Normal Rabbit Globulin Research Articles

Urinary excretion of nitrite and nitrate in experimental glomerulonephritis reflects systemic immune activation and not glomerular synthesis.

In immune-induced glomerulonephritis (gn), glomeruli (gl) synthesize nitric oxide (NO), and urinary nitrite (NO2-) excretion is increased. In mammals on a low nitrate (NO3-) diet, urinary NO3- is a measure of endogenous NO3- synthesis. Excretion is increased after administration of macrophage activators, reflecting induction of NO production. To determine whether increased urinary NO2- gn is due to glomerular synthesis we studied urinary NO2-/NO3- in accelerated nephrotoxic nephritis induced by preimmunization with rabbit immunoglobulin G (IgG), followed by rabbit anti-rat nephrotoxic globulin, and in control rats similarly preimmunized with rabbit IgG, but followed by normal rabbit serum. Both urinary NO2- and NO3- were increased by i.p. preimmunization with rabbit IgG (peak 463 +/- 171 nmol NO2-/60.3 +/- 9.4 nmol NO3-/24 h, P < 0.001 for both NO2- and NO3- compared with preimmunization levels). Repeat immunization with i.v. rabbit anti-rat nephrotoxic globulin (nephritic rats) or normal rabbit globulin (control rats) again increased urinary NO2- and NO3-. There was no statistically significant difference in urinary NO2- and NO3- levels between nephritic rats where globulin had nephrotoxic activity and the control rats injected with normal rabbit globulin, despite increased NO2- synthesis in ex vivo nephritic glomeruli after nephrotoxic globulin (7.9 +/- 1.9 nmol/2000 gl/48 h; controls 3.2 +/- 1.0 nmol/2000 gl/48 h). Thus neither urinary NO2- nor NO3- levels reflect local activation of the NO pathway in glomeruli. As reported for other stimulants, we show here that systemic stimulation with foreign antigen increased NO synthesis.

Development of Coated Tubes RIA for Serum T3 (Tri‐Iodothyronine) for Production Scale

A coating procedure that could provide immobilization of antibodies, with increased binding capacity, that is cost effective, simple, robust, and appropriate for production scale application, is described. This coating approach of T3 antibodies to the polystyrene tubes has been systematically investigated to determine its utility for the development of coated tube Radioimmunoassay (RIA) for T3 in human serum. Further, the results obtained by the developed coating procedure are found to be comparable with those obtained by the “gold standard,” the liquid phase RIA for T3. The coating procedure is completed in three major steps, each step involving an overnight incubation. The normal rabbit γ‐globulins are physically adsorbed onto the polystyrene tubes and incubated. After washing, a second antibody (goat anti‐rabbit antiserum) is added and incubated. To this antigen specific antibody is added (T3 antibody produced in rabbit) and further incubated. Finally, the non‐specific sites on the tubes are saturated by the blocking solution. The concentration of normal rabbit globulin, titers of second antibody and T3 antibody, and time required for coating are optimized to arrive at a suitable coating protocol. The coated tubes were evaluated for precision, reproducibility, and stability. Various parameters such as total reaction volume, incubation time and temperature, total number and volume of washings, concentration of 8‐anilino‐1‐naphthalene sulfonic acid (ANS), and quantity of tracer per tube are optimized to arrive at a suitable standard curve. The optimized assay is validated for the quality control parameters such as intra‐ and inter‐assay variations, recovery, and parallelism. The developed coated tubes assay had an assay range of 0.3–4.8 ng/mL with a sensitivity of 0.3 ng/mL at 90% B/B 0. Batch to batch variation in coating was <10%. The coated tubes were stable up to 1 year, which is adequate for production scale.

New cause for false-positive results with the Pastorex Aspergillus antigen latex agglutination test

The Pastorex Aspergillus antigen test for detection of Aspergillus galactomannan antigen in the sera of patients with invasive aspergillosis is used in many clinical laboratories. A serum sample contaminated with Penicillium chrysogenum gave a strongly positive reaction (1:128) which was heat stable, was not eliminated by pronase treatment, and was not detected by a normal rabbit globulin control. This observation was shown to be due to cross-reactions of the monoclonal antibody EB-A2 used by the kit with several airborne fungi likely to contaminate serum samples, including Penicillium chrysogenum, Cladosporium herbarum, Acremonium species, Alternaria alternata, Fusarium oxysporum, Wangiella dermatitidis, and Rhodotorula rubra.

