Studies on the Fate of Different 125I-DNP-HSA Conjugates in Mouse Peritoneal Cells, Spleen and Liver in Vivo, and in Mouse Peritoneal Cells in Vitro

The degree of uptake of DNP ¹²⁵I-HSA (= DNP *HSA) conjugates by mouse peritoneal exudate (PE) Cells in vivo and their retention in The- spleen and liver depended on the density of hapten per carrier molecule. Forty to 400 times more DNP₂₉*HSA and DNP₃₉*HSA were ingested by PE cells as compared to HSA alone. Significantly more DNP₂₉*HSA and DNP₃₉*HSA persisted in the spleen and liver after intraperitoneal injection than *HSA, DNP₆*HSA or DNP₁₁*HSA. *HSA, DNP₆*HSA, and DNP₁₁*HSA were rapidly catabolized after uptake in macrophages in vitro, but a small amount of these antigens was associated with the cells. About 50% of the *HSA was membrane-bound. *HSA conjugated with DNP₂₉ or DNP₃₉ was more rapidly catabolized by PE cells than when it was present in low-density hapten-carrier conjugates. More of the high-density conjugates were associated with the PE cells than was the case with the other conjugates, and again about 50% of the radioactivity was msembrane-bound. Thus, the degree of metabolism and persistence of *HSA in vitro was also related to the density of hapten on the carrier.     

Antigen in tissues: V. Effect of endotoxin on the fate of, and on the immune response to, serum albumin and to albumin—antibody complexes*

Bacterial endotoxin was injected into rat hind footpads together with bacterial flagellin and ¹²⁵I-labelled human serum albumin (HSA); the latter was used unmodified or heat denatured (H.HSA) or as an HSA—antibody complex. Endotoxin did not affect the trapping, retention nor localization of the labelled HSA in the popliteal and aortic lymph nodes, whether the antigen had been injected as HSA, H.HSA or as an HSA—antibody complex.

If endotoxin was injected at the same time as: (1) flagellin, there was an increased production of anti-flagellin antibody; and (2) H.HSA or HSA—antibody complex, detectable amounts of anti-HSA antibody were produced. When H.HSA and endotoxin were injected, the primary response was long lived yet the period of induction of antibody formation and of antigen persistence in the lymphoid tissues was short. If, during the primary antibody response to H.HSA, the animals were challenged with HSA, equally strong secondary antibody responses occurred with an HSA—antibody complex or with HSA alone.

The results were interpreted in terms of the tissue localization pattern of H.HSA (medullary macrophage) and HSA—antibody complex (medulla and lymphoid follicles). It was suggested that: (1) induction of antibody formation and priming of cells for a secondary antibody response might occur following localization of the antigen in the medulla and that antigen localization in the lymphoid follicles might not be a strict requirement for this; and (2) the follicular localization of antigen might be the preferential mechanism for the firing of a secondary antibody response.

     

In Vitro Regulation of the Anamnestic Antibody Response Upon Addition of Heterologous Antigens to Primed Lymphoid Cells

Alum-precipitated keyhole limpet hemocyanin (KLH) and polymerized human serum albumin (HSA) were injected into the hind foot pads of rabbits. Many months later the popliteal lymph nodes were removed and cell cultures prepared and incubated in media containing different concentrations of KLH or native HSA. These primed cells responded to some concentrations of KLH with the synthesis not only of homologous antibody but of heterologous IgG antibody to HSA. They also responded to some concentrations of HSA with the production of heterologous antibody to KLH as well as homologous antibody. In some experiments the addition of KLH induced the cells to synthesize as much antibody to HSA as did the addition of HSA itself. The nonspecific stimulation of the anamnestic antibody response to HSA required the cultures to contain KLH, HSA, KLH-reactive memory cells and, presumably memory cells reactive with HSA.

The suspension of KLH-and HSA-primed cells responded to other concentrations of KLH or egg albumin (EA) with inhibition of the antibody response to HSA. This non-specific inhibition did not require lymph node cells which had been primed by the previous injection of KLH or EA.

KLH and HSA did not cross-react with respect to immunogenicity in vitro or in vivo or reactivity with antibody in vitro.

