Relationship between kinetic stability and immunogenicity of HLA-DR4/peptide complexes

Immunodominant T cell epitopes from the autoantigen human cartilage glycoprotein 39 have previously been mapped in the context of HLA-DR*0401 and *0402, using mice expressing HLA-DR4 transgenes. We measured the dissociation rates of these epitopes from soluble recombinant DR*0401 and DR*0402 to assess the relationship between peptide/HLA-DR4 kinetic stability and immunogenicity. Experiments were performed at endosomal pH (5.5) and at cell surface pH (7), in the absence and presence of soluble recombinant HLA-DM (sDM). All (4/4) immunodominant peptide/HLA-DR complexes exhibit dissociation half-times of 1h to several days. In contrast, most (3/4) non-immunodominant complexes dissociate with half-times <30 min under at least one of these conditions. Interestingly, a complex which is stable except in the presence of HLA-DM at pH 5.5 is immunogenic only following peptide immunization, while a complex which is stable at acidic but not at neutral pH, is non-immunogenic following either whole protein or peptide immunization. These data indicate that kinetic stability of peptide/MHC complexes in vivo is a key determinant of immunogenicity.     

Experimental basis of enhancing the immunogenicity of influenza B virus by genetic recombination

Redistribution of the immunogenicity marker in the course of genetic recombination of influenza B virus was studied in animal experiments on virus strains differing in their ability to induce antibody formation following a single peroral or intraperitoneal immunization. Immunogenicity of influenza virus could be enhanced by recombination of a strain possessing a low activity with a highly immunogenic homotypic strain. Efficiency of the transfer of the immunogenicity marker depended on the properties of viruses used for recombination. Strains at a low passage level proved to be more prospective "donors of immunogenicity" than hyperattenuated thermosensitive viruses which were unsuitable for this purpose. There was no complete correlation between hemagglutinating activities of influenza B viruses and of the recombinant.     

Experimental demonstration of lateral cohesion in a layer of adsorbed protein and in layers of gold-antibody complexes bound to surface immobilised antigen

The spatial distribution of ferritin molecules adsorbed on quartz surfaces and the spatial distribution of antibody-coated colloidal gold particles over an antigen-coated surface was studied by electron microscopy. Ferritin molecules were found to initially adsorb in small clusters at 2.5 X 10(9) sites/cm2. The initial nucleation was followed by growth of the clusters. Gold antibody complexes were initially bound to the antigen-coated surface as single particles, and formation of clusters was a secondary event at higher particle densities. The results indicate that lateral cohesion between macromolecules may play a role in the stability of adsorbed layers of protein and surface immobilised antigen-antibody complexes.     

Uses and limitations in the demonstration of specific circulating immune complexes in patients with amoebiasis

A micro-enzyme linked immunosorbent assay (micro-ELISA) has been evaluated as a diagnostic test to detect amoebic antigen in polyethylene glycol (PEG) precipitated circulating immune complexes (CIC) in sera from patients with amoebiasis. The immune complexes were captured on rabbit anti-amoebic IgG-coated wells of microtitration plates and the complexed antigen was detected by enzyme linked antihuman immunoglobulins. A titre of greater than 160 for the immune complexes was considered to be of clinical significance. The immunoassay detected amoebic, antigen-specific CIC in 35 (94.5%) of 37 patients with confirmed amoebic liver abscess. Twenty (55.5%) of 36 clinically suspected cases of amoebic liver abscess had amoebic antigen-specific CIC and responded favourably to anti-amoebic chemotherapy. Only two (20%) of 10 cases of non-dysenteric symptomatic intestinal amoebic infection had amoebic antigen-specific CIC. One (10%) of 10 patients with non-amoebic intestinal disorders also had amoebic antigen in CIC. However, none of 15 cases of non-amoebic hepatic disorders that included hydatid disease, metastatic adenocarcinoma, hepatocellular carcinoma, cholecystitis and choledocal cyst, 13 cases of rheumatoid arthritis and 25 apparently healthy subjects had amoebic antigen in CIC. The levels of the amoebic antigen-specific CIC did not correlate (p greater than 0.05) with either the number of abscess(es) or lobe(s) of the liver involved. However, the levels of antigen-specific CIC were higher (p less than 0.01) in patients with a liver size of more than 5 cm below the right costal margin. Antigen-specific CIC levels tended to decline or disappear during 3-6 months following completion of therapy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Experimental cholestasis promotes the deposition of glomerular IgA immune complexes.