Recombinant Tumor Necrosis Factor Stimulates Interleukin-1 Production in Glomerular Cultures from Rats with Nephrotoxic Serum Nephritis

We have investigated the effect of recombinant tumor necrosis factor (r-TNF) on the release of interleukin-1 (IL-1) in glomerular cultures from rats with an accelerated autologous form of nephrotoxic serum nephritis (NTSN). Freshly isolated glomeruli from the NTSN rats were incubated for 24 h in the presence of r-TNF. When the glomeruli were activated by r-TNF substantial amounts of IL-1 could be detected in the supernatants as measured by the mouse thymocyte assay. The r-TNF-induced IL-1 activity was significantly higher in rats with NTSN than those in normal controls and the other control group, consisting of preimmunized rats (rabbit IgG), then given normal rabbit globulin instead of NTS. To avoid the effect of prostaglandins on the IL-1 assay, we cultured the glomeruli with addition of indomethacin and assayed IL-1 activity in the culture supernatants. This cyclooxygenase inhibitor augmented r-TNF-induced IL-1 production. Our data suggest that r-TNF can serve as a potent stimulator of IL-1 synthesis in glomerular cultures from rats with NTSN.

Interleukin-1 production by lipopolysaccharide-stimulated glomeruli from rats with nephrotoxic serum nephritis.

The objective of the present work was to characterize some aspects of interleukin-1 (IL-1) production by nephritic glomeruli after stimulation with bacterial lipopolysaccharide (LPS). Freshly isolated glomeruli from rats with an accelerated autologous form of nephrotoxic serum nephritis (NTSN) were incubated for 24 h in the presence of LPS. The modified NTSN was produced by an intravenous injection of nephrotoxic serum (NTS) into the rats which had been previously immunized with rabbit IgG and Freund's complete adjuvant. The glomerular cultures of the NTSN rats were found to release significantly increased amounts of IL-1 after LPS stimulation when compared to the values obtained with normal controls and the other control group, consisting of preimmunized rats (rabbit IgG), then given normal rabbit globulin instead of NTS. To block the effect of prostaglandins on the IL-1 assay, we cultured the glomeruli with the addition of indomethacin and assayed IL-1 activity in the culture supernatants. The use of indomethacin resulted in a further increase in IL-1 production. The administration of a rabbit anti-rat macrophage serum reduced the production of IL-1 activity in the NTSN rats. Our findings support the notion that at least in NTSN rats activated macrophages are present and probably account for their increased IL-1 activity. This description of IL-1 activity produced by LPS-stimulated nephritic glomeruli may introduce a new element to the early events leading to glomerular inflammation.

Production of Interleukin-1 in Macrophage Cultures from Rats with Nephrotoxic Serum Nephritis

To investigate the role of cell-mediated immunity in the pathogenesis of experimental glomerulonephritis, we examined interleukin-1 (IL-1) activity in the culture supernatant of lipopolysaccharide-stimulated peritoneal macrophages from rats with an accelerated autologous form of nephrotoxic serum nephritis (NTSN). Modified NTSN was produced by an intravenous injection of nephrotoxic serum (NTS) into the rats which had previously been immunized with rabbit IgG and Freund's complete adjuvant. It was found that the NTSN rats had significantly increased levels of IL-1 activity when compared to the values obtained with normal controls and the other control group, consisting of pre-immunized rats (rabbit IgG), then given normal rabbit globulin instead of NTS. The administration of a rabbit anti-rat macrophage serum reduced the production of IL-1 activity in the NTSN rats. In this experimental model IL-1 synthesis by peritoneal macrophages is present early in the disease process and may be an important mediator of glomerular immune injury.

Role of membrane immunoglobulin (Ig) crosslinking in membrane Ig-mediated, major histocompatibility-restricted T cell-B cell cooperation.

Resting murine B lymphocytes can present rabbit anti-Ig to T cell lines specific for normal rabbit globulin. The T cell-B cell interaction is major histocompatibility complex (MHC)-restricted, and leads to activation, proliferation, and differentiation of the resting B cell into an antibody-secreting cell. Efficient antigen presentation and B cell activation depends upon binding of rabbit globulin to (membrane) mIg. To investigate the role of mIg in this polyclonal model for a T cell-dependent primary antibody response, we determined whether crosslinking of mIg is required either for efficient antigen presentation, as measured by helper T cell activation, or for the B cell response to T cell help, since all the direct effects of anti-Ig on B cells require crosslinking of mIg. We found that monovalent Fab' fragments of anti-IgM or anti-IgD work as efficiently as their divalent counterparts. Therefore, a signal transduced through the antigen receptor seems not to be required when T cell help is provided by an MHC-restricted T helper cell recognizing antigen on the B cell surface. Moreover, rabbit globulin bound to class I MHC molecules in the form of anti-H-2K also results in efficient antigen presentation and T cell-dependent B cell activation. However, mIg still appears to be specialized for antigen presentation, since anti-Ig is presented about three- to fivefold more efficiently than anti-H-2K.

