Immune responses to Mycoplasma conjunctivae in alpine ibex, alpine chamois, and domestic sheep in Switzerland

The humoral immune response of three alpine chamois (Rupicapra rupicapra rupicapra), two alpine ibex (Capra ibex ibex) and three domestic sheep naturally affected with infectious keratoconjunctivitis (IKC), and four ibex and two sheep experimentally infected with Mycoplasma conjunctivae was analysed. In addition, the local immune response to M. conjunctivae was analysed using conjunctival washes from chamois and sheep. Immunoblot analysis of sera using whole cell antigens of M. conjunctivae revealed the major immunogenic proteins which had molecular masses of 175, 83, 68, 60, 50, 42, 36, and 33 kDa. Major antigens were found at 83, 68, 60, and 42 kDa in both sera and conjunctival washes from naturally infected animals of all three Caprinae species. In experimentally infected animals, antibodies to the 68 and 60 kDa antigens were dominant. Naturally infected animals showed much stronger immune reactions than those experimentally infected, and specific antibodies appeared 2 to 4 wk after experimental infection. To evaluate possible cross-reactions, whole cell antigen of M. conjunctivae was analysed by immunoblot against hyperimmune sera of closely related Mycoplasma spp. Antibodies to the 175, 73, 68, 60, and 33 kDa antigens appeared to be specific to M. conjunctivae. Cross-reactions mainly with 83, 50, and 42 kDa antigens were detected, in particular with M. ovipneumoniae and M. bovoculi hyperimmune sera, but also with antisera against M. capricolum capricolum and M. putrefaciens.     

Serodiagnosis of infection with verocytotoxin-producing Escherichia coli

Human sera (167) were screened for antibodies to lipopolysaccharide (LPS) prepared from strains of Verocytotoxin-producing Escherichia coli (VTEC) belonging to a range of serogroups, secreted proteins expressed by attaching and effacing VTEC, enterohaemolysin and H = 7 flagellar proteins. Twelve sera (about 7%) contained antibodies to the LPS of E. coli 05 (one), 026 (two), 0115 (two), 0145 (one), 0163 (one) and 0165 (five). Sera containing antibodies to the LPS of E. coli O26 and O145 also contained antibodies to secreted proteins of 100 and 40 kDa. An additional 34 sera, known to contain antibodies to the lipopolysaccharide of E. coli O157, were examined for antibodies to enterohaemolysin, H = 7 flagellar antigens and bacterial cell surface-associated proteins of 5, 6 and 22 kDa. Three sera contained antibodies to enterohaemolysin and one serum contained antibodies to flagellar proteins. Antibodies to membrane-associated proteins were not detected. It was concluded that enterohaemolysin, H = 7 flagellar proteins and the cell surface-associated proteins were unsuitable for use in immunoassays for providing evidence of infection with VTEC.     

Shared antigens between human malignant melanoma cells and Mycobacterium bovis (BCG).

This study was undertaken to investigate the antigenic relationships between human malignant melanoma cells and Mycobacterium bovis (BCG). Rabbits were immunized with sonicates of BCG or with malignant melanoma cells from different patients and the resulting antisera were tested for their capacity to bind radiolabeled soluble extracts prepared from BCG and melanoma cells. The binding of antibodies to radiolabeled antigens was studied by precipitation of radiolabeled antigen-antibody complexes by anti-rabbit immunoglobulin. Antibodies in sera from rabbits immunized with either BCG (anti-BCG) or melanoma cells (anti-melanoma) bound both the labeled BCG and melanoma antigens. Control antisera, from rabbits immunized with human acute or chronic lymphatic leukemia cells or with normal human spleen cells, did not bind significant amounts of radiolabeled BCG. Antibodies in sera from rabbits immunized with normal spleen cells bound small but significant amounts of radiolabeled melanoma antigens. Binding by anti-BCG and anti-melanoma to the radiolabeled antigens was studied before and after absorption of antisera with cells from human melanoma, leukemia, guinea pig hepatoma, and normal human spleen cells. Inhibition studies using unlabeled BCG extracts also were carried out. The absorption and inhibition studies confirmed that the binding reactions were specific and that antigens from five melanoma patients shared antigenic determinants with BCG.     

