The cellular architecture of the antimicrobial response network in human leprosy granulomas.

Granulomas are complex cellular structures composed predominantly of macrophages and lymphocytes that function to contain and kill invading pathogens. Here, we investigated the single-cell phenotypes associated with antimicrobial responses in human leprosy granulomas by applying single-cell and spatial sequencing to leprosy biopsy specimens. We focused on reversal reactions (RRs), a dynamic process whereby some patients with disseminated lepromatous leprosy (L-lep) transition toward self-limiting tuberculoid leprosy (T-lep), mounting effective antimicrobial responses. We identified a set of genes encoding proteins involved in antimicrobial responses that are differentially expressed in RR versus L-lep lesions and regulated by interferon-γ and interleukin-1ß. By integrating the spatial coordinates of the key cell types and antimicrobial gene expression in RR and T-lep lesions, we constructed a map revealing the organized architecture of granulomas depicting compositional and functional layers by which macrophages, T cells, keratinocytes and fibroblasts can each contribute to the antimicrobial response.

Corticosteroid therapy in borderline tuberculoid leprosy patients co-infected with HIV undergoing reversal reaction: a clinical study.

Background: Mycobacterium leprae and HIV cause infectious diseases of great concern for the public health care sector worldwide. Both are especially worrisome diseases when patients become co-infected and exhibit the expected clinical exuberance. The objective of this study was to evaluate episodes of reversal reaction (RR) and the effect of the use of corticosteroids on the treatment of borderline tuberculoid leprosy patients co-infected with the human immunodeficiency virus (HIV). Methods: This is a retrospective cohort study in which the clinical manifestations of the patients and their responses to corticosteroid therapy were observed. Variables were analysed during and after multidrug therapy between the first and last days of prednisone, which occurred up to a maximum of 6 months after initiating corticosteroid therapy. Results: A total of 22 HIV-positive and 28 HIV-negative cases were included. Loss of sensitivity and neural thickening were statistically significant while clinically ulcerated lesions were only observed in the co-infected group. Most patients were diagnosed with leprosy in the presence of RR and six patients manifested RR as an immune reconstitution inflammatory syndrome. On average, both groups received similar doses of corticosteroids (difference of 0·1 mg/kg/day).

Induction and treatment of anergy in murine leprosy.

Leprosy is a disease consisting of a spectrum of clinical, bacteriological, histopathological and immunological manifestations. Tuberculoid leprosy is frequently recognized as the benign polar form of the disease, while lepromatous leprosy is regarded as the malignant form. The different forms of leprosy depend on the genetic and immunological characteristics of the patient and on the characteristics of the leprosy bacillus. The malignant manifestations of lepromatous leprosy result from the mycobacterial-specific anergy that develops in this form of the disease. Using murine leprosy as a model of anergy in this study, we first induced the development of anergy to Mycobacterium lepraemurium (MLM) in mice and then attempted to reverse it by the administration of dialysable leucocyte extracts (DLE) prepared from healthy (HLT), BCG-inoculated and MLM-inoculated mice. Mice inoculated with either MLM or BCG developed a robust cell-mediated immune response (CMI) that was temporary in the MLM-inoculated group and long-lasting in the BCG-inoculated group. DLE were prepared from the spleens of MLM- and BCG-inoculated mice at the peak of CMI. Independent MLM intradermally-inoculated groups were treated every other day with HLT-DLE, BCG-DLE or MLM-DLE, and the effect was documented for 98 days. DLE administered at a dose of 1.0 U (1 × 10(6) splenocytes) did not affect the evolution of leprosy, while DLE given at a dose of 0.1 U showed beneficial effects regardless of the DLE source. The dose but not the specificity of DLE was the determining factor for reversing anergy.

Clinically unsuspected neuritic leprosy with caseation necrosis.