Inhibition of a mouse hepatoma by the alkylating agent Trenimon linked to immunoglobulins.

Trenimon was conjugated in active alkylating form to rabbit anti-mouse H6 hepatoma globulin (AHG) with retention of antibody activity. H6 hepatoma-inoculated mice were given various combinations of conjugates, free Trenimon, and unconjugated immunoglobulins in daily injections for 5 days. Linkage of Trenimon to immunoglobulins reduced systemic toxicity of the drug, with comparative retention of its antitumor activity. The antitumor action of Trenimon was potentiated by AHG irrespective of whether the drug was directly linked to AHG or free AHG was administered along with Trenimon linked to normal rabbit globulin (NRG). In vitro, Trenimon bound to AHG was less inhibitory to hepatoma cells than free Trenimon, but more inhibitory than Trenimon-NRG conjugates. There was no significant endocytosis of conjugates by the hepatoma cells. This suggests that unlike free Trenimon, the target molecules of Trenimon-immunoglobulin conjugates are not intracellular DNA but are located on the surface of the hepatoma cells.

Application of a radiometric ear assay for studies of adjuvant arthritis in rats.

A radioisotopic method, originally developed for measuring the cellular response in delayed hypersensitivity lesions in mice, has been evaluated in adjuvant arthritic rats. Focal accumulation of 5-iodo-2'-deoxyuridine-125I (125IUdR) at a site of antigen challenge (left pinna) was measured and expressed as increased radioactivity in the challenged (left) over the unchallenged (right) ear (L/R ear ratio). Immunologic specificity of the assay was established with anti-lymphocyte globulin (ALG)-treated arthritic rats. ALG significantly inhibited the 125IUdR L/R ear ratio; normal rabbit globulin had no effect on this parameter. A significant negative correlation was observed between the 125IUdR ear ratios and subjective arthritic scores in established adjuvant disease. Certain characteristics of the 125IUdR radiometric ear assay in rats were established in several types of experiments: disappearance of the isotope from blood, whole body irradiation studies in turpentine-injected rats, and cyclophosphamide pretreatment in a sheep erythrocyte antigenic system. The results of this study support the utility of the 125IUdR ear assay to quantify cellular accumulation at a site of antigen challenge in adjuvant arthritic rats and possibly other antigenic systems in this species.

Selective neutralization by antiinterferon globulin of macrophage activation by L-cell interferon, Brucella abortus ether extract, Salmonella typhimurium lipopolysaccharide, and polyanions

Addition of interferon (IF) inducers pyran copolymer, poly(I)-poly(C), an ether extract of Brucella abortus (Bru-Pel), or Salmonella typhimurium lipopolysaccharide (LPS) to cultures of peritoneal macrophages in vitro enhanced their cytotoxic activity for MBL-2 lymphoblastic leukemia cells. To evaluate the role of induced IF in the macrophage activation, highly specific rabbit anti-L-cell IF globulin was added to resting macrophage cultures at the same time as the macrophage-activating agents. Macrophage activation by these various biological and synthetic agents was totally neutralized by anti-IF globulin but not by normal rabbit globulin. Similarly, the anti-IF globulin inhibited the ability of chromatography-purified Newcastle disease virus-induced IF to render macrophages cytotoxic, and the degree of neutralization of IF titer corresponded with the inhibition of IF-induced macrophage-mediated cytotoxicity. In contrast, macrophage activation by concanavalin A-induced lymphokine, which contains an antigenically different IF, was not affected by high titers of the anti-L-cell IF antibodies. The results indicate that endogenously generated type I IF may play an important role in control of macrophage function.