Previous experimental and clinical studies support a role for the hepatobiliary system in the clearance of oligomeric IgA from serum, and alterations of this system have been associated with the deposition of IgA in the renal mesangium. The present studies in mice address the question of whether the mesangial deposition of IgA following cholestasis includes immune complexes. While bile duct ligation resulted in mesangial IgA deposition within several days in approximately 75% of animals, whether deliberately orally immunized, nonimmunized, or given injected immune complexes, mice that underwent sham operations had IgA deposits only if orally immunized. Moreover, mice that had been orally immunized or given injected immune complexes and whose bile ducts had been ligated contained deposits of specific IgA antibody and antigen. In the ligated mice some of the IgA was secretory IgA, as demonstrated by the presence of secretory component. Thus, bile duct ligation promotes the deposition of circulating IgA immune complexes, presumably by decreasing their clearance from serum, and gives rise to secretory IgA in the glomerular mesangium. The secretory immune system probably plays a role in the pathogenesis of idiopathic and cirrhosis-related human IgA nephropathy.     

Experimental Leishmania (L.) amazonensis leishmaniasis: characterization and immunogenicity of subcellular fractions

A technique developed in Trypanosoma cruzi biochemical studies was successfully used to fractionate Leishmania (Leishmania) amazonensis promastigotes. Ultrastructural analyses revealed a membrane fraction (MF) associated to subpellicular microtubules, a ribosomal-rich microsomal fraction (MicF), and a flagellar fraction (FF) free of associated membrane. All fractions proved to be immunogenic through delayed type hypersensitivity reaction assays. Therefore, a protocol was designed to test whether these fractions could elicit a protective response in mice infected by L. (L), amazonensis. The protocol consisted of a BCG injection (as cellular immunity inducer), followed by cyclophosphamide (once its cytotoxic effect is over, this immunosupressor can increase the number of circulating leukocytes), then an injection with one of the fractions followed by a challenge. When compared to infected control animals, mice injected with any of the fractions presented a smaller footpad swelling, especially those injected with MicF or FF. Macroscopically, immunized mice under modulation by BCG presented no swelling. Histopathological studies performed on day 120 revealed fewer amastigotes and more intense inflammation in lesions of MicF and FF injected mice. Animals injected with MF presented an intermediate pattern. Parasite quantification corroborated these results. The results show that all fractions are potent immunostimulators, but MicF and FF have the strongest protective ability.     

Immunohistochemical demonstration of keratins in the cysts of thyroid glands,parathyroid glands,and C-cell complexes of the dog

Cyst structures were often detected in and around thyroid glands of the dog. The present study revealed the frequency of occurrence, the light microscopic features, and the immunoperoxidase reactions to anti-keratin and anti-ldS-thyroglobulin antisera of each cyst located in parathyroid III, parathyroid IV, thymus IV, C-cell complexes, and thyroid parenchyma from 112 dogs. In each location, cysts showed characteristic features. In parathyroid III, the cysts were covered with single or pseudostratified epithelium composed of ciliated cells; whereas in parathyroid IV they were covered with keratinizing stratified squamous epithelium. In C-cell complexes, small cysts lined with small packed cells were predominant, and large cysts lined with single cuboidal cells or stratified squamous cells were also present. In thymus IV located in the close vicinity of parathyroid IV, cyst epithelium consisted of several types of cells showing variable futures. In thyroid parenchyma, there were several types of cysts: some were covered with ciliated columnar cells, and others were covered with two or multilayers of small packed cells or cuboidal cells. In spite of these differences in appearance of the cysts located in different tissues, all their epithelia were immunoreactive to the keratin antisera, except for small cysts in C-cell complexes, which were regarded as immature structures. Thus, the presence of keratin filaments in epithelial cells seems to be a characteristic feature of all cysts. The lumens of each cyst contained variable amounts of amorphous materials, which showed colloid-like, flocculent, foamy, and granular features and were periodic acid-Schiff-positive in variable degrees, from weak to intense. Although the lumenal contents of the cysts in parathyroid III revealed no immunoreactivity for 19S-thyrogIobulin, those in thyroid parenchyma, C-cell complexes, parathyroid IV, and thymus IV reacted strongly with the 19S-thyroglobulin antiserum.     

Experimental demonstration of pathogenic potential of Anisakis physeteris and Anisakis paggiae in Wistar rats