Transplantation in minature swine. IV. Chemical characterization of MSLA and Ia-like antigens

Immunochemical analyses of radioactively labeled lymphocyte antigens from miniature swine of three different homozygous major histocompatibility (MHC) types, AA, CC, and DD, have been performed. Anti-MHC sera were incubated with lentil lectin purified Nonidet P-40 swine lymphocyte extracts. Antigen-antibody complexes were then precipitated with protein A bearing staphylococci, eluted, and electrophoresed on sodium dodecyl sulfate polyacrylamide gels. Analysis of antigens from AA or DD cells revealed peaks of 42,000, 31,000, 25,000, and 11,000 dalton m.w. Platelet absorption of the anti-MHC sera yielded antibodies that only precipitated the intermediate m.w. molecules and lysed a subpopulation of swine peripheral blood lymphocytes, suggesting that these molecules were the miniature swine analogues of murine Ia antigens. Antibodies eluted from platelets lysed all lymphocyte populations and precipitated only the 42,000 and 11,000 dalton peaks, indicating that these molecules represent the analogue of murine H-2 histocompatibility antigens, containing a heavy chain and putative swine beta2-microglobulin.     

Common swine leucocyte antigen (SLA) haplotypes in NIH and Sinclair miniature swine have similar effects on the expression of an inherited melanoma

Mixed lymphocyte culture and serological typing of NIH and Sinclair miniature swine indicate that the two herds share a common SLA haplotype. NIH haplotype a (International Haplotype H10) appears identical to Sinclair haplotype B, which has significant effects on the penetrance of Sinclair swine cutaneous malignant melanoma (SSCM). Offspring of crosses between melanoma-bearing Sinclair swine homozygous for the B haplotype and non-melanoma NIH aa swine have tumour incidence identical to Sinclair melanoma BB× Sinclair non-melanoma BB offspring. Our results provide further support for the involvement of the swine leucocyte antigen (SLA) complex in the inheritance of SSCM, and identify a new source of non-tumour animals that have all of the genes for SSCM except those at the tumour-initiator locus.     

Identification of centromere proteins in different mammalian cells

The characterization of centromeric proteins is facilitated using anti-centromere antibodies present in the sera of patients with the CREST variant of scleroderma. We have employed these sera to determine whether or not those proteins are present in different mammalian species, as well as to study their tissue distribution. Here, we describe the immunofluorescent pattern and the proteins recognized by CREST sera in dividing and resting cells from mouse, rat, swine, hamster, rabbit, and man. In nuclear preparations from cultured cells, thymocytes and spermatozoa from these species, the antigens recognized by CREST sera are proteins of 18 to 20 kDa in all species tested, except in rat. Additionally, two peptides of 80 and 140 kDa were observed in human preparations. In contrast, a 50 kDa peptide is the primary protein detected by the sera in rat nuclei.     

Melanomas with concordant loss of multiple melanocytic differentiation proteins: immune escape that may be overcome by targeting unique or undefined antigens

Melanoma-reactive HLA-A*0201-restricted cytotoxic T lymphocyte (CTL) lines generated in vitro lyse autologous and HLA-matched allogeneic melanoma cells and recognize multiple shared peptide antigens from tyrosinase, MART-1, and Pmel17/gp100. However, a subset of melanomas fail to be lysed by these T cells. In the present report, four different HLA-A*0201⁺ melanoma cell lines not lysed by melanoma-reactive allogeneic CTL have been evaluated in detail. All four are deficient in expression of the melanocytic differentiation proteins (MDP) tyrosinase, Pmel17/gp100, gp75/trp-1, and MART-1/Melan-A. This concordant loss of multiple MDP explains their resistance to lysis by melanoma-reactive allogeneic CTL and confirms that a subset of melanomas may be resistant to tumor vaccines directed against multiple MDP-derived epitopes. All four melanoma lines expressed normal levels of HLA-A*0201, and all were susceptible to lysis by xenoreactive-peptide-dependent HLA-A*0201-specific CTL clones, indicating that none had identifiable defects in antigen-processing pathways. Despite the lack of shared MDP-derived antigens, one of these MDP-negative melanomas, DM331, stimulated an effective autologous CTL response in vitro, which was restricted to autologous tumor reactivity. MHC-associated peptides isolated by immunoaffinity chromatography from HLA-A1 and HLA-A2 molecules of DM331 tumor cells included at least three peptide epitopes recognized by DM331 CTL and restricted by HLA-A1 or by HLA-A*0201. Recognition of these CTL epitopes cannot be explained by defined, shared melanoma antigens; instead, unique or undefined antigens must be responsible for the autologous-cell-specific anti-melanoma response. These findings suggest that immunotherapy directed against shared melanoma antigens should be supplemented with immunotherapy directed against unique antigens or other undefined antigens, especially in patients whose tumors do not express MDP. Received: 31 October 1997 / Accepted: 4 August 1999     

