Boris Albini

Systemic Inflammation in Cardiovascular and Periodontal Disease: Comparative Study

ABSTRACT 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.

Anti-basement membrane antibodies and antigen--antibody complexes in rabbits injected with mercuric chloride.

Abstract 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.

Distribution and Engraftment Patterns of Human Tonsillar Mononuclear Cells and Immunoglobulin-Secreting Cells in Mice with Severe Combined Immunodeficiency: Role of the Epstein-Barr Virus

Tonsils seem to be the ideal source of 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.

Effect of alcohol on spleen cells and their functions in C57BL/6 mice

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.

Dissociation of immune complexes in tissue sections by excess of antigen.

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 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.

The Distribution of Immunocompetent Cells in the Compartments of the Palatine Tonsils in Bacterial and Viral Infections of the Upper Respiratory Tract

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.

Co-purification of soluble human galactosyltransferase and immunoglobulins.

Abstract 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.

Tissue distribution of mucosal antibody-producing cells specific for respiratory syncytial virus in severe combined immune deficiency (SCID) mice engrafted with human tonsils.

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.

Immune complexes in primary biliary cirrhosis contain mitochondrial antigens

Abstract 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.

Identification of Streptococcus pyogenes proteins that bind to rabbit kidney in vitro and in vivo

Proteins were extracted from the surface of a nephritogenic strain of Streptococcus pyogenes M12 and tested for binding to rabbit kidney using indirect immunofluorescence and enzyme-linked immunoassays. Streptococcal antigens bound in vitro in a fine linear pattern to basal laminae of glomeruli, Bowmans capsule, and tubules. Perfusion of rabbit kidney in vivo with streptococcal components resulted in focal and segmental fine granular staining of glomerular capillaries. Three streptococcal proteins (43, 31 and 9 kDa) were recovered from renal tissue that was pretreated in vitro with S. pyogenes extract. Streptococcal components bound in vitro to heparan sulfate, heparin, laminin and collagen IV but only weakly or not at all to fibronectin, bovine serum albumin or dextran sulfate. Affinity chromatography of bacterial extracts on heparin-agarose produced a 9 kDa streptococcal protein (pI 9.5) which bound to kidney basement membranes in vitro and in isolated perfused kidneys. Several additional strains of group A streptococci were found to contain the 9 kDa cationic protein. This bacterial protein, when released into the blood by the bacterium during infection, may contribute to the pathogenesis of streptococcus-associated nephritides in man.