Skin Lesions Of Leprosy Patients Research Articles

IL-22 in Mycobacterium leprae-induced Nerve injury.

The neurotropism of M. leprae is a unique characteristic that distinguishes it from other mycobacteria and is also the main cause of deformity in patients. However, the mechanism of peripheral nerve injury is still unclear. In the present study, immunohistochemistry and qPCR showed that the skin lesions of leprosy patients were significantly higher than those of other mycobacterium infected skin lesions, and IL-22 was concentrated around the cutaneous nerves. Further study found that IL-22 is involved in disease by regulating neuroinflammation and promoting apoptosis through the JAK-STAT pathway.

Gene Expression of Human Beta-Defensin-3 and Cathelicidin in the Skin of Leprosy Patients, Household Contacts, and Healthy Individuals from Indonesia.

Leprosy, a chronic infectious peripheral neuropathy, is caused by Mycobacterium leprae. This bacterium produces triacylated lipopeptides that can induce the immune system via the Toll-like receptor 2/1 (TLR 2/1) complex. Activation of TLR 2/1 produces proinflammatory cytokines and antimicrobial peptides (AMPs), including human beta-defensin-3 (HBD-3) and cathelicidin. To analyze differences in gene expression of HBD-3 and cathelicidin in the skin of leprosy patients, household contacts, and healthy individuals. An analytic observational study was conducted at the Outpatient Clinic of Dermatology and Venereology of Dr Mohammad Hoesin General Hospital, Palembang, Indonesia, from January 2021 to June 2022. In each group of 18 subjects, 72 samples were collected, including skin lesion in leprosy patients, normal skin in leprosy patients, household contacts, and healthy individuals. A comparison of HBD-3 and cathelicidin gene expression between the four groups was analyzed using Pearson Chi Square, Kruskal-Wallis, and Mann-Whitney Test. The median value of HBD-3 gene expression on skin lesion in leprosy patients was 260.61 (0.19-3734.10); normal skin in leprosy patients was 1.91 (0.01-151.17); household contacts skin was 7.93 (0.27-121.10); and healthy individuals' skin was 1.00 (1.00-1.00) is highly significant difference (p < 0.0001). The median value of cathelicidin gene expression on skin lesion in leprosy patients was 38.72 (0.28-1852.17); normal skin in leprosy patients was 0.48 (0.01-15.83); household contacts skin was 9.8 (0.04-128.0); and healthy individual skin was 1.00 (1.00-1.00), also highly significant difference (p < 0.0001). Gene expression of HBD-3 and cathelicidin increased in skin lesions of leprosy patients and household contacts.

The protective role of tissue-resident interleukin 17A-producing gamma delta T cells in Mycobacterium leprae infection.

Mycobacterium leprae is a kind of disease-causing bacteria and results in leprosy in human. Gamma delta (γδ) T cell is a T-cell subset that is presented in both human dermis and epidermis. These cells bridge innate and adaptive immune responses and play critical roles in regulating anti-microbial defense, wound healing, and skin inflammation. Here, we investigated skin resident γδ T cells in patients with leprosy. Our data showed that γδ T cells significantly accumulated in skin lesions of leprosy patients with tuberculoid (TT) form. IL-23 can predominantly stimulate dermal γδ T cells to produce interleukin 17 (IL-17), a cytokine which may lead to disease protection. These γδ T cells expressed a specific set of surface molecules, and majority of these cells were Vδ1+. Also, IL-23 can stimulate the expansion of dermal γδ T cells expansion. Moreover, our results revealed that the transcription factor RORγt was responsible for IL-17A expression in leprosy lesion. Therefore, these data indicated that IL-23-responsive dermal γδ T cells were the major resource of IL-17A production in the skin and could be a potential target in the treatment of leprosy.

A new paradigm for leprosy diagnosis based on host gene expression.

