Small-fibre neuropathy in leprosy the role of in vivo confocal microscopy: A cross-sectional study.

Background Leprosy (or Hansen's disease) continues to present considerable challenges regarding containment and early diagnosis. Leprosy is considered to be primarily a neural disease that first affects the sensory function of small fibres. Although the condition is well described in terms of clinical manifestations and histology, few studies have been undertaken to detect damage done to small-fibre sensory nerves. In vivo confocal microscopy is a useful tool for conducting a detailed evaluation of these structures, although its use in individuals affected by leprosy has still not been explored. Objective To evaluate in vivo confocal microscopy findings in Hansen's disease patients and their association with clinical variables relating to this disease. Method A cross-sectional case-series type study was carried out between October 2019 and May 2021, in Recife, Pernambuco, Brazil. Socio-demographic and clinical data were gathered from 21 patients with leprosy. The douleur neuropathique 4 neuropathic pain questionnaire was used to evaluate pain. In vivo confocal microscopy of the cornea was employed to evaluate the small-calibre fibres. Findings were compared with those for a control group of 23 healthy individuals. Results In relation to clinical parameters, 90.5% of the patients were classified as "multibacillary" according to the World Health Organization criteria, and 70% as dimorphic or borderline, in accordance with the Madrid classification. Around 52.4% had received a diagnosis after one year or less of living with the disease, while 95.2% presented alterations in small-fibre sensory function and 35% presented such alterations in the large fibre. Neuropathic pain was present in 81% of the patients. In vivo confocal microscopy found no statistically significant difference in mean age and distribution according to sex between the Hansen disease patients and the control group of healthy individuals. The median-of-means for dendritic cells and volume of sub-basal nerve fibres in the control group were used to test for normality. Both eyes of all leprosy patients examined contained higher number of dendritic cells than the median value and a volume of sub-basal nerve fibres lower than the mean. These differences were statistically significant (P < 0.001 and P < 0.001, respectively). Multibacillary individuals had a median number of dendritic cells two times that of paucibacillary individuals (P = 0.035). Limitations No association was found between the variables examined using in vivo confocal microscopy and clinical variables relating to small-fibre damage, the neuropathic pain questionnaire or alterations detected by the neurological examination. We believe, however, that Cochet-Bonnet esthesiometry of the cornea may have revealed such an association. Conclusion In vivo confocal microscopy is a useful diagnostic tool for detecting small fibre loss in individuals affected by leprosy and may constitute a useful addition to the range of tools available to help curb the effects of neuropathy in these patients.

Utility of photodynamic diagnosis plus reflectance confocal microscopy in detecting the margins of extramammary Paget disease.

BACKGROUND: Due to the clinically poorly delineated unclear margin of extramammary Paget disease, the recurrence rate after surgical resection is high. AIMS: To compare photodynamic diagnosis and photodynamic plus reflectance confocal microscopy diagnosis in determining the tumor margins in patients with extramammary Paget disease. METHODS: Thirty-six patients with histopathologically confirmed primary extramammary Paget disease between January 2017 to June 2018 were included in the study. The skin lesion margins were preoperatively observed by the naked eye and with photodynamic diagnosis and photodynamic diagnosis plus reflectance confocal microscopy and they were compared to the postoperative histopathological examination results. RESULTS: Among the 130 sections taken from 36 patients, 83 sections (63.8%, 83/130) had tumor margins beyond the macroscopic line with a distance of 3.5 ± 3.1mm and a median of 2.7mm. Forty-six sections (35.4%, 46/130) exceeded the photodynamic diagnosis marker line with a distance of 2.1 ± 1.7mm and a median of 1.5mm. Twenty seven sections (20.8%, 27/130) were obtained beyond the photodynamic diagnosis plus reflectance confocal microscopy marker line with a distance of 1.4 ± 1.2mm and a median of 0.9mm. LIMITATIONS: Photodynamic diagnosis and reflectance confocal microscopy detection can be used to observe only the superficial margin of the tumor and not the deep part. Moreover, reflectance confocal microscopy was not used alone as a control. CONCLUSION: In terms of determining the extramammary Paget disease margin invasively, photodynamic diagnosis and photodynamic diagnosis plus reflectance confocal microscopy were found superior to observations made with the naked eye, while photodynamic diagnosis plus reflectance confocal microscopy was superior to photodynamic diagnosis alone.

