Serum proteome of leprosy patients undergoing erythema nodosum leprosum reaction: regulation of expression of the isoforms of haptoglobin.

Validated proteome profile allows better understanding of disease progression, subtype classification, susceptibility patterns, and disease prognosis. Leprosy is a spectral disease, with clinically, histologically, immunologically, and bacteriologically distinguishable subtypes. In addition, a significant fraction of patients undergo immune mediated reactions even after multidrug therapy (MDT). Erythema nodosum leprosum (ENL) is an immune complex mediated reactional condition in leprosy, characterized by a systemic inflammatory condition afflicting borderline lepromatous (BL) and lepromatous leprosy patients (LL). In this study, we have analyzed serum proteome of leprosy patients undergoing ENL reactions and compared it with that of healthy noncontact controls. Depletion of albumin and immunoglobulin G (IgG) was optimized using Aurum serum protein mini kit (Bio-Rad), and then two-dimensional gel electrophoresis (2-DE) of these serum samples was performed. Differentially expressed proteins were identified by MALDI-TOF and MALDI-TOF MS/MS mass spectrometry. Significant increase in one of the isoforms of alpha2 chain of haptoglobin was observed in ENL condition. In addition, haptoglobin phenotype was determined for healthy controls and leprosy patients. Hp 0-0 phenotype was detected in 21.4% of the ENL patients undergoing treatment, which on follow up examination showed typable phenotype, thus showing a condition of acquired anhaptoglobinemia. Since ENL still remains a threat to leprosy disease management, the above findings may provide new insights in understanding the development and progression of this inflammatory condition.

Detection of phase variation in expression of proteins involved in hemoglobin and hemoglobin-haptoglobin binding by nontypeable Haemophilus influenzae.

Haemophilus influenzae can utilize different protein-bound forms of heme for growth in vitro. A previous study (I. Maciver, J. L. Latimer, H. H. Liem, U. Muller-Eberhard, Z. Hrkal, and E. J. Hansen. Infect. Immun. 64:3703-3712, 1996) indicated that nontypeable H. influenzae (NTHI) strain TN106 expressed a protein that bound hemoglobin-haptoglobin and was encoded by an open reading frame (ORF) that contained a CCAA nucleotide repeat. Southern blot analysis revealed that several NTHI strains contained between three and five chromosomal DNA fragments that bound an oligonucleotide probe for CCAA repeats. Three ORFs containing CCAA repeats were identified in NTHI strain N182; two of these ORFs were arranged in tandem. The use of translational fusions involving these three ORFs and the beta-lactamase gene from pBR322 revealed that these three ORFs, designated hgbA, hgbB, and hgbC, encoded proteins that could bind hemoglobin, hemoglobin-haptoglobin, or both compounds. Monoclonal antibodies (MAbs) specific for the HgbA, HgbB, and HgbC proteins were produced by immunizing mice with synthetic peptides unique to each protein. Both HgbA and HgbB were readily detected by Western blot analysis in N182 cells grown in the presence of hemoglobin as the sole source of heme, whereas expression of HgbC was found to be much less abundant than that of HgbA and HgbB. The use of these MAbs in a colony blot radioimmunoassay analysis revealed that expression of both HgbA and HgbB was subject to phase variation. PCR and nucleotide sequence analysis were used in conjunction with Western blot analyses to demonstrate that this phase variation involved the CCAA repeats in the hgbA and hgbB ORFs.

Haptoglobulin phenotypes in leprosy.

Haptoglobulin phenotyping was carried out in fifty controls and in thirty five leprosy patients. In controls the incidence Hp phenotypes 2-2, 2-1 and 2-1 (Mod) is 76%, 16% and 8% respectively. In leprosy patients, the incidence of phenotypes 2-2, 2-1, 1-1 and 0-0 is 77%, 11%, 3% and 9% respectively. The incidence of phenotype 2-2, 1-1 and 0-0 is more in leprosy patients than in controls and is significant (p less than 0.05). In none of the leprosy patients phenotype 2-1 (Mod) was recorded.