According to a model which was proposed, the KLH induced KLH-reactive T lymphocytes to produce soluble factor(s) which regulated the response of HSA-reactive B lymphocytes to HSA-specific determinants which persisted from the original injection.     

高渗盐溶液对失血性休克大鼠红细胞几何形状的影响

目的:采用微管吸吮技术,观察高渗盐溶液对失血性休克大鼠红细胞几何形状的影响。方法:Wistar大鼠随机分为0.9%NaCl(NS)、7.5%NaCl(HS)、5%NaCl-3.5%NaAc(HSA)3组。10min内放血使平均动脉压降至5.3kPa,维持90min。休克模型复制完成后,分别按4mL/kg体重静脉注入NS、HS、HSA,5min内输完。取血测定休克前、后及给药后的红细胞几何形状。结果:休克后红细胞表/体比值、球度指数无明显变化,红细胞直径较休克前下降非常显著。治疗后,HS组与NS、HSA组比较,表/体比值降低非常显著;HS组和HSA组球度指数非常显著高于NS组,HSA组球度指数非常显著低于HS组。HS组和HSA组红细胞直径非常显著低于NS组。结论:HS和HSA不利于失血性休克大鼠红细胞几何形状的改善。HSA对红细胞几何形状的不利影响低于HS。     

The influence of adjuvants on the immunological response of the chicken: I. Effects on primary and secondary responses of various adjuvants in the primary stimulus

The effect of various adjuvant procedures on the antibody response (first 21 days) for human serum albumin (HSA) has been studied in the chicken. The lack of effect on the circulating antibody level contrasts with their action in some mammals.

The administration of depôt-type adjuvants failed to increase the peak circulating antibody levels (8–12 days after injection of antigen) by comparison with control birds. However, the circulating antibody level declined more slowly in birds given HSA in a water-in-oil emulsion than in birds given HSA in saline.

The administration of endotoxin and `surface active' adjuvants also failed to increase the peak circulating antibody levels over that of control birds. In three experiments there was significant depression of peak antibody levels in birds given endotoxin adjuvant in comparison to control birds.

The administration of HSA in Freund's complete adjuvants containing Mycobacterium tuberculosis or Mycobacterium avium did not result in elevation of peak antibody levels compared to those of control birds given HSA in saline or HSA in a water-in-oil emulsion.

Experiments to determine the effect of adjuvants from each of the main groups on the establishment of immunological memory were performed. Chickens were given adjuvant with the primary injection of HSA. A second injection of HSA without adjuvant was given 56 days later. None of the adjuvants used produced an increase in the peak antibody level attained during the secondary response compared to control birds.

     

高渗氯化钠-醋酸钠溶液改善失血性休克大鼠微循环的作用

目的：研究高渗氯化钠－醋酸钠溶液对失血性休克大鼠微循环的作用。方法：ＳＤ大鼠随机分为７．５％ＮａＣｌ（ＨＳ）、５％ＮａＣＬ－３．５％ＮａＡｃ（ＨＳＡ）、０．９％ＮａＣｌ（ＮＳ）三组。休克模型完成后,分别按４ｍＬ／ｋｇ给药,观察脊斜肌微循环的变化。测定局部用药后,ＨＳ和ＨＳＡ对血管内径的直接作用,结果：ＨＳ较ＨＳＡ更为显著升高ＭＡＰ。给药５ｍｉｎ,ＨＳＡ、粪土中脊斜肌第三级细动静脉内径、血流速度、血     

In Vitro Regulation of the Anamnestic Antibody Response Upon Addition of Heterologous Antigens to Primed Lymphoid Cells

Alum-precipitated keyhole limpet hemocyanin (KLH) and polymerized human serum albumin (HSA) were injected into the hind foot pads of rabbits. Many months later the popliteal lymph nodes were removed and cell cultures prepared and incubated in media containing different concentrations of KLH or native HSA. These primed cells responded to some concentrations of KLH with the synthesis not only of homologous antibody but of heterologous IgG antibody to HSA. They also responded to some concentrations of HSA with the production of heterologous antibody to KLH as well as homologous antibody. In some experiments the addition of KLH induced the cells to synthesize as much antibody to HSA as did the addition of HSA itself. The nonspecific stimulation of the anamnestic antibody response to HSA required the cultures to contain KLH, HSA, KLH-reactive memory cells and, presumably memory cells reactive with HSA.