Diagnosis of subclinical extramammary Paget's disease with a combination of noninvasive photodynamic diagnosis and reflectance confocal microscopy.

Extramammary Paget's disease is a rare skin malignancy, and its diagnosis requires invasive biopsy and histopathological examination. Surgery is the standard treatment for extramammary Paget's disease patients; however, as incision boundaries and the depth of tumor cell infiltration are often unclear, the postoperative recurrence rate is high. We present a case in which we used photodynamic diagnosis in combination with reflectance confocal microscopy before surgery to detect an extramammary Paget's disease lesion that was located 3 cm away from the classical lesion. This secondary lesion exhibited a subclinical presentation, and it was eventually confirmed as an extramammary Paget's disease lesion by pathological examination. During detection using our technique, we delineated the boundaries of the extramammary Paget's disease lesion as a guide for surgical excision. The findings of our case demonstrate that photodynamic diagnosis combined with reflectance confocal microscopy can be used for the noninvasive diagnosis of subclinical extramammary Paget's disease and may be used to guide strategies for planning treatment and preventing relapse.

Novel Diagnostics for Kaposi Sarcoma and Other Skin Diseases in Resource-Limited Settings.

In resource-limited settings, point-of-care diagnostic devices have the potential to reduce diagnostic delays and improve epidemiologic surveillance of dermatologic conditions. We outline novel-point-of care diagnostics that have recently been developed for dermatologic conditions that primarily affect patients living in resource-limited settings, namely, Kaposi sarcoma, cutaneous leishmaniasis, leprosy, Buruli ulcer, yaws, onchocerciasis, and lymphatic filariasis. All of the technologies described in this article are prototypes, and some have undergone field testing. These devices still require validation in real-world settings and effective pricing to have a major impact on dermatologic care in resource-limited settings.

Distinct Roles of Th17 and Th1 Cells in Inflammatory Responses Associated with the Presentation of Paucibacillary Leprosy and Leprosy Reactions.

It is well established that helper T cell responses influence resistance or susceptibility to Mycobacterium leprae infection, but the role of more recently described helper T cell subsets in determining severity is less clear. To investigate the involvement of Th17 cells in the pathogenesis of leprosy, we determined the immune profile with variant presentations of leprosy. Firstly, IL-17A, IFN-γ and IL-10 were evaluated in conjunction with CD4+ T cell staining by confocal microscopy of lesion biopsies from tuberculoid (TT) and lepromatous leprosy (LL) patients. Secondly, inflammatory cytokines were measured by multiplex assay of serum samples from Multibacillary (MB, n = 28) and Paucibacillary (PB, n = 23) patients and household contacts (HHC, n = 23). Patients with leprosy were also evaluated for leprosy reaction occurrence: LR+ (n = 8) and LR- (n = 20). Finally, peripheral blood mononuclear cells were analysed by flow cytometry used to determine the phenotype of cytokine-producing cells. Lesions from TT patients were found to have more CD4+ IL-17A+ cells than those from LL patients. Higher concentrations of IL-17A and IL-1ß were observed in serum from PB than MB patients. The highest serum IFN-γ concentrations were, however, detected in sera from MB patients that developed leprosy reactions (MB LR+ ). Together, these results indicate that Th1 cells were associated with both the PB presentation and also with leprosy reactions. In contrast, Th17 cells were associated with an effective inflammatory response that is present in the PB forms but were not predictive of leprosy reactions in MB patients.

Jagged1 Instructs Macrophage Differentiation in Leprosy.