Tumor inhibition by chlorambucil covalently linked to antitumor globulin

The alkylating agent chlorambucil has been covalently bound to antitumor globulins directed against cell surface antigen(s) of the mouse EL 4 lymphoma. The resulting conjugates retained alkylating and antibody activities and were used to treat C57 BL 6 j mice after prior inoculation with the syngeneic EL 4 lymphoma. Forty per cent of mice treated with the covalent conjugate, 78% of mice treated with non-covalent conjugate and 50% of mice treated with chlorambucil followed by antitumor globulin survived tumor-free for more than 100 days. Untreated mice had a mean survival of 18 ± 1.3 days and the groups that received chlorambucil or antitumor globulin only, or chlorambucil covalently bound to normal rabbit globulin died within 30 days of tumor inoculation.

Enhancement of leukemogenesis in mice after prolonged administration of anti-interferon or normal rabbit globulin

The prolonged adminstration of rabbit anti-mouse L cell interferon globulin had a marked potentiating effect on Rauscher Murine Leukemia Virus (MuLV-R) infection in BALB/c mice, as shown by spleen size. Normal rabbit globulin had a lesser, but still significant, augmenting effect on splenic enlargement. It was possible to discriminate quantitatively between the non-specific enhancement of splenomegaly in MuLV-R infected mice due to antigenic stimulation with normal rabbit globulin and the effects due to elimination of endogenous interferon by specific antibodies. The difference in the spleen-enlarging activity between the anti-interferon IgG and normal rabbit IgG was found to be maximal 3-4 weeks after infection when potent, diluted anti-interferon IgG (58 microgram protein per dose) was used. It would appear that the endogenous interferon, even prodcued in undetectable amounts, plays an essential role in controlling infection with an oncogenic virus.

Tumor localization of 131-I-labeled antibodies by radionuclide inaging.

Intravenous injections of 131-I-labeled anti-EL4 lymphoma antibodies showed progressive localization of radioactivity in EL4 transplants but not in B16 melanoma in mice carrying both tumors. Normal rabbit globulin labeled with 131-I did not localize in either tumor and cleared more slowly from the internal organs. Metastatic localization of intravenous 131-I-labeled anti-tumor antibodies was also observed in 2 cancer patients.

Tumor localization of radiolabeled antibodies raised by a mouse plasma cell tumor

Antibodies raised in rabbits against a mouse plasma cell tumor were labeled with 131I and injected along with normal rabbit globulin labeled with 125I into plasmacytoma bearing mice and the degree of localization of the antibodies in the tumors determined. There was localization of the anti-tumor antibody above the background localization of the normal globulins. The localization apparently took place largely on the tumor cell surfaces since following cell fractionation experiments the fixation was found to be largely in the microsome fraction. The antibodies raised against the tumor also showed localization in liver, spleen and brain.

Synthesis of antihemophilic factor antigen by cultured human endothelial cells.

Antihemophilic factor (AHF, Factor VIII) antigen has been demonstrated in cultured human endothelial cells by immunofluorescence studies using monospecific rabbit antibody to human AHF. Control studies with cultured human smooth muscle cells and human fibroblasts were negative. By radioimmunoassay it was demonstrated that cultured human endothelial cells contain AHF antigen which is released into the culture medium. Cultured smooth muscle cells and fibroblasts did not have this property. Cultured endothelial cells incorporated radioactive amino acids into high molecular weight, AHF antigen-rich protein fractions prepared from the culture media, 7% of the radioactive amino acid counts incorporated into this material were precipitated by globulin prepared from rabbit anti-AHF whereas normal rabbit globulin precipitated only 1.5% of the counts. Although cultured endothelial cells actively synthesize AHF antigen, AHF procoagulant activity was not detected in the culture medium. Studies seeking a basis for the lack of procoagulant activity have not clarified this deficiency, but they have established that exogenous AHF procoagulant activity is not inactivated by the tissue culture system.

CYTOLOGICAL AND HISTOLOGICAL EVENTS FOLLOWING TREATMENT WITH ANTI‐THYMOCYTIC GLOBULIN IN MICE

The present experiment was undertaken in order to obtain additional information on the increased cell decay in lymphatic tissues after ATS‐treatment. After injection of rabbit‐anti‐mouse‐globulin G (ALG) in Balb/c mice an increased cell decay was found in spleens and mesenteric nodes by the use of the Nigrosin Dye Exclusion Test on unfixed cell suspensions. After one injection of ALG the decay increased to the same high level as seen after ATS‐injection, no further increase being found after 7 days of injections. One week after treatment was stopped, increased decay was still evident. In suspensions from the thymus the decay remained on normal level. Mice injected with normal rabbit globulin G showed no increased decay in the thymolymphatic system. The most prominent histological findings were depletion of the thymus dependent areas in lymphatic tissue and increased numbers of blast‐cells in the blood and efferent lymphatics after ALG‐injections. The possible ways in which treatment with anti‐lymphocytic antibodies may lead to increased decay in lymphatic tissue are discussed in connection with the other findings, and it is concluded that the most likely explanation is a shift in population from long‐lived thymus derived cells to shortlived cells in lymphatic tissue, and it is emphasized that the Nigrosin Dye Exclusion Test is a useful test in studies on lymphoid cell kinetics.

