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Background Connective tissue diseases (CTDs) are a heterogeneous group of disorders defined by the association of a variety of clinical manifestations with immunologic and other laboratory findings. Overlap of syndromes and aberrant findings appear rather frequently. Methods Sera of eight antinuclear antibody (ANA) negative, cases of subacute cutaneous lupus erythematosus (SCLE) with antibodies to Ro (SS-A) and a ninth case with clinical and laboratory signs of Sjogren’s syndrome and systemic lupus erythematosus (SLE) were tested for complement (C′) fixing antinuclear antibodies (C-ANAs). The ninth case was examined in depth by direct immunofluorescence (DIF) and a two-step “C + DIF” test of biopsies for C′ fixation to in vivo bound ANAs, as well as serum tests for C-ANA, ANA, and SCLE markers. Results Sera of five of the eight ANA negative, Ro(SS-A) positive SCLE cases had C-ANAs. The ninth case, a 50-year-old woman with clinical and laboratory signs of Sjogren’s syndrome and SLE, gave a strong positive C + DIF reaction in the skin biopsy for in vivo bound ANAs that fix C′, but negative ANAs and C-ANAs in routine serum tests; they revealed antimitochondrial antibodies. Serum tests on normal skin, however, revealed weak ANA and strong C-ANA reactions with in vitro fixed C′. Conclusions ANA negative cases of SCLE or Sjogren’s syndrome may have C-ANAs. A case with Sjogren’s syndrome and signs of SLE had both in vivo and in vitro C′ fixing ANAs. C-ANA tests can aid in the identification of such cases.

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.

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.

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.

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 mono-layers 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.

Tonsils seem to be the ideal source for lymphocytes seeding to the mucosa of the respiratory tract. The distribution and engraftment of human lymphocytes injected into mice with severe combined immunodeficiency (SCID) are not well understood. C.B-17 SCID mice were injected intraperitoneally with human tonsillar mononuclear cells (hu-TMC). The hu-TMC-SCID mouse chimeras were subsequently tested for the appearance and distribution of human lymphocytes tagged with H33342 and immunoglobulin-secreting cells in various systemic and mucosal immunocompetent tissues. This was done by fluorescence microscopy of tissue sections for cells supravitally stained before transfer and by an enzyme-linked immunospot assay using cells isolated from murine organs. Most importantly, engraftment of hu-TMC proved to be dependent on the presence of anti-Epstein-Barr virus (EBV) antibody in the donor. hu-TMC engrafted, in decreasing numbers, in the following systemic organs: peritoneal cavity, liver, spleen and bone marrow. Among mucosal tissues tested, hu-TMC were seen in lungs, but not in the intestines. The engraftment of hu-TMC in the lung was more extensive than that in the spleen. These studies demonstrate that hu-TMC engraft in a variety of murine tissues. The striking preference of hu-TMC for the lungs when compared to intestines suggests selective engraftment among distinct mucosal tissues. The hu-TMC-SCID mouse chimera promises to be a unique animal model to study human-mucosa-associated lymphoid cells and EBV-related lymphomagenesis and B cell tumor progression.

Epidemiological studies have implicated periodontal disease (PD) as a risk factor for the development of cardiovascular disease (CVD). These studies addressed the premise that local infection may perturb the levels of systemic inflammatory mediators, thereby promoting mechanisms of atherosclerosis. Levels of inflammatory mediators in the sera of subjects with only PD, only CVD, both diseases, or neither condition were compared. Subjects were assessed for levels of C-reactive protein (CRP), serum amyloid A (SAA), ceruloplasmin, α1-acid-glycoprotein (AAG), α1-antichymotrypsin (ACT), and the soluble cellular adhesion molecules sICAM-1 and sVCAM by enzyme-linked immunoabsorbent and/or radial immunodiffusion assays. CRP levels in subjects with either condition alone were elevated twofold above subjects with neither disease, whereas a threefold increase was noted in subjects with both diseases (P= 0.0389). Statistically significant increases in SAA and ACT were noted in subjects with both conditions compared to those with one or neither condition (P= 0.0162 and 0.0408, respectively). Ceruloplasmin levels were increased in subjects with only CVD (P= 0.0001). Increases in sVCAM levels were noted in all subjects with CVD (P= 0.0054). No differences in sICAM levels were noted among subject groups. A trend toward higher levels of AAG was noted in subjects with both conditions and for ACT in subjects with only PD. Immunohistochemical examination of endarterectomy specimens of carotid arteries from subjects with atherosclerosis documented SAA and CRP deposition in association with atheromatous lesions. The data support the hypothesis that localized persistent infection may influence systemic levels of inflammatory mediators. Changes in inflammatory mediator levels potentially impact inflammation-associated atherosclerotic processes.