Dapsone induced hypohaptoglobinemia in lepromatous leprosy patients.

Dapsone has been used in various dermatological disorders and in leprosy. One of the main side effects of dapsone therapy is anemia, mostly hemolytic. We aimed at finding the effect of dapsone therapy on serum haptoglobin levels which could be an indirect evidence for intravascular hemolysis, supported by secondary investigations such as liver functions (serum lactic dehydrogenase, alkaline phosphatase, bilirubin), blood hemoglobin levels, urinary excretion of urobilinogen, and erythrocytes. As in other infectious conditions, haptoglobins were raised in untreated lepromatous cases, compared to controls (p less than 0.05). Dapsone treatment of 100 mg daily for 14 days brought down the haptoglobin level significantly as compared to the untreated cases and the controls (p less than 0.05). An elevated alkaline phosphatase and lactate dehydrogenase indicate some liver dysfunction following dapsone therapy. A significant drop in blood hemoglobin level and a concomitant increase in serum bilirubin, urinary excretion of urobilinogen, and a significant fall in the serum hemoglobin binding capacity (haptoglobin level) following treatment with dapsone are quite suggestive of mild intravascular hemolysis.

Haptoglobin phenotypes in leprosy.

Serum haptoglobin phenotypes were studied in 80 patients with leprosy classified according to the criteria of Ridley and Jopling. The distribution of phenotypes was: 2-2, 65%; 2-1, 27.5%; 1-1, 1.25%; and 0-0, 6.25%. This distribution was not significantly different from the controls except for the phenotype 0-0 (p less than 0.02). Thus, although this genetic marker did not correlate with the occurrence of the variety of disease, it is possible that leprosy caused inhibition of haptoglobin synthesis and therefore an apparent increased frequency of the 0-0 phenotype. Evidence for such a secondary anhaptoglobinemia was available in one case.

Cross-reactions between serum proteins and water soluble liver tissue antigens of the nine-banded armadillo (Dasypus novemcinctus Linn.) and man.

Cross-reactions between serum proteins and water soluble liver antigens of the nine-banded armadillo (Dasypus novemcinctus Linn.) and man were studied by crossed immunoelectrophoresis (CIE). Armadillo serum tested with rabbit antiserum against human serum proteins gave twelve components in CIE. Nine of these cross-reacting proteins were identified and showed partial identity with the corresponding human proteins. The electrophoretic mobility of alpha 2-macroglobulin and Gc-globulin differed in the two species. An ultrasonicate of normal armadillo liver gave twenty-eight anodic and eight cathodic components in CIE. By absorption experiments with armadillo serum, twenty of the former and seven of the latter were shown to be liver tissue components. A combination of CIE and crossed-line immunoelectrophoresis (CLIE) revealed the presence of twelve anodic and six cathodic liver tissue components cross-reacting with man. A cathodic armadillo liver antigen called (CALA-17) showed partial identity with that of man both in tandem and fused rocket immunoelectrophoresis. The implications of the findings are discussed in relation to the use of armadillo-grown M. leprae for skin testing and other purposes in man.

Genetic markers and leprosy in South African negroes. Part I. Serum protein polymorphisms.

The phenotype frequencies of the serum protein polymorphisms Hp, Gc, Tf, Gm and Inv were determined on a sample of 250 South African Negroes with leprosy. These results were compared with data derived from 918-977 (depending on the polymorphism tested) healthy Negro controls of similar geographical and ethnic origin, in order to determine whether or not any association existed between specific phenotypes and the occurrence of leprosy. The data derived from the present study were also compared with those of similar comparative analyses on African and non-African populations. Because of the contradictory results between samples with regard to the polymorphisms Hp, Gc and Inv, an association of any of these phenotypes with leprosy appears to be highly improbable. With regard to the polymorphisms Tf and Gm, however, such associations cannot be ruled out. The questions arising from the results are discussed.