IL-12 and IL-18 synergistically induce the bactericidal activity of murine peritoneal cells against M. leprae.

We examined the effect of IL-12 and IL-18 on bactericidal activities of mouse peritoneal cell (PC) against Mycobacterium leprae (M. leprae). We demonstrated that IL-12 and IL-18 synergistically induced the NO-dependent bactericidal activity of PC by stimulating Natural Killer (NK) cells and T-cells through IFN-gamma production. IL-12 and IL-18 induced host cell death through NK-cells and T-cells. Therefore. IL-12 and IL-18 play an important role on direct killing of intracellular M. leprae and on indirect killing of them through inducing host cell death.

Comparison of polymerase chain reaction, immunohistochemistry and conventional histopathology in the diagnosis of early leprosy in Sichuan Province of China.

46 formalin-fixed, paraffin-embedded skin biopsy specimens, which were clinically suspected or diagnosed as early leprosy, were retrieved from the files of Sichuan, China from 1997 to 1999. All of them were examined by polymerase chain reaction (PCR) using the primers amplifying the 130 base-pair fragment of the gene from the 16S ribosomal RNA of Mycobacterium leprae, hematoxylin and eosin (H&E) staining, modified Fite-Faraco technique for M. leprae and immunostaining with the antiserum against the PGL-1, LAM-B, S-100 protein using ABC method. PCR was positive for 27 (58.7%) of 46 specimens. In 13 (28.3%) among them, only PCR signals were positive for M. leprae and all other test were negative. AFB was positive for 7 (15.2%) of 46, PGL-1 was positive for 17 (36.9%) of 46, LAM-B was positive for 10 (21.7%) of 46. Early epithelioid cells granuloma was detected in 4 (8.7%) patients (TT 3, BT 1), macrophage granuloma was detected in 1 (2.2%) patient (BL), S-100 protein staining showed early nerve granuloma for 4 (8.7%) of 46, peripheral nerve inflammatory infiltration for 11 (23.9%) of 46. Comparison PCR with other method showed statistically significant difference. PCR have an advantage over microscopic examination in detecting M. leprae in biopsy specimens negative for acid-fast bacilli.

An epidemiological study on Mycobacterium leprae infection and prevalence of leprosy in endemic villages by molecular biological technique.

One of the most important unsolved questions in epidemiology of leprosy is the highly uneven geographic distribution of the disease. There are many hyperendemic "pockets" in endemic countries. Little is known about the reasons why leprosy is hyperendemic in these areas. We conducted, therefore, a series of epidemiological studies on Mycobacterium leprae infection and prevalence of leprosy in North Maluku district, Maluku Province, Indonesia where leprosy is highly endemic. It was found that considerable number of general inhabitants are seropositive to various mycobacterial antigens and 27% of the villagers were carrying leprosy bacilli on their surface of nasal cavity. These results suggested the importance of M. leprae in the residential environment in infection of the leprosy bacillus and the resulting transmission of the disease. Based on these observations, we conclude that new preventive measures are essential for global elimination of leprosy in addition to early diagnosis and multidrug therapy (MDT).

[Present situation of leprosy in highly endemic area of tropical Asia--a seroepidemiological study of Mycobacterium leprae infection in general inhabitants].

One of the most important unsolved problems in epidemiology of leprosy is the heterogeneous geographic distribution of the disease. There are highly endemic area called "Pocket" in the endemic countries. Little is known why leprosy is so endemic in the area. We conducted, therefore, an epidemiological study on M. leprae infection and distribution of leprosy bacilli in the environment by using serological and molecular biological techniques. It was found that considerable number of general inhabitants in the pocket are infected with leprosy bacilli and more than 20% of the villagers are carrying M. leprae on the surface of the nasal cavity; suggesting that leprosy bacilli in the residential environment play an important role in high prevalence of leprosy in the endemic area. New preventive measures such as chemoprophylaxis, in addition to MDT, will be needed for global elimination of the disease.

Transepidermal elimination of lepromatous granuloma: a mechanism for mass transport of viable bacilli.

