Bacillus Calmette-Guérin (BCG) vaccine generates immunoregulatory cells in the cervical lymph nodes in guinea pigs injected intra dermally.

This work demonstrates the presence of immune regulatory cells in the cervical lymph nodes draining Bacillus Calmette-Guérin (BCG) vaccinated site on the dorsum of the ear in guinea pigs. It is shown that whole cervical lymph node cells did not proliferate in vitro in the presence of soluble mycobacterial antigens (PPD or leprosin) despite being responsive to whole mycobacteria. Besides, T cells from these lymph nodes separated as a non-adherent fraction on a nylon wool column, proliferated to PPD in the presence of autologous antigen presenting cells. Interestingly, addition of as low as 20% nylon wool adherent cells to these, sharply decreased the proliferation by 83%. Looking into what cells in the adherent fraction suppressed the proliferation, it was found that neither the T cell nor the macrophage enriched cell fractions of this population individually showed suppressive effect, indicating that their co-presence was necessary for the suppression. Since BCG induced granulomas resolve much faster than granulomas induced by other mycobacteria such as Mycobacterium leprae the present experimental findings add to the existing evidence that intradermal BCG vaccination influences subsequent immune responses in the host and may further stress upon its beneficial role seen in Covid-19 patients.

Development of LepReact, a defined skin test for paucibacillary leprosy and low-level M. leprae infection.

The persistence of new leprosy cases in endemic areas such as India, Brazil, Bangladesh, and the Philippines has encouraged studies of chemoprophylaxis among contacts of patients. Epidemiological screening tools to enable early detection of infected individuals in endemic populations would be critical to target individuals most in need of intervention. Despite decades of attempts, however, there still are no tests available for the early detection of low-level infection with Mycobacterium leprae. In this report, we describe the development of a leprosy skin test using M. leprae-specific antigens. We selected the chimeric LID-1 fusion protein, formulated to achieve maximum performance at a minimal dose, as a skin test candidate based on its ability to elicit delayed-type hypersensitivity (DTH) reactions in M. leprae immune guinea pigs in a sensitive and specific manner, i.e., with no cross-reactivity observed with other mycobacterial species. Importantly, evaluations in armadillos indicated that intradermal inoculation of formulated LID-1 could distinguish uninfected from M. leprae-infected animals manifesting with symptoms distinctly similar to the PB presentation of patients. Together, our data provide strong proof-of-concept for developing an antigen-specific skin test to detect low-level M. leprae infection. Such a test could, when applied with appropriate use of chemo- and/or immunoprophylaxis, be instrumental in altering the evolution of clinical disease and M. leprae transmission, thus furthering the objective of zero leprosy.

Long-lasting endothelium-dependent relaxation of isolated arteries caused by an extract from the bark of Combretum leprosum.

OBJECTIVE: To describe and to characterize the relaxing effect of an extract of the bark of Combretum leprosum on isolated arterial rings from different animals. METHODS: Rings (3 to 4mm) from rabbit, rat, or porcine arteries rings were suspended in an organ bath (Krebs, 37°C, 95%O2/5%CO2) to record isometric contractions. After the stabilization period (2 to 3 hours) contractions were induced by the addition of phenylephrine (0.1 to 0.3µM) or U46619 (10 to 100nM), and Combretum leprosum extract was added on the plateau of the contractions. Experiments were performed to determine the potency, duration, reversibility, and to get insights on the potential mechanism involved in extract-induced relaxations. RESULTS: In all rings tested, Combretumleprosum extract (1.5µg/mL) was able to cause relaxations, which were strictly endothelium-dependent. In rabbit or rat thoracic aorta rings, the relaxations were reversed by vitamin B12a or L-NG-nitroarginine. In porcine right coronary arteries and rabbit abdominal aorta, extract caused both L-NG-nitroarginine-sensitive and L-NG-nitroarginine-resistant relaxations. In rabbit thoracic aorta, the extract was relatively potent (EC50=0.20µg/mL) and caused relaxations; intriguingly the endothelium continued to produce relaxing factors for a long period after removing the extract. The magnitude of extract-induced relaxations was significantly reduced in the absence of extracellular Ca2+; in addition, the TRPs channels blocker ruthenium red (10µM) was able to revert extract-induced relaxations. Phytochemical analyses indicated that the extract was rich in polyphenol-like reacting substances. CONCLUSIONS: Combretum leprosum extract contains bioactive compounds capable of promoting Ca2+-dependent stimulation of endothelial cells which results in a prolonged production of relaxing factors.