In 1988, two groups of investigators reported the successful adoptive transfer of nonneoplastic human cells into mice with severe combined immunodeficiency (SCID).1–3 These mice originated from the CB-17 lcr strain by a spontaneous autosomal recessive mutation4 affecting the VDJ recombinase system.5 SCID mice lack functional B and T cells, but have a normal arsenal of macrophages and NK cells. The survival of human immunoreactive cells in SCID mice allows for the study of the human immune system in an animal model. Thus the human immune system is available for experimental protocols feasible up to now only for the study of animal immune systems. SCID mice engrafted with human immunologically reactive cells were called “human-mouse chimeras,” “SCID-hu mice” and, hu-SCID mice. These mice are of interest for the study of maturation and differentiation of human hematopoietic cells,6 tumor immunology,7,8 human immune responses to infectious agents, neoplasms and autoantigens,9,10 and drug effects on the human immune system. The hu-SCID mouse also seems an exciting model for the direct study of components of human mucosa-associated lymphoid tissue (MALT), their interactions, functions, and traffic in vivo.11,12

The tissue binding properties of streptococcal lipoteichoic acid (LTA) were studied using normal and passively immunized BALB/c mice. After intraperitoneal injection in non-immunized mice, 3H-LTA concentrations in blood, heart, kidney and liver were highest between 24 and 30 h post-injection. LTA deposits in heart remained high for the next 24 h, whereas other tissue levels decreased. Constant amounts of 3H-LTA were detected in urine throughout the 48 h period. In passively immunized mice, the amount of tissue deposition of 3H-LTA was inversely proportional to the ratio of antibodies to LTA. Autoradiography revealed focal deposits of 3H-LTA in heart, kidney and liver. These observations indicate that LTA, released by streptococci growing at remote body sites, can be carried by the blood to internal organs where it can accumulate and participate in pathogenesis.

One of the major problems using man-mouse chimeras is still the difficulty to demonstrate unequivocally engraftment of human cells in murine tissues. Using supravital labelling of human leukocytes with the Hoechst dye H33342, it was possible to demonstrate directly their engraftment and to assess their distribution in the tissues of the severe combined immunodeficiency (SCID) mice. The human cells can be traced for a period of 4-5 weeks. In contrast to earlier reports, combined marker and labelled-cell studies suggest that T-cell surface marker CD3 with reported specificity for human lymphocytes are indeed found, also in man-mouse chimeras, only on human cells. The ratio of B and T cells of human origin changes significantly after transfer into SCID mice and differs among various SCID tissues. The simple staining procedure using the supravital nuclear dye H33342 opens new possibilities for the study of cellular interactions and host responses of the human immunoreactive cells in an increasingly well-characterized animal model.

Mice with severe combined immunodeficiency were inoculated intraperitoneally with 50 x 10(6) human tonsillar mononuclear cells (hu-TMCs) from Epstein-Barr virus (EBV) antibody seropositive or seronegative human subjects. Between 5 and 11 weeks later, 29.4% (10/34) of mice injected with hu-TMCs from EBV seropositive donors, but none of 34 animals receiving hu-TMCs from EBV seronegative donors, developed intraabdominal and/or intrathoracic tumours (P, 0.002). By means of in situ hybridization using alpha satellite DNA from human chromosome 17, all tumours produced after cell transfer from EBV seropositive donors were identified to be of human origin. Histologically the tumours resembled large cell lymphomas; the EBV genome was detected by in situ hybridization and EBV nuclear antigen by immunofluorescence in these tumours. The tumours were poly- or oligoclonal, and stained for human IgG and IgM, and less frequently IgA and IgD. Serum levels of human immunoglobulin in animals developing human tumours were significantly higher than in reconstituted mice without tumours and the sera exhibited polyoligo- or monoclonality in immunoelectrophoresis. Human interleukin 6 was detected in the serum of six of 10 animals with human lymphomas, but not in any animals without human lymphoma.

