Survival and prognostic factors in bullous pemphigoid: A retrospective cohort study.

Background Bullous pemphigoid is the most common subepidermal autoimmune blistering disease. Till now, the reported prognostic factors in bullous pemphigoid vary considerably. Aims The purpose of this study was to determine the overall survival rate and prognostic factors in bullous pemphigoid. Methods We conducted a retrospective cohort study on newly diagnosed bullous pemphigoid patients between July 2001 and November 2019 in a referral unit for autoimmune blistering skin diseases in Romania. Results One hundred forty-eight patients were included in the study. The Kaplan-Meier overall survival rates at 1, 3, 5 and 10 years were respectively 74.2% (95% confidence interval, 67.5-81.6%), 53.4% (45.7-62.2%), 43.6% (35.9-53%) and 31.3% (23.5-41.7%). The median follow-up among survivors was 48 months (interquartile range: 11-150). Ninety (60.8%) patients died during the follow-up period; of them, 38 (42.2%) had active disease at the time of death. Advanced age, neurological diseases, valvular heart disease, malignancies, use of statins, skin infections and extensive cutaneous involvement were linked to poorer outcomes, while the use of topical corticosteroids was associated with increased overall survival. Limitations This study lacks a control cohort to validate the obtained results. It was conducted in a retrospective manner in a single centre. In addition, indirect immunofluorescence microscopy was not performed in all patients. Conclusion Beyond ageing and neurological comorbidities, the prognosis of bullous pemphigoid patients was significantly influenced by the presence of skin infections, valvular heart disease, use of statins and extensive cutaneous involvement. Topical corticosteroid treatment was associated with increased survival in these patients.

Efficacy of fluorescent microscopy versus modified Fite-Faraco stain in skin biopsy specimens of leprosy cases - a comparative study.

BACKGROUND: Histopathological examination of skin remains the cornerstone in the diagnosis of leprosy. At a few centers, fluorescent microscopy has been found to be useful in detecting more acid-fast bacilli (AFB) compared to modified Fite-Faraco staining but is sparsely documented. Hence, we studied the sensitivity of fluorescent microscopy and modified Fite-Faraco stain in the detection of Mycobacterium leprae in tissue sections. METHODS: Patients attending our outpatient department during January 2019 to June 2020 with the clinical features of leprosy were examined, and the diagnosis was confirmed by histopathology after informed consent. Tissue sections were stained by fluorescent stain and modified Fite-Faraco stain. Bacillary index was calculated for each case. RESULTS: Forty patients were recruited after following the inclusion and exclusion criteria. AFB were demonstrated in 20 patients by modified Fite-Faraco stain and in 27 patients with fluorescent stain. The sensitivity of fluorescent staining method (67.5%) was higher than modified Fite-Faraco stain (50%). Bacillary index was increased in 26 out of 40 cases by the fluorescent staining compared to the modified Fite-Faraco staining. Chi-square value was 69.3 and P value was 0.000, indicating a statistically significant correlation. LIMITATIONS: Fluorescent microscope is expensive, and trained people are needed to identify the bacilli. CONCLUSION: Fluorescent staining is more sensitive than modified Fite-Faraco staining in the detection of AFB in tissue sections. The bacilli detected per field were high with the fluorescent staining compared to the modified Fite-Faraco method.

Laboratory diagnosis of leprosy: Two staining methods from bacilloscopy and rapid ml flow test.

Background: The diagnosis of leprosy is based on the characteristic signs and symptoms of the disease, subsidized by laboratory tests. When positive, the bacilloscopy closes the diagnosis for leprosy. Phenolic glycolipid-I, or PGL-I, is a molecule in the bacillus cell wall that confers a greater immune response. The ML Flow test is an immunochromatographic test for the detection of anti-PGL-I IgM in human blood or serum. Methods: A prospective study with data collection and biological materials in patients with suspected leprosy from August 2020 to May 2021. For microscopy, intradermal smears were stained with Auramine O, and after reading under a fluorescence microscope, reviewed by Ziehl-Neelsen. The ML flow test was performed according to the Bührer-Sékula protocol. To assess the agreement between the methods, the Kappa index was estimated. Results: Of the 94 suspected leprosy patients, 31 (32.9%) were diagnosed with leprosy. There was moderate agreement between the results of the ML Flow and Auramine O tests (Kappa = 0.58) and substantial agreement between the ML Flow and Ziehl-Neelsen microscopy (Kappa = 0.72). In paucibacillary cases, serology was positive in 100% of patients. Conclusions: This study concluded that the Ziehl-Neelsen technique remains the best option for standard leprosy staining, and the ML flow test is more positive among the three techniques evaluated and can be an effective tool in the early diagnosis of leprosy cases.

