Altered Actinobacteria and Firmicutes Phylum Associated Epitopes in Patients With Parkinson's Disease.

Recent evidence suggests that inflammation was participated in the pathogenesis of PD, thus, to understand the potential mechanism of gut microbiota in the pathogenesis of Parkinson's disease (PD), we performed a metagenomic analysis of fecal samples from PD patient and controls. Using a two-stage metagenome-wide association strategy, fecal DNA samples from 69 PD patients and 244 controls in three groups (comprising 66 spouses, 97 age-matched, and 81 normal samples, respectively) were analyzed, and differences between candidate gut microbiota and microbiota-associated epitopes (MEs) were compared. In the study, 27 candidate bacterial biomarkers and twenty-eight candidate epitope peptides were significantly different between the PD patients and control groups. Further, enriched 4 and 13 MEs in PD were positively associated with abnormal inflammatory indicators [neutrophil percentage (NEUT.1), monocyte count/percentage (MONO/MONO.1), white blood cell count (WBC)] and five candidate bacterial biomarkers (c_Actinobacteria, f_Bifidobacteriaceae, g_Bifidobacterium, o_Bifidobacteriales, p_Actinobacteria) from Actinobacteria phylum, and they were also positively associated with histidine degradation and proline biosynthesis pathways, respectively. Additionally, enriched 2 MEs and 1 ME in PD were positively associated with above inflammatory indicators and two bacteria (f_Lactobacillaceae, g_Lactobacillus) from Firmicutes phylum, and they were also positively associated with pyruvate fermentation to propanoate I and negatively associated with isopropanol biosynthesis, respectively. Of these MEs, two MEs from GROEL2, RPSC were derived from Mycobacterium tuberculosis, triggered the T cell immune response, as previously reported. Additionally, other candidate epitope peptides derived from Mycobacterium tuberculosis and Mycobacterium leprae may also have potential immune effects in PD. In all, the altered MEs in PD may relate to abnormalities in immunity and glutamate and propionate metabolism, which furthers our understanding of the pathogenesis of PD.

Epitope imprinting of Mycobacterium leprae bacteria via molecularly imprinted nanoparticles using multiple monomers approach.

Mycobacterium leprae causes endemic disease leprosy which becomes chronic if not treated timely. To expedite this 'timely diagnosis', and that also at an early stage, here an attempt is made to fabricate an epitope-imprinted sensor. A molecularly imprinted polymer nanoparticles modified electrochemical quartz crystal microbalance sensor was developed for sensing of Mycobacterium leprae bacteria through its epitope sequence. Multiple monomers, 3-sulphopropyl methacrylate potassium salt, benzyl methacrylate and 4-aminothiophenol were utilized to imprint this bacterial epitope. Imprinted nanoparticles were electropolymerized on gold coated quartz electrode. The sensor was able to show specific binding towards the blood samples of infected patients, even in the presence of 'matrix' and other plasma proteins such as albumin and globulin. Even other peptide sequences, similar to epitope sequences only with two amino acid mismatches were also unable to show any binding. Sensor withstood analytical tests viz. selectivity, specificity, matrix effect, detection limit (0.161 nM), quantification limit (and 0.536 nM), reproducibility (RSD 2.01%). Hence a diagnostic tool for bacterium causing leprosy is successfully fabricated in a facile manner which will broaden the clinical access and efficient population screening can be made feasible.

Monitoring and detection of leprosy patients in Southwest China: A retrospective study, 2010-2014.

More than 100 counties, mainly in southwest China, report incidence rates of leprosy >1/100,000. The current study analysed the epidemiology of leprosy in southwest China to improve our understanding of the transmission pattern and improve control programs. 207 counties were selected in southwest China. Leprosy patients and their household contacts were recruited. The data from the medical interview and the serological antileprosy antibody of the leprosy patients were analysed. A total of 2,353 new cases of leprosy were interviewed. The distribution of leprosy patients was partly associated with local natural and economic conditions, especially several pocket areas. A total of 53 from 6643 household contacts developed leprosy, and the incidence rate of leprosy in the household contacts was 364/100,000 person-years. We found that NDO-BSA attained higher positive rates than MMP-II and LID-1 regardless of clinical types, disability and infection time in leprosy patients. By means of combination of antigens, 88.4% patients of multibacillary leprosy were detected, in contrast to 59.9% in paucibacillary leprosy. Household contacts should be given close attention for the early diagnosis, disruption of disease transmission and precise control. Applications of serology for multi-antigens were recommended for effective coverage and monitoring in leprosy control.