Spleen cells from C57BL/6 mice maintained on alcohol containing liquid diet for two weeks were evaluated for different immune functions. On an average, 22% fewer spleen cells were recovered from alcohol-fed mice when compared to cells from control animals. In alcohol-fed mice, the relative frequency of B cells increased, whereas total T cells including CD4 + cells decreased significantly. Alcoholic mice, when challenged with poly(rI) poly(rC), produced significantly less interferon than control mice. In vitro production of interferon alpha and gamma by the spleen cells of alcoholic mice was reduced by 67–90%. No significant differences were seen in the level of natural killer cell activity in spleen cells of control and alcoholic mice. These results suggest that chronic alcohol intake can result in not only changes in the number of immune cells, but more importantly affect their biological functions such as their ability to produce interferons.

SUMMARY Groups of C.B-17 SCID mice were reconstituted intraperitoneally with human tonsillar mononuclear cells (hu-TMC) from children seropositive for antibody to respiratory syncytial virus (RSV) and subsequently challenged intraperitoneally with inactivated RSV or sham-immunized. The synthesis and the distribution characteristics of human antibody to RSV in various murine tissues were studied using an enzyme-linked immunospot assay (ELISPOT). No specific antibody was observed in sham-immunized animals. In contrast, mice engrafted with hu-TMC exhibited the appearance of specific human antibody secreting cells (hu-ASC) after i.p. immunization with inactivated RSV. RSV-specific hu-ASC were detected only in animals engrafted with cells from donors seropositive for antibodies to Epstein-Barr virus. Hu-TMC engrafted mice showed RSV-specific IgM and, in lower numbers, IgG hu-ASC in several tissues including the lungs. Numbers of RSV-specific IgA hu-ASC were low, however, and detected only in the lung. No RSV-specific hu-ASC were detected in the intestine. These data demonstrate for the first time that hu-TMC-SCID chimeras respond to immunization with viral antigen. Furthermore, the results suggest that hu-TMC engraft in lungs but not in the intestinal tissue.

1 DEVELOPMENT OF LYMPHOMAS IN HUMAN ORIGIN IN MICE WITH SEVERE COMBINED IMMUNODEFICIENCY (SCID): ROLE OF EPSTEIN-BARR VIRUS (EBV) AND INTERLEUKIN-6 (IL-6)

The paired radiolabel technique was used to quantitate the amount of bovine serum albumin (BSA) deposited in the form of immune complexes in renal, pulmonary, splenic, hepatic, and cardiac tissues in rabbits with experimentally induced systemic chronic serum sickness (CSS). 24 h after injection, the concentrations of 125I-BSA found in the lungs, the spleen, and the kidneys of rabbits with systemic CSS induced by high doses of BSA (HDCSS) were in the same range (12.21, 10.93, and 12.68% × 10––3 injected dose/g tissue, respectively). Immunofluorescence studies demonstrated granular deposits of BSA, rabbit IgG, and C3 in the basement membranes of these organs. In rabbits during the phase between early and late proteinuric phases of CSS, 125I-BSA was present in the lung (16.85% × 10––3 ID/g tissue) and, in smaller amounts, in the kidneys, the spleen, and the liver. By immunofluorescence, immune reactants were found in phagocytic cells only. In rabbits with CSS induced by low doses of BSA (LDCSS), considerable amounts of antigen (22.72% × 10––3 ID/g tissue) were found in the kidney in late proteinuric phase, but not in other organs. In rabbits with early proteinuric phase of CSS, BSA was deposited in the kidneys (5.60% × 10––3 ID/g tissue) and, albeit in smaller amounts, in the lungs and the spleen (3.25 and 2.15% × 10––3 ID/g tissue, respectively). By immunofluorescence, scattered minimal deposits of BSA were seen in glomeruli and in pulmonary and splenic phagocytic cells. In both early and late proteinuric phases of CSS the heart did not appear to contain significant amounts of BSA. These quantitative data confirm that in rabbits with early or late proteinuric phases of CSS, BSA is deposited in the kidney and further show that comparable amounts of BSA are present in the lung and in the spleen of rabbits in the late proteinuric phase of HDCSS.