Serration pattern analysis as a practical adjunct tool for categorization of subepidermal autoimmune blistering diseases.

BACKGROUND: Serration pattern analysis helps in the classification of subepidermal autoimmune blistering disorders; more precisely, it helps to differentiate epidermolysis bullosa acquisita from other subepidermal autoimmune blistering disorders. Most of the published reports of this tool have come from a single center. OBJECTIVES: The objectives of the study were to study the utility of serration pattern analysis in classifying subepidermal autoimmune blistering disorders. METHODS: Seventy five cases of subepidermal autoimmune blistering disorders were enrolled in this prospective study. A three millimeter punch biopsy was taken from the perilesional skin or mucosa for direct immunofluorescence; indirect immunofluorescence was carried out using salt-split skin. Subclassification of subepidermal autoimmune blistering disorders was done based on direct immunofluorescence, indirect immunofluorescence on salt-split skin, indirect immunofluorescence using knockout skin and serration pattern analysis findings. RESULTS: Indirect immunofluorescence was positive in 68 cases; 14 cases showed a dermal staining pattern while the rest showed either an epidermal or a combined pattern. All patients with epidermal or combined staining patterns showed "n" serrated pattern on direct immunofluorescence. Nine patients with dermal staining on indirect immunofluorescence also revealed an "n" serration pattern on direct immunofluorescence indicating the diagnosis of anti-p200 pemphigoid, and the rest showed a "u" serrated pattern. Three patients with negative indirect immunofluorescence showed "u" serration on direct immunofluorescence while the rest showed "n" serration. LIMITATIONS: ELISA and immunoblotting could not be performed due to resource constraints. CONCLUSION: Based on indirect immunofluorescence and serration pattern analysis, classification of the majority of patients with subepidermal autoimmune blistering disorders was possible in our study. Pattern recognition is a cost-effective tool and can be easily learnt. It is recommended to be practiced in all laboratories where facilities for advanced immunological diagnosis are unavailable.

Anti-P 200 pemphigoid - The most common floor binding subepidermal autoimmune bullous disease in a tertiary care center in south India.

BACKGROUND: The pemphigoid group of diseases may present clinically and immunologically in a very similar fashion. Indirect immunofluorescence microscopy with readily available salt-split human skin in a BIOCHIP™ helps to classify these conditions as those with either with roof binding or floor binding of immunoreactants. Epidermolysis bullosa acquisita, anti-laminin 332 pemphigoid and anti-p200 pemphigoid show floor binding, while in the most frequent type of pemphigoid disease, bullous pemphigoid, epidermal side staining pattern is seen on salt-split skin Aims: The aim of the study was to detect the target antigens in sub-epidermal bullous diseases. METHODS: Forty patients with bullous pemphigoid diagnosed by lesional histopathology and direct immunofluorescence microscopy were re-evaluated by a BIOCHIP™ mosaic containing both tissue substrates and recombinant target antigens. Sera with floor pattern staining on salt-split skin were further evaluated by immunoblotting with dermal extract. RESULTS: Five patients with floor staining had anti-p200 pemphigoid. LIMITATIONS: We could not perform serration pattern analysis of direct immunofluorescence in our patients. CONCLUSION: Histopathology and direct immunofluorescence microscopy cannot differentiate between various entities of pemphigoid diseases. A multivariant approach using a BIOCHIP™ mosaic including salt-split skin followed by immunoblotting with dermal extract helps to identify the target antigen.