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.

Pathogen-specific epitopes as epidemiological tools for defining the magnitude of Mycobacterium leprae transmission in areas endemic for leprosy.

During recent years, comparative genomic analysis has allowed the identification of Mycobacterium leprae-specific genes with potential application for the diagnosis of leprosy. In a previous study, 58 synthetic peptides derived from these sequences were tested for their ability to induce production of IFN-γ in PBMC from endemic controls (EC) with unknown exposure to M. leprae, household contacts of leprosy patients and patients, indicating the potential of these synthetic peptides for the diagnosis of sub- or preclinical forms of leprosy. In the present study, the patterns of IFN-γ release of the individuals exposed or non-exposed to M. leprae were compared using an Artificial Neural Network algorithm, and the most promising M. leprae peptides for the identification of exposed people were selected. This subset of M. leprae-specific peptides allowed the differentiation of groups of individuals from sites hyperendemic for leprosy versus those from areas with lower level detection rates. A progressive reduction in the IFN-γ levels in response to the peptides was seen when contacts of multibacillary (MB) patients were compared to other less exposed groups, suggesting a down modulation of IFN-γ production with an increase in bacillary load or exposure to M. leprae. The data generated indicate that an IFN-γ assay based on these peptides applied individually or as a pool can be used as a new tool for predicting the magnitude of M. leprae transmission in a given population.

Rational design and evaluation of a multiepitope chimeric fusion protein with the potential for leprosy diagnosis.

Despite the reduction in the number of leprosy cases registered worldwide as a result of the widespread use of multidrug therapy, the number of new cases detected each year remains stable in many countries. This indicates that Mycobacterium leprae, the causative agent of leprosy, is still being transmitted and that, without an earlier diagnosis, transmission will continue and infection will remain a health problem. The current means of diagnosis of leprosy is based on the appearance of clinical symptoms, which in many cases occur after significant and irreversible nerve damage has occurred. Our recent work identified several recombinant antigens that are specifically recognized by leprosy patients. The goal of the present study was to produce and validate the reactivity of a chimeric fusion protein that possesses the antibody binding properties of several of these proteins. The availability of such a chimeric fusion protein will simplify future test development and reduce production costs. We first identified the antibody binding regions within our top five antigen candidates by performing enzyme-linked immunosorbent assays with overlapping peptides representing the amino acid sequences of each protein. Having identified these regions, we generated a fusion construct of these components (protein advances diagnostic of leprosy [PADL]) and demonstrated that the PADL protein retains the antibody reactivity of the component antigens. PADL was able to complement a protein that we previously produced (the leprosy IDRI [Infectious Disease Research Institute] diagnostic 1 [LID-1] protein) to permit the improved diagnosis of multibacillary leprosy and that had a good ability to discriminate patients with multibacillary leprosy from control individuals. A serological diagnostic test consisting of these antigens could be applied within leprosy control programs to reduce transmission and to limit the appearance of leprosy-associated disabilities and stigmatizing deformities by directing treatment.

Indirect immunofluorescence to demonstrate lichen planus specific antigen (LPSA) in lichen planus.

BACKGROUND: Current evidence suggests that lichen planus is an immunological disease. Cytotoxic CD8+ cells in the lesional epidermis recognize a unique antigen called lichen planus specific antigen. This antigen could be demonstrated by indirect immunofluorescence using the patient's serum and autologous lesional skin. AIM: To study indirect immunofluorescence pattern in lichen planus, among Indian patients. METHODS: Twenty-five consecutive patients with the clinical diagnosis of lichen planus were enrolled in the study. Direct immunofluorescence was done in all patients. Indirect immunofluorescence using lesional skin as substrate was done in all 25 patients and five patients with other dermatoses. RESULTS: A specific fluorescence pattern corresponding to the distribution of lichen planus specific antigen was observed in the stratum spinosum and granulosum in 22 (88%) patients. It was absent from other parts of the epidermis, dermis and in patients with other dermatoses. CONCLUSION: Indirect immunofluorescence is a useful adjuvant test in lichen planus, particularly in atypical cases.

Distinguishing features of anti-beta2 glycoprotein I antibodies between patients with leprosy and the antiphospholipid syndrome.

