Study of desmoglein 1 and 3 antibody levels in relation to disease severity in Indian patients with pemphigus.

OBJECTIVES: To conduct a cross-sectional study to compare Dsg1 and Dsg3 antibody levels independently with severity of disease activity in pemphigus vulgaris (PV) and pemphigus foliaceus (PF). METHODS: Blood samples from 44 patients with pemphigus (PV-38, PF-6) were analyzed using ELISA. The severity of skin and mucosal disease was graded using a score from 0 to 3. RESULTS: A statistically significant correlation between increase in Dsg 3 antibody titres with severity of oral involvement and Dsg 1 titres with severity of skin involvement was found in both PV and PF patients (p < 0.01). However, we were unable to demonstrate a relationship between increased titres of Dsg1 and Dsg 3 antibodies with oral and skin involvement respectively. CONCLUSION: This study suggests that the severity of skin and oral disease in pemphigus is determined by the quantities of Dsg1 and Dsg3 antibodies respectively.

Prospective study of serological conversion as a risk factor for development of leprosy among household contacts

Although the prevalence of leprosy has decline over the years, there is no evidence that incidence rates are falling. A method of early detection of those people prone to develop the mosth infectious form of leprosy would contribute to breaking the chain of transmission. Prophylactic treatment of serologically idenfified high-risk contacts of incidend patients should be an operationally feasible approach for routine control programs. In addition, classification of high-risk household contacts will allow control program resources to be more focused. Is this prospective study, we examined the ability of serology used for the detection of antibodies to phenolic glycolipid I of Mycobacterium leprae to identify those household contacts of multibacillary leprosy patients who had the highest risk of developing leprosy. After the start of multidrug therapy for the index case, a new case of leprosy developed in one in seven of the 178 households studied. In households where new cases appeared, the seropositivity rates were significantly higher (P<0.001) than those in households without new cases. Seropositive household contacts had a significantly higher risk of developing leprosy (relative hazard adjusted for age and sex [aRH], 7.2), notably multibacillary leprosy (aRH=24), than seronegative contacts

Detection of S-100 antigen and anti ceramide antibody in sera of leprosy patients with and without reaction.

Levels of anticeramide antibodies and S-100 antigen in leprosy patients with and without reaction are compared in this study. The increase in levels of IgM anti ceramide antibody in the tuberculoid group of patients with reaction, when compared to those without reaction, is significant (P < 0.05). Similarly, significant increase (P < 0.01) was observed in the borderline group with reaction. No significant change in anti ceramide antibody level was observed in the lepromatous group of patients with and without reaction. Mean levels of S-100 were slightly lower in all three groups of patients with reaction, but the differences were not statistically significant.

Antibodies to 65Kd heat-shock protein were elevated in rheumatoid arthritis.

Antibodies to 65Kd heat-shock protein (hsp) of mycobacterium leprae were measured by enzyme-linked immunosorbent assay (ELISA) in the three immunoglobulin classes in paired sera and synovial fluids of patients with rheumatoid arthritis (RA). Titers of anti-hsp antibody were expressed by optical density (OD) values for sera or indexes (OD values divided by amounts of immunoglobulin in each class) for synovial fluids and for their paired sera. Indexes of anti-hsp antibody were higher in synovial fluids than those in sera at 15/18 for IgG, 17/18 for IgA and 16/18 for IgM class. These results suggest the participation of anti-hsp antibodies to synovitis in RA.

Value of ELISA using A60 antigen in the serodiagnosis of tuberculosis.