Single Crystal FLIM Characterization of Clofazimine Loaded in Silica-Based Mesoporous Materials and Zeolites.

Clofazimine (CLZ) is an effective antibiotic used against a wide spectrum of Gram-positive bacteria and leprosy. One of its main drawbacks is its poor solubility in water. Silica based materials are used as drug delivery carriers that can increase the solubility of different hydrophobic drugs. Here, we studied how the properties of the silica framework of the mesoporous materials SBA-15, MCM-41, Al-MCM-41, and zeolites NaX, NaY, and HY affect the loading, stability, and distribution of encapsulated CLZ. Time-correlated single-photon counting (TCSPC) and fluorescence lifetime imaging microscopy (FLIM) experiments show the presence of neutral and protonated CLZ (1.3-3.8 ns) and weakly interacting aggregates (0.4-0.9 ns), along with H- and J-type aggregates (<0.1 ns). For the mesoporous and HY zeolite composites, the relative contribution to the overall emission spectra from H-type aggregates is low (<10%), while for the J-type aggregates it becomes higher (~30%). For NaX and NaY the former increased whereas the latter decreased. Although the CLZ@mesoporous composites show higher loading compared to the CLZ@zeolites ones, the behavior of CLZ is not uniform and its dynamics are more heterogeneous across different single mesoporous particles. These results may have implication in the design of silica-based drug carriers for better loading and release mechanisms of hydrophobic drugs.

Model Protein Adsorption on Polymers Explained by Hansen Solubility Parameters.

Hansen solubility parameters (HSP) theory has been successful in explaining the wettability of organic solvents on polymer surfaces and miscibility of different polymers. Here, we demonstrate that the amount of bovine serum albumin (BSA) protein adsorption on different polymer surfaces can also be explained by HSP. Interestingly, the HSP of the adsorbed BSA proteins calculated from the protein adsorption data is different than the HSP of native BSA protein itself. The HSP of the adsorbed BSA proteins are more hydrophobic than the native BSA protein. This observation suggested adsorbed BSA proteins are partially denatured and exposed their hydrophobic core toward the polymer surfaces. These results highlight a new strategic direction to understand interaction of protein with a surface: a theoretical approach that compliments experimental approach. The model in this study could be used to predict the amount of BSA adsorption on a polymer or any other solid surface, if the HSP of that surface is known. Further, the model can serve as a prescreen method to identify surfaces that are problematic at the outset and inform subsequent empirical studies to select packaging that will have the least adsorption for the specific biologic application.

Epidermolysis bullosa acquisita and anti-p200 pemphigoid as major subepidermal autoimmune bullous diseases diagnosed by floor binding on indirect immunofluorescence microscopy using human salt-split skin.

BACKGROUND: Subepidermal autoimmune bullous diseases are a diverse group of diseases with overlapping clinical and immunopathological features. Indirect immunofluorescence microscopy on artificially split skin helps to classify these conditions into those with staining on the epidermal side of the split ("roof-binding") and those with staining on the dermal side ("floor-binding"). Epidermolysis bullosa acquisita is the prototype of "floor-binding" subepidermal autoimmune bullous diseases. However, not all floor-binding sera are associated with epidermolysis bullosa acquisita. AIM: The aim of this study was to evaluate the clinical and immunological profile of patients with floor-binding subepidermal autoimmune bullous disease by indirect immunofluorescence microscopy and to identify the target antigens in them. METHODS: Ten patients who showed a floor-binding pattern were studied with regard to their clinical and immunopathological characteristics. Target antigens were identified by modified indirect immunofluorescence microscopy using recessive dystrophic epidermolysis bullosa skin, enzyme linked immunosorbent assay, and immunoblotting. RESULTS: Diagnosis of epidermolysis bullosa acquisita was confirmed in six patients. Three patients with an inflammatory subepidermal autoimmune bullous disease mimicking bullous pemphigoid reacted with a 200 kDa protein on immunoblotting with dermal extract, as is characteristic of anti-p200 pemphigoid. One serum showed both roof and floor binding, and reacted with the BP180 antigen. LIMITATION: We could not perform serration pattern analysis in our patients. CONCLUSION: In this study, we report three cases of anti-p200 pemphigoid from India. These cases, though indistinguishable clinically from bullous pemphigoid, revealed a floor-binding pattern on indirect immunofluorescence using salt-split skin.

