Pathobiology : journal of immunopathology, molecular and cellular biology •
This study describes the presence of alpha-galactosyl epitopes on 12 cervical biopsy samples with features of human papillomavirus infection (HPV) and different stages of cervical intraepithelial neoplasia. An avidin-biotin-peroxidase assay with a monoclonal antibody recognizing gal(alpha 1-->3)gal residues was strongly positive in 5 of 12 cases. None of the controls stained (p = 0.02). Immunostaining was intense in the areas with the highest viral load (koilocytes and keratinocytes) and absent in malignant foci. Immunostaining was also absent in normal exo- and endocervical epithelium of 12 controls with no features of HPV infection. A faint background staining in cases and controls was evident, but similar. These initial findings suggest that alpha-galactosyl epitopes are expressed in cervical squamous cells infected with HPV, turning them vulnerable to lysis by natural anti-alpha-galactosyl antibodies.
The American journal of tropical medicine and hygiene •
Humoral immune responses were studied in 118 Venezuelan patients with either active mucocutaneous (MCL) or localized cutaneous leishmaniasis (LCL). Most patients had elevated antibody levels to the six promastigote oligosaccharide residues studied: galactosyl(alpha 1-2)galactose, galactosyl(alpha 1-3)galactose, galactosyl(alpha 1-6)galactose, galactosyl(alpha 1-3)mannose, galactofuranosyl(beta 1-3)mannose, and galactocerebroside. Significantly higher antibody levels were found in patients with MCL against galactosyl(alpha 1-3)galactose and Leishmania tropica glycoinositol phospholipid (GIPL)-1, GIPL-2, and GIPL-3 compared with patients with LCL. For both clinical forms of American cutaneous leishmaniasis (ACL), the most reactive antigen was galactosyl(alpha 1-3)galactose, with elevated levels found in 63% and 79% of MCL and LCL patients, respectively. In patients with MCL and LCL, no significant relationship was found between antibody levels against a given oligosaccharide residue and clinical parameters such as age, leishmanin diameter, number of skin lesions, or time of evolution. It is noteworthy that 33% and 15% of MCL and LCL patients, respectively, did not have elevated antibody levels against the six different oligosaccharide residues studied. This suggests the presence of a subpopulation of non-humoral immunoreactive ACL patients. The relationship between abnormal levels of oligosaccharide antibodies and the final outcome of the disease remains to be established.
The American journal of tropical medicine and hygiene •
An antibody reactive with the galactosyl(alpha 1-2)galactose [gal(alpha 1-2)gal] epitope was characterized in human sera by enzyme-linked immunosorbent assay, red blood cell (RBC) and laminin absorption, and oligosaccharide inhibition. This antibody was found evenly distributed between the IgG and IgM classes and was present at high titers in the serum of all normal adults studied, but in 75% of children less than three years of age, it was observed at the lower limit of detection, and gradually increased to adult levels by the age of six. Although this antibody bound to gal(alpha 1-3)gal-linked synthetic antigens, it did not bind to the same residues present in rabbit, rat, and guinea pig RBC or in murine laminin or nidogen. These latter results, plus the fact that antigen-antibody binding was strongly blocked by gal(alpha 1-2)gal but not by methyl-alpha-galactopyranoside or melibiose, suggest that this antibody is indeed different from anti-gal(alpha 1-3)gal antibody. Anti-gal(alpha 1-2)gal antibody levels were significantly elevated in 66% of patients with chronic chagasic cardiomyopathy, but were not elevated in patients with different clinical forms of leishmaniasis, Trypanosoma rangeli-infected patients, or in patients with 15 other infectious and inflammatory diseases. Gal(alpha 1-2)gal antibodies did not absorb to intact T. cruzi parasites, but absorbed strongly to trypomastigote and epimastigote sonicates, suggesting some masking of reactive epitopes.(ABSTRACT TRUNCATED AT 250 WORDS)
Bretaña A, Avila JL, Contreras-Bretaña M, Tapia FJ
Experimental parasitology •
Patients with Chagas' disease or different clinical forms of leishmaniasis (cutaneous or visceral) have elevated galactosyl alpha (1-3)galactose antibodies. Using colloidal gold immunocytochemistry--monoclonal antibody gal-13 (specific for lipid-linked galactosyl alpha (1-3)galactose residues) and anti-nidogen antibodies and lectin cytochemistry (Bandeiraea simplicifolia IB4), both techniques specific for demonstrating galactosyl alpha (1-3)galactose residues--we have found terminal disaccharide residues on the Trypanosoma cruzi external surface of Vero cell-derived trypomastigotes but not in intact epimastigotes (although disrupted epimastigotes strongly stained), in the lips of the flagellar pocket, and on the parasitic side exactly opposite to the flagellar pocket in amastigote and promastigote forms of American Leishmania. These results resemble those obtained using anti-laminin antibodies in both trypanosomatids. In addition, results obtained with anti-nidogen antibodies seem to recognize in Trypanosoma cruzi and American Leishmania culture forms another different unknown terminal disaccharide. These results confirm the presence of terminal galactosyl alpha (1-3)galactose residues in both trypanosomatids, and that rabbit anti-laminin antibodies are indeed also recognizing galactosyl alpha (1-3)galactose residues as demonstrated for human circulating antibody. The presence of abundant galactosyl alpha (1-3)galactose residues on Trypanosomatid family members suggests a specific unknown role in parasite physiology for this terminal disaccharide.