Long-lasting endothelium-dependent relaxation of isolated arteries caused by an extract from the bark of Combretum leprosum / Extrato das cascas de Combretum leprosum causa relaxamento dependente de endotélio de longa duração em artérias isoladas

Objective To describe and to characterize the relaxing effect of an extract of the bark of Combretum leprosum on isolated arterial rings from different animals.Methods Rings (3 to 4mm) from rabbit, rat, or porcine arteries rings were suspended in an organ bath (Krebs, 37°C, 95%O2/5%CO2) to record isometric contractions. After the stabilization period (2 to 3 hours) contractions were induced by the addition of phenylephrine (0.1 to 0.3µM) or U46619 (10 to 100nM), and Combretum leprosum extract was added on the plateau of the contractions. Experiments were performed to determine the potency, duration, reversibility, and to get insights on the potential mechanism involved in extract-induced relaxations.Results In all rings tested, Combretumleprosum extract (1.5μg/mL) was able to cause relaxations, which were strictly endothelium-dependent. In rabbit or rat thoracic aorta rings, the relaxations were reversed by vitamin B12a or L-NG-nitroarginine. In porcine right coronary arteries and rabbit abdominal aorta, extract caused both L-NG-nitroarginine-sensitive and L-NG-nitroarginine-resistant relaxations. In rabbit thoracic aorta, the extract was relatively potent (EC50=0.20µg/mL) and caused relaxations; intriguingly the endothelium continued to produce relaxing factors for a long period after removing the extract. The magnitude of extract-induced relaxations was significantly reduced in the absence of extracellular Ca2+; in addition, the TRPs channels blocker ruthenium red (10µM) was able to revert extract-induced relaxations. Phytochemical analyses indicated that the extract was rich in polyphenol-like reacting substances.ConclusionsCombretum leprosum extract contains bioactive compounds capable of promoting Ca2+-dependent stimulation of endothelial cells which results in a prolonged production of relaxing factors.

Objetivo Descrever e caracterizar os relaxamentos induzidos por um extrato das cascas de Combretum leprosum em anéis de artérias de diferentes espécies de animais.Métodos Anéis (3 a 4mm) de artérias de coelho, rato e porco foram montados em cubas para órgão isolado (Krebs, 37°C, 95%O2/5%CO2) para registro das contrações isométricas. Após um período de estabilização (2 a 3 horas), as contrações foram induzidas com fenilefrina (0,1 a 0,3µM) ou U46619 (10 a 100nM); no platô dessas contrações, adicionamos o extrato Combretum leprosum. Diferentes protocolos foram realizados para determinar potência, duração, reversibilidade e mecanismo dos relaxamentos induzidos pelo extrato.Resultados Em todas as preparações testadas, o extrato de Combretum leprosum (1,5µg/mL) provocou relaxamentos dependentes de endotélio. Em aorta torácica de coelho ou rato, os relaxamentos foram revertidos pela vitamina B12a ou L-NG-nitro-arginina. Em anéis de aorta abdominal de coelho e de artérias coronárias de porco, o extrato causou relaxamentos sensíveis e resistentes à L-NG-nitro-arginina. Em aorta torácica de coelho, o extrato foi relativamente muito potente (EC50=0,20μg/mL) e quando causou relaxamentos; intrigantemente o endotélio continuou a produzir fatores relaxantes por um longo período após remoção do extrato. A magnitude dos relaxamentos induzidos pelo extrato foi significativamente reduzida em ausência Ca2+ extracelular; ademais, o vermelho de rutênio (10μM), um bloqueador de canais TRPs, foi capaz de reverter os relaxamentos induzidos pelo extrato. Análises preliminares indicaram que o extrato continha compostos com reatividade química semelhante à polifenóis.Conclusão O extrato de Combretum leprosum contem compostos bioativos capazes de promover estimulação dependente de Ca2+ das células endoteliais a qual resulta numa produção prolongada de fatores relaxantes.

The challenge of producing skin test antigens with minimal resources suitable for human application against a neglected tropical disease; leprosy.