The suspension of KLH-and HSA-primed cells responded to other concentrations of KLH or egg albumin (EA) with inhibition of the antibody response to HSA. This non-specific inhibition did not require lymph node cells which had been primed by the previous injection of KLH or EA.

KLH and HSA did not cross-react with respect to immunogenicity in vitro or in vivo or reactivity with antibody in vitro.

According to a model which was proposed, the KLH induced KLH-reactive T lymphocytes to produce soluble factor(s) which regulated the response of HSA-reactive B lymphocytes to HSA-specific determinants which persisted from the original injection.     

Bystander suppression of the immune response to human serum albumin in rats fed ovalbumin.

Bystander suppression of delayed-type hypersensitivity (DTH) and the antibody response to human serum albumin (HSA) were studied in young normal rats and in young rats made partially tolerant to ovalbumin (OVA) by feeding an OVA-containing diet for 4 weeks from weaning. At 2 months of age, the animals were intracutaneously immunized with a mixture of OVA and HSA in Freund's complete adjuvant (FCA) at one site of the back, or separately at two different sites on the back. All rats made orally tolerant to OVA showed a significantly reduced IgE and IgG anti-OVA antibody production and DTH response to OVA, compared to the controls. OVA-fed rats subsequently immunized with a mixture of OVA + HSA had significantly lower IgE and DTH responses to HSA than the controls. When rats were immunized with OVA and HSA at two different sites, however, there was no difference in the response to HSA between the OVA-fed rats and the control rats, which rules out the possibility of shared epitopes between the antigens. Ear-challenge with the mixture of OVA + HSA gave a significantly lower DTH reaction in the tolerant rats immunized with a mixture of the antigens, compared to the control rats. However, suppression of the DTH reaction was not seen when tolerant and control rats were immunized with HSA alone and challenged with the mixture of OVA + HSA in one ear. These results present evidence that young rats orally tolerant to one antigen show a suppressed T-cell and antibody response to an unrelated antigen, provided that the two antigens are given in a mixture during the inductive phase. There was no evidence for bystander suppression of the T-cell response at the effector site.     

Globulin-Enriched Protein Supplements Shorten the Pre-Compaction Mitotic Interval and Promote Hatching of Murine Embryos

PROBLEM: To determine whether Synthetic Serum Substitute (SSS), which contains human globulins in addition to Human Serum Albumin (HSA), is superior to HSA alone as a protein supplement for embryo culture. METHOD: Development of mouse zygotes to eight-cell/compacting morulae and to hatching/hatched blastocysts was assessed in Human Tubal Fluid (HTF) medium containing either SSS or HSA. RESULTS: Although there was no difference in the overall blastocyst rate at 120 h in HTF+SSS versus HTF+HSA, significantly more embryos at 54 h were at the eight-cell/compacting morula stage in HTF+SSS. At 120 h, there were more hatching/hatched blastocysts in HTF+SSS, and hatching correlated with SSS concentration. Addition of isolated globulins to HSA significantly stimulated the number of hatching/hatched blastocysts. Hatching could be “rescued” by transfer of embryos grown in HTF+HSA to globulin-containing media and prevented by removal of globulins as late as the compacted morula stage (54 h). CONCLUSIONS: SSS is superior to HSA alone for embryo culture. The stimulatory effects on mitosis and hatching may be mediated directly by globulins or by other components in the globulin-enriched fraction.     

Restoration of the specific immunological reactivity of tolerant rabbits by conjugated antigens

Immunization of HSA-tolerant animals with sulphanil-HSA induces the formation of antibodies that react with native HSA. To test whether the anti-HSA response does actually represent complete restoration of the original reactivity, the antibodies produced following immunization of tolerant animals with sulphanil-HSA were compared to those produced by normal rabbits immunized with the native antigen. Gel-diffusion analysis of the precipitins disclosed that the patterns of the two types of antibody were not completely identical. The antibodies of the `restored' tolerant rabbits were directed mainly towards an antigenic determinant of the native HSA, to which normal rabbits immunized with HSA did not respond. When the `restored' rabbits were challenged with native HSA after a prolonged rest period, the antibodies formed thereafter were completely identical with anti-HSA produced by normal HSA-immunized rabbits. Thus, complete restoration of the original immunological reactivity to HSA was achieved following an intermediary stage of atypical anti-HSA elicited by the conjugated protein.

