Invasion of human microvascular endothelial cells by Mycobacterium leprae through Mce1A protein.

In patients with lepromatous leprosy, Mycobacterium leprae is often observed inside the human microvascular endothelial cells (HMVEC) surrounding Schwann cells (SC) at the site of lesions in the peripheral nerves. Based on this observation, it is considered that the nasal mucous may be the invasion pathway for M. leprae and HMVEC serve as an important reservoir for the bacteria before they invade SC. In light of previous research which revealed that Mce1A protein mediates bacterial invasion into nasal epithelial cells and HMVEC, we conducted a study to determine whether the invasion of M. leprae into HMVEC can be suppressed by blocking the Mce1A protein. In this study, we analyzed bacterial invasive activity by adding recombinant Escherichia coli, which express the active region (InvX:72 a.a.) of Mce1A protein on their external membrane, into cultured HMVEC, using the adhesin involved in the diffuse adherence mechanism. The number of bacteria that invaded into the cells was then measured by a colony counting method. The active region of Mce1A was divided into four sections, and hyperimmune antisera was prepared for each section for analyzing the inhibitory effect against invasion. The invasive activity was suppressed by antibodies against InvX regions 1-24 a.a., 25-46 a.a. and 58-72 a.a. This suggests that the InvX regions 1-24 a.a., 25-46 a.a. and 58-72 a.a. of Mce1A protein play an important role in the invasion of M. leprae into HMVEC and that it may be possible to suppress entry of M. leprae in HMVEC with antibodies against these regions.

False positive reaction of the immunohistochemistry technique using anti-BCG polyclonal antibodies to identify Mycobacterium leprae in wild nine-banded armadillos.

The authors studied 66 wild nine-banded armadillos from Brazil. The ear samples were collected and Ziehl-Neelsen or Fite-Faraco stains were performed, as well as immunostaining using polyclonal BCG antibody, to avaluate the presence of the Mycobacterium leprae. The AFB were not detected by the Ziehl-Neelsen or Fite-Faraco staining, neither immunoexpression of the BCG marker. However, many normal structures from the ears of the nine-banded armadillos, such as condrocytes, condroblasts, fibroblasts and endothelial cells, and Gram positive bacteria cocci, showed false positive reaction by the BCG marker. The authors discuss the use of the immunohistochemical studies with the polyclonal BCG antibody to identify M. leprae antigens in wild armadillos.

Dipstick assay to identify leprosy patients who have an increased risk of relapse.

Classification of leprosy patients into paucibacillary (PB) and multibacillary (MB) determines the duration of treatment; misclassification increases the risk of relapse because of insufficient treatment if an MB patient is classified as PB. We explored the possibility of using a simple dipstick assay based on the detection of antibodies to the Mycobacterium leprae-specific phenolic glycolipid-I (PGL-I) as a tool for classification of patients into PB and MB for treatment purposes. The sensitivity of the dipstick test for detection of MB patients was 85.1%, the specificity 77.7%. We found that of the 71 dipstick negative PB patients 25 (35.2%) were clinically cured at the end of treatment, compared with only two (9.5%) of the 21 dipstick positive PB patients. Of 170 patients in the study population, nine (5.3%) relapsed within the 5-year follow-up period. Seven were MB patients, all dipstick positive. Two PB patients relapsed, one was dipstick negative and one was dipstick positive. Dipstick positivity is a risk factor for the future development of relapses, especially in those groups of patients who had received a shorter-than-usual course of treatment and the dipstick can be used as an additional, simple tool for classification of patients and for identification of those patients who have an increased risk of relapse.

[The isolation of monoclonal antibodies to strains of Mycobacterium leprae passed through laboratory animals]. / Poluchenie monoklonal'nykh antitel k shtammam mikobakterii lepry, passiruemym na laboratornykh zhivotnykh.

Hybridoma synthetizing monoclonal antibodies (mAB) IIIE4 has been obtained in hybridization of myeloma P3-X63-Ag8. 653 cells and splenocytes from BALB/c mice immunized with M. leprae passaged on rats. The mAB specificity evaluated by enzyme immunoassay using ultrasonic disintegrates of M. leprae obtained from human, Dasypus novemcinctus, rat lepromas as well as mycobacteria of 7 species and E. coli indicated that the mAB reacted only with mycobacteria passaged on the rats. They had weak cross reactivity with M. avium.

Identification of mycobacterial antigens for "ELISA" serology in the diagnosis of leprosy and tuberculosis.

