- Volume 73 , Number 3
- Page: 222–5
Serologic recognition of low molecular weight mycobacterial protein fractions in lepromatous patients with type ii reactions (enL)
To the Editor:
Hansen's disease is a mycobacterial infection that produces physical disabilities. The progression of the disease is slow and indolent but in some cases there are changes in the immunological status with the development of acute episodes represented by reactional states. Many of these reactional episodes occur after treatment has been finalized and, therefore, it is important to clarify whether they constitute relapses. We wished to determine if specific patterns of serologic recognition of mycobacterial proteins were associated with Type 2 reactional states in lepromatous patients. Serum samples were taken from 12 adult patients, mean age of 43 ± 16 yrs, with a predominance of males (80% M and 20% F), who were undergoing a Type 2 reactional episode (erythema, nodosum leprosum, ENL). These sera were divided in two groups of six sera each: sera from Group I were antibody-positive to phenolic glycolipid (PGL-I), the other six (Group II) were negative. ENL reactions were characterized using histopathological criteria, including the presence of undifferentiated macrophages and relatively abundant PMNs, with or without acid-fast bacilli. The group of six patients that gave negative reactions for antibodies to PGL-I (Group II) had completed multidrug therapy; they presented an average of six episodes of ENL. Of the six patients in Group I with detectable antibodies to PGL-I, two were still being treated. ENL reactions were less frequent in Group I (average 4 episodes).
Soluble component fractions were obtained by an electroelution technique from Mycobacterium leprae soluble extract (MLSA) and Mycobacterium bovis soluble extract (MbSA) (5, 6). The soluble extracts were obtained by rupturing purified bacilli with the French Press (2). The extracts contain cytosol proteins as well as proteins freed from the cell walls. Insoluble material was eliminated by centrifugation. Protein concentration was determined by the BCA method (7).
Starting with a 10% SDS-PAGE preparative gel under dissociating and denaturing conditions, 1 mg of MLSA and MbSA was resolved in polypeptides of different mobilities (see The Figure), which were fractionated by electroelution in a mini BIORAD® 65-1256 electroelutor, according to the instructions provided by the manufacturer.
The Figure. OE, original extract (M. bovis). F1, F2. different electro-eluted fractions MW: molecular weight standards. (A, Coomassie brillant blue stain; B, Western blot with LL serum.) The typical ladder of eluted fractions corresponding to their molecular weights is shown, with successively smaller proteins from F1 to F12.
Twelve electroeluted fractions were obtained for both the MLSA and the MbSA antigens. ELISA tests were used to evaluate activity with the pooled sera, using IgG antibodies specific for the Fc gamma chain (Sigma A0170) as the second antibody (4).
A clear difference in recognition was seen between the two groups of sera studied. In the ELISA tests with both MLSA and MbSA electroeluted fractions, we saw an immunodominant recognition of proteins with a relative mobility of 30 kDa, corresponding to Fraction 9 (see The Table). There was also serologic recognition of low molecular weight MLSA proteins (less than 30 kDa) in patients in group I which was not observed in Group II.
In preliminary studies we previously reported a clear difference between the IgG antibody levels directed towards soluble mycobacterial proteins (Mycobacterium bovis MbSA and Mycobacterium leprae MLSA) in an ENL active group (n = 4) as compared with the non-active group (n = 4) (3). In the ENL active patients we found IgG antibody levels towards MbSA and MLSA of 0.535 ± 0.24 and 0.731 ± 0.32, respectively, as compared with the non-active patients, whose values towards the same total proteins were zero. In this study using the electroelution technique we were able to demonstrate the immunodominant antigens found in patients in an ENL reactional state.
Many authors have shown a decrease of IgM antibodies directed towards phenolic glycolipid (PGL-I), which is an M. leprae structural component (1) in these reactional patients. To examine this, we separated the reactional patients in two groups, according to their PGL-I positivity. IgM antibodies against native PGL-I were measured in an enzyme linked immunosorbent assay using the method described previously (8).
In addition to the immunodominant recognition towards proteins with a 30 kDa relative mobility, both with MbSA and MLSA, we also saw that the recognition in Group I involves a larger number of protein fractions, including low molecular weight proteins (<30 kDa), compared to the patients in Group II.
We have recently increased the number of multibacillary patients (n = 70), and there have been no significant differences in the Mycobacterium leprae 30 kDa protein antibodies between patients who had Type II reactions and those who did not. In this larger group of 70 multibacillary patients, nine presented ENL reactions and the other 61 did not. Of the nine with ENL, eight (89%) gave positive reactions to the 30 kDa protein, average optical density 0.8816. Of the 61 remaining patients, 42 (69%) gave positive reactions to the 30 kDa protein, average OD 0.5885. This difference was not statistically significant, p = 0.42, but the observation suggests a trend toward stronger reactivity in patients with ENL. The sera of newly diagnosed multibacillary patients reacted with other peptides of both higher and lower molecular weights. In this population of 70 patients, 62.6% were in treatment and presented bacillary indices of less than 2+. Reactivity was strongly associated with bacillary load. Reactivity to the 10 kDa protein of M. leprae was lower in treated patients than in new cases (unpublished data).
In conclusion, both patients who had ENL as well as those who did not responded to the 30 kDa peptide of M. leprae, but the reactions tended to be stronger in the former group. Additional more detailed studies will be necessary to detect a clear marker for ENL, using individual proteins of the 85B complex or specific peptide sequences of other proteins that might discriminate between patients with or without reactional phenomena.
Acknowledgment. This research was supported by grant number S1-2001000859 from Fondo Nacional Ciencias y Tecnología (FONACIT) Caracas, Venezuela.
—Elsa María Rada, M.Sc.,
Laboratorio Leprología y Patología Experimental
Instituto de Biomedicina,
Caracas, Venezuela
—Edgar A. Zambrano, Ph.D.,
Laboratorio de Bioquímica de Parásitos,
Instituto de Biomedicina,
Caracas, Venezuela
—Nacarid Aranzazu, M.D.,
Clinical Section M.D., Instituto de Biomedicina
Caracas, Venezuela
—Jacinto Convit, M.D.,
Director, Instituto de Biomedicina
Caracas, Venezuela
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