The Dihydroxy Metabolite of the Teratogen Thalidomide Causes Oxidative DNA Damage.

Thalidomide [α-(N-phthalimido)glutarimide] (1) is a sedative and antiemetic drug originally introduced into the clinic in the 1950s for the treatment of morning sickness. Although marketed as entirely safe, more than 10 000 babies were born with severe birth defects. Thalidomide was banned and subsequently approved for the treatment of multiple myeloma and complications associated with leprosy. Although known for more than 5 decades, the mechanism of teratogenicity remains to be conclusively understood. Various theories have been proposed in the literature including DNA damage and ROS and inhibition of angiogenesis and cereblon. All of the theories have their merits and limitations. Although the recently proposed cereblon theory has gained wide acceptance, it fails to explain the metabolism and low-dose requirement reported by a number of groups. Recently, we have provided convincing structural evidence in support of the presence of arene oxide and the quinone-reactive intermediates. However, the ability of these reactive intermediates to impart toxicity/teratogenicity needs investigation. Herein we report that the oxidative metabolite of thalidomide, dihydroxythalidomide, is responsible for generating ROS and causing DNA damage. We show, using cell lines, the formation of comet (DNA damage) and ROS. Using DNA-cleavage assays, we also show that catalase, radical scavengers, and desferal are capable of inhibiting DNA damage. A mechanism of teratogenicity is proposed that not only explains the DNA-damaging property but also the metabolism, low concentration, and species-specificity requirements of thalidomide.

Experimental Infection of Rhodnius prolixus (Hemiptera, Triatominae) with Mycobacterium leprae Indicates Potential for Leprosy Transmission.

Leprosy is a chronic dermato-neurological disease caused by infection with Mycobacterium leprae. In 2013 almost 200,000 new cases of leprosy were detected around the world. Since the first symptoms take from years to decades to appear, the total number of asymptomatic patients is impossible to predict. Although leprosy is one of the oldest records of human disease, the mechanisms involved with its transmission and epidemiology are still not completely understood. In the present work, we experimentally investigated the hypothesis that the mosquitoes Aedes aegypti and Culex quinquefasciatus and the hemiptera Rhodnius prolixus act as leprosy vectors. By means of real-time PCR quantification of M. leprae 16SrRNA, we found that M. leprae remained viable inside the digestive tract of Rhodnius prolixus for 20 days after oral infection. In contrast, in the gut of both mosquito species tested, we were not able to detect M. leprae RNA after a similar period of time. Inside the kissing bug Rhodnius prolixus digestive tract, M. leprae was initially restricted to the anterior midgut, but gradually moved towards the hindgut, in a time course reminiscent of the life cycle of Trypanosoma cruzi, a well-known pathogen transmitted by this insect. The maintenance of M. leprae infectivity inside the digestive tract of this kissing bug is further supported by successful mice footpad inoculation with feces collected 20 days after infection. We conclude that Rhodnius prolixus defecate infective M. leprae, justifying the evaluation of the presence of M. leprae among sylvatic and domestic kissing bugs in countries endemic for leprosy.

Experimental infection of Rhodnius prolixus (Hemiptera, Triatominae) with Mycobacterium leprae indicates potential for leprosy transmission