Anticardiolipin (ACA), anti-beta2 glycoprotein I (beta2GPI), and antiprothrombin antibodies of IgG and IgM classes were quantitated by enzyme-linked immunosorbent assays in 176 untreated leprosy patients across the histopathological spectrum. Positivity rates ranged from 21% (IgG ACA) to 30% (IgM anti-prothrombin) versus 4% in healthy controls (p <10(-2) to 10(-3)). Levels of IgM anti-beta2GPI and IgG ACA were significantly higher in lepromatous leprosy and multibacillary patient subgroups. IgG3 was the most common subclass reactive to both beta2GPI and prothrombin in selected high-titer leprosy sera, unlike antibodies from patients with the antiphospholipid syndrome (APS) largely restricted to IgG2. In leprosy patients, but not in the APS control group, there was no statistical correlation between ACA and anti-beta2GPI antibody levels. Likewise, a large fraction of anti-beta2GPI positive sera (36/45 and 28/44 for IgG and IgM, respectively) were unreactive in the standard ACA assay. Most assayed anti-beta2GPI antibodies from leprosy patients showed (i) ability to recognize both human and bovine beta2GPI immobilized on non-irradiated polystyrene plates, (ii) concentration-dependent inhibition of binding by cardiolipin, and (iii) relatively high avidity binding to fluid-phase beta2GPI, thereby differing from those found in APS. Finally, the location of the major epitopic region on the beta2GPI molecule targeted by autoantibodies was different in leprosy and APS, as assessed by direct binding to domain I- and V-deleted mutants and competition with the mouse monoclonal antibody 8C3, directed at domain I. Thus, leprosy-related antiphospholipid antibodies comprise persistent IgG and IgM anti-beta2GPI that differ from APS-related ones with respect to IgG subclass, avidity and epitope specificity, possibly reflecting distinct pathophysiological significance.

HLA-DRB1 leprogenic motifs in nigerian population groups.

Amino acid residues involved in the peptide binding groove of HLA-DRB1 alleles were examined in three Nigerian ethnic groups with leprosy (n = 287) and 170 controls to determine the role of DRB1 alleles in disease outcome with Mycobacterium leprae. Nine positively charged motifs and two others with neutral charge to the binding groove were detected. These motifs occurred more frequently in leprosy (leprogenic) than was expected by chance (P < 0.0001). In contrast, five motifs with net negative or 'modified' neutral charges to the pocket were negatively associated with leprosy. We conclude that clinical outcome of infection with M. leprae is largely determined by a shared epitope in DRB1 alleles marked by several motifs. These motifs occur in otherwise normal DRB1 alleles, characterized by net positive or neutral charges in the binding groove. We hypothesize that these polarities cause poor binding of DRB1 to M. leprae. On presentation, the signal via the T cell receptor results in muted cell-mediated immunity. The resulting response translates to various forms of leprosy depending on degree of charge consonance between M. leprae and host DRB1 allele. Other factors within or without the HLA complex, such as the T cell receptor repertoire, may also influence the resulting disease.

Dominant recognition of a cross-reactive B-cell epitope in Mycobacterium leprae 10 K antigen by immunoglobulin G1 antibodies across the disease spectrum in leprosy.

Mycobacterium leprae-specific immunoglobulin G1 (IgG1) antibodies in patients with leprosy show a direct correlation with bacterial load (rho=0.748; P<0002) suggesting that IgG1 B-cell responses may be surrogate markers of disease progression. To investigate if this upregulation was a general feature of IgG1 responses to all M. leprae (ML) antigens, we analysed responses to several recombinant purified ML heat-shock proteins (HSP). Three recombinant HSPs (ML10 K, ML 18 K and ML 65 K) were tested for their ability to induce various IgG subclasses in patients with either the lepromatous (LL/BL, n=26) or tuberculoid form (BT/TT, n=39) of the disease as well as in healthy households (HC, n=14) and endemic controls (EC=19). Our major findings were: (1) selective augmentation of IgG1 antibody responses to ML10 K; (2) recognition of a restricted number of epitopes across the disease spectrum and healthy controls by IgG1 antibodies; (3) dominant recognition of cross-reactive epitopes which were common to both ML and MT 10 K. This response was not related to contamination with endotoxin. Epitope mapping using 15-mer overlapping peptides spanning the ML 10 000 MW revealed an immunodominant IgG1 binding peptide (aa41-55) in patients as well as healthy controls. This peptide is a shared epitope with M. tuberculosis 10 K suggesting that postswitched IgG1 B cells recognizing this epitope rather than naive B cells are being expanded.