True incidence of leprosy and its impact on transmission will not be understood until a tool is available to measure pre-symptomatic infection. Diagnosis of leprosy disease is currently based on clinical symptoms, which on average take 3-10 years to manifest. The fact that incidence, as defined by new case detection, equates with prevalence, i.e., registered cases, suggests that the cycle of transmission has not been fully intercepted by implementation of multiple drug therapy. This is supported by a high incidence of childhood leprosy. Epidemiological screening for pre-symptomatic leprosy in large endemic populations is required to facilitate targeted chemoprophylactic interventions. Such a test must be sensitive, specific, simple to administer, cost-effective, and easy to interpret. The intradermal skin test method that measures cell-mediated immunity was explored as the best option. Prior knowledge on skin testing of healthy subjects and leprosy patients with whole or partially fractionated Mycobacterium leprae bacilli, such as Lepromin or the Rees' or Convit' antigens, has established an acceptable safety and potency profile of these antigens. These data, along with immunoreactivity data, laid the foundation for two new leprosy skin test antigens, MLSA-LAM (M. leprae soluble antigen devoid of mycobacterial lipoglycans, primarily lipoarabinomannan) and MLCwA (M. leprae cell wall antigens). In the absence of commercial interest, the challenge was to develop these antigens under current good manufacturing practices in an acceptable local pilot facility and submit an Investigational New Drug to the Food and Drug Administration to allow a first-in-human phase I clinical trial.

Identification of mimotopes of Mycobacterium leprae as potential diagnostic reagents.

BACKGROUND: An early diagnostic test for detecting infection in leprosy is fundamental for reducing patients' sequelae. The currently used lepromin is not adequate for disease diagnosis and, so far, no antigen to be used in intradermoreaction has proved to be sensitive and specific for that purpose. Aiming at identifying new reagents to be used in skin tests, candidate antigens were investigated. METHODS: Random peptide phage display libraries were screened by using antibodies from leprosy patients in order to identify peptides as diagnostic reagents. RESULTS: Seven different phage clones were identified using purified antibodies pooled from sera of leprosy patients. When the clones were tested with serum samples by ELISA, three of them, 5A, 6A and 1B, allowed detecting a larger number of leprosy patients when compared to controls. The corresponding peptides expressed by selected phage clones were chemically synthesized. A pilot study was undertaken to assess the use of peptides in skin tests. The intradermal challenge with peptides in animals previously sensitized with Mycobacterium leprae induced a delayed-type hypersensitivity with peptide 5A (2/5) and peptide 1B (1/5). In positive controls, there was a 3/5 reactivity for lepromin and a 4/5 reactivity of the sensitized animals with soluble extract of M. leprae. CONCLUSIONS: The preliminary data suggest that may be possible to develop reagents with diagnostic potential based on peptide mimotopes selected by phage display using polyclonal human antibodies.

Expression of CXCL10 (IP-10) and CXCL11 (I-TAC) chemokines during Mycobacterium tuberculosis infection and immunoprophylaxis with Mycobacterium indicus pranii (Mw) in guinea pig.

Mycobacterium indicus pranii (earlier known as Mycobacterium w) has been used as an immunmodulatory agent in leprosy and tuberculosis by mediating the release of various cytokines and chemokines. CXCL10 (IP-10) and CXCL11 (I-TAC) chemokines are involved in T-cell migration and stimulation of natural killer cells in Mycobacterium tuberculosis infection. In this study, the effect of heat killed M. indicus pranii (alone and in conjunction with chemotherapy) on disease progression was determined by colony forming units (CFUs) in guinea pig lung following their aerosol infection and the expression levels of CXCL10 and CXCL11 were studied by quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR) and in situ RT-PCR. Four groups of animals included; infection only (Rv), immunoprophylaxis (RvMw), chemotherapy (RvCh) and combination of immunoprophylaxis with chemotherapy (RvChMw). In the group where immunoprophylaxis was given in combination with chemotherapy, the CFU counts reduced significantly at 4th week post-infection as compared to animals that received immunoprophylaxis or chemotherapy alone. At the same time, all groups of animals had elevated expression of CXCL 10 which was significantly high only in animals that received Mw with or without chemotherapy. Unlike to CXCL 10, study demonstrated suppressed expression CXCL 11 in both immunoprophylaxis as well as chemotherapy groups that became up-regulated in synergistic response of immunoprophylaxis and chemotherapy. Taken together, data indicates that the expression of CXCL10 and CXCL11 positively correlates with anti-tubercular treatment (at least with combination of immunoprophylaxis and chemotherapy). Therefore, prior immunization with Mw appears to be a good immunomodulator for release of chemokines and augments the effect of chemotherapy.

