STUDY ON NUDE MICE INOCULATED WITH MYCOBACTERIUM LEPRAE BY MULTIPLE ROUTES

Immune-deficient nude mice were inoculated with nude mouse derived Mycobacteriurn leprae by multiple routes (intravenously. subcutaneously at the foot pads and ears). The results showed that these inoculated animals were capable of producing a great number of Mycobacterium leprae to a level 10(11-12) per gram of tissue. and were detected histopathologically to have heavy leproniatous lesions. The dissemination of the infection was found particularly in sites with lower body temperature.The organisms have a partiality to striated muscles and peripheral nerves. The authors suggest that experimental leprosy in nude mice is a very useful tool for leprosy resarch. especially in countries without armadillos. Compared with the single-route inoculation reported previously. multiple-route inoculation is more available.

Rapid Identification of Mycobacterium Leprae by Polymerase Chain Reaction-restriction Fragment Length Polymorphism Analysis of the Heat Shock Protein 65 Gene from Skin Specimens

INTRODUCTIONLeprosy caused by Mvcobacterium leprae （M. leprae）, is a chronic granulomatous disease affecting the skin and peripheral nervous system, which is transmitted through direct contact with nontreated or inadequate treatment patients. Diagnosis of leprosy depends on the clinical signs and symptoms and slit skin smear positivity. However, it＇s sometimes similar with other granulomatous disease caused by mycobacterial infection. Early stage leprosy is difficult to diagnose by clinical criterion alone because the sensitivity of acid-fast bacilli staining is quite low. The polymerase chain reaction-restriction fragment length polymorphism （PCR-RFLP） shows the great advantage in rapid identification and diagnosis for early cases and has a differentiation between leprosy and nonleprosy cases.

Three-dimensional models of antigens with serodiagnostic potential for leprosy:An in silico study

BACKGROUND Leprosy is a disease caused by Mycobacterium leprae(M.leprae),an intracellular pathogen that has tropism and affects skin and nervous system cells.The disease has two forms of presentation:Paucibacillary and multibacillary,with different clinical and immunological manifestations.Unlike what occurs in the multibacillary form,the diagnostic tests for the paucibacillary form are nonspecific and not very sensitive,allowing the existence of infected individuals without treatment,which contributes to the spread of the pathogen in the population.To mitigate this contamination,more sensitive diagnostic tests capable of detecting paucibacillary patients are needed.AIM To predict the three-dimensional structure models of M.leprae antigens with serodiagnostic potential for leprosy.METHODS In this in silico study,satisfactory templates were selected in the Protein Data Bank(PDB)using Basic Local Alignment Search Tool to predict the structural templates of ML2038,ML0286,ML0050,and 85B antigens by comparative modeling.The templates were selected according to general criteria such as sequence identity,coverage,X-ray resolution,Global Model Quality Estimate value and phylogenetic relationship;Clustal X 2.1 software was used in this analysis.Molecular modeling was completed using the software Modeller 9v13.Visualization of the models was made using ViewerLite 4.2 and PyMol software,and analysis of the quality of the predicted models was performed using the QMEAN score and Z-score.Finally,the three-dimensional moels were validated using the MolProbity and Verify 3D platforms.RESULTS The three-dimensional structure models of ML2038,ML0286,ML0050,and 85B antigens of M.leprae were predicted using the templates PDB:3UOI(90.51%identity),PDB:3EKL(87.46%identity),PDB:3FAV(40.00%identity),and PDB:1F0N(85.21%identity),respectively.The QMEAN and Z-score values indicated the good quality of the structure models.These data refer to the monomeric units of antigens,since some of these antigens have quaternary structure.The validation of the models was performed with the final three-dimensional structure-monomer(ML0050 and 85B antigens)and quaternary structures(ML2038 and ML0286).The majority of amino acid residues were observed in favorable and allowed regions in the Ramachandran plot,indicating correct positioning of the side chain and absence of steric impediment.The MolProbity score value and Verify 3D results of all models indicated a satisfactory prediction.CONCLUSION The polarized immune response against M.leprae creates a problem in leprosy detection.The selection of immunodominant epitopes is essential for the development of more sensitive serodiagnostic tests,for this it is important to know the three-dimensional structure of the antigens,which can be predicted with bioinformatics tools.