Attempts to break down natural tolerance to RSA by treatment with sulphanil-RSA failed to give any evidence of an autoimmune elimination of RSA. To test whether this inability to break tolerance to RSA was due to the presence of an excess of native protein, animals that had been made tolerant to HSA were immunized with a mixture of sulphanil-HSA and native HSA. Under such conditions, the termination of acquired tolerance was inhibited. The possible relevance of these observations to the cellular basis of immunological tolerance is discussed.

     

Phylogenetic origin of human chromosomes 7, 16, and 19 and their homologs in placental mammals

The origin of human chromosomes (HSA) 7, 16, and 19 was studied by comparing data obtained from chromosome banding, chromosome painting, and gene mapping in species belonging to 11 orders of placental mammals (Eutherians). This allowed us to propose the reconstruction of their presumed ancestral forms. The HSA7 homologs were composed of two parts, the largest forming an acrocentric. The smallest formed one arm of a small submetacentric; the other arm was composed of sequences homologous to the short arm of HSA16 (HSA16p). The sequences homologous to the long arm of HSA16 (HSA16q) were associated with sequences homologous to the long arm of HSA19 (HSA19q) and formed another submetacentric. From their origin, these chromosomes underwent the following rearrangements to give rise to current human chromosomes: centromeric fission of the two submetacentrics in ancestors of all primates (approximately 80 million years ago); fusion of the HSA19p and HSA19q sequences, originating the current HSA19, in ancestors of all simians (approximately 55 million years ago); fusions of the HSA16p and HSA16q sequences, originating the current HSA16 and the two components of HSA7 before the separation of Cercopithecoids and Hominoids ( approximately 35 million years ago); and finally, pericentric and paracentric inversions of the homologs to HSA7 after the divergence of orangutan and gorilla, respectively. Thus, compared with HSA16 and HSA19, HSA7 is a fairly recent chromosome shared by man and chimpanzee only.     

Interference of oral immunization with the intestinal absorption of heterologous albumin

Rats were immunized with human serum albumin (HSA) by a single intragastric administration of 200 mg of HSA. Two weeks later their capacity to absorb a similar intragastric test dose of HSA was found to be greatly impaired, the concentrations of HSA in mesenteric venous serum having been reduced to 33 %, 25 % and 60 % of those in similarly tested but unprimed animals, respectively 1, 2 and 3 hours after the intragastric test dose. Rats given antigen (HSA) together with intestinal secretions from intragastric immunized rats also showed a striking decrease in intestinal absorption of a test dose, compared to controls given the antigen together with nonimmune intestinal secretions. From these data it is concluded that local immunization of the gut impairs its capacity to absorb the corresponding antigen, and that this effect is largely due to the failure to absorb antigen bound to secretory antibodies.     

Characterisation of the binding of digitoxin and acetyldigitoxin to human serum albumin by high-performance affinity chromatography

Zonal elution and high-performance affinity chromatography were used to examine interactions of the drugs digitoxin and acetyldigitoxin with the protein human serum albumin (HSA). This was done by injecting small amounts of digitoxin and acetyldigitoxin onto an immobilized HSA column in the presence of mobile phases that contained various concentrations of digitoxin, acetyldigitoxin or other solutes as competing agents. A fixed concentration of beta-cyclodextrin was also present in the mobile phase as a solubilising agent. It was found that digitoxin and acetyldigitoxin each had strong interactions at a single common binding site on HSA, but with slightly different equilibrium constants for this region. Neither compound showed any competition with warfarin or L-tryptophan, which were used as probes for binding at the warfarin-azapropazone and indole-benzodiazepine sites of HSA. These results confirmed the presence of a separate binding region on HSA for digitoxin-related compounds.     