Leprosy is a devastating disease caused by Mycobacterium leprae. It includes a spectrum of clinicopathological lesions. Neuritic leprosy with caseation necrosis (abscess) manifesting as a soft tissue mass is a relatively rare presentation of leprosy. Here, the authors report their experience with three patients with neuritic leprosy. The patients presented with swellings in the right ulnar nerve, the right great auricular nerve, and the temporal branch of the right sixth cranial nerve. The clinical impression was that of tumorous masses. Gross examination of the biopsy specimens revealed caseous necrotic materials. Further histological evaluation disclosed tuberculoid granulomas with extensive caseation necrosis. Stains for acid-fast bacilli were positive in the third case. A comparison between the caseation encountered in the tuberculoid neurotic leprosy and the neurolysis of lepromatous neurotic leprosy has been also discussed. The findings here emphasize "mass lesion with necrosis" as a possible clinical presentation of the neuritic leprosies. The clinicopathologic features were addressed and the relevant literature was reviewed.

Interleukin-4 regulates the expression of CD209 and subsequent uptake of Mycobacterium leprae by Schwann cells in human leprosy.

The ability of microbial pathogens to target specific cell types is a key aspect of the pathogenesis of infectious disease. Mycobacterium leprae, by infecting Schwann cells, contributes to nerve injury in patients with leprosy. Here, we investigated mechanisms of host-pathogen interaction in the peripheral nerve lesions of leprosy. We found that the expression of the C-type lectin, CD209, known to be expressed on tissue macrophages and to mediate the uptake of M. leprae, was present on Schwann cells, colocalizing with the Schwann cell marker, CNPase (2',3'-cyclic nucleotide 3'-phosphodiesterase), along with the M. leprae antigen PGL-1 in the peripheral nerve biopsy specimens. In vitro, human CD209-positive Schwann cells, both from primary cultures and a long-term line, have a higher binding of M. leprae compared to CD209-negative Schwann cells. Interleukin-4, known to be expressed in skin lesions from multibacillary patients, increased CD209 expression on human Schwann cells and subsequent Schwann cell binding to M. leprae, whereas Th1 cytokines did not induce CD209 expression on these cells. Therefore, the regulated expression of CD209 represents a common mechanism by which Schwann cells and macrophages bind and take up M. leprae, contributing to the pathogenesis of leprosy.

Colonization by Helicobacter pylori of leprosy patients in Spain: immunomodulation to low molecular weight antigens of H. pylori.

We studied the prevalence of Helicobacter pylori in patients with leprosy and the effects of co-infection on the immune response to Helicobacter antigens in the polar groups of leprosy (lepromatous and tuberculoid). We showed that there is no difference in the prevalence of H. pylori in patients with leprosy as compared to a non-leprosy population. We also demonstrated that the immune response to low molecular weight H. pylori antigens (35, 26 and 19 kDa) differs in patients with lepromatous as compared to those with tuberculoid leprosy. In lepromatous leprosy, we show that there is a higher prevalence of the 35 and 26 kDa antigens, but a lower prevalence of the 19 kDa antigen. These immunological results are consistent with previous histopathological studies illustrating a more severe gastrointestinal inflammation in lepromatous patients; importantly, a response to the 35 kDa antigen is recognized as a marker for the development of ulcerative disease.

Appropriately Selected Nerve in Suspected Leprous Neuropathy Yields High Positive Results for Mycobacterium leprae DNA by Polymerase Chain Reaction Method.