Leprosy is a chronic dermato-neurological disease caused by infection with Mycobacterium leprae. In 2013 almost 200,000 new cases of leprosy were detected around the world. Since the first symptoms take from years to decades to appear, the total number of asymptomatic patients is impossible to predict. Although leprosy is one of the oldest records of human disease, the mechanisms involved with its transmission and epidemiology are still not completely understood. In the present work, we experimentally investigated the hypothesis that the mosquitoes Aedes aegypti and Culex quinquefasciatus and the hemiptera Rhodnius prolixus act as leprosy vectors. By means of real-time PCR quantification of M. leprae 16SrRNA, we found that M. leprae remained viable inside the digestive tract of Rhodnius prolixus for 20 days after oral infection. In contrast, in the gut of both mosquito species tested, we were not able to detect M. leprae RNA after a similar period of time. Inside the kissing bug Rhodnius prolixus digestive tract, M. leprae was initially restricted to the anterior midgut, but gradually moved towards the hindgut, in a time course reminiscent of the life cycle of Trypanosoma cruzi, a well-known pathogen transmitted by this insect. The maintenance of M. leprae infectivity inside the digestive tract of this kissing bug is further supported by successful mice footpad inoculation with feces collected 20 days after infection. We conclude that Rhodnius prolixus defecate infective M. leprae, justifying the evaluation of the presence of M. leprae among sylvatic and domestic kissing bugs in countries endemic for leprosy.

Apoptosis-inducing activity of clofazimine in macrophages.

Clofazimine is a riminophenazine compound which has been used for the treatment of leprosy since the 1960s. Although the drug is effective in the management of leprosy reactions because of its anti-inflammatory activity, the mechanism leading to the cessation of inflammation is not well understood. In the present study, it was shown that clofazimine exhibits apoptosis-inducing activity in macrophages. When human monocyte-derived macrophages were cultured in vitro in the presence of clofazimine, the cells exhibited a marked decrease in metabolic activity and showed shrinkage in cell size, indicating cell death. Nuclear condensation and fragmentation were also observed by Giemsa and Hoechst 33248 stains. The endonuclease inhibitor ZnCl(2) inhibited the clofazimine-induced cell death. Significant enhancement of caspase-3 activity was observed in clofazimine-treated macrophages and THP-1 cells. Collectively, these results suggest the apoptosis-inducing activity of clofazimine in macrophages, which may also be responsible for the antibacterial properties of clofazimine.

Diagnostic significance of colloid body deposition in direct immunofluorescence.

BACKGROUND: Colloid bodies (CB) in direct immunofluorescence (DIF) studies are usually found in interface dermatitis. Furthermore, CB can be found in various skin diseases and even in normal skin. AIM: To evaluate the diagnostic value of CB deposits in DIF studies. METHODS: From 1996-2007, data from 502 patients where DIF studies showed immunoreactants at CB were enrolled. The definite diagnoses of these patients were based on clinical, histopathological and immunofluorescent findings. The results of DIF studies were analyzed. RESULTS: Immunoreactants at CB were detected in 44.4%, 43.8%, 4.2%, 3.8%, and 2.2% of interface dermatitis, vasculitis, autoimmune vesiculobullous disease, panniculitis, and scleroderma/morphea, respectively. The most common immunoreactant deposit of all diseases was Immunoglobulin M (IgM). Brighter intensity and higher quantity of CB was detected frequently in the group with interface dermatitis. CONCLUSIONS: Immunoreactant deposits at CB alone can be found in various diseases but a strong intensity and high quantity favor the diagnosis of interface dermatitis. CB plus dermoepidermal junction (DEJ) deposits are more common in interface dermatitis than any other disease. Between lichen planus (LP) and discoid lupus erythematosus (DLE), CB alone is more common in LP; whereas, CB plus DEJ and superficial blood vessel (SBV) is more common in DLE. The most common pattern in both diseases is CB plus DEJ. The quantity and intensity of CB in LP is higher than in DLE.

Background noise of infection for using ANCA as a diagnostic tool for vasculitis in tropical and developing countries.