Transcriptional profiling is a powerful tool to investigate and detect human diseases. In this study, we used bulk RNA-sequencing (RNA-Seq) to compare the transcriptomes in skin lesions of leprosy patients or controls affected by other dermal conditions such as granuloma annulare, a confounder for paucibacillary leprosy. We identified five genes capable of accurately distinguishing multibacillary and paucibacillary leprosy from other skin conditions. Indoleamine 2,3-dioxygenase 1 (IDO1) expression alone was highly discriminatory, followed by TLR10, BLK, CD38, and SLAMF7, whereas the HS3ST2 and CD40LG mRNA separated multi- and paucibacillary leprosy. Finally, from the main differentially expressed genes (DEG) and enriched pathways, we conclude that paucibacillary disease is characterized by epithelioid transformation and granuloma formation, with an exacerbated cellular immune response, while multibacillary leprosy features epithelial-mesenchymal transition with phagocytic and lipid biogenesis patterns in the skin. These findings will help catalyze the development of better diagnostic tools and potential host-based therapeutic interventions. Finally, our data may help elucidate host-pathogen interplay driving disease clinical manifestations.

Expression of matrix metalloproteinase-9 in skin lesions of leprosy patients: the difference between paucibacillary and multibacillary cases

Expression of matrix metalloproteinase-9 in skin lesions of leprosy patients: the difference between paucibacillary and multibacillary cases

Evaluation of nerve growth factor serum level for early detection of leprosy disability.

Introductionthis research aimed to analyze nerve growth factor (NGF) contents as diagnostic tools for early disability in leprosy patients and the cut-off point value.Methodsresearch samples consisted of 79 leprosy patients with disability grade 0 or 1 who met the clinically approved inclusion criteria. The age of patients ranged from 14 to 50 years. For both sample groups, blood serum was collected to determine NGF concentration. NGF level was analyzed by enzyme-linked immunosorbent assay (ELISA) according to the manual guide of the kit insert from Cussabio®. Statistical analysis used SPSS 17 software for Windows. A comparison was performed with the Student's t-test and the NGF concentration cut-off point was determined using a receiver operating characteristic (ROC) curve.Resultsthe research result demonstrated that NGF concentration in multibacillary leprosy with disability grade 0 was higher than in grade 1. Leprosy with disability grade 0 had an NGF content reaching 100.46 pg/mL, while those with grade 1 had a lower concentration of NGF at 30.56 pg/mL. The higher disability grade indicated a lower NGF concentration in the blood serum. Based on the ROC analysis result, the NGF cut-off was shown to be 81.43 pg/mL. This result indicated that low NGF in nerve and skin lesions of leprosy patients contributes to early peripheral nerve malfunction due to Mycobacterium leprae infection.Conclusionthese results prove that NGF can be used as a marker of early disability in leprosy, with the cut-off value at 81.43 pg/mL.

The inflammasome in leprosy skin lesions: an immunohistochemical evaluation.

ObjectiveLeprosy is a chronic infectious disease presenting with a spectrum of clinical manifestations that correspond to the type of immune response that develops in the host. Factors that may be involved in this process include inflammasomes, cytosolic proteins responsible for the activation of caspase 1, IL-1β and IL-18 secretion, and induction of a type of death called pyroptosis.Patients and methodsWe evaluated the expression of inflammasome markers (nucleotide-binding oligomerization domain-like receptor containing pyrin domain 1 [NLRP1], nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 [NLRP3], caspase 1, IL-1β, and IL-18) by immunohistochemistry in 43 samples of skin lesions of leprosy patients from the groups indeterminate (I) leprosy (13 patients), tuberculoid (TT) leprosy (15 patients), and lepromatous leprosy (LL; 15 patients).ResultsThe evaluated markers were most upregulated in LL lesions, followed by lesions of TT leprosy and I leprosy. Differences were statistically significant between the I leprosy and LL leprosy forms and between the I leprosy and TT leprosy forms. Positive and significant correlations were found between IL-18 and caspase 1 in LL (r=0.7516, P=0.0012) and TT leprosy (r=0.7366, P=0.0017). In I leprosy, correlations were detected between caspase 1 and IL-1β (r=0.6412, P=0.0182), NLRP1 and IL-18 (r=0.5585, P=0.473), NLRP3 and IL-18 (r=0.6873, P=0.0094), and NLRP1 and NLRP3 (r=0.8040, P=0.0009).ConclusionThe expression of inflammasome markers in LL lesions indicates the ineffectiveness of this protein complex in controlling the infection. Caspase 1 may be involved in the pyroptotic cell death in the lepromatous form of the disease. Inflammasomes may act together in the initial phase of I leprosy; this phenomenon may influence the clinical outcome of the disease.