Increasing evidence suggests that in Chagas' disease chronic-phase pathology is autoimmune in nature. There are at least two nonexclusive explanations for the generation of autoimmunity in Chagas disease: a) infection with the parasite perturbs immunoregulation, leading to loss of tolerance for self-antigens; b) immune recognition of T. cruzi antigens is crossreactive with selected mammalian antigens, leading to autoimmunity (molecular mimicry). Through this latter mechanism, T. cruzi antigens that share epitopes with mammalian nervous tissue may drive autoreactive B- or T-cell clones to expand and cause autoimmune lesions in chronic chagasic patients. Several different antigens sharing this characteristic have been studied, as for example the 160-kDa flagellum-associated surface protein (Fl-160), which has a nervous tissue crossreactive epitope composed by twelve amino acids. Additionally, it has been demonstrated that a trypomastigote stage-specific 85kDa surface glycoprotein (Gp85) has terminal galactosyl(alpha 1-3)galactose terminal residues, which are reactive with chronic chagasic sera. Common glycolipid antigens have also been reported, as for example galactocerebroside, sulfogalactocerebroside and sulfoglucuronylcerebroside, all of them specifically present at high concentrations in mammalian nervous system and in T. cruzi trypomastigotes. Chronic chagasic patients produce elevated levels of antibodies against these three glycolipid antigens. They also do against terminal galactosyl(alpha 1-3)galactose residues present on several acid and neutral glycolipids common either to nervous system or parasite. These antibodies are powerful lytic for circulating T. cruzi trypomastigotes. Another common strongly immunogenic residues are galactosyl(alpha 1-2)galactose, galactosyl(alpha 1-6)galactose and galactofuranosyl(beta 1-3)mannose residues present on several glycoinositolphospholipids (GIPL), against which chronic chagasic patients have elevated levels of specific antibodies. In brief, very specific host-parasite relationships existing only in Chagas' disease may explain the particular peripheral nervous tissue damage seen in acute or chronic stages of this disease. This specificity could depend either on invasion of autonomic ganglia by T. cruzi trypomastigotes and modification of nervous cell surface structures by some of the several mechanisms of acquired molecular mimicry.
The glycoinositol phospholipid (GIPL) profiles of American Leishmania spp. (L. mexicana and L. braziliensis), Leishmania donovani, and American Trypanosoma spp. (T. cruzi and T. rangeli) were compared. The major GIPLs in these parasites include tetraglycosyl-, pentaglycosyl-, and hexaglycosylphosphatidylinositol. These were partially identified by their comigration by high-performance thin-layer chromatography with purified L. major GIPLs, gas-liquid chromatography of the monosaccharides released after aqueous HF treatment, N-acetylation and methanolysis, sensitivity to exoglycosidases, and antibody absorption on several specific natural haptens. Members of the genus Leishmania have two other highly polar glycolipids, while the T. rangeli glycolipid profile was quite different from those of other kinetoplastids that were studied. On a weight basis, the glycan core of L. major GIPL-1 is the most reactive, followed by GIPL-3 and GIPL-2. Antibodies to the core glycans of GIPL-1, GIPL-2, and GIPL-3 were present at a low titer in the serum of every normal individual studied, while elevated GIPL-2 antibody levels were present in 80 to 100% of T. cruzi-, T. rangeli-, or L. donovani-infected patients, with lower values being found for GIPL-3 (30 to 60%) and GIPL-1 (30 to 50%). Except for GIPL-2 antibodies, which were mainly located on immunoglobulin G (IgG) and IgM, GIPL-1 and GIPL-3 antibodies were mainly distributed in IgM, with lower reactivity present in IgG. Antigen-antibody binding was very selectively blocked with Gal(alpha 1-3)Man, or Gal(beta 1-4)Man, Gal(alpha 1-3)Gal, and Gal(alpha 1-6)Gal for GIPL-1, GIPL-2, and GIPL-3 antibodies, respectively.