Identification of Mycobacterium leprae DNA by polymerase chain reaction (PCR) is a reliable and an affordable method to confirm leprosy. DNA from 87 nerve samples (61 from paraffin blocks and 26 fresh samples) was extracted. Mycobacterium leprae DNA was amplified by PCR from 80/87 (92%) specimens. Patients were seen over a period of 11 years (2007-2019), and leprosy was diagnosed based on clinical and characteristic histopathology findings. The clinical diagnostic possibilities were as follows: leprous neuropathy in 73/80 (91.3%), mononeuritis multiplex of unknown etiology in four (5.0%), vasculitic neuropathy in two (2.5%), and distal symmetric sensory motor neuropathy in one (1.3%). The biopsied nerves were as follows: superficial radial = 34 (42.6%), dorsal cutaneous branch of ulnar = 19 (23.8%), sural = 18 (22.5%), and superficial peroneal = 9 (11.3%), and corresponding neurological deficits were recorded in 77 (96.3%) cases. The histopathological diagnoses in total group were as follows: (borderline tuberculoid (BT) = 52, tuberculoid (TT) = 8, borderline lepromatous (BL) = 8, borderline borderline (BB) = 3, nonspecific inflammation = 3, healed/fibrosed = 4, and axonopathy = 2). Acid fast bacilli (AFB) was demonstrated in 11 (13.7%) samples. For comparison, 31 clinically and histopathologically defined non-leprous disease control nerves (inherited neuropathy = 20, vasculitis = 8, and nutritional neuropathy = 3) subjected to PCR were negative for M. leprae DNA. In most instances, there are multiple thickened peripheral nerves in suspected cases of leprosy, but neurological deficits pertaining to the thickened nerve are not as widespread. The current findings emphasize the importance of selecting the most appropriate nerve for biopsy to obtain a positive PCR result. We infer that clinical, histopathological, and PCR tests complement each other to help achieve a definitive diagnosis of leprosy particularly in pure neuritic leprosy and in leprous neuropathy with negative skin smears/biopsy.

IL-26 contributes to host defense against intracellular bacteria.

IL-26 is an antimicrobial protein secreted by Th17 cells that has the ability to directly kill extracellular bacteria. To ascertain whether IL-26 contributes to host defense against intracellular bacteria, we studied leprosy, caused by the obligate intracellular pathogen Mycobacterium leprae, as a model. Analysis of leprosy skin lesions by gene expression profiling and immunohistology revealed that IL-26 was more strongly expressed in lesions from the self-limited tuberculoid compared with expression in progressive lepromatous patients. IL-26 directly bound to M. leprae in axenic culture and reduced bacteria viability. Furthermore, IL-26, when added to human monocyte-derived macrophages infected with M. leprae, entered the infected cell, colocalized with the bacterium, and reduced bacteria viability. In addition, IL-26 induced autophagy via the cytoplasmic DNA receptor stimulator of IFN genes (STING), as well as fusion of phagosomes containing bacilli with lysosomal compartments. Altogether, our data suggest that the Th17 cytokine IL-26 contributes to host defense against intracellular bacteria.

Highly Reduced Genome of the New Species Mycobacterium uberis, the Causative Agent of Nodular Thelitis and Tuberculoid Scrotitis in Livestock and a Close Relative of the Leprosy Bacilli.

Nodular thelitis is a chronic enzootic infection affecting dairy cows and goats. The causative agent was recently shown to be related to the leprosy-causing bacilli Mycobacterium leprae and Mycobacterium lepromatosis In this study, the genome of this pathogen was sequenced and analyzed. Phylogenomic analyses confirmed that the pathogen present in nodular thelitis and tuberculoid scrotitis is a distinct species related to the leprosy bacilli and Mycobacterium haemophilum Because the pathogen was originally isolated from a bovine udder, it was named "Mycobacterium uberis" The genome of "M. uberis" is only 3.12 Mb in length, which represents the smallest mycobacterial genome identified so far but which is close to that of leprosy bacilli in size. The genome contains 1,759 protein-coding genes and 1,081 pseudogenes, indicative of extensive reductive evolution and likely the reason that M. uberis cannot be grown axenically. The pseudogenization and genome reduction in M. uberis seem to have been to some extent independent from the results determined for the genomes of the leprosy bacilli.IMPORTANCEM. uberis is an emerging skin pathogen in dairy animals. Its genome underwent massive reduction and gene decay, leading to a minimal set of genes required for an obligatory intracellular lifestyle, which highly resembles the evolution of the leprosy agents M. leprae and M. lepromatosis The genomic similarity between M. uberis and the leprosy bacilli can help in identifying key virulence factors of these closely related species or in identifying genes responsible for the distinct differences between thelitis or scrotitis and leprosy with respect to clinical manifestations. Specific DNA markers can now be developed for quick detection of this pathogen.