In Situ complement activation and T-cell immunity in leprosy spectrum: An immunohistological study on leprosy lesional skin.

Mycobacterium leprae (M. leprae) infection causes nerve damage and the condition worsens often during and long after treatment. Clearance of bacterial antigens including lipoarabinomannan (LAM) during and after treatment in leprosy patients is slow. We previously demonstrated that M. leprae LAM damages peripheral nerves by in situ generation of the membrane attack complex (MAC). Investigating the role of complement activation in skin lesions of leprosy patients might provide insight into the dynamics of in situ immune reactivity and the destructive pathology of M. leprae. In this study, we analyzed in skin lesions of leprosy patients, whether M. leprae antigen LAM deposition correlates with the deposition of complement activation products MAC and C3d on nerves and cells in the surrounding tissue. Skin biopsies of paucibacillary (n = 7), multibacillary leprosy patients (n = 7), and patients with erythema nodosum leprosum (ENL) (n = 6) or reversal reaction (RR) (n = 4) and controls (n = 5) were analyzed. The percentage of C3d, MAC and LAM deposition was significantly higher in the skin biopsies of multibacillary compared to paucibacillary patients (p = <0.05, p = <0.001 and p = <0.001 respectively), with a significant association between LAM and C3d or MAC in the skin biopsies of leprosy patients (r = 0.9578, p< 0.0001 and r = 0.8585, p<0.0001 respectively). In skin lesions of multibacillary patients, MAC deposition was found on axons and co-localizing with LAM. In skin lesions of paucibacillary patients, we found C3d positive T-cells in and surrounding granulomas, but hardly any MAC deposition. In addition, MAC immunoreactivity was increased in both ENL and RR skin lesions compared to non-reactional leprosy patients (p = <0.01 and p = <0.01 respectively). The present findings demonstrate that complement is deposited in skin lesions of leprosy patients, suggesting that inflammation driven by complement activation might contribute to nerve damage in the lesions of these patients. This should be regarded as an important factor in M. leprae nerve damage pathology.

Apoptotic activity and Treg cells in tissue lesions of patients with leprosy

In order to understand the apoptotic response and the participation of Treg cells in the spectral clinical evolution of leprosy, this study evaluated the immunohistochemical expression of caspase-3 and FoxP3 in skin lesions of leprosy patients with the polar forms of the disease. Forty-nine patients with a confirmed diagnosis of the disease were selected, including 27 with the TT form and 22 with the LL form. Quantitative analysis of caspase-3 immunostaining showed a higher expression of this mediator in the LL form (3.409 ± 0.6517 cells/mm2; p = 0.0001). Immunostaining for the transcription factor FoxP3 was higher in the LL form (3.891 ± 0.9294 cells/mm2; p = 0.0001). A moderate correlation between the two markers was observed in the TT form (r = 0.5214; p = 0.005). It can be concluded that Treg cells and apoptosis play an effective role for the host defense response, inducing mechanisms involved in the activation of cascades that interfere with the control of the immune response and cell homeostasis.

Clinical and immunological evaluation after BCG-id vaccine in leprosy patients in a 5-year follow-up study.

IntroductionThe use of bacillus Calmette–Guérin (BCG) has long been considered a stimulus for immune reactivity in leprosy household contacts. Probably, the combination of multidrug therapy with BCG could facilitate the clearance of leprosy bacilli in the host, reduce relapse rates, and shorten the duration of skin-smear positivity.MethodsTo investigate the mechanism of action of BCG, a study involving 19 leprosy patients, eleven multibacillary (MB) and eight paucibacillary, was performed to assess the in vitro production of interleukin (IL)-10, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, IL-6, and IL-17 in the supernatant of peripheral blood mononuclear cells, before and 30 days after inoculation with BCG intradermally (BCG-id). Peripheral blood mononuclear cells isolated by Ficoll–Hypaque gradient were cultivated with Concanavalin-A (Con-A), lipopolysccharides (LPS), or BCG. The supernatant was collected for ELISA quantification of cytokines. The immunohistochemistry of IFN-γ, IL-1, IL-10, IL-12, transforming growth factor (TGF)-β, and TNF-α was carried out in biopsies of skin lesions of leprosy patients before and 30 days after inoculation of BCG-id. These patients were followed up for 5 years to assess the therapeutic response to multidrug therapy, the occurrence of leprosy reactions, and the results of bacterial index and anti-PGL-1 serology after the end of treatment.ResultsThe results showed increased production of cytokines after BCG-id administration in MB and paucibacillary leprosy patients. There was statistically higher levels of TNF-α (P = 0.017) in MB patients and of IL-17 (P = 0.008) and IFN-γ (P = 0.037) in paucibacillary patients. Immunohistochemical staining, especially for TNF-α, was more intense in biopsies of MB leprosy patients taken after BCG-id administration, probably for induction of innate human immunity. The clinical evaluation suggests that BCG-id is able to induce a more effective therapeutic response, with reduction of the number and the intensity of leprosy reactions.ConclusionThese results suggest that BCG-id induces activation of the initial phase of immunocellular activity: innate human immunity (increase in TNF-α, IL-12 and macrophage activation). Therefore, we conclude that the use of BCG-id could be indicated as an adjuvant to multidrug therapy in treatment of leprosy patients.