Determinant capture by MHC class II DR3 during processing of mycobacteria leprae 65kD heat shock protein by human B cells.

Using T cell immunoblotting we have characterised the immunogenic fragments derived from the Mycobacteria Leprae 65kD heat shock protein that become associated with MHC class II DR3 during processing by a human B cell line. After 5 h incubation with antigen, a peptide of approximately 12kD (approximately 110 amino acids) was the only major fragment found associated with the class II MHC. The association of this oligopeptide was abolished if an excess of a synthetic peptide representing the minimal epitope was included in the culture or when cells were incubated at 4 degrees C. This suggests that the generation of this moiety is dependent on cell metabolism and that its binding to MHC is specific. This large fragment may represent an intermediate in the processing pathway, directly demonstrating the role of MHC in determinant capture during antigen degradation.

A novel mycobacterial antigen relevant to cellular immunity belongs to a family of secreted lipoproteins.

The gene sequence of a novel 24.1 kDa Mycobacterium tuberculosis protein was identified within the Sanger Centre (UK) M. tuberculosis genome database (cosmid MTCY24G1) by searching with a 126 bp DNA sequence isolated from a genomic M. leprae lambda gt11 library with M. leprae reactive human T cell clones as probes. The 24.1 kDa antigen is common to the vaccine strain Mycobacterium bovis BCG, as well as Mycobacterium leprae. The 699 bp open reading frame encodes a 233 amino acid long precursor protein with a signal peptide sequence for secretion and a consensus motif for lipid conjugation, which suggests that the mature protein is an exported lipoprotein antigen. The molecular mass of the mature protein antigen from M. leprae sonicate was shown to correspond to the deduced size of the M. tuberculosis protein by T cell Western analysis. Homology searches revealed two other similarly sized hypothetical secreted mycobacterial lipoproteins within the M. tuberculosis genome database.

Cross-recognition by T cells of an epitope shared by two unrelated mycobacterial antigens.

The molecular mimicry represented by cross-recognition of determinants shared by unrelated antigens by antibodies or T cells is of broad immunological interest. In this study, we analyzed the cross-recognition by CD4+ T cells of a peptide epitope shared by two mycobacterial proteins of diverse sequence, represented by the 19-kDa antigen of Mycobacterium tuberculosis and the 28-kDa antigen of Mycobacterium leprae. This epitope was immunodominant with respect to the 19-kDa antigen, but cryptic in relation to the 28-kDa antigen. The cross-reactive epitope cores were identified by Pepscan window analysis and found to be eight residues long in both antigens (residues 69-76 and 127-134). Alignment of these octameric sequences revealed two identical and five conservatively related amino acids. Within the epitope core, two residues (73Asn and 76Ile) were identified as critical for recognition on the basis of inhibition of the cross-reactive T cell proliferative response using singly substituted analog peptides. These results suggest that T cell cross-reactive epitopes can exist in proteins with apparently not more than random levels of sequence homology. Their potential for unsuspected cross-sensitization may play a role in the maintenance of T cell memory, in the pathogenesis of autoimmune diseases and possibly in a wide range of host immune responses to infectious pathogens.

Detection of antibodies against phenolic glycolipid-1 (PGL-1), 35-kDa and 30-40-kDa components of Mycobacterium leprae in the cerebrospinal fluid of of leprosy patients.

The involvement of the central nervous system (CNS) in lepromatous leprosy (LL) patients was investigated; 33 patients were examined clinically in detail and upper motor neuron involvement was observed in eight and lower motor neuron in three of these patients. Anti-Mycobacterium leprae antibodies could be detected in the CSF by PGL-1 enzyme-linked immunosorbent assay (ELISA) and monoclonal antibody (MAb) based competitive assays against defined epitopes on the 35-kDa protein and 30-40-kDa polysaccharide (lipoarabinomannan) antigens with MAbs MLO4 and ML34, respectively. Antibodies against PGL-1 and 35-kDa protein were observed in more subjects than antibodies against the 30-40-kDa antigen. Some correlation was observed between the upper motor neuron signs and antibody positivity for 35-kDa and PGL-1 antigens in the CSF of these patients.

Antibody reactivity to mycobacterial 65 kDa heat shock protein: relevance to autoimmunity.