A 35-year-old male with lepromatous leprosy showed significant progression of the disease on initial examination. Along with typical lepromatous skin lesions, many scar-forming lesions were present, mainly on his extremities. Some lesions showed erosive surfaces. From clinicopathological findings, these lesions were suspected to be due to the partial excretion of intradermal lepromatous granulomata by 'transepidermal elimination'. Increased local volume, which might be due mainly to rapidly growing lepromatous infiltration before chemotherapy, is suspected of triggering this phenomenon. There is no doubt that many fresh Mycobacterium leprae were included in these excretions. After the initiation of chemotherapy, no new scar-forming lesions were observed.

Pathological investigation of armadillos infected with Mycobacterium leprae.

An infection experiment with M. leprae was carried out using 20 nine-banded armadillos. As a result, the development of leprous lesions and a marked multiplication of AFB were confirmed in a high rate of 13 out of 15 cases (86.8%) in the inoculated groups. These changes were found to be progressing at post mortem of one case even with the shortest life period for 7.5 months and were very serious in one case with the longest life period for 33 months, suggesting the continuation of symptoms, though it is an expression neglecting the individual difference in susceptibility to leprosy. Among infected viscera with AFB, the most conspicuous lesions were found in the liver and spleen. The developed lesions were found in the lung, stomach and kidney which had been never seen in HD in human cases, and so, which may characterize armadillos' leprosy. The change in the peripheral nerve was not so severe when compared with that in HD in human cases. This difference will remain as a future pathological problem to be solved.

A serologic Mycobacteria leprae gelatin particle agglutination (MLPA) in the diagnosis of leprosy: comparison with conventional enzyme-linked immunoassay and bacterial index.

A gelatin particle agglutination test (MLPA) for the detection of anti-phenolic glycolipid-1 (PGL-1) antibodies was compared with the slit skin smear method in the diagnosis of leprosy. MLPA and BI tests showed a good agreement rate of 88.1% and MLPA and ELISA tests showed an excellent agreement rate 96.2%. This MLPA test is simple and reliable, it will be very convenient for the medical practitioners, it would be of great benefit for leprosy patient as well because this test would look like a routine blood examination compared with slit skin smear method which is widely known diagnostic tool for leprosy.

[Uveitis in leprosy patients].

We examined 24 dermatologically cured leprosy patients with ongoing uveitis (UV+) and 22 age and type matched controls (UV-) to study the late phase leprous UV. All patients have been skin smear negative for more than 10 years. The history of chemotherapy, 5 years before and after a accomplishing bacterial negativity, was evaluated and represented by "SCORE". It was found that anti-PGL-I and anti-LAM-B antibodies were significantly higher in UV+ group compared to the controls. The mean SCORE of chemotherapy in UV+ group was significantly lower than in the controls. Iris pearls were seen in 10 cases or 42% out of 24 UV+ patients. No iris pearls were seen in control group. These results suggest that insufficient chemotherapy and consequent incomplete elimination of bacilli are the risk factors for leprous UV in the quiescent stage of the disease.

Immunopathological stain of lipoarabinomannan-B (LAM-B) for diagnosis of leprosy.

We developed an immunopathological staining of LAM-B antigen in formalin-fixed paraffin-embedded tissues, and compared it with, PGL-I immunostaining, Fite Faraco's stain and periodic acid carbol pararosaniline (PACPR) stain. Out of the total 28 leprosy cases, 27 were positive to LAM-B immunostaining while 23 were positive to PGL-I stain. Fite's stain was positive in 21 cases while PACPR stain was positive in 24 cases. In scrofuloderma, LAM-B antigen was observed only in the granuloma while no other positive findings were noted with other stains. Normal skin did not give any positive findings with any of the stains. Other dermatoses showed no positive findings to any of the stains tested. LAM-B staining was observed in the nerve even in the absence of bacilli in leprosy tissues. Presence of LAM-B in the cutaneous nerves is helpful in discriminating leprosy from other mycobacterioses. Considering the high sensitivity of LAM-B and the predilection of M. leprae for the nerves, we concluded that LAM-B staining can be a useful new tool in the prompt diagnosis of leprosy, especially in suspected or early cases.

[Modification of Harada's method for rapid staining of mycobacteria].

Harada employed periodic acid-carbol pararosaniline and periodic acid-methenamine silver stain for demonstrating chromophobic bacilli which do not get stained with conventional carbol fuchsin or counter stain. This staining method takes considerable time for complete oxidation with periodic acid. We have succeeded in reducing the oxidation time by using hydrogen peroxide treatment prior to periodic acid and with the use of acidified sodium hydrogen sulfite treatment before carbol pararosaniline stain. We also found that in methenamine silver stain, combined use of semicarbazide and microwave treatment can shorten the whole staining time up to four hours without losing ito sensitivity.