Antineutrophil cytoplasmic antibody (ANCA) is often used in the laboratory to confirm paucicellular vasculitis like Wegener's granulomatosis, Churg Strauss syndrome or polyarteritis nodosa in the presence of suggestive clinical features. In tropical countries, tuberculosis, leprosy and, occasionally, malaria can produce clinical features similar to a vasculitic illness and all the three infections are known to be associated with auto antibodies. We tested 318 patients suffering from malaria, tuberculosis or leprosy for ANCA positivity. ANCA positivity was found in 19%, 32% and 30% of malaria, tuberculosis and leprosy patients (Pradhan V, Badakere S, Shankarkumar V, Iyer Y, Ghosh K, Karnad D, Indian J Malariol, 39:51-59, 2002; Pradhan V, Badakere S, Ghosh K, Pawar A, Indian J Med Sci, 58:283-288, 2004a; Pradhan V, Badakere S, Shankarkumar V, Lepr Rev, 75:50-56, 2004b), respectively, raising the possibility that ANCA positivity with clinical features suggestive of vasculitis in tropical countries may even be related to the background noise of this seropositivity caused by one of these three infections rather than confirming the diagnosis of paucicellular vasculitis. Hence, one should be careful about the background noise of ANCA positivity caused by these infections while diagnosing a vasculitic illness.

M. leprae inhibits apoptosis in THP-1 cells by downregulation of Bad and Bak and upregulation of Mcl-1 gene expression.

BACKGROUND: Virulent Mycobacterium leprae interfere with host defense mechanisms such as cytokine activation and apoptosis. The mitochondrial pathway of apoptosis is regulated by the Bcl-2 family of proteins. Expression of Fas ligand and apoptotic proteins is found in leprosy lesions and M. leprae has been shown to activate pro-apoptotic Bcl-2 genes, Bak and Bax. However, the mechanism by which M. leprae modulates apoptosis is as yet unclear. We investigated expression of apoptotic genes in THP-1 monocytes in response to infection by M. leprae and non-pathogenic M. bovis BCG. RESULTS: M. leprae did not induce apoptosis in THP-1 cells, while BCG induced a significant loss of cell viability by 18 h post-infection at both (multiplicity of infection) MOI-10 and 20, with an increase by 48 h. BCG-induced cell death was accompanied by characteristic apoptotic DNA laddering in cells. Non-viable BCG had a limited effect on host cell death suggesting that BCG-induced apoptosis was a function of mycobacterial viability. M. leprae also activated lower levels of TNF-alpha secretion and TNF-alpha mRNA expression than BCG. Mycobacterium-induced activation of apoptotic gene expression was determined over a time course of infection. M. leprae reduced Bad and Bak mRNA expression by 18 h post-stimulation, with a further decrease at 48 h. Outcome of cell viability is determined by the ratio between pro- and anti-apoptotic proteins present in the cell. M. leprae infection resulted in downregulation of gene expression ratios, Bad/Bcl-2 mRNA by 39% and Bak/Bcl-2 mRNA by 23%. In contrast, live BCG increased Bad/Bcl-2 mRNA (29 %) but had a negligible effect on Bak/Bcl-2 mRNA. Heat killed BCG induced only a negligible (1-4 %) change in mRNA expression of either Bak/Bcl-2 or Bad/Bcl-2. Additionally, M. leprae upregulated the expression of anti-apoptotic gene Mcl-1 while, BCG downregulated Mcl-1 mRNA. CONCLUSION: This study proposes an association between mycobacterium-induced apoptosis in THP-1 cells and the regulation of Bcl-2 family of proteins. M. leprae restricts apoptosis in THP-1 cells by downregulation of Bad and Bak and upregulation of Mcl-1 mRNA expression.

Debaryomyces hansenii, a highly osmo-tolerant and halo-tolerant yeast, maintains activated Dhog1p in the cytoplasm during its growth under severe osmotic stress.

The HOG pathway is an important mitogen-activated protein kinase (MAPK) signal transduction pathway in Saccharomyces cerevisiae that mediates adaptation of cells to hyper-osmotic stress. Activation of this pathway causes rapid but transient, phosphorylation of the MAPK Hog1p. Phosphorylated Hog1p is rapidly transported to the nucleus that results in the transcription of target genes. The HOG pathway appears to be ubiquitous in yeast. Components of HOG pathway have also been identified in Debaryomyces hansenii, a highly osmotolerant and halotolerant yeast. We have studied activation of HOG pathway in D. hansenii under different stress conditions. Our experiments demonstrated that the pathway is activated by high osmolarity, oxidative and UV stress but not by heat stress. We have provided evidence, for the first time, that D. hansenii maintains phosphorylated Dhog1p in the cytoplasm during its growth under severe osmotic stress.