Studies on circulating gonococcal antibodies and antigens.

One hundred human sera obtained from acute gonococcal disease and 100 sera from nongonococcal diseases or healthy persons were concentrated four times and examined for the presence of circulating gonococcal antigens and antibodies by means of a counterimmunoelectrophoresis (CIE) and delayed hypersensitivity assay. Antibodies reacting with cytoplasmic gonococcal antigens were detected by CIE in 92% of sera received from patients suffering from acute gonococcal disease. Gonococcal antigens were found in the concentrated sera of 82.3% of patients on the basis of dermal reactions observed upon injections of these sera into the skin of rabbits sensitized with disrupted gonococci; 51.8% of the patients' sera gave delayed hypersensitivity reactions in rabbits sensitized with cytoplasmic antigens of N. gonorrhoeae. Control sera from healthy people and those with non-gonococcal diseases did not react with any of the preparations tested.     

Human antibody responses to Brugia malayi antigens in brugian filariasis.

Human antibody responses to Brugia malayi antigens were studied with sera from a Brugia endemic area in South India. Patients with clinical filariasis had significantly higher IgE and lower IgG4 levels to adult worm antigens than people with asymptomatic microfilaraemia. Intermediate antibody levels were observed in endemic normals. A majority of sera from each clinical group contained IgG antibodies to surface antigens of infective larvae (L3) by IFAT. IgG immunoblot studies did not reveal group differences in L3 antigen recognition. IgE antibodies bound to a subset of antigens bound by IgG. IgE antibodies in sera from clinical filariasis patients preferentially bound to L3 antigens at 200, 97, 68 and 58 kDa compared with sera from microfilaria carriers. These results are consistent with prior studies of antibody responses in filariasis and add new information on the targets of IgG and IgE antibodies to L3 antigens in brugian filariasis.     

Acanthocheilonema viteae: vaccination of jirds with irradiation-attenuated stage-3 larvae and with exported larval antigens

Jirds (Meriones unguiculatus) were immunized with irradiated (35 krad) stage-3 larvae (L3) of Acanthocheilonema viteae. The induced resistance against homologous challenge infection and the antibody response of the animals were studied. Immunization with 3, 2, or 1 dose of 50 irradiated L3 induced approximately 90% resistance. Immunization with a single dose of only 5 irradiated L3 resulted in 60.8% protection while immunization with a single dose of 25 L3 induced 94.1% protection. The protection induced with 3 doses of 50 irradiated L3 did not decrease significantly during a period of 6 months. Sera of a proportion, but not all resistant jirds, contained antibodies against the surface of vector derived L3 as defined by IFAT. No surface antigens of microfilariae or adult worms were recognized by the sera. Vaccinated animals had antibody responses against antigens in the inner organs of L3 and in the cuticle and reproductive organs of adult worms as shown by IFAT. Immunoblotting with SDS-PAGE-separated L3 antigens and L3-CSN revealed that all sera contained antibodies against two exported antigens of 205 and 68 kDa, and against a nonexported antigen of 18 kDa. The 205-kDa antigen easily degraded into fragments of 165, 140, 125, and 105 kDa which were recognized by resistant jird sera. Various antigens of adult worms, but relatively few antigens of microfilariae, were also recognized. To test the relevance of exported antigens of L3 to resistance, jirds were immunized with L3-CSN together with a mild adjuvant. This immunization induced 67.7% resistance against challenge infection and sera of the immunized animals recognized the 205- and 68-kDa antigens of L3.     