Invasion of periodontal tissues by different bacterial morphotypes has been reported in human periodontitis; however, limited information is available as to prevalence, localization and the bacterial species involved. The present study determined prevalence and gingival localization of Actinobacillus (Haemophilus) actinomycetemcomitans in periodontal lesions of juvenile periodontitis patients. Thirty-five gingival biopsies were obtained from 12 juvenile periodontitis patients at the time of periodontal therapy. One additional control biopsy was obtained from each of two adult periodontally healthy subjects, one adult periodontitis patient and one periodontally healthy monkey (Macaca fosibolius). The biopsies were carefully processed to avoid mechanical introduction of bacteria into the tissues and were examined using light and electron microscopy. Rabbit antisera specific for the three A. actinomycetemcomitans serotypes were used for immunofluorescence microscopic localization of A. actinomycetemcomitans antigens in the gingival sections. Immunofluorescence microscopy showed A. actinomycetemcomitans specific antigens in the gingival tissues of 11 of the 12 juvenile patients examined. None of the control specimens showed evidence of A. actinomycetemcomitans antigens in the gingival connective tissue. One specimen from a periodontally healthy subject and the monkey biopsy, however, showed A. actinomycetemcomitans antigens in bacterial plaque on the surface of the crevicular epithelium. Transmission electron microscopic examination showed microcolonies of small gram-negative rods in the connective tissue, as well as single bacterial cells between collagen fibers and in areas of cell debris. In addition to these extracellular bacterial cells, evidence of bacterial cells was also found within gingival connective tissue phagocytic cells. The data from the present study suggest that the gingival tissue in juvenile periodontitis lesions harbors A. actinomycetemcomitans.

The rat small intestinal epithelial cell (enterocyte) has an average life span of 48h. Undifferentiated stem cells in the lower crypt region undergo division and differentiation into enterocytes as the cell moves on a basement membrane up the villus to reach the tip and be extruded into the lumen. The mechanism of this movement and relationship to basement membrane synthesis and/or renewal is unknown. This problem is discussed in regard to our findings suggesting that the major contribution to basement membrane synthesis and renewal may not be from the enterocyte.

Three groups of adult male Nya:(FG) rabbits were treated intrarectally, 3 times a week for 7 months, with 1 ml of fresh homologous semen, 20 ml of colonic enema, or 20 ml of enema followed by 1 ml of previously frozen semen. A control group was untreated. The results of several in vitro tests of humoral and cellular response showed that chronic intrarectal insemination leads to an abrogation of the immune response, expressed differently in the two groups of rabbits receiving semen. A consistently depressed blastogenic stimulation in response to T-cell mitogens was observed only in the animals receiving fresh semen. Neither group produced antilymphocyte antibodies. Anti-seminal-fluid and antispermatozoa antibodies were detected after 3 months of treatment in two animals receiving enema and semen and one animal receiving fresh semen. Circulating immune complexes were detected after 2 months of treatment in animals receiving enema and semen. These immune complexes contained predominantly IgM and were specific for seminal antigens. Assay of the cellular response to a T-cell-dependent antigen showed, in the animals receiving enema and semen, a pronounced decrease in the indirect IgA and IgG plaque-forming cells response with practically no effect on the IgM response. These results, taken together with those of the preceding paper, suggest that in intrarectally inseminated rabbits the functional capabilities of the immune system may deteriorate without histologic or morphologic changes in the anorectal mucosa of relevant lymphoid tissues.

Proliferative responses of normal (not immunized intentionally) spleen cells from inbred strains of mice to co-stimulation with phorbol ester (PMA) and calcium ionophore A23187 were studied. Striking differences in the magnitude of the responses of spleen cells and splenic T cells from various strains were observed. It appeared that these differences reflected mainly differences in the inducibility of the expression of the gene for the a chain of the IL 2 receptor (IL 2R) by phorbol ester. Formal genetic analysis suggested that the differences in response to phorbol ester and calcium ionophore are controlled by two independent genes with the alleles controlling good response being dominant. The differences in the inducibility of the IL 2R gene seemed to be controlled by alleles of a single gene. At least one of the putative genes may be a regulatory element affecting the gene for the a chain of IL 2R. The results may have a practical significance for devising more efficient procedure(s) to generate LAK cells used for tumor immunotherapy.

Preparations of human malignant effusion galactosyltransferase activity purified according to previously published techniques using enzyme-specific affinity chromatography consistently produced antibodies directed toward immunoglobulins with no detectable antigalactosyltransferase. Double immunodiffusion analysis of the antigen showed the presence of both IgG and IgA. Affinity chromatography with anti-human IgG-Sepharose and anti-human serum-Sepharose resulted in a 48,000-fold purification of galactosyltransferase activity with no detectable IgG by radioimmunoassay. Immunization of rabbits with this preparation produced antibodies directed against galactosyltransferase activity and minimal anti-Ig. The persistence of immunoglobulins during the purification of soluble galactosyltransferase activity through two enzyme-specific affinity chromatographic steps suggests an association of immunoglobulins with galactosyltransferase activity.

