488^ International Journal of Leprosy^ 1992

Humoral Response to Nerve Glycolipid Antigen inSera of Leprosy Patients

To THE EDITOR:Leprosy in humans is essentially a disease

of the peripheral nerves. The histopatho-logical demonstration of nerve invasion byMycobacterium leprae or the presence of in-flammatory granuloma in and around thenerve is considered essential for the diag-nosis of leprosy. However, the exact mech-anism underlying leprous neuritis is poorlyunderstood. Clinical findings such as the in-volvement of nerves remote from the siteof the infection or the absence of an etio-logical agent in the granulomas of tuber-culoid or borderline tuberculoid leprosycases suggest immunological involvementof the peripheral nerves as one of the pos-sible mechanism(s). Thus, apart from in-vestigating immune responses to Al. Lyraeantigens in the sera of the leprosy patients,recent focus has been on the different com-ponents of the peripheral nerve as an im-mune target (3• J. 8 • 9 ). Having known thepotential immunological role of glycolipidantigens in the pathogenesis of the Guillain-Barre syndrome—an inflammatory, demy-elinating peripheral neuropathy ('')—we at-tempted to detect antineural antibodies toglycolipid antigens of normal human nervesin the sera of leprosy patients as well as inthe sera of patients with neural disordersother than leprosy.

Serum samples. A total of 158 serumsamples—normal healthy individuals, 51;leprosy patients, 88 (typed according toRidley Jopling classification, '); neural dis-orders other than leprosy, 19—were testedfor antineural glycolipid antibodies at 1:200dilution using 1.5% bovine serum albumin(BSA) in phosphate buffered saline (PBS)containing 1.5% normal rabbit serum (NRS)as a diluent.

Nerve antigen. The source of the nerveantigen was cauda equina collected at au-topsy from a 26-year-old female with nohistory of any neurological disorder. Theconnective tissue and epineurium were re-moved, and the tissue stored at —70°C untilfurther use. Thin sections (approximately15-pm thick) were cut using a freezing mi-crotome (cryostat) and then processed for

glycolipid antigen extraction according tothe method of Ariga, et al.('). The lipid con-tent was determined and 1 pg per 50 pl wasused for coating in an ELISA system.

ELISA procedure. The ELISA proce-dure was essentially that of Ilyas, et al.( 5 )with minor modifications as follows: onehalf of the wells of the microliter polysty-rene plates (Immulon 2; Dynatech Inc.,Chantilly, Virginia, U.S.A.) were coated with50 pl of methanol; the remaining half werecoated with 50 pl of nerve glycolipid antigen(NGA) in methanol for 18 hr at room tem-perature (RT). After blocking the unreactedsites with 3% BSA in PBS for 2 hr at RT,100 pl of the diluted serum samples, as de-scribed earlier, were added in duplicate tothe methanol- as well as the antigen-coatedwells. The plates were incubated for 5 hr atRT, drained, washed four times with PBSand peroxidase-labeled rabbit antihumanIgM (1:1000 in 10% NRS; Dakopatts, Glos-trup, Denmark) was added. After incuba-tion for 1 hr at 37°C, the wells were drained,washed four times with PBS, and the en-zyme-linked immune complex was quan-titated using H,0,-0-phenylene diamine asa substrate (0.4 mg/ml in citrate phosphatebuffer, pH 5.0) at 490 nm using a Mini-reader II (Dynatech). The difference in theoptical density (OD) obtained with the an-tigen-coated wells and methanol-coatedwells was calculated with normal as well aspatient sera. The cut-off value (A OD at 490mm) of 0.15 (0.035 ± 0.039 S.D., normalsera) was used for screening the sera posi-tive.

Results. The overall percent positivityobtained with the 88 leprosy patients eval-uated was 23.86% (21/88), and all of thembelonged to the lepromatous or borderlinelepromatous or purely borderline type ofleprosy classification, thus giving a percentpositivity of 31.3 (21/67) in the leproma-tous leprosy group. The antibodies to NGAwere absent in tuberculoid-borderline tu-berculoid (TT-BT) type of leprosy as wellas in 25 patients with neural disorders otherthan leprosy (Fig. 1). An attempt was madeto correlate the presence of anti-PGL-I an-

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FIG. 1. Reactivity to nerve glycolipid antigen ofsera from healthy normal individuals and patients withvarious types of leprosy along with other neuropathies.LL = Lepromatous; BL = borderline lepromatous; BB= borderline; BT = borderline tuberculoid; TT = tu-berculoid leprosy.

tibodies with anti-NGA antibodies in thesepatients (Fig. 2). As is evident, an overallpoor correlation (r = 0.37) was obtainedbetween the two types of antibodies. Thisobserved discordance could be due to thesmaller size and heterogeneous group of pa-tients [tuberculoid to lepromatous (TT toLL) type] with varying treatment status,from no treatment to monodrug therapy/multidrug therapy for varying periods oftimes. It is worthwhile to note that in ourseries the untreated group tends to showlower/negligible immunoreactivity to nervelipid antigen in spite of having a high bac-terial load, while the treated group of pa-tients tends to have higher levels of im-munoreactivity. The absence of antibodiesto NGA in the early untreated leprosy casessuggests the possible role of cytoplasmic an-tigen (released during treatment) in nervereactions as postulated by Barnetson, etal. ( 2).