[The comparative cultural-biochemical and immunochemical identification of laboratory strains of cultured mycobacteria from leprous patient lesions and from soil]. / Sranitel'naia kul'tural'no-biokhimicheskaia i immunokhimicheskaia identifikatsiia laboratornykh shtammov kul'tiviruemykh mikobakterii iz leproznykh porazhenii bol'nykh i pochvy.

The paper presents the results of studied of cultural biochemical and immunochemical properties of the cultured mycobacteria isolated from patients with leprous lesions of (M.01, M.011) and the soil (M. lufu) in compared to other well-known mycobacteria and between them. M.01, M.011, and M. lufu differ from other representatives of the genus Mycobacterium, but they are close to each other in their cultural biochemical and immunochemical properties, antigenic composition, and protein spectra. The findings are an additional characteristic of the biological properties of M.01, M.011, and M. lufu.

Ribosomal protein L7 included in tuberculin purified protein derivative (PPD) is a major heat-resistant protein inducing strong delayed-type hypersensitivity.

The tuberculin purified protein derivative (PPD) is a widely used diagnostic antigen for tuberculosis. It consists of more than 100 denatured proteins in a culture filtrate of a heated culture of Mycobacterium tuberculosis. In two-dimensional electrophoretic analysis of PPDs from M. tuberculosis and M. bovis BCG, most proteins were diffusely separated and could not be seen as spots because of denaturation, whereas a few proteins showed relatively clear spots, indicating heat resistance. Two such proteins corresponded to ribosomal proteins L7 and L12. The mixture of these proteins L7/L2 induced a strong delayed-type hypersensitivity reaction. Another protein showing a clear spot was a GroES analogue, but this did not induce delayed-type hypersensitivity. There were a few other unidentified proteins. It is well known that L7 and L12 are encoded by the same gene and that they differ from each other only by an acetylic post-translational modification that occurs at the N-terminus of L12 converting it to L7 in Escherichia coli. L12, but not L7, was found in two-dimensional electrophoresis of BCG ribosomes, although we found two proteins corresponding to L7 and L12 in PPDs and a native culture filtrate of BCG. We compared the delayed-type hypersensitivity reaction elicited by L7/L12 derived from a culture filtrate of BCG and L12 derived from BCG ribosomes. L7/L12 from the culture filtrate could induce delayed-type hypersensitivity, but L12 from ribosomes could not, indicating that L7 was attributable to the induction of delayed-type hypersensitivity. The activity of L7/L12 was heat resistant. Neither glycosylation nor phosphorylation of L7/L12 from a culture filtrate could be detected. The acetylation at N-terminal of L12 was essential for the delayed-type hypersensitivity activity.

Hanseníase experimental murina: inoculaçäo do Mycobaterium leprae via intranasal / Murine experimental hansen's disease: intranasal inoculation of the mycobacterium leprae

Considerando as vias aéreas superiores como a mais provável porta de entrada do M.leprae no organismo, no presente estudo avaliou-se o comportamento do bacilo inoculado experimentalmente via intranasal. Assim, camondongos suiços foram inoculados por instilaçäo nasal com M.leprae e sacrificados em diferentes períodos de tempo. Em cada cada sacrifício os pulmöes foram removidos e submetidos ao lavado broncoalveolar (LBA) e análise histopatológica. O LBA foi avaliado quanto ao número de bacilos recuperados e número total e diferencial de células inflamatórias; foi ainda semeado em meios de cultivo Lowenstein-Jensen e Middlebrook 7H9 e 7H11. No LBA foram encontrados bacilos até o 6§ mês de inoculaçäo, näo houve crescimento de micobactérias nas culturas. O número total de células, linfócitos e neutrófilos do LBA obtido de camundongos infectados foi maior que o do grupo controle até o 6§ mês da experimentaçäo. A análise histopatológica revelou pequenos infiltrados celulares distribuídos pelo parênquima pulmonar e ao redor dos vasos até o 6§ mês de sacrifício; näo foram observadas lesöes de disseminaçäo. Os resultados obtidos revelam que a inoculaçäo de M. leprae, via intranasal, em camundongos Suíços, näo levou ao desenvolvimento da doença nem a multiplicaçäo dos bacilos; assim estudos aidcionais envolvendo outras linhagens de camundongos poderäo ser úteis para determinar a importância da via intranasal na infecçäo hansênica

Mycolactone: a polyketide toxin from Mycobacterium ulcerans required for virulence.

