Tissue suspension agglutination: a simple method to screen species-specific and organ-specific reactions.

Agglutination tests with preparations of parenchymatous organs were developed. The tissue suspensions were dried at room temperature after they had been spread as a very thin layer on a glass plate, or otherwise, they were lyophilized. The dried preparations were pulverized and then prepared as stable suspensions in saline. The agglutination test was conducted on a slide by mixing one drop of the tested serum at a convenient dilution with one drop of tissue powder suspension. Agglutination in the form of readily discernible clumps could be assessed after 1-10 min. By means of this procedure, species-specific reactions were studied using suspensions of kidneys of various species. Organ-specific reactions were noted with suspensions of brain and thyroid. Agglutination of thyroid powder was observed with rabbit anti-rabbit thyroid sera as well as with many, albeit not all, sera of patients with Hashimoto's disease.

"Heterophile" antigen in sera of human recipients of renal allografts.

It was noted that many sera of patients with renal allograft produce distinct precipitation lines in gel diffusion tests with about 20% of infectious mononucleosis sera. The antibodies in infectious mononucleosis sera were of IgM isotope, but, interestingly, they could be removed by guinea pig kidney homogenate, which indicated that the reactions studied were of the Hanganutziu-Deicher rather than of the Paul-Bunnell type. This contention was strengthened by the fact that positive transplantation sera reacted also with standard serum with Hanganutziu-Deicher antibodies. Thus far, the presence of the antigen in the transplantation sera could not be related to the clinical status of the patients, however, the antigen was noted primarily in those sera that did not contain heterophile transplantation antibodies. It was proposed that the antigen detected in the transplantation sera was an altered tissue antigen released from the grafted organ. Besides, interactions between two serum samples from the same patient were noted in immunodiffusion tests. These reactions occurred very seldom and were unrelated to heterophile transplantation antigens or antibodies.

Morphological studies on avian spinal cord chimeras.

Spinal cord chimeras were constructed by orthotopic grafting of quail embryonal neutral folds, neural crest and neural tube into chicken embryos. The spinal cord xenografts were accepted for varying lengths of time, but most chimeras eventually rejected the quail transplant. This was associated with perivenular cuffing and demyelination with preservation of most neurons, as well as clinical neurological symptoms. Twenty-four chimeras were studied to delineate the time of first appearance of glial deposits of immunoglobulin and to identify the subpopulations of T cells in spinal cord infiltrates. The results suggested that deposits of immunoglobulins on glial elements preceded inflammatory cell infiltration. The perivenular cuffs consisted predominantly of T cells and showed a preponderance of CD8- over CD4-positive cells (CD4/CD8 ratios around 0.6). Further, CD4+ cells were found almost exclusively in the central portions of the infiltrate, with the periphery consisting almost only of CD8+ cells. The diffuse cellular infiltrate of the parenchyme contained T and plasma cells. The T cells were almost exclusively CD8+. Plasma cells were seen only at the outer borders of the cuffs and dispersed throughout the quail-derived spinal cord tissue. It seemed that rejection of quail-derived melanocytes in feathers ('quail-like feathers'), described by us earlier, often preceded neurological symptoms and showed a histopathological pattern comparable to spinal cord lesions, i.e., predominantly perivascular cuffing. In preliminary studies, enhancement of disease by immunization with quail organ suspension and decreased intensity of disease by combined immunosuppressive treatment with FK 506 and cycylophosphamide were suggested. The data presented here are compatible with the hypothesis that rejection of CNS quail tissue by chimeras is preceded in the periphery by rejection of melanocytes in segments of skin and in feathers, and that the spinal cord rejection relies on xenoantibodies and on cytotoxic as well as delayed hypersensitivity-type T cells. Finally, these data strengthen the analogy between the histopathologic presentation and immune effector composition of the xenograft rejection lesions in the chimeras and the plaques seen in patients with multiple sclerosis.

Antibodies to quail erythrocytes in quail-chicken spinal cord chimeras.