An immunoglobulin M antibody reactive with galactosyl(alpha 1-3)mannose [Gal(alpha 1-3)Man] residues present on phospholipids extracted from Leishmania mexicana and L. braziliensis was found to be present in high titer in the serum of every normal individual studied. Periodate oxidation, acid hydrolysis, or acetylation suppressed immunoreactivity, suggesting that an oligosaccharide chain was responsible for antibody binding. Interaction occurs only with alpha-Gal terminal residues, since treatment of purified glycophospholipids with alpha-galactosidase but not with beta-galactosidase abolished it. Antibody bound to galactosyl(alpha 1-3)galactose-linked synthetic antigens but did not bind to the same residues present in rabbit, rat, and guinea pig erythrocytes or in murine laminin. Antigen-antibody binding was strongly blocked with Gal(alpha 1-3)Man and Gal(beta 1-4)Man. These results plus inhibition studies with several oligosaccharides suggest that they are indeed different from antibodies against the galactosyl(alpha 1-3)galactose residue. Anti-Gal(alpha 1-3)Man antibody values were significantly elevated in 89% of patients with diffuse cutaneous leishmaniasis, 84% of patients with localized cutaneous leishmaniasis, 69% of patients with mucocutaneous leishmaniasis, and 44 and 62% of patients with Trypanosoma cruzi or T. rangeli infection, respectively, but not in patients with 15 other different infectious and inflammatory diseases. Anti-Gal(alpha 1-3)Man antibody readily absorbed to American Leishmania and Trypanosoma culture forms, suggesting a surface membrane localization of reactive epitope. Gal(alpha 1-3)Man-bearing glycophospholipid was easily extracted from American Leishmania promastigotes and T. cruzi trypomastigotes as well as from American Trypanosoma culture forms. The possibility that this antibody arises against parasitic glycophospholipid-linked Gal(alpha 1-3)Man terminal residues is proposed.
Using rabbit erythrocyte-derived neutral glycosphingolipids enriched for a ceramide pentasaccharide as the antigen, we detected elevated anti-galactosyl-alpha(1-3)galactose (anti-G alpha G) antibody levels in 76% of children with active visceral leishmaniasis (kala-azar [KA]) and in 42% of clinically cured patients with KA who had been treated about 5 years previously with meglumine antimonate (30 mg/kg in a series of 15 daily injections). The long-term persistence of elevated G alpha G antibodies was also found in 56% of children living in the same geographic zone who, at the time of the initial clinical examination, had fever and evident splenomegaly with hyperglobulinemia but a negative bone marrow aspirate for leishmanial bodies. Five years after antimonate treatment, these clinically cured children with presumptive KA were studied serologically. Their mean G alpha G antibody values were slightly lower than those in patients with active KA but were still abnormal. Using different biochemical and immunological approaches, we found that elevated G alpha G antibodies present in patients with KA bound specifically to glycoconjugates with an alpha(1-3)-terminal galactose residue. G alpha G antibodies were mainly distributed between immunoglobulin classes G and M in patients with active KA and in antimonate-treated patients with clinically cured KA. The possibility of the existence of remnant living parasites or the persistence of inserted G alpha G epitopes in parasitized macrophages was proposed as a mechanism to explain the long-term persistence of abnormal G alpha G antibodies in patients apparently cured of KA.
Using rabbit erythrocyte-derived neutral glycosphingolipids enriched for a defined ceramide pentasaccharide as antigens, we have detected elevated anti-galactosyl-alpha(1-3)galactose (anti-G alpha G) antibody values in patients with American cutaneous leishmaniasis (ACL), chronic Chagas' disease, and Trypanosoma rangeli infections compared with normal subjects or with patients suffering from any of 15 other infectious diseases. The specificity of the G alpha G antibodies was determined by inhibition enzyme-linked immunosorbent assays, which revealed that several alpha-galactosyl- but not beta-galactosyl-bearing sugars blocked absorption of G alpha G antibodies to the specific antigen used. G alpha G antibodies were mainly distributed between immunoglobulin classes G and M in three Kinetoplastida infections studied, with a lower increase in reactivity detected in immunoglobulin A. Absorption of highly reactive G alpha G antibodies with purified murine laminin and nidogen, two basement membrane proteins, almost abolished G alpha G reactivity, suggesting the identity of anti-G alpha G with laminin and nidogen antibodies previously reported as elevated in Kinetoplastida infections. In ACL, G alpha G antibodies were detected in 71% of patients having skin lesions with a clinical evolution time of 0.5 month. This percentage increased with the time of evolution of skin lesions, reaching 93% in lesions older than 3 months, and tended to decrease inversely to the induration diameter in the skin leishmanin test. It is proposed that similar epitopes may exist on kinetoplast protozoa and that the determination of G alpha G antibodies may be a highly sensitive assay for the detection of humoral responses to Kinetoplastida infections.