Reactivity to the mycobacterial 65 kDa heat shock protein (HSP 65) has been implicated in the pathogenesis of adjuvant arthritis in the rat, and may be involved in the pathogenesis of rheumatoid arthritis or other autoimmune diseases in humans. Accordingly this study sought quantitative or qualitative differences in the antibody reactivity to HSP 65 between normal controls, patients with the multisystem autoimmune diseases, rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) and patients with the mycobacterial infections, tuberculosis (TB) and leprosy. Levels of antibodies to recombinant HSP 65 in serum were measured by ELISA in normal subjects and in patients with RA, SLE, TB or leprosy. Antibody reactivity was examined by Western blotting using polypeptide fragments of HSP 65 derived by recombinant DNA techniques, or by digestion with trypsin or cyanogen bromide (CNBr). Reactivity to a synthetic peptide, the adjuvant arthritis T-cell epitope of HSP 65 (180-188), was tested by ELISA. High levels of antibodies to full length recombinant HSP 65 from Mycobacterium bovis were present in all the groups tested. By Western blot analysis, most reactivity with intact HSP 65 was retained in a 32 kDa tryptic fragment, judged by sequencing and size estimations to represent amino acid residues 118- approximately 388. This sequence included a major T-cell epitope for adjuvant arthritis (180-188), but these nine amino acids were not essential for B-cell reactivity since most sera also reacted with residues 188-540 which lack the T-cell epitope. Moreover, the 180-188 synthetic peptide was unreactive by ELISA, and did not inhibit reactivity with the intact recombinant HSP 65. In conclusion, most individuals had antibodies to mycobacterial HSP 65, presumably resulting from previous bacterial infections. The magnitude of the response was unrelated to the occurrence of systemic autoimmune disease, and the pattern of antibody reactivity with recombinant and proteolytic fragments of HSP 65 suggests that the major B-cell epitope is conformational and consists of discontinuous regions of the molecule.

T and B cell responses to mycobacterial 65-kDa heat-shock protein in sheep infected with maedi visna virus.

Sheep infected with maedi visna virus were tested for immune reactivity to recombinant HSP65 and tuberculin PPD from mycobacteria. The results showed that both naturally and experimentally infected animals had elevated IgM but not IgG or IgA antibodies to HSP65 from Mycobacterium leprae or M. bovis. In experimentally infected animals, the elevated IgM antibodies appeared in blood from about 3 to 4 weeks postinfection. Increased T cell proliferative responses to HSP65 and PPD were also found in both naturally and experimentally infected sheep. The T cell responses to HSP65 were substantially inhibited by antibodies to ovine major histocompatibility complex class II molecules, indicating that the responses were class II restricted. Increased expression of a putative HSP65 molecule was observed in synovial membranes from sheep infected with maedi visna virus and goats infected with the related, caprine arthritis encephalitis virus. The results thus show that lentivirus infection induces T and B cell anti-HSP65 immune responses and suggest that synovial inflammation may be due, at least in part, to T and B cell recognition of HSP65-like molecules expressed in joints.

Recognition of Mycobacterium leprae recombinant 18,000 MW epitopes by IgG subclasses in leprosy.

IgG subclasses are known to be differentially regulated by cytokines (elaborated by activated T cells), which act as growth factors and immunoglobulin switch factors on B cells. In leprosy, we have previously shown that IgG subclass antibodies to a purified recombinant antigen of Mycobacterium leprae (18,000 MW) are restricted to IgG1 and IgG3 across the disease spectrum. The only significant difference observed was that lepromatous patients with low to undetectable T-cell responses showed a strong correlation between IgG1 and IgG3 (P < 0.001) antibodies while tuberculoid patients who showed strong T-cell responses did not show such a correlation. To examine if these differences were related to T-cell-mediated class switching in tuberculoid leprosy patients, we have studied epitope recognition by IgG1 and IgG3 using a panel of synthetic peptides spanning the 18,000 MW molecule in an enzyme-linked immunosorbent assay (ELISA). In lepromatous patients there was little similarity in peptide recognition by IgG1 and IgG3, with IgG1 recognition being restricted to a single dominant carboxy-terminal peptide while the IgG3 antibodies recognized a diverse set of peptides in the N-terminal half of the 18,000 MW molecule. In tuberculoid patients both IgG1 and IgG3 antibody showed recognition of similar peptides in the N-terminal half of the 18,000 MW molecule. Our results therefore support the hypothesis that immunoglobulin class switching is occurring in tuberculoid but not in lepromatous patients.