Detection of Mycobacterium leprae DNA in skin lesions of leprosy patients by PCR may be affected by amplicon size

Despite the high sensitivity and specificity of PCR for infectious disease diagnostics, it has presented low sensitivity for Mycobacterium leprae DNA detection in the tuberculoid pole (TT and BT) of leprosy. In order to demonstrate the effect of amplicon size on the efficacy of PCR detection of M. leprae DNA in skin lesions of leprosy patients, two pairs of primers targeting the M. leprae genomic DNA, RLEP3 (X17153), were used to amplify fragments of 372 and 130-bp until their PCR end-points were reached after 40 reaction cycles. Skin biopsies of leprosy lesions in 110 non-treated patients were used for bacilloscopy index (BI) analysis and PCR tests. The 130-bp fragment was detected in 73.6% of samples (81/110), and classified as TT (40%), BT (55.5%), and 100% of BB, BL and LL. The 372-bp fragment was detected in 52.7% and classified as TT (13.3%), BT (33.3%), BB (64.7%), BL (83.3%), and LL (95.2%). The BI of biopsies was positive in 39.1% of samples, classified as TT (0%), BT (2.2%), BB (64.7%), BL (91.6%), and LL (95.2%). The shorter amplicon (130-bp) has improved diagnosis by 20.9 and 34.5% in relation to the 372-bp fragment and the BI, respectively, and has shown a superior sensitivity (73.6%), specificity (100%) and accuracy (86.2%). The 130-bp amplicon could not detect % of positive BI of biopsies in BT cases. Therefore, for confirmatory diagnosis, we propose the use of PCR detection of the 130-bp genomic target, especially when the tuberculoid pole forms are considered, which has reached 51.6% of positivity in this group.

Cytokine expression and regulation in skin lesions of leprosy patients in type 1 reactions

Cytokine expression and regulation in skin lesions of leprosy patients in type 1 reactions

Study of gene probes in childhood leprosy.

The objective was to detect nucleic acids of M. leprae in skin lesions of leprosy patients and study the effect of treatment on these nucleic acids, using r-RNA gene probes, using a cross sectional study. The study was carried out at Department of Paediatrics, S.N. Medical College, Agra and Department of Microbiology, Central JALMA Institute for leprosy, Agra. The study included 32 cases of leprosy less than 16 years of age, divided into 3 groups viz. without treatment (12 cases), in middle of treatment (11 cases) and at the end of treatment (9 cases). All cases were subjected to a detailed history and thorough clinical examination. All of them had smear examination and lepromin test done and their skin biopsies were subjected to gene detection. Nucleic acids were isolated from skin biopsies of all cases by standard procedure. After dot blotting of these nucleic acids, they were hybridised with radioactive (p32) r-RNA probes. The results were interpreted after getting the X-ray films processed with background signals from controls. Majority of cases were between 13-16 years of age. As age advanced, the disease moved from tuberculoid end of spectrum towards lepromatous end (p < 0.05). Majority of paucibacillary (PB) cases were lepromin positive while majority of multibacillary (MB) cases were lepromin negative (p < 0.05). In specimens of untreated cases, 50% of PB specimens and 87.5% of MB specimens were positive for r-RNA probes. In multibacillary type 100% smear positive specimens and 67% smear negative specimens were positive for r-RNA probes. In patients during the middle of treatment positivity for r-RNA decreased and 20% of PB specimens and 16.6% MB specimens were positive. At the end of treatment (1 year for PB cases and 2 years for MB cases), the results of r-RNA were negative, which indicated that the treatment regimens used in the study were effective. This study supports the usefulness of r-RNA probes as a diagnostic and therapeutic tool in childhood leprosy.