HLA and leprosy in the pre and postgenomic eras.

Leprosy has intrigued immunologists for many decades. Despite minimal genetic variation between Mycobacterium leprae isolates worldwide, two completely different forms of the disease can develop in the susceptible human host: localized, tuberculoid, or paucibacillary leprosy, which can heal spontaneously, and disseminating, lepromatous, or multibacillary leprosy, which is progressive if untreated. The questions which host factors regulate these very different outcomes of infection, by what mechanisms, and whether these can be used to combat disease remain unanswered. Leprosy has been one of the very first human diseases in which human leukocyte antigen (HLA) genes were demonstrated to codetermine disease outcome. Jon van Rood was among the earliest researchers to recognize the potential of this ancient disease as a human model to dissect the role of HLA in disease. Decades later, it is now clear that HLA molecules display highly allele-specific peptide binding capacity. This restricts antigen presentation to M. leprae-reactive T cells and controls the magnitude of the ensuing immune response. Furthermore, specific peptide/HLA class II complexes can also determine the quality of the immune response by selectively activating regulatory (suppressor) T cells. All these factors are believed to contribute to leprosy disease susceptibility. Despite the global reduction in leprosy disease prevalence, new case detection rates remain invariably high, demonstrating that treatment alone does not block transmission of leprosy. Better tools for early detection of preclinical M. leprae infection, likely the major source of unidentified transmission, therefore is a priority. Newly developed HLA-based bioinformatic tools now provide novel opportunities to help combat this disease. Here, we describe recent work using HLA-DR peptide binding algorithms in combination with recently elucidated genome sequences of several different mycobacteria. Using this postgenomic HLA-based approach, we were able to identify 12 candidate genes that were unique to M. leprae and were predicted to contain T cell epitopes restricted via several major HLA-DR alleles. Five of these antigens (ML0576, ML1989, ML1990, ML2283, ML2567) were indeed able to induce significant T cell responses in paucibacillary leprosy patients and M. leprae-exposed healthy controls but not in most multibacillary leprosy patients, tuberculosis patients, or endemic controls. 71% of M. leprae-exposed healthy controls that did not have antibodies to the M. leprae-specific phenolic glycolipid-I responded to one or more M. leprae antigen(s), highlighting the potential added value of these unique M. leprae proteins in diagnosing early infection. Thus current state-of-the-art HLA immunogenetics can provide new tools for specific diagnosis of M. leprae infection, which can impact our understanding of leprosy transmission and can lead to improved intervention.

Isolation of mycobacterium leprae from untreated borderline tuberculoid, mid-borderline and indeterminate cases using the mouse foot pad technique--a study of 209 cases.

Using the mouse foot pad (MFP) system, isolation of Mycobacterium leprae was attempted in 209 skin biopsies obtained from 114 borderline tuberculoid (BT), 62 mid borderline (BB) and 33 indeterminate (1) untreated cases. Unequivocal growth in the foot pads of mice was seen in 100 (47.8%) cases. Of these 100 cases that showed growth in the mouse foot pad system, in 20 cases acid fast bacilli (AFB) were detected in small numbers (1 + ) in either smear or homogenate. The remaining 80 (42%) cases were negative for AFB in both smear and homogenate. The occurrence of viable bacilli and percentage take at 12 months was highest in BB (76 and 86%) followed by BT (38 and 75%) and I (30% and 52%) cases. In most of the BT (65%) and I (60%) cases, the first peak was seen only at 12 months. These results confirm that viable bacilli can be isolated and expanded from a good proportion of negative BT-BB cases using immunocompetent Swiss White mice.