Vitiligo-related pigment cell differentiation antigens are expressed on malignant melanoma cells following phenotypic reversion induced by contact inhibitory factor

Most vitiligo sera contain antibodies to surface antigens on pigmented human melanocytes but not to human or mouse amelanotic melanoma cells. A density-dependent line of hamster amelanotic melanocytic cells (FF) produces a diffusible factor (CIF) which restores contact inhibition of growth as well as several other normal phenotypic characteristics to hamster, murine, and human melanoma cells. The ability of CIF to induce the expression of a phenotypic characteristic of pigmented human melanocytic cells, i.e., the vitiligo-related surface antigens, on hamster and mouse amelanotic melanoma cells was investigated. Vitiligo and normal sera were reacted with CIF-treated and untreated hamster and mouse amelanotic melanoma cells for both indirect-immunofluorescence assays and ELISA. Immunofluorescence testing showed that about 80% of hamster and mouse melanoma cells had pigment-cell antigens (in the absence of pigmentation) in a granular surface pattern after, but not prior to, CIF-induced morphologic reversion and confluent growth. Less than 5% of the control hamster and mouse melanoma cells expressed such antigens at confluence. These results were confirmed by ELISA. Metabolic-labeling studies with 35S-methionine showed that the vitiligo antigens were synthesized by the CIF-treated melanoma cells. The slowing of melanoma cell proliferation in isoleucine-deficient medium failed to elicit the expression of vitiligo antigens. Since antigen appearance following phenotypic reversion occurred without pigment induction, it is concluded that vitiligo-related surface antigens and pigmentation are distinct aspects of a differentiated function which may be non-coordinately expressed. The expression of pigment-cell differentiation antigens on amelanotic melanoma cells is an additional feature of the pleiotypic trans-species response to CIF.     

Demonstration of B700 cross-reactive antigens on human and other animal melanomas

B700 is a melanoma-associated antigen originally detected by immunologic and biochemical criteria; it is expressed by several murine melanomas but is not detectable on any normal murine cells, or on murine nonmelanoma neoplasms. We have used antibodies raised against purified B700 to study the presentation of B700 and B700 crossreactive molecules on the surfaces of melanoma cells of various species and origins. The antibodies are shown to bind to all the melanoma cells tested, including five different murine melanoma lines (S91, JB/RH, JB/MS, K1735, and B16), three different B16 sublines (F1, F10, and BL6), three human, one hamster, and two swine melanoma cell lines. These results suggest the candidacy of B700-like molecules as "pan-melanoma" antigens.     

Schistosoma japonicum: Immunological response to circulating polysaccharide antigens in rabbits with a light infection

To study the detectability of circulating polysaccharide antigens and the immunological response to such antigens in rabbits with a light Schistosoma japonicum infection, sera of five rabbits infected with 50 cercariae were studied up to 29 weeks post infection (p.i.). While one rabbit developed no worm burden, the other rabbits developed low worm burdens (4 to 16 worms). In the sera of these rabbits, the only polysaccharide antigen demonstrable with immunoelectrophoresis (IEF), was the circulating anodic antigen (CAA). With the enzyme-linked immunosorbent assay (ELISA), CAA was detectable from 5 to 6 weeks p.i. in the sera of the two rabbits with the highest number of worm couples. The lowest CAA level which was detectable in unconcentrated sera from which serum proteins had been removed was 125 ng CAA/ml, corresponding with a worm burden of 4.5 worm/kg body wt. During the entire infection, CAA-specific immune complexes were only demonstrable in very low concentrations. Antibodies against polysaccharide antigens were assessed with immunofluorescent antibody (IFA) on Rossman's fixed sections of adult worms, with the ELISA, and with IEF. Specific IgA, IgG, and IgM antibodies were detectable from 2 to 3 weeks p.i. with IFA and ELISA. These early antibodies were shown to be directed against gut-associated antigens, while antibodies against parenchyma-associated antigens were found later in the infection. With IEF, antibodies against two trichloroacetic acid (TCA)-soluble antigens were detectable, including the major, S. japonicum-specific antigen 2.     