Mycobacterium ulcerans is the causative agent of Buruli ulcer, a severe human skin disease that occurs primarily in Africa and Australia. Infection with M. ulcerans results in persistent severe necrosis without an acute inflammatory response. The presence of histopathological changes distant from the site of infection suggested that pathogenesis might be toxin mediated. A polyketide-derived macrolide designated mycolactone was isolated that causes cytopathicity and cell cycle arrest in cultured L929 murine fibroblasts. Intradermal inoculation of purified toxin into guinea pigs produced a lesion similar to that of Buruli ulcer in humans. This toxin may represent one of a family of virulence factors associated with pathology in mycobacterial diseases such as leprosy and tuberculosis.

Molecular and immunological analyses of the Mycobacterium avium homolog of the immunodominant Mycobacterium leprae 35-kilodalton protein.

The analysis of host immunity to mycobacteria and the development of discriminatory diagnostic reagents relies on the characterization of conserved and species-specific mycobacterial antigens. In this report, we have characterized the Mycobacterium avium homolog of the highly immunogenic M. leprae 35-kDa protein. The genes encoding these two proteins were well conserved, having 82% DNA identity and 90% identity at the amino acid level. Moreover both proteins, purified from the fast-growing host M. smegmatis, formed multimeric complexes of around 1000 kDa in size and were antigenically related as assessed through their recognition by antibodies and T cells from M. leprae-infected individuals. The 35-kDa protein exhibited significant sequence identity with proteins from Streptomyces griseus and the cyanobacterium Synechoccocus sp. strain PCC 7942 that are up-regulated under conditions of nutrient deprivation. The 67% amino acid identity between the M. avium 35-kDa protein and SrpI of Synechoccocus was spread across the sequences of both proteins, while the homologous regions of the 35-kDa protein and the P3 sporulation protein of S. griseus were interrupted in the P3 protein by a divergent central region. Assessment by PCR demonstrated that the gene encoding the M. avium 35-kDa protein was present in all 30 M. avium clinical isolates tested but absent from M. intracellulare, M. tuberculosis, or M. bovis BCG. Mice infected with M. avium, but not M. bovis BCG, developed specific immunoglobulin G antibodies to the 35-kDa protein, consistent with the observation that tuberculosis patients do not recognize the antigen. Strong delayed-type hypersensitivity was elicited by the protein in guinea pigs sensitized with M. avium.

Lipids from Mycobacterium leprae cell wall suppress T-cell activation in vivo and in vitro.

The influence of Mycobacterium leprae cell wall lipids on lymphocyte functions has been investigated in vivo (delayed-type hypersensitivity) and in vitro. The inflammatory response has been earlier evaluated by the mouse footpad oedema model and the delipidated mycobacteria evoked a mild but significant inflammatory response. Herein a higher level of hypersensitivity reaction was observed with delipidated bacilli than with the intact mycobacteria. The lipids obtained from the extract of M. leprae external cell wall were used to prepare liposomes, which have not been shown to be toxic to lymphocytes. The method of lipidic extraction and the sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the lipid fraction did not reveal any trace of proteins. Thin-layer chromatography of this extract detected four different bands with an apolar character, suggestive of mycolic and fatty acids. These same M. leprae liposomes potently suppressed lymph node cells, as well CD4+ and CD8+ T-cell lines, and an antigen-specific T-cell clone (T 4-9) proliferation, even under potent stimulus. Cholesterol-choline liposomes, unrelated to M. leprae liposomes, used as a control in the biological assays showed no significant effect on lymphoblastic activity, which points to the specificity of M. leprae lipids. These data demonstrated that M. leprae cell wall lipids induce immune suppression in mice without causing any membrane alteration in T cells as assessed throughout kinetic studies in vitro. This fact is closely related to the down-regulating effect induced by M. leprae lipids which we have previously observed in macrophage functions in vivo and in vitro. Although this lipidic fraction showed a suppressive action on T lymphocytes in vitro (proliferation) and in vivo (delayed-type hypersensitivity), its possible significance in the establishment of a specific immune response to M. leprae must be further investigated.