Leprae reaction resembling rheumatologic disease as presenting feature of leprosy.

Leprosy is a chronic granulomatous infection caused by Mycobacterium leprae with predominant involvement of skin and nerves. We present a 70-year-old man with leprosy whose initial presentation resembled rheumatologic disease, due to leprae reaction. He presented with an 8-week history of worsening neuropathic pain in the right forearm, associated with necrotic skin lesions on his fingers that had ulcerated. Physical examination revealed two tender necrotic ulcers at the tip of the right middle finger and the dorsal aspect of the left middle finger. The patient had right wrist tenosynovitis and right elbow bursitis. Apart from raised inflammatory markers, the investigations for infection, connective tissue disease, vasculitis, thromboembolic disease and malignancy were negative. During the fourth week of hospitalization, we noticed a 2-cm hypoesthetic indurated plaque on the right inner arm. Further examination revealed thickened bilateral ulnar, radial and popliteal nerves. A slit skin smear was negative. Two skin biopsies and a biopsy of the olecranon bursa revealed granulomatous inflammation. He was diagnosed with paucibacillary leprosy with neuritis. He responded well to multidrug therapy and prednisolone; his symptoms resolved over a few weeks. This case illustrates the challenges in diagnosing a case of leprosy with atypical presentation in a non-endemic country.

In situ demonstration of Mycobacterium leprae antigens in leprosy lesions using monoclonal antibodies.

Cryostat sections of skin and nerve lesions of leprosy were stained with monoclonal antibodies recognising Mycobacterium leprae antigens and indirect immunofluorescence. In both the tuberculoid and lepromatous lesions, PGL1, 55-65-kDa, 17-kDa protein antigens and cross-reactive non-protein antigens were present. 65-kDa antigens were seen mainly in the skin lesions of lepromatous leprosy. The infiltrates in both the skin and nerve granulomas of tuberculoid and lepromatous leprosy showed membranous staining with monoclonal antibodies recognising PGL1 and 55-65-kDa antigens. Bacilli in the lesions and the cells in the lymph node granulomas of patients with tuberculosis or the infiltrates in the lesions of tinea corporis or sections of normal skin did not show any staining with these monoclonal antibodies. These results confirm that M. leprae antigens are present and are expressed on the infiltrating cells of leprosy lesions.

Criteria for diagnosis of pure neural leprosy.

The clinical diagnosis of pure neural leprosy (PNL) remains a public health care problem mainly because skin lesions-the cardinal features of leprosy-are always absent.Moreover, the identification of the leprosy bacillus is not easily achieved even when a nerve biopsy can be performed. In an attempt to reach a reliable PNL diagnosis in patients referred to our Leprosy Outpatient Clinic, this study employed a variety of criteria. The nerve biopsies performed on the 67 individuals whose clinical, neurological, and electrophysiological examination findings strongly suggested peripheral neuropathy were submitted to M. leprae identification via a polymerase chain reaction (PCR). Mononeuropathy multiplex was the most frequent clinical and electrophysiological pattern of nerve dysfunction, while sensory impairment occurred in 89% of all cases and motor dysfunction in 81%. Axonal neuropathy was the predominant electrophysiological finding, while the histopathological nerve study showed epithelioid granuloma in 14% of the patients, acid fast bacilli in 16%, and nonspecific inflammatory infiltrate and/or fibrosis in 39%. PCR for M. leprae was positive in 47% of the nerve biopsy samples (n=23). PCR, in conjunction with clinical and neurological examination results, can be a powerful tool in attempting to identify and confirm a PNL diagnosis.

Macular lesions in leprosy: a clinical, bacteriological and histopathological study.