Homotypic interaction of the heat-stable antigen is not responsible for its co-stimulatory activity for T cell clonal expansion

The heat-stable antigen (HSA) is an important co-stimulatory molecule on antigen-presenting cells (APC). However, the receptor on T cells that receives the co-stimulatory signal from HSA has not been identified. Because the HSA is transiently expressed on T cells after the T cell receptor/CD3 complex is engaged, and because it can bind to itself in a homotypic fashion, it has been proposed that homotypic interaction of HSA is responsible for its co-stimulatory activity. Here we test this hypothesis using mice that have a targeted mutation of the HSA gene, as well as novel transgenic mice that constitutively express HSA on T cells. We show that HSA-deficient T cells remain responsive to co-stimulation by HSA. Furthermore, constitutive expression of HSA does not enhance T cell response to co-stimulatory by HSA. Taken together, our results demonstrate that homotypic interaction of HSA is not responsible for co-stimulation mediated by HSA expressed on APC.     

Surface charge distribution is a determinant of antigen deposition in the renal glomerulus: studies employing ''charge-hybrid'' molecules.

The deposition of antigens and immune complexes (IC) in the renal glomerulus is charge-dependent. The demonstration that molecules of net anionic charge, but with discrete positively charged regions, exhibit affinity for the glomerular basement membrane (GBM) extends this concept. Charge hybrid (polar) molecules were constructed by covalently coupling small polycations (lysozyme or linear poly-L-lysine chains with a mean of 17 and 20 residues) to larger polyanions (ovalbumin or human serum albumin (HSA]. Although the products were of overall net anionic charge they still bound to glomerular structures. Immunofluorescence studies performed after i.v. injection of the samples into rats revealed that HSA:poly-L-lysine had the highest affinity. Radioisotopic measurements showed uptake of HSA:poly-L-lysine to be a function of the number of lysine residues; binding of HSA:poly-L-lysine20 was 2.5 times higher than HSA:poly-L-lysine17 (P less than 0.01). Prior injection of a small competing polycation (polyethyleneimine 1200) reduced uptake of HSA:poly-L-lysine by 75%, indicating the charge-based nature of the interaction. HSA:poly-L-lysine20 alone was effectively eliminated from the glomeruli within 72 h. Administration of HSA:poly-L-lysine followed by anti-HSA antibody induced immune complex formation in the capillary wall, giving rise to a granular immunofluorescence pattern and discrete subendothelial and subepithelial deposits. Molecules with polar structure do occur naturally and may contribute to immune complex formation in glomerulonephritis.     

Anaphylactoid reactions to infusions of plasma protein and human serum albumin Role of aggregated proteins and of stabilizers added during production

Six patients suffering from anaphylactoid reactions after infusion of pasteurized plasma (PP) or human serum albumin (HSA) were investigated. Clinical symptoms ranged from urticaria and hypotension to cardiac arrest. Immunoglobulin levels, especially of IgA, were normal, as were concentrations of complement factors C3, C4 and factor B. In skin and lymphocyte transformation tests patients, with the exception of one severely allergic to protein, did not react to the monomeric pure HSA. Five out of six patients reacted against HSA aggregates and three patients to the HSA modified by caprylate added as stabilizer during commercial HSA production. It is concluded that the anaphylactoid reactions developing after PP or HSA infusion result from a non-specific reaction to protein aggregates and in some cases possibly from a specific immune response to the caprylate-modified HSA.     

Structure and function changes of oxidized human serum albumin: physiological significance of the biomarker and importance of sampling conditions for accurate measurement

Human serum albumin (HSA) exists in both reduced and oxidized forms, and the percentage of oxidized albumin increases in several diseases; however, little is known regarding the pathological and physiological significance of oxidation due to poor characterization of the precise structural and functional properties of oxidized HSA. Here, we characterize both structural and functional differences between reduced and oxidized HSA. Using LC-ESITOFMS and FTMS analysis, we determined that the major structural change in oxidized HSA in healthy human plasma is a disulfide-bonded cysteine at the thiol of Cys34 of reduced HSA. Based on this structural information, we prepared standard samples of purified HSA, e.g. nonoxidized (intact purified HSA which mainly exists in reduced form), mildly oxidized and highly oxidized HSA. Using these standards, we demonstrated several differences in functional properties of HSA, including protease susceptibility, ligand-binding affinity and antioxidant activity. From these observations, we conclude that an increased level of oxidized HSA may impair HSA function in a number of pathological conditions. In addition, we determined blood and plasma sampling conditions for accurate measurement of the oxidized albumin ratio in plasma using EST-TOFMS screening.     