Advances in the pathogenic, genetic and immunological studies of leprosy

Leprosy is an infectious granulomatous disease caused by Mycobacterium leprae that affects the skin and can lead to deformity by damaging peripheral nerves.Although leprosy is no longer an incurable disease,its epidemic has not been well controlled because of its unclear routes of transmission and the lack of an effective vaccine.Moreover,leprosy has long been an ideal disease model for the study of genetics and immunology of infectious diseases due to its strong genetic predisposition and immune-dependent spectrum of clinical manifestations.Here,we review the latest and important findings of the pathogenesis of leprosy.Recent studies have shown that the highly conserved M.leprae is zoonotic,which further complicates the ambiguous transmission of M.leprae.Genetically,genome-wide association studies of leprosy have reported dozens of susceptibility genes,most of which are immune-related,and thus systematically elucidate the immunogenetic basis of the disease.Immunologically,plenty of novel mechanisms of host defense against intracellular bacterial infection and the modulation of host immunity by M.leprae have been depicted.Despite these great achievements,there are still gaps between pathogenic biology,genetics,and immunology of leprosy,limiting our in-depth understanding of leprosy pathogenesis.Further efforts,such as multi-omics data integration and the development of viable animal models for immunogenetic studies are urgently needed to accelerate advances in the precise prevention and treatment of leprosy.

Functional biomarker signatures of circulating T-cells and its association with distinct clinical status of leprosy patients and their respective household contacts

Background: Leprosy is a chronic infectious disease classified into two subgroups for therapeutic purposes:paucibacillary(PB)and multibacillary(MB),closely related to the host immune responses.In this context it is noteworthy looking for immunological biomarkers applicable as complementary diagnostic tools as well as a laboratorial strategy to follow-up leprosy household contacts.Methods:: The cross-sectional study enrolled 49 participants,including 19 patients and 30 healthy controls.Peripheral blood mononuclear cells(PBMC)were isolated and incubated in the presence of Mycobacterium leprae bacilli.The cells were prepared for surface(CD4+and CD8+)and intracytoplasmic cytokine staining(IFN-γ,IL-4 and IL-10).Multiple comparisons amongst groups were carried out by ANOVA,Kruskal–Wallis,Student T or Mann–Whitney test.Comparative analysis of categorical variables was performed by Chi-square.Functional biomarker signature analysis was conducted using the global median values for each biomarker index as the cut-off edge to identify the proportion of subjects with high biomarker levels.Results: The cytokine signature analysis demonstrated that leprosy patients presented a polyfunctional profile of T-cells subsets,with increased frequency of IFN-γ+T-cell subsets along with IL-10+and IL-4+from CD4+T-cells,as compared to health Controls(Venn diagram report).Moreover,statistical analysis was carried out using parametric or non-parametric variance analysis followed by pairwise multiple comparisons,according to the data normality distribution.L(PB)displayed a polyfunctional profile characterized by enhanced percentage of IFN-γ+,IL-10+and IL-4+produced by most T-cell subsets,as compared to L(MB)that presented a more restricted cytokine functional profile mediated by IL-10+and IL-4+T-cells with minor contribution of IFN-γproduced by CD4+T-cells.Noteworthy was that HHC(MB)exhibited enhanced frequency of IFN-γ+T-cells,contrasting with HHC(PB)that presented a cytokine profile limited to IL-10 and IL-4.Conclusions: Our data demonstrated that L(PB)displayed enhanced percentage of IFN-γ+,IL-10+and IL-4+as compared to L(MB)that presented functional profile mediated by IL-10+and IL-4+T-cells and HHC(MB)exhibited enhanced frequency of IFN-γ+T-cells,contrasting with HHC(PB).Together,our findings provide additional immunological features associated with leprosy and household contacts.These data provide evidence that biomarkers of immune response can be useful complementary diagnostic/prognostic tools as well as insights that household contacts should be monitored to access putative subclinical infection.

A comprehensive research agenda for zero leprosy

Background: Leprosy control achieved dramatic success in the 1980s–1990s with the implementation of short course multidrug therapy,which reduced the global prevalence of leprosy to less than 1 in 10000 population.However,a period of relative stagnation in leprosy control followed this achievement,and only limited further declines in the global number of new cases reported have been achieved over the past decade.Main text In 2016,major stakeholders called for the development of an innovative and comprehensive leprosy strategy aimed at reducing the incidence of leprosy,lowering the burden of disability and discrimination,and interrupting transmission.This led to the establishment of the Global Partnership for Zero Leprosy(GPZL)in 2018,with partners aligned around a shared Action Framework committed to achieving the WHO targets by 2030 through national leprosy program capacity-building,resource mobilisation and an enabling research agenda.GPZL convened over 140 experts from more than 20 countries to develop a research agenda to achieve zero leprosy.The result is a detailed research agenda focusing on diagnostics,mapping,digital technology and innovation,disability,epidemiological modelling and investment case,implementation research,stigma,post exposure prophylaxis and transmission,and vaccines.This research agenda is aligned with the research priorities identified by other stakeholders.Conclusions: Developing and achieving consensus on the research agenda for zero leprosy is a significant step forward for the leprosy community.In a next step,research programmes must be developed,with individual components of the research agenda requiring distinct expertise,varying in resource needs,and operating over different timescales.Moving toward zero leprosy now requires partner alignment and new investments at all stages of the research process,from discovery to implementation.