This study was set in a general, university hospital in the Canary Islands, with the objective to evaluate an enzyme-linked immunosorbent assay (ELISA) using A60 in the serodiagnosis of tuberculosis. IgG antibody to A60 was determined in 205 patients with active disease [149 culture-positive patients with pulmonary tuberculosis (positive sputum smear 94, negative sputum smear 55) and 56 patients with extrapulmonary tuberculosis], 20 patients with inactive disease, 22 patients with lepromatous leprosy, and 51 controls. The mean levels of anti-A60 antibodies were significantly higher in patients with active disease as compared with controls or patients with inactive disease. Differences were also found between tuberculous patients with pulmonary and extrapulmonary disease. In patients with pulmonary disease, significant differences were detected between smear-positive and smear-negative patients. The overall sensitivity of the test (cutoff 240 ELISA units) was 52.2%. The highest sensitivity was found among smear-positive patients with pulmonary tuberculosis (67%) and the lowest among those with extrapulmonary tuberculosis (32.1%). We conclude that ELISA for the measurement of IgG antibody to Mycobacterium tuberculosis antigen A60 could be of interest, specially in smear-negative cases and extrapulmonary tuberculosis.

Immune complexes and antibody levels in blisters over human leprosy skin lesions with or without erythema nodosum leprosum.

Erythema nodosum leprosum (ENL) is a serious complication of lepromatous leprosy, affecting skin and peripheral nerves in a large percentage of these patients, and is presumed to result from spontaneous immunologic changes. Its pathogenesis is poorly understood, although histopathologic features have suggested immune complex (IC)-mediated injury. Abundant circulating antibody is present but no convincing correlation has been established between circulating IC and ENL. We have examined cutaneous leprosy lesions in vivo using blisters induced by prolonged gentle suction to determine whether or not IC are demonstrable in lesions with or without ENL, using an IC assay based on monoclonal rheumatoid factor binding. We also examined whether antibodies involved in such IC are produced locally or reach the skin via the circulation. Surprisingly large quantities of IC were found in ENL lesions, and in some cases the quantities were significantly higher than in matching serum. Total IgG, IgA, and IgM in the skin were not higher than expected, however. Attempts to demonstrate increases in intracutaneous levels of specific anti-Mycobacterium leprae antibodies were unsuccessful. This is the first report of the demonstration of IC in suction blister fluid. The results indicate that large quantities of IC may be present in cutaneous leprosy lesions and are consistent with the hypothesis that they are formed in situ when circulating antibody combines with antigen in the skin. The nature of the antigen in these IC remains undefined.

Antibody to a 63 kilodalton insect protein in ankylosing spondylitis.

Ankylosing spondylitis (AS) is associated with antibodies to a heat shock puff on drosophila chromosomes. This observation was investigated by immunoblotting using extracts of the Schneider insect cell line and HeLa cells, before and after heat shock. An insect protein of 63 kilodaltons (but no equivalent human protein) was recognised by 21 (46%) of 46 serum samples from patients with AS, one of two patients with Reiter's syndrome, four (7%) of 60 patients with systemic lupus erythematosus, and two (4%) of 50 control subjects, but not by serum samples from patients with rheumatoid arthritis (RA). Previous heat shock did not appear to affect the strength of reaction, but ML-30, a monoclonal antibody to the mycobacterial 65 kilodalton heat shock protein (hsp65), also recognised an insect protein of 63 kilodaltons by immunoblotting. Antibodies to recombinant mycobacterial hsp65 were measured by enzyme linked immunosorbent assay (ELISA) in serum samples from patients with AS and RA. IgA binding to hsp65 was increased in 41% of AS and 19% of RA serum samples, but there was no correlation with detection of antibody to the insect 63 kilodalton protein.

Antibodies to Mycobacterium tuberculosis-specific epitopes in lepromatous leprosy.

Sera from patients with leprosy or tuberculosis and healthy subjects have been analysed for the presence of antibodies to four species-specific mycobacterial epitopes, four different viruses and five autoantigens. Antibodies to the Mycobacterium leprae-specific 35-kD protein and phenolic glycolipid I epitopes were not present in patients with active pulmonary tuberculosis. In contrast, antibody levels to species-specific epitopes of the 38-kD and 14-kD antigens M. tuberculosis were significantly elevated in patients with lepromatous leprosy. Neither of the two antigens is cross-reactive with M. leprae at the B cell level. However, it was considered that cross-reactive helper T cells could recall the response of M. tuberculosis-specific memory B cells, which had been primed through prior self-healing tuberculous infection. As an alternative explanation, the possible role of polyclonal B cell stimulation was considered. This seemed unlikely, however, since: (i) antibody levels to autoantigens, except anti-smooth muscle, were not elevated, and (ii) antibody levels to four distinct viruses, unlike those to all mycobacterial epitopes, showed no correlation with titres, to M. tuberculosis-specific epitopes.