Anisakis morphotype I is the principal etiologic agent of human anisakiasis, with differences in pathogenicity found between the Anisakis simplex s.s. and A. pegreffii species; however, the role of morphotype II larvae in this illness is not well understood. The purpose of this study is to verify the ability of morphotype II larvae to invade tissues via the experimental infection of Wistar rats, an animal model which simulates infection in humans. In the in vivo assay, 7.1 % (4/56 L3 morphotype II) showed pathogenic potential, defined as the capacity of the larvae to cause lesions, attach to the gastrointestinal wall or penetrate it. Two of these larvae, one of A. physeteris and one of A. paggiae, penetrated the stomach wall and were found within the abdominal cavity, with the first one producing a small lesion with blood vessel breakage. The majority of the L3 larvae of morphotype II were found in the intestine (51.8 %; 29/56) with the caecum being the least frequent location (8.9 %; 5/56). In contrast, 44.0 % (11/25) of the morphotype I larvae demonstrated pathogenic potential. Isoenzyme electrophoresis, PCR-RFLP of ITS1-5.8 s-ITS2 and PCR-sequencing of the cox2 mitochondrial gene were used to identify these larvae as A. physeteris (42.9 %), A. paggiae (30.3 %) and A. brevispiculata (1.8 %). Although the morphotype II larvae of A. physeteris and A. paggiae have lower pathogenic potential than morphotype I larvae of A. simplex s.s. (93 and 91 % lower, respectively), they may still be implicated in human anisakiasis, as they are capable of attaching to and penetrating the gastrointestinal wall of animals, demonstrating a similar pathogenicity to that of A. pegreffii. The techniques used for the identification of species reveal a great genetic heterogeneity of A. paggiae and A. physeteris, suggesting the existence of sibling species.     

An electron microscopic demonstration of immune complexes of hepatitis b e-antigen using colloidal gold as a marker

Immune complexes of the hepatitis B e-antigen (HBeAg) could be labeled and thus visually identified in the electron microscope by using antibody to HBeAg (anti-HBe) tagged with colloidal gold particles. Circulating immune complexes of HBeAg were detected in sera from patients with acute hepatitis B infections as well as from asymptomatic carriers of hepatitis B surface antigens (HBsAg). Sera positive for rheumatoid factor frequently contained mixed aggregates in which immune complexes of HBsAg were closely bound to immune complexes of HBeAg.     

Experimental granulomas induced by mycobacterial immune complexes in rats

After BCG were complexed with homologous anti- BCG serum IgM was found to be firmly bound to the organisms. Preparations were made at antigen/antibody equivalence and at two-fold and four-fold antibody excess. They were injected subcutaneously into rats. At equivalence the mixtures provoked rapidly developing necrotic destructive lesions containing many bacilli, Injection of mixtures at antibody excess caused the rapid formation of epithelioid cell granulomas without necrosis and containing few bacilli. Compared with the injection of BCG alone, injection of comparable numbers of IgM-complexed bacilli at equivalence led to more rapid necrosis but also more rapid resolution of the granulomas which followed. Delayed-type skin reactions to PPD took longer to develop after injection of complexed BCG, usually as long as 4 weeks. The delay varied directly with the degree of antibody excess. This failure to detect cell mediated immunity was reflected in the histology of the draining lymph nodes which differed strikingly from that seen after injection of BCG alone. In animals injected with bacilli in excess antibody, epithelioid cell granulomas formed and viable bacilli were apparently eliminated before skin reactions to PPD developed. It is concluded that circulating immunoglobulin, perhaps IgM in particular, is likely to play a significant role in the pathogenesis of tuberculosis and similar diseases and that the relative ratio of antigen to antibody within the lesions may be crucial in influencing the balance between tissue destruction and healing.     

Reflections on the immunogenicity of therapeutic proteins

Therapeutic proteins are now established as a major class of medication with proven efficacy in treating a wide range of diseases. The administration of such proteins to patients can lead to the development of antibodies that are able to bind and eventually neutralise the protein administered. Many advances have been made in understanding the immunogenicity of therapeutic proteins, but opinions are often conflicting. Although it is important to understand more about the antibodies generated against therapeutic proteins, the need for future research must not be neglected, so that other relevant factors can be identified and addressed to maximise treatment benefits for patients.     

Experimental pulpal immunization. I. Route of application and demonstration of immune response.

The experimental exposure of the pulpal room of rabbit teeth made it possible to apply an ovalbumin agar gel deposit into the pulpal room and subsequently investigate the immune response. Diffusion of antigen from the agar into environment was proven. After 3 weekly applications 5 rabbits showed hemagglutinating anti-ovalbumin titers between 1:128 and 1:512, while 7 rabbits receiving 10 weekly applications had titers between 1:8000 and 1:128,000. Control rabbits which received the agar deposit alone showed no antibody response. Control rabbits which obtained 3 identical weekly doses of ovalbumin in PBS by subcutaneous injection showed an identical immune response as animals immunized via the pulpal room. Specificity of antibodies was ascertained by passive hemagglutination with control antigens and hemagglutination inhibition. Intradermal injection of ovalbumin in 3 rabbits which obtained pulpal immunization, induced an Arthus reaction. The precipitating property of ovalbumin antibodies and their identity with defined ovalbumin antisera was proven by gel diffusion. The results obtained show, that antigens present in the pulpal room provide a strong immunogenic stimulus.