Mycobacterium leprae DNA Content, Cellular and Cytokine Patterns in Skin Lesions of Leprosy Patients undergoing Multidrug Therapy (MDT)

Skin biopsies from untreated and MDT-treated patients were examined for infiltrating cells and cells producing the cytokines TNF-α, IFN-γ, and IL-1β using immunohistochemistry. Biopsy specimens from untreated tuberculoid leprosy patients were characterized by the presence of cells producing TNF-α, IFN-γ, and IL-1β and of subepidermal Langerhans cells. These cells were rarely found or completely absent in biopsies of untreated lepromatous leprosy patients, but tended to increase under MDT. In a short-term therapy trial for three months with brodimoprim, dapsone, and rifampicin, 12 patients were monitored by follow-up biopsies. Semiquantitative PCR for mycobacterial DNA revealed two groups of patients: one group in which mycobacterial DNA in follow-up biopsies remained constant and a second group in which a decrease of mycobacterial DNA during therapy was noted. Immunophenotyping in these follow-up biopsies revealed that in the latter group IFN-γ-positive cells and Langerhans cells were present and γδ T cell receptor-positive cells tended to decrease during therapy. In contrast, in patients whose mycobacterial DNA did not change during therapy, these phenotypical manifestations were not observed. We therefore, conclude that assessment of mycobacterial DNA in combination with phenotyping of infiltrating cells and determination of cytokine patterns may be useful tools in establishing criteria for the effectiveness and duration of MDT in patients with leprosy.

Selective expansion of human gamma delta T cells by monocytes infected with live Mycobacterium tuberculosis.

Gamma delta (gamma delta) T cell receptor (TCR) expressing T cells comprise 3% of human peripheral blood lymphocytes, yet their role in the immune response remains largely unknown. There is evidence both in humans and in animal models that these cells participate in the immune response to mycobacterial antigens. In mice, exposure to mycobacterial antigens leads to the expansion of gamma delta T cells in draining lymph nodes and lungs. In humans, gamma delta T cell lines with reactivity to mycobacterial antigens have been derived from synovial fluid of a rheumatoid arthritis patient, skin lesions of leprosy patients, and peripheral blood of a healthy tuberculin reactor. Very little is known, however, about the factors which induce human gamma delta T cells to expand. In studies comparing the human T cell response to live and heat-killed Mycobacterium tuberculosis (MT), we have found that monocytes infected with live MT are very effective inducers of human gamma delta T cell expansion. After 7 d of exposure to live MT, gamma delta T cells were greatly increased in all healthy tuberculin reactors (PPD+) tested and frequently were the predominant T cell population. In contrast, heat-killed MT or purified protein products of MT induced a CD4+, alpha beta TCR+ T cell response with very little increase in gamma delta T cells. Furthermore, a similar selective induction of gamma delta T cells was observed when monocytes infected with live Salmonella were used to stimulate T cells. Heat-killed Salmonella, like heat-killed MT, induced a predominantly CD4+ alpha beta TCR+ T cell response. These findings suggest that human gamma delta T cells are a major reactive T cell population during the early stages of infection with living intracellular bacteria and are therefore likely to exert an important role in the initial interaction between host and parasite.

In vitro synthesis of anti-mycobacterial antibodies in biopsies from skin lesions of leprosy patients.

To demonstrate local synthesis of anti-Mycobacterium leprae antibodies, biopsies from skin lesions of leprosy patients were cultured in vitro in a medium containing 14C-labeled lysine and isoleucine, and the culture fluids were analyzed by crossed immunoelectrophoresis with intermediate gel and autoradiography. The results show that anti-M. leprae antibodies were synthesized in vitro in the biopsies from the skin lesions of leprosy patients and that the specificity of the locally produced antibodies varied from patient to patient.