Anti-neural antibodies in leprosy sera: further characterization of the antigens.

Sera or plasmas from 129 leprosy patients were tested by immunoblotting for antibodies that bound to proteins in a Triton-insoluble fraction enriched in neural intermediate filaments (IF fraction) from human or bovine spinal cord. Sixty samples (47%) showed positive staining of proteins at 35 kDa, 42 kDa or both. The presence of these antibodies appeared to be evenly distributed across the spectrum of disease. The frequency of these antibodies in samples from 12 healthy Ethiopians was similar to that in the leprosy group. Similar antibodies were found in only three of 28 samples from U.S. patients with neurologic diseases and in seven of 35 normal U.S. sera. Sera from U.S. tuberculosis patients stained multiple bands in the 50-30 kDa region of the blots; 11 of 16 stained bands corresponding to the 35 kDa or 42 kDa bands along with a number of other bands in this region. The 35 kDa and 42 kDa antigens do not appear to be breakdown products of neural filaments or glial fibrillary acidic protein, since antibodies to these proteins do not react with the 35 kDa or 42 kDa antigen. Further, the staining pattern with the leprosy sera is unchanged following Ca2+-mediated proteolysis of the IF-enriched fraction. The two antigens differ from one another in isoelectric point: the pI of the 35 kDa antigen is 5.9, and the pI of the 42 kDa antigen is 4.8. Staining of the immunoblots with antibodies against a number of known neural antigens failed to identify the 35 kDa and 42 kDa antigens. The 42 kDa antigen appears to be a component of axolemma, since 42 kDa-positive leprosy sera stained a protein with identical migration in preparations of bovine peripheral nervous system and human central nervous system axolemma. In some sera, antibodies reacting with the 35 kDa antigen were adsorbed by D-O bovine serum albumin, a synthetic analogue of the terminal disaccharide portion of the phenolic glycolipid 1 of Mycobacterium leprae. Antibodies to the 42 kDa antigen were not removed by this treatment.

Use of anti-M. leprae phenolic glycolipid-I antibody detection for early diagnosis and prognosis of leprosy.

Untreated patients suffering from tuberculoid, lepromatous and indeterminate leprosy, their domiciliary contacts, and healthy controls, all living in Guadeloupe, West Indies, were tested by an ELISA for detecting IgM antibodies to the terminal disaccharide of the phenolic glycolipid-I antigen of Mycobacterium leprae. On most subjects, a Mitsuda test was also performed. A large majority of the tuberculoid patients and healthy subjects were Mitsuda positive. The seropositivity rate reached 44% among tuberculoid patients, and 6% among healthy subjects, with low antibody levels. Lepromatous patients were all Mitsuda negative and seropositive, with antibody production varying from low levels, as seen in tuberculoid patients, to much higher levels. Indeterminate leprosy patients included 62% Mitsuda-positive subjects and 54% seropositive subjects with a large dispersion of antibody levels. Comparing the results of the Mitsuda test to those of the ELISA by factorial analysis allowed us to define several subgroups among this population: some (25%) showed a "lepromatous-like" immune status (Mitsuda negative, seropositive); others (54%) exhibited "tuberculoid-like" profiles (Mitsuda positive without antibodies or with low antibody levels). "Lepromatous-like" cases were significantly older than "tuberculoid-like" patients. A group of subjects (17%) was Mitsuda negative and seronegative, thus displaying a true "indeterminate" immune profile, which had not been seen in other forms of the disease and had been observed in only 2 out of 51 healthy controls. A large majority of contacts was Mitsuda positive, with 33% of them being seropositive, indicating that the prevalence of M. leprae infection greatly exceeds that of overt leprosy in this population.(ABSTRACT TRUNCATED AT 250 WORDS)

Trypsin-treated and coomassie blue-stained epimastigote antigen in a microagglutination test for Chagas' disease.