Multi-organ involvement and especially extraintestinal manifestations have suggested an immune complex-mediated pathogenesis of ulcerative colitis and Crohn’s disease. Using various techniques controversial data have been reported on the incidence and levels of circulating immune complexes and their correlation to clinical presentation. Sera of 131 patients with inflammatory bowel disease (78 Crohn’s disease, 53 ulcerative colitis) representing a wide spectrum of disease activity, treatment and presence or absence of extraintestinal manifestations were tested for circulating immune complexes using Raji cell indirect immunofluorescence assay, Raji cell radioimmunoassay, C1q solid phase assay and polyethylene glycol precipitation coupled with measurements of optical density and subsequent immunoelectrophoresis or radial immunodiffusion. Circulating immune complexes in low concentrations were observed in a small number of patients with inflammatory bowel disease, the frequency and concentrations being slightly higher in patients with Crohn’s disease than in those with ulcerative colitis. No association of concentrations of circulating immune complexes with disease activity or presence of extraintestinal manifestations could be demonstrated. These data do not support the claim for a major role of circulating immune complexes in the pathogenesis of inflammatory bowel disease.

New Zealand rabbits were injected three times a week with 2 mg of mercuric chloride (HgCl2) (MC) to test the hypothesis that toxic agents release autologous antigens, generating antibody response and tissue injury. After 2 weeks of injections the rabbits developed anti-basement membrane antibodies binding to renal and extrarenal basement membranes and to the peri- and endo-mysium of skeletal muscles. Glomerular histology appeared normal. Membranous glomerulonephritis developed 1–2 months later with granular subepithelial deposits containing rabbit IgG and C3. Similar deposits were present in the basement membranes of other organs. Raji cell tests for immune complexes in the sera were negative in the initial stages and positive in the late stages of the disease. Radioactive MC was found in the cytoplasm of renal tubular, intestinal, and hepatic epithelial cells but not in glomerular deposits. Renal eluates and selected sera reacted with the basement membranes and the collagen matrix in normal or collagenase-digested sections of renal or extrarenal normal rabbit tissues. This reactivity was reduced or abolished by washing tissue sections in PBS or by absorption of renal eluates with whole kidney homogenates. The findings are consistent with the hypothesis that MC induces a biphasic disease characterized first by the production of antibodies to basement membranes and the extracellular collagen matrix and subsequently by antigen-antibody complexes formed in situ and in the circulation and presumably containing soluble polysaccharide components of the collagen matrix.

Serum antibodies to five cow's milk proteins (alpha-casein, bovine serum albumin, beta-lactoglobulin A and B, and alpha-lactalbumin) were investigated in young patients with inflammatory bowel disease (56 Crohn's disease, 24 ulcerative colitis). IgG antibodies against bovine serum albumin, beta-lactoglobulin A and beta-lactoglobulin B were higher in Crohn's disease patients than in those with ulcerative colitis or the controls. IgG anti-bovine serum albumin antibodies were higher in those Crohn's disease patients who had higher scores of disease activity. Finally, IgA antibodies to alpha-casein were higher in patients with Crohn's disease and ulcerative colitis when compared to controls. These findings may be due to increased uptake of dietary antigens or enhanced immunological response occurring in Crohn's disease patients.

Immune complexes (IC) present in the glomeruli of rabbits with chronic serum sickness (CSS) and in patients with systemic lupus erythematosus (SLE), idiopathic membranous nephropathy (IMN), and acute poststreptococcal glomerulonephritis (PSGN) were analyzed by incubation with antigenic preparations. The efficacy of these preparations to dissolve IC was assayed by comparison of results of direct immunofluorescence tests performed with the kidney tissues before and after incubation with antigenic preparations. The FITC-conjugated antisera used in these tests were specific for IgG, C3, and – in the case of CSS – for the eliciting antigen, bovine serum albumin (BSA). During the acute proteinuric phase of CSS in rabbits, incubation of tissue sections with BSA alone led to complete dissolution of IC. In many rabbits with late phase proteinuria, however, tissues had to be incubated with both BSA and aggregated fraction II of rabbit serum. In all biopsy specimens from patients with IMN, and in some specimens from patients with PSGN and SLE, aggregated fraction II of human serum resulted in complete or incomplete dissolution of IC. On the other hand incubation of tissues with excess DNA in SLE or with streptococcal antigens in PSGN did not lead to dissolution of IC. These studies suggest significant participation of antibodies to aggregated immunoglobulins (i.e., rheumatoid factors or rheumatoid-like factors) in IC found in the above-mentioned diseases. Other antigen-antibody systems, however, may also contribute to the deposits in the glomerulonephritides studied.