Among 150 untreated patients of leprosy, 19 had only macular lesions; three were of the indeterminate type, and eight each were of the tuberculoid and the borderline types, according to the Indian Association of Leprologists (IAL, 1981) classification. The clinical, bacteriological, and histopathological parameters of these 19 patients were studied both before and after six months of WHO Multi Drug Therapy (MDT/1982). A single macule was present in seven (36.84%) patients. In twelve (63.16%), two or more were seen. In eighteen (94.74%), one or more peripheral nerves were enlarged. The size of the macules varied from 5 to 15 cm, and there were no changes seen even after treatment. In most (94.74%) of the patients, the macules were hypopigmented. The surfaces were rough and dry in seven (36.84%) but smooth in the other twelve (63.16%). The margins were well defined in the seven (36.84%) patients with single macules but ill defined in the other twelve (63.16%). After six months of antileprosy treatment, the single macules showed some resolution. Slit skin smear examination was negative in all cases before and after treatment. Clinico-histopathological correlations were seen in only six (31.58%) patients; the clinical diagnoses were indeterminate and tuberculoid leprosy in three (15.79%) patients each. In the indeterminate group, the clinico-histopathological correlation was 100%; it was 37.50% in the tuberculoid group. There were no correlations between the clinical and histopathological parameters in thirteen (68.42%) cases. After six months of treatment, the histopathology became nonspecific in all patients. The lepromin test was positive in six (31.58%) patients; four were of the tuberculoid group and one each from the indeterminate and borderline leprosy groups. Hence, although macular lesions can be seen throughout the leprosy spectrum, it is difficult to correlate their clinical, bacteriological and histopathological parameters.

Generalized annular borderline tuberculoid leprosy and update in management of Hansen's disease.

We describe a patient with widespread borderline tuberculoid leprosy and significant peripheral nerve involvement. Despite the presence of widespread lesions, Fite stains and polymerase chain reaction studies were initially negative. We discuss the diagnosis and treatment of leprosy including recent changes in treatment regimens and duration.

[Edema in leprosy: the clinical and therapeutic aspects]. / Edema na hanseníase: aspectos clínicos e terapêuticos.

Edema, which is commonly described as a symptom of reactional states, may occur during the course of leprosy. Both diagnosis and adequate treatment measures are often difficult to achieve and failure to do so may result in permanent damage to the lower limbs. In a one-year follow-up study of leprosy patients--ten multibacillary and one paucibacillary--who had been submitted to a clinical protocol for diagnosis and pathological classification, a clinical pattern of localized and/or systemic edema was observed. Among these patients, five simultaneously presented other symptoms related to reactional states, 4 were diagnosed as Type I, and one as Type II. On the other hand, while three of the patients did not present reaction at the time when edema was diagnosed, they did develop some aspects of reactional disease later on (two had neuritis e one had Type I reaction). The edemas that preceded or were associated with reactional episodes showed clinical regression as a result of specific treatment against reactions (corticosteroids and/or pentoxifylline and/or thalidomide) in the absence of another treatment normally used for edemas. Although these data need to be confirmed by controlled studies, they strongly suggest that immunological mechanisms are involved in the physiopathology of edema in leprosy.

An attempt to produce experimental tuberculoid leprosy in the nine-banded armadillo.

In an attempt to produce experimental tuberculoid leprosy, three nine-banded armadillos, two with borderline tuberculoid lepromin reaction, and one with tuberculoid lepromin reaction, were chosen. They were injected subcutaneously in a four square centimetre area in the abdominal skin with saline suspension of 6.5 x 10(7) M. leprae. Induration of skin at the injected site appeared in 24 hours and persisted for 6 months in one and for 18 months in the other two animals. Histopathological examination of the infected site at 6 weeks, 18 and 20 months showed progressively decreasing granulomatous inflammation; but the cutaneous nerves were uninvolved. Autopsy examination of the three animals failed to show disseminated disease. Since there was no evidence of nerve involvement, experimental transmission of tuberculoid leprosy to armadillos could not be established in this study.