Insights into regulation of human Schwann cell proliferation by Erk1/2 via a MEK-independent and p56Lck-dependent pathway from leprosy bacilli.

Activation of extracellular signal-regulated kinase (Erk) 1/2, which plays a critical role in diverse cellular processes, including cell proliferation, is known to be mediated by the canonical Raf-mitogen-activated protein kinase kinase (MEK) kinase cascade. Alternative MEK-independent signaling pathways for Erk1/2 activation in mammalian cells are not known. During our studies of human primary Schwann cell response to long-term infection of Mycobacterium leprae, the causative organism of leprosy, we identified that intracellular M. leprae activated Erk1/2 directly by lymphoid cell kinase (p56Lck), a Src family member, by means of a PKCepsilon-dependent and MEK-independent signaling pathway. Activation of this signaling induced nuclear accumulation of cyclin D1, G1/S-phase progression, and continuous proliferation, but without transformation. Thus, our data reveal a previously unknown signaling mechanism of glial cell proliferation, which might play a role in dedifferentiation as well as nerve regeneration and degeneration. Our findings may also provide a potential mechanism by which an obligate intracellular bacterial pathogen like M. leprae subverts nervous system signaling to propagate its cellular niche for colonization and long-term bacterial survival.

Subcellular localization of mycobacteria in tissues and detection of lipid antigens in organelles using cryo-techniques for light and electron microscopy.

The survival of intracellular pathogens within a host is determined by microbial evasion, which can be partially attributed to their subcellular trafficking strategies. Microscopic techniques have become increasingly important in understanding the cell biology of microbial infections. These recently developed techniques can be used for the subcellular localization of antigens not only in cultured cells but also within tissues such as Mycobacterium tuberculosis in lung and Mycobacterium leprae in skin. High-resolution immunofluorescence microscopy can be used in combination with cryo-immunogold electron microscopy using consecutive cryo-sections on the same tissue block forming a direct connection between the two microscopy techniques. The detection of mycobacterial lipid antigens in situ at an ultrastructural level is currently a challenge, but new modifications can be used to address this. These methods might be of interest to microbiologists and cell biologists who study host-pathogen interactions.

Axonal spherical bodies in the peripheral nerves of leprosy patients.

Spherical bodies, roughly 10 micro m in diameter, which have not been reported before, were found in the peripheral nerve axons of specimens collected during post-mortem examination of leprosy patients. These bodies were found in the fascicles of all peripheral nerves of the extremities examined (median, radial, ulnar, peroneal and sciatic nerves). Their incidence was not related to the type of leprosy. The area immediately below the thickened perineurium, a feature associated with leprosy, often showed a large number of spherical bodies. When observed under a transmission electron microscope, the spherical lesions often showed a lamellar structure, although some of them were amorphous. No structure resembling organelles was seen within the bodies. Observation with the merge technique showed a clearly lamellar structure in most of the spherical bodies. These bodies and the surrounding myelin sheaths were partially polarized. The axonal spherical bodies observed in our study seem to represent lesions gradually formed due to glycoprotein denaturation over long periods of time and to be associated with leprosy-caused thickening of the perineurium of peripheral nerves.

Langerhans cells utilize CD1a and langerin to efficiently present nonpeptide antigens to T cells.

Langerhans cells (LCs) constitute a subset of DCs that initiate immune responses in skin. Using leprosy as a model, we investigated whether expression of CD1a and langerin, an LC-specific C-type lectin, imparts a specific functional role to LCs. LC-like DCs and freshly isolated epidermal LCs presented nonpeptide antigens of Mycobacterium leprae to T cell clones derived from a leprosy patient in a CD1a-restricted and langerin-dependent manner. LC-like DCs were more efficient at CD1a-restricted antigen presentation than monocyte-derived DCs. LCs in leprosy lesions coexpress CD1a and langerin, placing LCs in position to efficiently present a subset of antigens to T cells as part of the host response to human infectious disease.