Employing a series of monoclonal antibodies directed against T cell subsets using the avidin-biotin complex method as the immunoperoxidase technique and using fluorescein-conjugated antisera directed against the major immunoglobulins, we have studied the distribution of immunocompetent cells in sections of tonsils from 21 patients with various inflammatory diseases of the tonsils, including Streptococcal tonsillitis, recurrent tonsillitis and tonsillitis associated with infectious mononucleosis. The following conclusions can be made in regard to our study. The percentage of T cells decreases in all compartments of the tonsils with increasing episodes of tonsillitis as well as with infectious mononucleosis and Streptococcal tonsillitis. Similarly, the percentage of HLA-DR positive cells decreases with increasing episodes of tonsillitis and is statistically significant in the mantle zone. The percentage of IgM B cells and IgD B cells tends to increase in the extrafollicular zone and decrease in the mantle zone with increasing episodes of tonsillitis as well as with increasing age. The percentage of IgG and IgA plasma cells is highest in children who have had 3-5 episodes of tonsillitis, but markedly decreases in the follicle and extrafollicular compartments in patients who have had more than 5 episodes of tonsillitis. FACS analysis of B cells in the tonsils and peripheral blood show a marked decrease in IgD in both the tonsil and the peripheral blood and a significant increase of IgG in the peripheral blood. These findings may suggest late clonal expansion of B cells in recurrent tonsillitis and Streptococcal tonsillitis. Finally, the distribution of immunocompetent cells in recurrent tonsillitis, Streptococcal tonsillitis and tonsillitis associated with infectious mononucleosis appears to be independent of age.

Only few reports are available on the consequences of chronic oral administration of low doses of mercuric chloride (HgCl2). Forty Brown-Norway rats received 150 μg HgCl2/100 g body weight 3 times a week by gavage or by i.m. injection with 100 μg twice per week. After 2 weeks of oral HgCl2 administration, the rats lost weight and hair. Phases of proteinuria were observed in weeks 5–8 and then continuously from week 12 until the end of the experiment at week 39. Antibodies binding to renal, intestinal, and vascular basement membrane developed after 2 weeks; circulating immune complexes were detectable in increasing titers starting at week 3. There were linear deposits of IgG, IgM, and IgA in the glomerular basement membrane and tubular basement membrane, and along the intestinal basement membrane. After week 11, the first granular immune deposits were observed in renal and intestinal basement membranes. Light microscopy showed thickening of glomerular basement membrane, mesangial matrix, and tubular basement membrane. In addition, interstitial nephritis was observed in some animals. Interestingly, kidney involvement was as severe in the orally as the i.m.-treated animals.

The sensitivity and specificity of immunohistological staining procedures employing horseadish peroxidase labeled antibody were evaluated with the aid of chessboard titrations. Two indirect staining systems, detecting antinuclear antibody and pemphigus intercellular antibody, were examined with reference to indirect immunofluorescence. In both systems, chessboard titration results showed that plateau titers of serum antibodies appeared to be a function of label content. The plateau endpoints reflected the anti-immunoglobulin concentrations of conjgates. With a conjugate of molar enzyme to protein ratio (E/P) of 1.1, the sensitivity of indirect staining appeared to be equivalent to that of immunofluorescent staining performed with a conjugate with molar fluorescein to protein ratio of 4.8. Sensitivity decreased sharply with conjugates of low E/P rations. Three methods of assaying the peroxidase content of conjugates were evaluated for reproducibility and sensitivity in relation to staining properties.

Immunohistologic studies were performed on autopsy tissues of 2 patients with systemic lupus erythematosus. All tissues examined--the kidney, lung, spleen, liver, intestine, peritoneum, and choroid plexus--contained immune deposits. Antinuclear antibody concentration in immunoglobulin G eluted from lung and spleen tissue was higher than in serum immunoglobulin G. These findings support the assumption that in systemic lupus erythematosus the renal as well as the extrarenal lesions can be attributed to vascular deposition of immune complexes.