A microagglutination test using trypsin-treated and Coomassie blue-stained Trypanosoma cruzi epimastigote antigen was adapted for the diagnosis of Chagas' disease. When incorporated in the test, 2-mercaptoethanol treatment of chagasic sera had no influence on antibody titer. In contrast, titers in sera from patients with visceral leishmaniasis, African trypanosomiasis, and autoimmune disorders, subjected to similar treatment, showed remarkable decline. Accordingly, a lower cut-off point for Chagas' disease serological negativity could be taken resulting in a higher sensitivity (95.6%); the specificity was 94.7%. Similar specificities were obtained with Leishmania donovani chagasi and L. d. donovani antigens applied to homologous visceral leishmaniasis and heterologous Chagas' sera. Of 316 nonchagasic sera, only 3 with leptospirosis and 1 with leprosy showed seropositive titers prior to and after 2-mercaptoethanol treatment.

Leishmania major: solid phase radioimmunoassay for antibody detection in human cutaneous leishmaniasis.

A radioimmunoassay for the quantitative determination of antileishmanial antibody in sera from patients suffering from cutaneous leishmaniasis was developed. The assay, using as antigen either the soluble fraction from freeze-thawed sonicated Leishmania major (LRC-L137) promastigotes or a carbohydrate-lipid containing fraction obtained by extraction with hexane-isopropanol, was shown to be sensitive and reproducible. The sera of 95 patients were examined. These were from patients with cutaneous leishmaniasis (26 from the Jordan Valley and 13 from Sinai), kala-azar (9), malaria (24), schistosomiasis (10), toxoplasmosis (5), and leprosy (8); controls were 37 normal human sera. No significant antigen dependent differences were observed using sera from cutaneous leishmaniasis patients, although differences in the immunological response were observed between the two populations of these patients. Antileishmanial activity was not detected in sera from patients with malaria, schistosomiasis, or toxoplasmosis. Although sera from leprosy patients crossreacted with the carbohydrate-lipid containing fraction, it was nevertheless more strain specific than freeze thawed sonicated L. major.

Evaluation of the micro enzyme-linked immunosorbent assay (ELISA) for antibodies in American visceral leishmaniasis: antigen selection for detection of infection-specific responses.

This study was designed to evaluate the ELISA for diagnosis of American visceral leishmaniasis (AVL) using antigen prepared from different Leishmania isolates and from a strain of Trypanosoma cruzi. Two Leishmania donovani chagasi isolates from Bahia and Maranhão (both states of northern Brazil), one L. donovani from Sudan, one L. mexicana amazonensis isolate, and one T. cruzi isolate were used. A total of 375 sera were tested, including 119 from AVL patients, 96 from nonleishmaniasis hospitalized patients, 20 from healthy persons, 30 from patients with mucocutaneous leishmaniasis, 28 from patients with Chagas' disease, 20 from patients with tuberculosis, 21 from leprosy patients, 27 from schistosomiasis patients and 14 from patients with systemic mycoses. The antigens prepared from L. d. chagasi (Bahia) and L. m. amazonensis showed the highest sensitivity (98% and 99%, respectively) for detecting antibodies in sera from AVL patients. However, the specificity of L. d. chagasi (Bahia) antigen was better than that of L. m. amazonensis (96% vs. 86%). Comparison among the three L. donovani isolates demonstrated that the antigen prepared with the isolate from the same area where the sera originated yielded higher mean absorbance than the others. By using spectrophotometric absorbance values it was possible to use a single dilution of serum (between 1/100-1/400) since a clear separation was seen between AVL patients and controls. No patients with the other diseases who were tested gave positive results. We suggest that ELISA can be a very convenient, sensitive, and specific test for diagnosis of AVL when soluble antigen, preferably from an isolate from the test area, is used.