL) cholesterol, was studied in rabbits. In rabbits immunized twice with bovine serum albumin (BSA) with an interval of about 6 weeks, a significant decrease in plasma HDL-cholesterol ( P < 0.01) and in the HDL/total cholesterol ratio ( P < 0.02) was found 6 days after the second BSA injection. As a result of repeated bleedings commenced 6 days after the second BSA injection, the changes in plasma triglycerides (TG) and total cholesterol (TC) were similar to those found previously in non-immunized rabbits. In contrast to the findings in non-immunized rabbits, no significant decrease in HDL-cholesterol was found as a consequence of the bleedings, but a negative correlation ( P < 0.05) between plasma TG and HDL-cholesterol appeared. It is concluded that immune complexes may affect plasma HDL-cholesterol concentrations and thereby possibly also the development of atherosclerosis.

Cerebrospinal fluid and the choroid plexus during acute immune complex disease

During acute immune complex disease in the rabbit, immune deposits could be detected in the choroid plexus, as well as the kidney, in a majority of the animals sacrificed from 9 to 14 days after the injection of bovine serum albumin (BSA). During this time there was a good correlation between deposits in the choroid plexus and increases in cerebrospinal fluid (CSF) albumin and IgG. A relatively unchanged CSF IgG to albumin ratio was observed, while the CSF to serum albumin level was increased, suggesting an alteration in the blood—CSF barrier permeability to serum proteins. In addition, the temporal appearance of BSA and anti-BSA in the CSF of these animals mirrored their appearance in the serum and in one animal immune complexes could be detected in the CSF. The significance of these results is discussed in relation to the pathogenesis of central nervous system (CNS) disease in systemic lupus erythematosus (SLE).

Fever induced in rabbits by intravenous injection of bovine serum albumin.

1. Bovine serum albumin injected into conscious rabbits produced a dose-dependent fever of short latency and duration of at least 2 1/2 hr. 2. The fever was not due to contamination by other proteins nor due to contamination by bacterial endotoxin. 3. The fever following albumin injection had characteristics similar to endogenous pyrogen fever. 4. Some traditional ways of distinguishing bacterial endotoxin fever from other types of fever need to be reassessed.

OCULAR ANTI-INFLAMMATORY AND SYSTEMIC IMMUNOSUPPRESSIVE EFFECTS OF TOPICALLY APPLIED FLUOROMETHOLONE

Anti-inflammatory and systemic immunosuppressive effects of topically applied steroidal and nonsteroidal anti-inflammatory agents on experimental immunogenic uveitis in rabbits were studied. The onset of the immunogenic uveitis induced by a single intravitreous injection of bovine serum albumin (BSA) was significantly correlated with the appearance of anti-BSA antibody in the serum. A significant correlation was also found between the maximum serum level of antibody and the maximum severity of uveitis. Suspensions of fluorometholone, dexamethasone or indomethacin were applied topically twice a day for a month to eyes injected with BSA. Ocular inflammation was suppressed by fluorometholone and dexamethasone, fluorometholone having the greater anti-inflammatory activity. The circulating antibody titer was also suppressed by both these steroids, but conversely fluorometholone had the lower systemic immunosuppressive activity. Indomethacin did not suppress the uveitis or circulating antibody production. The results of these investigations indicate that the anti-inflammatory effect of the steroid fluorometholone surpasses the immunosuppressive effect, unlike usual anti-inflammatory steroids.

Delayed Type Hypersensitivity to Bovine Serum Albumin and to Lipid-Conjugated Bovine Serum Albumin in Mice

Delayed type hypersensitivity (DTH) to bovine serum albumin (BSA) and to lipid-conjugated BSA were studied comparatively. Unlike the case of BSA with which no DTH can be detected with native antigen, injection of butyric-conjugated BSA (Bu-BSA) in sensitized mice provokes a typical DTH for an early and limited period. Alum-precipitated Bu-BSA (Al-Bu-BSA) provokes from the beginning a stronger DTH which persists a much longer period.

Experimental Chronic Serum Sickness in Rats

This article describes a method of immunization that produces chronic serum sickness in rats within a relatively short time. Fisher rats, which were immunized subcutaneously three times with bovine serum albumin (BSA) in adjuvant, responded with high titers of antibodies to BSA. 2 weeks after the third subcutaneous immunization, daily increasing amounts of BSA were injected either intraperitoneally or intravenously. When an intravenous dose of 2mg of BSA was reached, the rats were given daily intravenous injections of BSA for several weeks. This procedure, which avoided death from anaphylaxis, induced severe proliferative glomerulonephritis in all the rats and produced deposition of antigen-antibody complexes in many other organs besides the kidney. This highly reproducible model of experimental chronic serum sickness in inbred animals may have applications for the study of the mechanisms of immune complex disease.

Acute inflammation following intraperitoneal injection of antigen into actively sensitised rats.

Rats actively sensitised to bovine serum albumin (BSA) in Freund's complete adjuvant were challenged by intraperitoneal injection of BSA. By washing out the peritoneal cavities at different times it was possible to divide the changes that occurred into two phases. The first was characterised by the release of histamine and slow reacting substance of anaphylaxis (SRS-A) with an associated extravasation of plasma proteins. The second phase was characterised by an increase in neutrophilic polymorphonuclear cells in the peritoneal cavity. Extracellular enzyme activites were also investigated. The kinetics of these events were studied and are discussed in terms of the possible mechanisms involved.