Using an immunoblotting assay (ImBA), several immuno-crossreactive antigenic components (ImCRAC-myc) have been identified in the whole sonicates of M. bovis-BCG, and M. tuberculosis (Mtb) and M. leprae (ML) whereby the sera of 100% lepromatous leprosy (L-Lep) reacted to 29/33 KD doublet and that of 100% tuberculoid leprosy (T-Lep) reacted to 64 KD bands. The antigens upon purification from Mtb Sonicates were used in a direct ELISA to measure antibody isotypes in the sera from L-Lep, T-Lep, healthy Lep. contacts (Lep. c), normal Dutch controls (N) and tuberculosis (TB) patients. A significantly high IgG titre to the doublet 29/33 KD and to 64 KD were observed among L-Lep and T-Lep patients respectively in comparison to sera from other groups of individuals. In certain cases of L-Lep patients, raised IgM titre to either or both to 29/33 KD doublet and 64 KD were also found. On the other hand, consistantly but significant high IgA-antibody titre to cell wall (CW), cytosol (cyt) and P90 fractions of Mtb distinguished clearly the TB patients from Lep groups, normals (NN) and Lep-c. It appeared that such antibody reactivity of TB sera might be directed to the groups of 58-60, 38-40, 18-20 and 14 KD antigens of mycobacteria e.g. Mtb. On the basis of the present observations we conclude that the measurement of class specific antibody response to the panel of these antigens could diagnose differentially between Lep, TB and NN/Lep-c among the population at large in an endemic area.

Demonstration of PGL I antigens in skin biopsies in indeterminate leprosy patients: comparison with serological anti PGL I levels.

The survey of histological findings in leprosy patients from 1985 to March 1988 has been carried out at the Pasteur Institute in Noumea. Histologically, according to Ridley Jopling criteria, 82 patients were classified, 14 as T.T., 12 as B.T., 2 as B.B., 7 as B.L., 4 as L.L.s., 24 as L.L.p. and 19 as Indeterminate leprosy. Histological examination of tissues sections, using a monoclonal anti-PGL I antibody showed PGL I antigen in histiocytic cells of the infiltrate and more rarely in Langerhans cells in 7 cases of indeterminate leprosy. The Ziehl staining method revealed the presence of alcoholo resistant bacilli in only one case. For 10 patients, histological findings were compared with serological results. In 3 cases, the diagnosis was confirmed by the 2 techniques (immunohistology and serology). In 2 cases by only immunohistology or by serology and in 3 cases the diagnosis was not confirmed by either methods. These results showed the interest of the immunohistological and serological methods in indeterminate leprosy, specially in children. A study of household contacts may be of interest.

Evaluation of a phenolglycolipid antigen (PGL-Tb 1) from M. tuberculosis in the serodiagnosis of tuberculosis: comparison with PPD antigen.

Sera from 38 tuberculous patients and 62 healthy controls (31 PPD skin test positive and 31 negative) were assayed, by enzyme-linked immunosorbent assay (ELISA), to test the activity of IgG and IgM antibodies against purified protein derivative (PPD) antigen and a phenolglycolipid antigen (PLG-Tb 1) isolated and purified from Mycobacterium tuberculosis strain Canetti. Using PPD antigen, the sensitivity and specificity were respectively, 50 and 93.5% for IgG and 71.1 and 59.7% for IgM antibody activity. Against PGL-Tb 1 antigen, IgG had a sensitivity of 94.7% and the specificity was 96.8%, for IgM antibody they were 65.8% and 75.8% respectively. The ELISA using PGL-Tb 1 antigen could be a useful way to develop a rapid technique to aid in the diagnosis of tuberculosis.

Isolation of recombinant mycobacterial antigens by an automatic and generally applicable purification method for beta-galactosidase fusion proteins.

An automated two-dimensional chromatographic method has been developed for the isolation and concentration of recombinant fusion proteins with beta-galactosidase. The system consists of an immunoaffinity column with anti-beta-galactosidase antibodies as ligand, followed by an anion-exchange column. It was used for the purification and concentration of recombinant fusion proteins from Mycobacterium tuberculosis and M. leprae. Small amounts of crude lysates of Escherichia coli were loaded stepwise onto the immunoaffinity column with intermittent washing, elution and re-equilibration. After several cycles the eluate was passed through the anion-exchanger. Using an immunoaffinity gel of 5-ml volume and the anion-exchanger Mono Q HR 5/5, from 10 ml of crude E. coli lysate (containing up to 50 mg of protein) up to 100 micrograms of recombinant protein in a 2-ml volume could be isolated overnight.

IgA and IgM antibodies against Mycobacterium leprae in cord sera and in patients with leprosy: an indicator of intrauterine infection in leprosy.

A solid-phase radioimmunoassay was developed for demonstration and quantification of IgA and IgM anti-M. leprae antibodies. IgA and IgM anti-M. leprae antibodies were demonstrated in a lepromatous serum pool, in various amounts in individual patients with lepromatous leprosy, and in lower concentration in tuberculoid leprosy and non-leprosy controls. IgA and IgM anti-M. leprae antibodies were demonstrated in cord sera from babies of mothers with leprosy. The reliability of fetal IgA and IgM antibody synthesis as an indicator of intrauterine infection in leprosy is discussed.