Mechanisms underlying binding of immune complexes to macrophages.

The mechanism of binding of immune complexes to macrophages was investigated using purified antibody and haptens of different valences. Antibody alone bound to macrophages; enhancement of binding occurred when polyvalent and divalent haptens were present at equivalence but did not occur in great antigen excess. Monovalent hapten did not increase the binding of antibody at any concentration ratio tried, though it inhibited the enhancement due to oligovalent hapten. Ultracentrifuged normal rabbit globulin also inhibited the binding of complexes indicating the presence of exposed binding sites on the uncomplexed molecules. Complexes bound more strongly than antibody alone as determined from elution studies. These results support the hypothesis that the enhancement of antibody binding to macrophages in the presence of antigen is due to increased energy of binding resulting from summation of individual binding sites already exposed on the antibody molecules. It was also possible, by saturating the macrophages with gamma globulin, to estimate the number of binding sites per cell; this was calculated to be approximately 2 million per alveolar macrophage.

Density of BAI Strain A Avian Leukosis Virus-Antibody Complexes Relative to Antigen-Antibody Ratio

Summary Immune complexes of differing antibody-virus ratios were prepared with rabbit anti-BAI strain A avian leukosis virus and the homologous antigen by means of the quantitative precipitin reaction. These complexes were centrifuged to their equilibrium densities on potassium tartrate gradients and the results were compared with the analogous properties of free virus and normal rabbit γ globulin centrifuged under the same conditions. The equilibrium density of the complexes was greater than that of the free virus and was directly proportional to the antibody-virus ratio. Normal rabbit globulin was not sedimented significantly under the same experimental conditions.

The Effect of Immunologically Induced Lymphopenia on Antibody Formation

Summary The immunologic responsiveness of rats was studied following selective destruction of the lymphocyte mass by means of rabbit anti-rat lymphocyte globulin (ALG). ALG pre-treatment of rats before antigenic stimulation by sheep erythrocytes completely suppressed antibody formation. Repeated immunization of these animals well beyond the period of induced peripheral lymphocytopenia resulted in some antibody production but of only the IgM type. By contrast, administration of the soluble antigen KLH after pre-treatment with ALG caused marked enhancement of antibody production in comparison with control groups pre-treated with saline and normal rabbit globulin. Some possible mechanisms of such enhanced response were discussed.

Electron Microscope Observations on the Cuticle and Submicroscopic Binding of Antibody in Ascaris suum Larvae

An electron microscope study of the cuticles of secondand third-stage Ascaris suum larvae did not reveal the presence of submicroscopic pores. Ferritin-conjugated immune rabbit globulin was bound to the surface of the cuticle of these larvae. Ferritin-labeled normal rabbit globulin was observed on the cuticles of some secondbut not third-stage larvae. Preliminary electron microscope studies of second-stage larvae isolated from the peritoneal cavity of immune mice after an intraperitoneal injection showed that the peritoneal cells adhered to the cuticles in 4 hr. The cells were mainly macrophages and appeared to be in direct contact with the cuticles of the larvae but no apparent morphologic alteration in the larvae was observed. Fluorescent antibody studies have shown that the cuticles of several nematode species are antigenic (Crandall et al., 1963; Taffs and Voller, 1963; Baratawidjaja et al., 1963). It has been suggested that at least some of the fluorescent antibody binding might be due to antigens within the helminth which pass through submicroscopic pores and adhere to the cuticle (Taffs and Voller, 1963; Jackson, 1963). This investigation was undertaken to determine by electron microscopy whether pores are present in the cuticle of Ascaris suum larvae and to localize the submicroscopic antibody-binding sites on the larvae with ferritinlabeled immune globulin. A preliminary electron microscope study showing the cellular reaction to larvae isolated from the peritoneal cavity of immune mice is