Quail-chicken spinal cord chimeras are a model for temporary acceptance followed by rejection of xenografts and also for demyelinating lesions of the central nervous system. The antiglobulin test with quail erythrocytes was employed to detect antibodies in sera of quail-chicken spinal cord chimeras. Sera of all 46 chimeras tested gave positive results. In virtually all instances, antibodies were detected within 10 weeks after hatching and they persisted for all the observation time up to 8 months. The antibodies detected in these tests were directed against species antigens of the quail. They were apparently identical with xenoantibodies described in a previous study, which were detected by indirect immunofluorescence with quail tissue sections; on the other hand, mixed agglutination tests with quail embryonal cell monolayers employed previously had detected a broader spectrum of antibodies that did the antiglobulin tests with quail erythrocytes. The antiglobulin test with quail erythrocytes seems the most cost-efficient and convenient test to monitor xenoantibody formation in this animal model.

Streptococcus-mutans-induced nephritis in rabbits: rheumatoid factors and nephritogenicity.

The pathogenesis of streptococcus-induced nephritides (SIN) involves immune complex-mediated inflammation; however, specific mechanisms are still poorly understood. Using preparations of two strains of Streptococcus mutans (SM) in attempts to induce SIN in rabbits, one preparation was strongly and the other virtually not nephritogenic. The non-nephritogenic preparation provided a negative control for our studies. Streptococcal components were present in circulating immune complexes (CIC) as well as in tissue-bound immune complexes (TIC), especially early in the disease. CIC and TIC also contained rheumatoid factors (RF), which tended to predominate in late stages of the disease. The nephritogenic and the non-nephritogenic preparations of SM shared the same major tissue-binding components and induced similar titers of antimicrobial antibodies, but differed significantly in their ability to induce CIC and RF. It is proposed that kidney-binding microbial components, antimicrobial antibodies and high serum concentration of RF are necessary and sufficient determinants for the pathogenesis of SIN in this rabbit model.

Interaction of antibody with Forssman antigen in guinea pigs. A mechanism of adaptation to antibody- and complement-mediated injury.

Forssman antigen is a glycosphingolipid with antigenic specificity determined by extra-membrane haptenic sugars similar to blood group antigens and antigens that are the main barrier to xenogeneic organ transplantation. Herein, we describe the localization of Forssman antigen in guinea pig lungs and kidneys and the consequences of its interaction with antibodies in vitro and in vivo (Forssman reaction). Exposure of cultured guinea pig aortic endothelial cells to Forssman antibodies induced rapid redistribution of antigen-antibody complexes at the cell surface, followed by shedding that occurred by blebbing of plasma membrane as vesicles or fragments, and was associated with disappearance of antigen from the cell surface (antigenic modulation). Guinea pigs surviving frequent intravenous infections of increasing amounts of antibodies, for a total of 20 to 40 lethal doses, developed a partial or complete adaptation to generalized Forssman reaction, and adaptation was associated with partial or complete modulation of Forssman antigen at the surface of the pulmonary and, in minor degree, renal endothelial and epithelial cells. These findings support the hypothesis that modulation of endothelial carbohydrate antigens contributes to adaptation of highly vascularized organs exposed to tolerable levels of allo- or xenoantibodies.

Role of late complement components in experimental autoimmune thyroiditis.

Availability of a line of rabbits deficient in the sixth complement component (C6-D) made it possible to evaluate the role of the terminal complement complex (TCC) in the development of experimental autoimmune thyroiditis (EAT) of the rabbit. Immunization with saline extract of homologous thyroid, known to be composed predominantly of thyroglobulin, led in normocomplementemic (NC) rabbits to severe thyroiditis, with cellular infiltrates occupying 50-95% of the thyroid, and to minimal or moderate thyroiditis, with 1-35% of thyroids infiltrated in C6-D rabbits. Cellular infiltrates consisted predominantly of mononuclear cells with appreciable numbers of granulocytes. Destruction of thyroid follicles was extensive and diffuse in NC rabbits, but it was only minimal and focal in C6-D rabbits. Immunohistology revealed in both groups of rabbits deposits of IgG and C3 along follicular basal laminae. In addition, NC rabbits showed deposition of C6 and MAC in thyroid follicles. These results suggested that TCC is necessary for the development of fully expressed, severe EAT; simultaneously, however, they showed that a significantly reduced EAT can develop without TCC. Administration of NC but not of C6-D rabbit serum to C6-D rabbits resulted in a significant increase in the severity of EAT. It was also shown that C6-D rabbits have "normal" T-cell activity, since they developed experimental autoallergic encephalomyelitis as readily as NC rabbits. Therefore, it is likely that development of EAT is indeed impaired by the C6 deficiency in rabbits. The requirement for TCC observed in this study may be relevant to the understanding of the pathogenesis of Hashimoto's thyroiditis, in which thyroid tissue was recently shown to contain TCC deposits.