Our efforts in demonstrating the presenceof antibodies to whole nerve sonicate in thesera of leprosy patients using SDS-PAGEelectrophoresis showed a pattern compa-rable to that seen with normal sera. Similarresults of equal binding of sera from normaland leprosy patients to peripheral nerve hasbeen reported by Ghaswala, et al.(4 ).

The studies reported in this communi-cation indicate that a specific nerve glyco-lipid component appears to be involved inthe immune mechanism. Further charac-terization of the antigen along with its utility

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FIG. 2. A scatter diagram showing the correlationbetween anti-natural disaccharide (of phenolic glyco-lipid-I) bovine serum albumin (ND-BSA) antibody andanti-nerve glycolipid antigen-antibody reactivity in serafrom leprosy patients.

for monitoring the course of the disease isin progress.

—Rekha V. Sahasrabudhe, B. Pharm.Acworth Leprosy Hospital Research Society

—Subhada R. Dandekar, M.Sc.Damayanti H. Shah, Ph.D.

Radiation Medicine Center (BARC)Tata Memorial Center AnnexParel, Bombay 400012, India

—Subhada S. Pandya, M.B.B.S.Sharad S. Naik, M.Sc.Evelyn Sequeira, B.Sc.

Acworth Leprosy Hospital Research SocietyWadala, Bombay 400031, India

—R. Ganapati, M.B.B.S., D.V.D.Bonthay Leprosy ProjectSion-ChunabhattiBombay 400022, India

Reprints requests to Dr. Shah.

REFERENCES1. ARIGA, T., KOHR1YAMA, T. and FREDDO, L. Char-

acterization of sulphated glucoronic acid contain-ing glycolipids reacting with IgM M-proteins inpatients with neuropathy. J. Biol. Chem. 262 (1987)848-853.

2. BARNETSON, R. ST.C., MUNE, G., PEARSON, J. M.H. and KRONVALL, G. Cell mediated and humoralimmunity in "reversal reactions." Int. J. Lepr. 44(1976) 267-274.

3. EUSTIS-TURF, E. P., I3ENJAMINS, J. A. and LEE-FORD, M. J. Characterization of anti-neural an-tibodies in the sera of leprosy patients. J. Neu-roimmunol. 10 (1986) 313-330.

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490^ International Journal of Leprosy^ 1992

4. GHASWALA, P. S., MISTRY, N. F. and ANTIA, N.H. Serum antibodies of normals and leprosy pa-tients show equal binding to peripheral nerve. Int.J. Lepr. 57 (1989) 690-692.

5. ILYAS, A. A., QUARLES, R. H., MACINTOSH, T. D.,DOBERSON, M. J., TRAPI', B. D., DALAKAS, M. C.and BRADY, R. 0.IgM in human neuropathy re-lated to paraproteinemia binds to a carbohydratedeterminant in the myelin associated glycoproteinand to a ganglioside. Proc. Natl. Acad. Sci. U.S.A.81 (1984) 1225-1229.

6. PRAKASH, 0., SREEVATSA, P. and SENGUPTA, U.An attempt to demonstrate anti-nerve antibodiesin leprosy sera using rabbit nerve as an antigen.Int. J. Lepr. 58 (1990) 129-130.

7. RIDLEY, D. F. and JOPLING, W. H. Classificationof leprosy according to immunity; a five-groupsystem. Int. J. Lepr. 34 (1966) 255-273.

8. THOMAS, B. M. and MUKHERJEE, R. Antineuralantibodies in sera of leprosy patients. Clin. Im-munol. Immunopathol. 57 (1990) 420-429.

9. VEMURI, N. and MUKHERJEE, R. Immunoreactiv-ity of nerve lipid antigens in leprosy. J. Clin. Lab.Anim. 5 (1991) 157-161.

10. Yu, R. K., ARICA, T., KOIIRIYAMA, T., KUSUNOKI,S., MEDA, Y. and MIYATANI, N. Autoimmunemechanisms in peripheral neuropathics. Ann.Neurol. 27 Suppl. (1990) S30—S35.