Studies of immunological activity of Aspergillus flavus Link. III. Presence of antibodies against fungal antigens in the sera of persons from selected groups

The aim of this study was to determine the occurrence of antibodies against antigens of A. flavus (APP, AEM, AS, API), A. fumigatus and A. candidus. One hundred and fifty two sera of individuals connected with industrial environment were tested, in which A. flavus was permanently isolated: 339 sera of healthy controls-blood donors of city of Poznań, and 24 sera of patients with confirmed or suspected Aspergillosis were also included in the study. The sera were tested for a presence of specific antibodies by immunoprecipitation in 1% agar gel, by using inactivated sera and above mentioned antigens. In a group of people having permanent contact with A. flavus, antibodies to antigens derived from this genus were present in 4.6% of individuals while against A. fumigatus antigens in 0% and A. candidus 0.7%. In blood donors group 5 times lower percentage of sera having anti-A. flavus antibodies was found and a complete lack of detectable antibodies for other two genera. The results of the studies of patient sera indicate a necessity of broadening a set of fungal antigens used for an investigation of this type of sera. Antibodies against A. flavus were found in three patients and for A. fumigatus in 7 patients. One patient had antibodies for both genera and two patients had antibodies against A. flavus lacking antibodies against A. fumigatus. The results of this study indicate that antigens of A. flavus should be included into serodiagnosis of Aspergillosis.     

Membrane-associated antigens of human malignant melanoma IV: Changes in expression of antigens on cultured melanoma cells

Summary Established melanoma cell lines were cultured for one passage (approximately 1 week) in different lots of fetal calf and new born calf sera and then tested against a panel of previously positively reacting sera from melanoma patients and polyspecific HL-A alloantisera. Using indirect immunofluorescence the cells showed varying degrees of reactivity ranging from positive to negative reactions depending on the supplementing serum in the culture medium. When standardized culture conditions were used and the cells were tested by immune adherence at several weeks intervals against panels of sera from melanoma patients, from tumor patients other than melanoma, from pregnant women, and from normal donors, most of the sera reacted identical, but some sera not only had changed quantitatively but also qualitatively from a negative to a positive reaction and vice versa indicating a shift in the spectrum of expressed antigens. When single cell clones from a cell line were isolated and tested against a panel of antisera, striking differences in reactivity were observed suggesting that the shift in the spectrum of expressed antigens was due to the outgrowth of dominating subclones with antigen patterns different from the previously dominating subclones. This conclusion was further supported by experiments in which a weakly positive reacting serum was employed to separate a cell line into positively and negatively reacting sublines. Unit gravity sedimentation and density gradient sedimentation were used in order to separate rosetted from non-rosetted tumor cells which had been prepared by immune adherence. It is concluded that cultured cell lines are in a dynamic state and that differentiation is one of the major mechanisms accounting for a change in antigen expression.     

Serological Diagnosis of Classical Swine Fever

Antibody levels in post-infection sera from a pig inoculated with a low virulent strain of classical swine fever virus (Hannover 62) and in sera from two pigs inoculated with another low virulent strain (Spielbach 66) and from an in-contact pig were assayed by complement fixation and immunofluorescence using classical swine fever virus (ALD strain) and bovine virus diarrhoea virus (UG 59 strain) as antigens. The complement fixation test used was modified by addition of a preparation of porcine Glq to the complement and by mercaptoethanol treatment of the immune serum before use. The mercaptoethanol treatment of the immune serum resulted in complete elimination of a haemolytic prozone often seen with porcine immune sera. In the sera from the inoculated animals complement-fixing antibodies appeared earlier than neutralizing antibodies. A few weeks after inoculation there was a correlation between the presence of complement-fixing and neutralizing antibodies. During the entire observation period of 13 weeks it was not possible to demonstrate complement-fixing or neutralizing antibodies in serum from a pig exposed to infection by contact with the two pigs inoculated with the Spièlbach 66 strain of classical swine fever virus.     