Transfer of experimental autoimmune thyroiditis by in situ perfusion of thyroids with immune sera.

The role of autoantibodies in the pathogenesis of autoimmune thyroiditis (AT) still remains poorly understood. Serum transfer experiments in experimental AT (EAT) and spontaneous AT (SAT) animal models frequently did not succeed or only resulted in minor thyroid lesions. The inconsistent results may have arisen because of technical problems inherent in serum transfer, the major of which is to obtain high enough concentrations of autoantibodies over long enough periods at the potential site of tissue injury. An attempt was made to circumvent this hurdle by repeated in situ perfusions of the rabbit thyroid via the superior thyroid artery. In situ perfusion of rabbit thyroids with high-titered homologous sera reactive with saline thyroid extract indeed led to thyroiditis characterized by granular deposits of rabbit IgG and C3 in the thyroid follicular basal laminae, cellular infiltrates consisting of mononuclear cells and granulocytes, destruction of the thyroid follicular architecture, and focal fibrosis. Perfusion with control sera lacking thyroid-reactive antibodies did not lead to thyroid lesions. These results demonstrate that humoral antibodies can induce severe AT comparable to actively induced thyroiditis.

Glomerular lesions induced in the rabbit by physicochemically altered homologous IgG.

Immunization of rabbits with physicochemically altered homologous or even autologous IgG induces formation of antibodies combining with IgG of rabbit and of foreign species. Cardiac but not renal lesions were reported in such animals. This study examined the nephritogenic potential of the immune response to cationized or heat-aggregated homologous IgG of b9 or b4 allotype in rabbits of the b4 allotype. Rabbits injected with either b9 or b4 cationized IgG produced antibodies reactive with rabbit and human IgG and with histones; they also developed abnormal glomerular deposits of IgG b4 and C3 corresponding to alterations of the glomerular basement membranes (GBM). Rabbits injected with either b9 or b4 aggregated IgG developed antibodies reactive with rabbit and human IgG and abnormal glomerular deposits of IgG b4 and C3 in the GBM and in the mesangium with subendothelial and mesangial electron-dense deposits. Some rabbits in both groups had proliferative and exudative glomerulonephritis and proteinuria. The results showed that immunization of rabbits with physicochemically altered homologous IgG induces an immune response to rabbit and human IgG and to histones as well as glomerular deposits of autologous IgG and C3 and other glomerular lesions.

Studies on avian spinal cord chimeras. I. Neurological and histological manifestations.

Spinal cord chimeras were produced by replacing a small fragment of neural tube of a 2-day-old White Leghorn chicken embryo with a similar fragment from a Japanese quail embryo. The embryo mortality was 61%, and 72% of hatched birds were 'cripples' and had to be sacrificed within 5 days after hatching. Forty-nine chimeras, 10.9% of the total number of operated embryos, were alive for more than 3 weeks. For at least 17 days after hatching, all birds behaved like normal chicks, and the grey quail-like feathers were the only manifestations of their chimerism. Initial neurological symptoms of unsteady walking and drooping of the wings were noted in all birds except for 1 that died an accidental death before it became sick. Advanced symptoms characterized by paralysis of the legs forcing the bird to lie on its side were noted in 40 birds. The chimeras could be divided into two groups, each consisting of 24 birds. The short-survival (SS) chimeras of the first group became terminally ill and had to be sacrificed within 3 months. The long-survival (LS) chimeras of the second group showed more protracted disease, in that only 16 of them showed symptoms of the advanced disease, and the majority showed partial or complete recovery. Ten of the LS birds were kept alive for more than 8 months. Furthermore, many LS chimeras lost their grey feathers. The hallmarks of neurohistological manifestations were mononuclear cell infiltrates, demyelinization with preservation of axons and scar formation. These lesions were restricted to the quail fragment of the spinal cord except for 2 birds in which distant cellular infiltrates were observed. Direct immunofluorescence tests for chicken IgG were positive in spinal cords of most SS chimeras but only of some LS chimeras.