SummaryThe effect of prolonged immunization of mice with bovine serum albumin (BSA) or ovalbumin (OA) on the growth of the transplanted, methylcholanthrene-in-duced fibrosarcoma was studied. When immune complexes appeared in the kidneys and in the circulation, mice were injected subcutaneously with various numbers of syngeneic tumor cells.Tumor developed within 17 days in all mice immunized with BSA and injected with 104, 103 and 102 tumor cells and control mice injected with 104 and 103, whereas only 3 of 10 control mice injected with 102 tumor cells developed tumors at that time. All mice immunized with OA and injected with 105, 104 and 103 tumor cells and control mice injected with 105 and 104 developed tumor within 12 days after injection of the tumor cells, whereas in the control group injected with 103 tumor cells, only 6 of 12 mice did so. Weight of the tumors from the mice immunized with BSA or with OA was significantly greater than that of tumors from control mice.

Summary: The distribution of B and T lymphocytes and macrophages in the middle ear fluid (MEF) was studied in groups of children with otitis media associated with serous, seromucinous, or mucoid effusions in the middle ear. The subpopulations of T and B lymphocytes and macrophages were identified in the MEF by employing the techniques of spontaneous erythrocyte rosetting (E), membrane immunofluorescence, erythrocyte-amboceptor-complement (EAC) and erythrocyte-amboceptor (EA) rosette formation, respectively. A significant number of B and T lymphocytes or macrophages were detected in all specimens; however, certain differences were observed in their relative distribution in different types of effusions. Serous MEF was characterized by a cellular response associated predominantly with macrophages, although T lymphocytes were also seen frequently. No B lymphocytes were seen in such exudates. The seromucinous MEF contained B and T lymphocytes and macrophages in approximately similar proportions. Significantly, however, the mucoid effusion consisted mainly of B lymphocytes. Only macrophages and no T cells were observed in these effusions. The cytologic findings reported here may be helpful in a more definitive classification of middle ear effusions. These data may suggest a possible role of the different components of cellular immunity in the development of middle ear effusions. Speculation: E rosette-forming cells predominate in the serous and seromucinous effusions of otitis media. EAC rosette-forming cells appeared as a predominant cell type in mucoid effusions. The different types of effusions in otitis media may represent the various stages of the same inflammatory process. The differences in the types of immunocompetent cells observed may reflect the nature of the underlying immune response in serous, seromucinous, or mucoid forms of otitis media.

Sera of patients with lepromatous leprosy were studied for the presence of a variety of antibodies and immune complexes (IC). The frequencies of heterophile, Hanganutziu-Deicher and Forssman antibodies were 61 and 43%, respectively, which were significantly higher than those in other diseases. The frequency of antibodies to cardiolipin was 89% and the frequency of rheumatoid factor was 34%. Circulating IC were demonstrated in 54% of the patients’ sera by Raji-cell test and in 43% by anti-antibody inhibition test. Analyses of immunoglobulin classes of IC revealed that IgG was predominant in IC of patients with lepra reaction (LR) and IgM in patients without LR. Immune deposits were found in and between cells of dermis in skin biopsy specimens of patients with LR.

Chickens of the Obese strain (OS) of white Leghorns display an hereditary, spontaneous autoimmume thyroiditis (SAT) beginning within the first few weeks of life and characterized by hypothyroidism, severe mononuclear cell infiltration of the thyroid glands, and circulating thyroglobulin autoantibodies. The fact that this disease occurs in an avian species is of great advantage for various immunologic and embryologic studies. In contrast to most experimentally induced autoimmune diseases SAT is B-dependent. This observation led to a new concept for the classification of autoimmune diseases into T- and B-dependent types with intermediate forms. This classification may be relevant to the proper diagnosis and selective immunosuppressive treatment of this group of immunopathologic conditions. Data are presented which favor both a primary thyroid alteration and an aberration of the immunological self-recognition mechanism as factors underlying the development of SAT.

There is evidence that autologous antigens may have a role in the pathogenesis of various types of nephritides spontaneously developing in animals and in man. Data available suggest the involvement of antigens of glomerular basement membrane (GBM), cell nuclei, thyroid, and, possibly, brush border of proximal tubules, and tumors (Wilson and Dixon, 1976). The mechanisms of autoimmunization, however, are not well understood.

Serum antibodies to five cow's milk proteins, alpha-casein, bovine serum albumin (BSA), beta-lactoglobulin A and B (BLG-a, BLG-b) and alpha-lactalbumin (ALA) were investigated in young patients with inflammatory bowel disease, 56 with Crohn's disease (CD), 24 with ulcerative colitis (UC). IgG antibodies against BSA and BLG-a and -b were higher in Crohn's disease patients as compared to those with ulcerative colitis and controls. The IgG anti-BSA were higher in the group of CD patients with higher score of disease activity. Additionally, IgA antibodies to alpha-casein were higher in CD and UC compared to control. These findings may be due to increased uptake of dietary antigens or enhanced immunological response occurring in CD patients.