Antibodies to vesicular stomatitis virus in populations of feral swine in the United States

From 1979 to 1985, 941 feral swine (Sus scrofa) from 53 locations in 15 states were serologically tested for antibodies to vesicular stomatitis virus (VSV). Antibodies to New Jersey serotype VSV were present in 75 swine from five locations in Arkansas, Florida, Georgia, and Louisiana. Within these populations, antibody prevalences ranged from 10 to 100%. No antibodies to Indiana serotype were detected.     

Expression of MAGE-C1/CT7 and MAGE-C2/CT10 predicts lymph node metastasis in melanoma patients

MAGE-C1/CT7 and MAGE-C2/CT10 are members of the large MAGE family of cancer-testis (CT) antigens. CT antigens are promising targets for immunotherapy in cancer because their expression is restricted to cancer and germ line cells and a proportion of cancer patients presents with immune responses against CT antigens, which clearly demonstrates their immunogenicity. This study investigates the expression of MAGE-C1/CT7 and MAGE-C2/CT10 in primary and metastatic melanoma. Immunohistochemical staining of tissue microarrays that consisted of 59 primary malignant melanomas of the skin, 163 lymph node and distant melanoma metastases and 68 melanoma cell lines was performed. We found MAGE-C1/CT7 expression in 15 out of 50 (24%) primary melanomas and 15 out of 50 (24%) cell lines, whereas MAGE-C2/CT10 was detected in 17 out of 51 (33%) primary melanomas and 14 out of 68 (17%) cell lines. MAGE-C1/CT7 and MAGE-C2/CT10 were both detected in 40% of melanoma metastases. Patients with MAGE-C1/CT7 or MAGE-C2/CT10 positive primary melanoma had significantly more lymph node metastases (p = 0.005 and p<0.001, resp.). Prediction of lymph node metastasis by MAGE-C1/CT7 and MAGE-C2/CT10 was independent of tumor cell proliferation rate (Ki67 labeling index) in a multivariate analysis (p = 0.01). Our results suggest that the expression of MAGE-C1/CT7 and MAGE-C2/CT10 in primary melanoma is a potent predictor of sentinel lymph node metastasis.     

Approaches to augmenting the immunogenicity of the ganglioside GM2 in mice: purified GM2 is superior to whole cells

The gangliosides GM2, GD2, and GD3 are differentiation antigens largely restricted to cells of neuroectodermal origin. They are expressed on most melanomas, astrocytomas, and neuroblastomas and have been shown to function as effective targets for monoclonal antibodies. In previous studies, we have immunized melanoma patients and mice with a series of melanoma cell vaccines containing these antigens, but have observed only occasional antibody responses. We report here the results of experiments in which an irradiated whole cell vaccine shown previously to be optimal was compared with a series of vaccines containing purified GM2. Mice were pretreated with low dose cyclophosphamide (Cy), or not, and were immunized twice with syngeneic melanoma cells (JB-RH) known to contain 60 micrograms of GM2 or with vaccines containing 50 micrograms of purified GM2. Serum was obtained at regular intervals and was tested by immune adherence, complement dependent cytotoxicity, and protein A assays on the JB-RH cell line. The whole cell vaccine, GM2 alone, GM2 incorporated into complete Freund's adjuvant, and GM2 attached to E. coli were all minimally immunogenic. GM2 attached to Salmonella minnesota or BCG, and GM2 attached to certain liposome preparations containing monophosphoryl lipid A, were found to be moderately immunogenic. GM2 attached to the R595 mutant of Salmonella minnesota was found to be significantly more immunogenic. Pretreatment with Cy significantly increased the immunogenicity of this vaccine. The specificities of selected sera were tested in inhibition assays and were limited to GM2. Antibodies produced after immunization were generally exclusively IgM and mediated potent complement-dependent cytotoxicity on JB-RH cells. These results identify R595 as the most effective adjuvant tested for augmenting the immunogenicity of GM2 and show that with regard to antibody production, purified tumor antigen presented optimally can be more immunogenic than optimally presented whole tumor cells containing the same amount of antigen.