Studies on avian spinal cord chimeras. II. Immune response of the chicken host to the graft of quail tissue.

A previous study described clinical and pathological manifestations observed in 49 chimeras produced by replacing a small fragment of the neural tube of a chicken embryo by a similar fragment from a Japanese quail embryo. Predominant antibodies in sera of these birds were directed against xenogeneic antigens which are devoid of organ specificity and which are present in quail tissues but absent from chicken tissues. Mixed agglutination test with quail cell monolayers detected such antibodies in sera of all chimeras tested. In most instances, positive reactions were observed already in the 4th week after hatching; they persisted in all birds throughout the observation period of up to 8 months. Some of the detected antibodies were directed against saline-nonextractable surface antigens of quail cells. Enzyme immunoassay with quail organ extracts, agglutination of quail tissue particles, and indirect immunofluorescence test with quail organ sections were positive with sera of many, but not all, chimeras. CNS-specific antibodies, apparently autoantibodies, were detected in serum samples of only one chimera, using enzyme immunoassay with extract of chicken brain and indirect immunofluorescence test with sections of chicken spinal cord. Eluates from lesional spinal cord contained antibodies to non-organ-specific quail antigens but not to CNS-specific antigens. Cerebrospinal fluids of many chimeras had antibodies to quail antigens, but no evidence for antibody formation within the CNS was obtained. Cell-mediated immunity could be demonstrated in all chimeras tested by means of lymphocyte proliferation test after stimulation by quail organ extract. It was concluded that pathological events in the studied chimeras have been most likely mediated primarily by humoral immune responses to non-organ-specific quail antigens.

Antibodies for trypsinized human red blood cells in human sera.

During studies of agglutination of trypsinized human red blood cells (RBC) by anti-Rh sera, it was noticed that some human sera without Rh antibodies agglutinated these erythrocytes. Of 933 normal and pathological sera subsequently screened, 116 produced such agglutination. The agglutinins were of IgM nature and were directed against an antigen exposed or created by tryptic digestion of RBC. They reacted equally well with autologous as with allogeneic trypsinized RBC and were different from T agglutinins and from Paul-Bunnell antibodies. The mode of formation of these antibodies remains unknown.

Glomerulonephritis in NZW kidneys grafted into NZB/W mice.

After left nephrectomy, 3 10-week old NZB/W mice received orthotopic grafts of kidneys from parental NZW mice of the same age. At autopsy conducted at the age of 33-38 weeks, glomerulonephritis of similar extent was noted in the recipients' own and in the grafted kidneys. Also, very similar granular deposits of immunoglobulins and complement were demonstrated in these kidneys. It was concluded that the absence of glomerulonephritis in NZW mice cannot be attributed to the refractoriness of their kidney to this disease.

Studies on immunological tolerance induced in mice by kidney allografts.

(C57BL/6 x A/J)F1 murine recipients of DBA/2 kidney allografts developed tolerance to DBA/2 tissues, which was measured by observation of growth of a DBA/2 tumor. Eleven, 14 and 18 days after inoculation, the size of the tumor was considerably larger in kidney-grafted than in nongrafted animals. Still, the susceptibility of grafted animals to the tumor did not equal the susceptibility of the DBA/2 mice syngeneic with the tumor. Removal of the renal graft left the recipients with significant tolerance which, however, was weaker than that of the mice in which the kidney graft was left undisturbed. In parabiosis experiments, it was noted that the size of the DBA/2 tumor was equal in the partner which received the DBA/2 kidney graft and in the partner which was not grafted. These experiments rather clearly showed that the tolerance studied was of an 'infectious' type and that it was apparently transferred by some suppressing factor(s) present in the circulation.

Humoral transplantation antibodies play a role in protracted rejection of murine renal allografts.

Murine renal allografts were studied using (C57BL/6J x A/J)F1 mice as recipients and DBA/2 mice as donors. In this strain combination, protracted rejection was noted in that the circulation was maintained in the graft for over 10 weeks. In all grafts examined after 3 weeks, mononuclear cell infiltrates were noted; in addition, all grafts had immune deposits, apparently containing transplantation antibodies, in glomeruli, tubuli and vessels. These results stressed the role of humoral immunity in protracted renal allograft rejection.