Large doses of cadmium, gold, or mercury salts produce acute toxic lesions, e.g., renal tubular necrosis (Gritzka and Trump, 1968). Administration of low doses of heavy metals may induce immunologically mediated diseases. Thus, membranous nephropathy has been described in humans in association with administration of mercury-containing compounds (Mendema et al., 1963). During the last decade, several animal models of chronic mercuric chloride-induced, immunologically mediated diseases have been established (Bariety et al., 1971; Roman-Franco et al., 1976, 1978; Sapin et al., 1977; Albini and Andres, 1982). In these models, pathogenesis involves autoimmunization to connective tissue components and development of immune complex-mediated pathology.

Circulating immune complexes (IC) are often detected in patients with primary biliary cirrhosis (PBC). To investigate the nature of the antigen(s) present in IC, sera of 15 patients were incubated with an excess of the suspected antigens. The addition of mitochondrial antigens resulted in dissociation of IC in sera of 6 of 15 patients with PBC but not of 5 patients with alcoholic hepatitis nor 5 patients with rheumatoid arthritis. These data indicate that in some patients with PBC, circulating IC contain mitochondrial antigens.

The possible role of circulating immune complexes (IC) in the production of gastrointestinal lesions was studied in rabbits with chronic serum sickness (CSS) induced by multiple daily injections of bovine serum albumin (BSA). All rabbits generating a marked antibody response developed IC glomerulonephritis. In approximately 50% of these rabbits granular deposits of BSA, rabbit IgG, and C3 were also found in the gastrointestinal tract. The immune deposits in the gastrointestinal tract were mainly present in the vessel walls, close to the intestinal glands and the surface epithelium, and between the smooth muscle cells. This was accompanied by slight to moderate edema of the mucosa and the submucosa and mild infiltration of inflammatory cells. Electron-dense deposits were found in a pattern corresponding to that observed for BSA, rabbit IgG, and C3. Degranulated neutrophils, basophils, and mast cells were noticed in the interstitium. The presence in the same areas of granular deposits of BSA, IgG, and C3, corresponding to electron-dense deposits, suggests that the deposits contain BSA-anti-BSA complexes. These findings show that in rabbits with CSS circulating IC may localize and induce injury in the gastrointestinal tract.

Recent immunohistological studies have suggested that Actinobacillus actinomycetemcomitans is present in the gingival tissues in juvenile periodontitis lesions. The present study examined tissue bound A. actinomycetemcomitans by bacterial culture and immunohistological demonstration of antigen in tissue. A total of 14 periodontitis lesions were examined. Eleven biopsies were obtained from gingiva adjacent to A. actinomycetemcomitans infected pockets, while the remaining three control biopsies were obtained from gingiva adjacent to pockets where subgingival A. actinomycetemcomitans infection could not be detected. Each biopsy was hemisected, one half was used for immunofluorescence microscopic examination while the other half was processed for culture of A. actinomycetemcomitans. The latter section was surface-disinfected, repeatedly washed and then minced to release bacteria from within the tissues. Aliquots from the serial washings and the minced tissue suspension were cultured on medium selective for A. actinomycetemcomitans. Surface disinfection and serial washings gradually decreased cultivable A. actinomycetemcomitans in the washings aliquots. Following tissue disruption, an increase in colony-forming units of A. actinomycetemcomitans was seen from eight of the 11 test biopsies. This bacterium could not be detected in washings or minced tissue suspensions from the control biopsies obtained from lesions in which subgingival A. actinomycetemcomitans was previously not detected. A positive correlation was seen between the presence of A. actinomycetemcomitans antigens in the gingival biopsies and; (1) A. actinomycetemcomitans colony-forming units released from the minced tissues (r = 0.90, p = 0.000), as well as; (2) the colony-forming units from the periodontal pocket (r = 0.62, P = 0.017).(ABSTRACT TRUNCATED AT 250 WORDS)

CHICKENS of the Obese strain (OS) spontaneously develop severe autoimmune thyroiditis during the first weeks of life, with clinical signs of hypothyroidism1,2. Serological tests reveal both circulating and bound thyroglobulin autoantibodies3. The lymphoid infiltration of the thyroid gland is characterised by a predominance of plasma cells and the formation of a large number of germinal centres1. In view of the histological and serological features, the spontaneous autoimmune thyroiditis of OS chickens can be considered as an appropriate animal model for human Hashimoto thyroiditis2.