Demonstration of PGL-I & LAM-B antigens in paraffin sections of leprosy skin lesions.

An investigation on the demonstration of PGL-I and LAM-B antigens in thirty-four paraffin embedded skin biopsies taken from leprosy patients who covered the whole spectrum of the disease and in four control specimens was carried out. Neither the PGL-I antigen nor the LAM-B antigen was demonstrated in the normal skin specimens that were used as negative control; and only the LAM-B antigen appeared in the tuberculosis specimens in which the PGL-I antigen was negative. The PGL-I antigen was demonstrated on thirty-three leprosy samples except one TT sample and the LAM-B antigen, on all samples by immunochemical staining technique. The antigens were identified as intracytoplasmic bacillary staining, in solitary, granular as well as debris patterns; and as soluble antigenic staining, in vacuolar or amorphous pattern. In LL and BL cases, the antigens were detected predominantly from macrophages and peripheral nerves in all five staining patterns; in BB cases, from macrophages mostly in the granular as well as debris patterns, from the nerves in the vacuolar pattern; while in TT and the majority of BT cases, they were mainly from nerve remnants inside the granuloma in the vacuolar or amorphous staining pattern. In addition, it is interesting to note that the immunochemical staining was able to differentiate the foamy change from the hydropic degeneration. We also found that the antigens distributed in arrector pili muscles and the walls of muscular vessels were obviously related to the unmyelinated nerve fibers innervating the smooth muscle cells.(ABSTRACT TRUNCATED AT 250 WORDS)

The use of improved silver impregnating staining method in the differential diagnosis of tuberculoid leprosy.

A comparative study on the usefulness of Kawatsu's silver impregnating staining method compared with S-100 protein ABC technique for the differential diagnosis of tuberculoid leprosy was carried out. The results of neurohistological examination obtained from both methods were almost the same. It can be said that Kawatsu's method is useful and cost-effective, and thus suitable for practical use in developing countries.

Susceptibility of severe combined immunodeficient (SCID) mice to Mycobacterium leprae: multiplication of the bacillus and dissemination of the infection at early stage.

Inoculation of M. leprae were made into the both hind feet at a dose of 4.8 x 10(6) bacilli per foot in order to determine the susceptibility to M. leprae of SCID mice which is severely deficient in both T- and B cell immunity. SCID mice was found to have an extremely high susceptibility to M. leprae, and the progress of infection observed in the SCID mice were shown a rapid systemic spread of infection at the all over the tissues as well as the growth of the leprosy bacilli at the site of inoculation. Therefore, SCID mice can be used as a suitable multibacillary model for the study of leprosy.

Histo-bacteriological investigation on borderline tuberculoid leprosy.

The multi-sections, which were stained by Fite method, of skin biopsies taken from twelve active BT cases were examined under the guidance of special stains for demonstration of nerve components. All cases were AFB positive. Bacilli were found in infiltrated nerves in 11 cases, of which, in 7 cases, bacilli were detected in nerve fragments within epithelioid cell granuloma. And bacilli were seen in arrector pili muscles in 2 cases. No bacilli were detected in other sites. Since the survival of M. leprae in nerves is one of the reasons causing relapse, this paper suggests that it would be better to treat active BT cases with multibacillary regimen recommended by WHO even though smear-negative.

Novel gelatin particle agglutination test for serodiagnosis of leprosy in the field.

We developed a novel gelatin particle agglutination test (MLPA) for the serodiagnosis of leprosy; this test is especially useful for clinical practice and epidemiological surveys of leprosy in countries in which the disease is endemic. The antigen used in the test is the chemically synthesized trisaccharide moiety of Mycobacterium leprae-specific phenolic glycolipid I. MLPA is a simple and easy technique having sensitivity and specificity comparable to those of the conventional indirect enzyme-linked immunosorbent assay. The new technique was found to be useful for monitoring of chemotherapy and predictive diagnosis of high-risk individuals in contact with persons with leprosy and may be useful for the prediction of relapse. We are now preparing to supply a quality-controlled ready-to-use MLPA kit for leprosy control in countries in which leprosy is endemic.