A major T-cell-inducing cytosolic 23 kDa protein antigen of the vaccine candidate Mycobacterium habana is superoxide dismutase.

This study describes the purification and immunochemical characterization of a major 23 kDa cytosolic protein antigen of the vaccine candidate Mycobacterium habana (TMC 5135). The 23 kDa protein alone was salted out from the cytosol at an ammonium sulfate saturation of 80-95%. It represented about 1.5% of the total cytosolic protein, appeared glycosylated by staining with periodic acid/Schiff's reagent, and showed a pl of approximately 5.3. Its native molecular mass was determined as approximately 48 kDa, suggesting a homodimeric configuration. Immunoblotting with the WHO-IMMLEP/IMMTUB mAbs mc5041 and IT61 and activity staining after native PAGE established its identity as a mycobacterial superoxide dismutase (SOD) of the Fe/Mn type. The sequence of the 18 N-terminal amino acids, which also contained the binding site for mc5041, showed a close resemblance, not only with the reported deduced sequences of Mycobacterium leprae and Mycobacterium tuberculosis Fe/MnSODs, but also with human MnSOD. In order to study its immunopathological relevance, the protein was subjected to in vivo and in vitro assays for T cell activation. It induced, in a dose-related manner, skin delayed hypersensitivity in guinea-pigs and lymphocyte proliferation in BALB/c mice primed with M. habana. Most significantly, it also induced lymphocyte proliferative responses, in a manner analogous to M. Ieprae, in human subjects comprising tuberculoid leprosy patients and healthy contacts.

Immunogenicity and protection studies with recombinant mycobacteria and vaccinia vectors coexpressing the 18-kilodalton protein of Mycobacterium leprae.

The activation of antigen-specific T lymphocytes is essential for the control of leprosy infection in humans and experimental animals. T cells recognize a variety of protein antigens from Mycobacterium leprae, including the 18-kDa protein, which is limited in distribution among mycobacteria and which is absent from Mycobacterium tuberculosis and the vaccine strain, Mycobacterium bovis BCG. Adjuvant preparations of mycobacterial protein antigens have had limited protective efficacy for experimental infections in animals. Since recombinant vectors may elicit more effective T-cell responses than adjuvant preparations, recombinant vaccinia virus (VV18) and M. bovis BCG (BCG18) vectors expressing the 18-kDa protein of M. leprae were prepared. Both VV18 and BCG18 stimulated anti-18-kDa protein antibody and lymphocyte proliferative responses. Sequential immunization with VV18 followed by BCG18 induced higher levels of specific immunoglobulin G2a antibodies than immunoglobulin G1 antibodies, in contrast to immunization with VV18 or BCG18 alone. The protective efficacy of immunization with VV18 from a challenge with BCG18 was examined in two murine models of mycobacterial infection. After intravenous challenge, mice immunized with recombinant vaccinia virus exhibited lower initial levels of replication and earlier clearance of BCG18 from their spleens than mice immunized with vaccinia virus expressing an unrelated protein. After footpad infection in a dissemination model, there was earlier clearance of BCG18 from specifically immunized mice. However, immunization of mice with VV18 did not prevent a productive mycobacterial infection.

Analysis of vaccines prepared from armadillo-derived M. leprae; results of an inter-laboratory study coordinated by the World Health Organization. Immunology of Mycobacterial Diseases (IMMYC) Steering Committee, World Health Organization.

Preparations of armadillo-derived Mycobacterium leprae used in vaccine trials were analyzed using a combination of morphological, chemical and immunological criteria. When compared to more recent preparations, vaccine lots prepared in 1984 and 1985 were found to contain fewer intact bacilli and lower amounts of M. leprae antigens. These differences may be characteristic of the original preparations, or alternatively, may have arisen during prolonged storage. The early vaccine lots were those used in the recently published Venezuela trial.