Localization of a protein antigen in the chicken spleen. Effect of various manipulative procedures on the morphogenesis of the germinal centre.

The time course of the localization of a protein antigen human serum albumin (HSA) into the chicken spleen after intravenous injection is analysed. Localization within seconds to the region surrounding the Schweigger-Seidel sheaths is accomplished by HSA complexes with chicken anti-HSA or by heat aggregated HSA. The localization of soluble HSA has to await the synthesis of sufficient chicken anti-HSA to accomplish localization to the same white pulp sites in the spleen at 25-30 hours after injection. By the use of complexes of HSA-anti-HSA in ten times antigen excess, the time for localization of HSA withing germinal centres was accelerated as compared with soluble HSA, so that newly formed centres containing antigen-bearing dendritic ells were seen at 48 hours instead of 72 hours after use of soluble HSA. Neonatally bursectomized and irradiated (Bx+Irr.) birds fail to localize HSA into germinal centres or to dendritic cells within the white pulp. Heat-aggregated human gamma-globulin (HGG) injected intravenously into Bx+Irr. birds rapidly localizes within seconds to the periphery of Schweigger-Seidel sheaths and at 24 hours can be seen attached to the surface of typical dendritic cells throughout the white pulp. Hence, heat-aggregated HGG can localize to dendritic cells in the absence of specific antibody. However, such localization to dendritic cells in Bx+ Irr. birds is not followed by segregation of the aggregated HGG-bearing dendritic cells within germinal centres--a further stage in the process which is presumed to require B cells and/or specific antibody. Localization of heat-aggregated HGG to white pulp dendritic ells was prevented by treatment with pepsin sufficient to destroy the ability of aggregated HGG to activate guinea-pig complement. Similary, in vivo decomplementation with a purified anticomplementary fraction (CoF) from the venom of Naja naja resulted in failure of intravenously injected HSA to localize to white pulp dendritic cells and failure of subsequent germinal centre formation. However, such decomplementation did not prevent the localization of aggregated HGG to white pulp dendritic cells. These facts are discussed in the light of hypotheses concerning germinal centre formation and the homeostasis of the antibody response in the bird.     

Studies on antigenic competition: Effect of antigen dose on the immune response of mice injected simultaneously with human serum albumin and ferritin

Human serum albumin (HSA) and ferritin in Freund's incomplete adjuvant (FIA) were injected simultaneously, intraperitoneally into mice in various dose/ratios. The degree of suppression of the primary and secondary immune responses to a small dose of HSA was dependent on the dose of ferritin injected simultaneously. A totally suppressed primary response was associated with a secondary response which only attained the level of a primary response to HSA in FIA.

Similar results were obtained in experiments in which a constant dose of ferritin and varying amounts of HSA were injected simultaneously in FIA into mice.

Suppression of the immune response to alum-precipitated HSA in the presence of soluble ferritin was not as striking as when the antigens were injected with FIA. Nevertheless, suppression of the primary response was again associated with a reduced secondary response.

The results are discussed in the context of the possible mechanism(s) of antigenic competition.

     

Induction and maintenance of the antibody response by different forms of human serum albumin.

Various forms of human serum albumin (HSA) were compared in their ability to induce and maintain the antibody response. In an in vitro model system, antibody synthesis was induced spontaneously during the maintenance phase of the immune response--presumably by persisting antigen. When different lymph nodes (LN) of the same rabbit were primed simultaneously with different forms of HSA, the spontaneous responses obtained in cell cultures prepared from LN primed with high molecular weight forms of HSA were greater than the responses obtained in cell cultures prepared from similar LN primed with lower molecular weight forms of HSA. This difference in response was consistent regardless of the method employed in antigen preparation and persisted for many months. The in vitro results indicating that the antibody response would be maintained at a higher level in animals immunized with high molecular weight forms of antigen and that the method of preparation would not be of major significance were confirmed in vivo in a mouse system.