As circulating monocytes enter the site of disease, the local microenvironment instructs their differentiation into tissue macrophages (MΦ). To identify mechanisms that regulate MΦ differentiation, we studied human leprosy as a model, since M1-type antimicrobial MΦ predominate in lesions in the self-limited form, whereas M2-type phagocytic MΦ are characteristic of the lesions in the progressive form. Using a heterotypic co-culture model, we found that unstimulated endothelial cells (EC) trigger monocytes to become M2 MΦ. However, biochemical screens identified that IFN-γ and two families of small molecules activated EC to induce monocytes to differentiate into M1 MΦ. The gene expression profiles induced in these activated EC, when overlapped with the transcriptomes of human leprosy lesions, identified Jagged1 (JAG1) as a potential regulator of MΦ differentiation. JAG1 protein was preferentially expressed in the lesions from the self-limited form of leprosy, and localized to the vascular endothelium. The ability of activated EC to induce M1 MΦ was JAG1-dependent and the addition of JAG1 to quiescent EC facilitated monocyte differentiation into M1 MΦ with antimicrobial activity against M. leprae. Our findings indicate a potential role for the IFN-γ-JAG1 axis in instructing MΦ differentiation as part of the host defense response at the site of disease in human leprosy.

Mycobacterial tlyA gene product is localized to the cell-wall without signal sequence.

The mycobacterial tlyA gene product, Rv1694 (MtbTlyA), has been annotated as "hemolysin" which was re-annotated as 2'-O rRNA methyl transferase. In order to function as a hemolysin, it must reach the extracellular milieu with the help of signal sequence(s) and/or transmembrane segment(s). However, the MtbTlyA neither has classical signals sequences that signify general/Sec/Tat pathways nor transmembrane segments. Interestingly, the tlyA gene appears to be restricted to pathogenic strains such as H37Rv, M. marinum, M. leprae, than M. smegmatis, M. vaccae, M. kansasii etc., which highlights the need for a detailed investigation to understand its functions. In this study, we have provided several evidences which highlight the presence of TlyA on the surface of M. marinum (native host) and upon expression in M. smegmatis (surrogate host) and E. coli (heterologous host). The TlyA was visualized at the bacterial-surface by confocal microscopy and accessible to Proteinase K. In addition, sub-cellular fractionation has revealed the presence of TlyA in the membrane fractions and this sequestration is not dependent on TatA, TatC or SecA2 pathways. As a consequence of expression, the recombinant bacteria exhibit distinct hemolysis. Interestingly, the MtbTlyA was also detected in both membrane vesicles secreted by M. smegmatis and outer membrane vesicles secreted by E. coli. Our experimental evidences unambiguously confirm that the mycobacterial TlyA can reach the extra cellular milieu without any signal sequence. Hence, the localization of TlyA class of proteins at the bacterial surface may highlight the existence of non-classical bacterial secretion mechanisms.

IL-27 Suppresses Antimicrobial Activity in Human Leprosy.

The mechanisms by which intracellular pathogens trigger immunosuppressive pathways are critical for understanding the pathogenesis of microbial infection. One pathway that inhibits host defense responses involves the induction of type I interferons and subsequently IL-10, yet the mechanism by which type I IFN induces IL-10 remains unclear. Our studies of gene expression profiles derived from leprosy skin lesions suggested a link between IL-27 and the IFN-ß induced IL-10 pathway. Here, we demonstrate that the IL-27p28 subunit is upregulated following treatment of monocytes with IFN-ß and Mycobacterium leprae, the intracellular bacterium that causes leprosy. The ability of IFN-ß and M. leprae to induce IL-10 was diminished by IL-27 knockdown. Additionally, treatment of monocytes with recombinant IL-27 was sufficient to induce the production of IL-10. Functionally, IL-27 inhibited the ability of IFN-γ to trigger antimicrobial activity against M. leprae in infected monocytes. At the site of disease, IL-27 was more strongly expressed in skin lesions of patients with progressive lepromatous leprosy, correlating and colocalizing with IFN-ß and IL-10 in macrophages. Together, these data provide evidence that in the human cutaneous immune responses to microbial infection, IL-27 contributes to the suppression of host antimicrobial responses.

T-cell regulation in lepromatous leprosy.

Regulatory T (Treg) cells are known for their role in maintaining self-tolerance and balancing immune reactions in autoimmune diseases and chronic infections. However, regulatory mechanisms can also lead to prolonged survival of pathogens in chronic infections like leprosy and tuberculosis (TB). Despite high humoral responses against Mycobacterium leprae (M. leprae), lepromatous leprosy (LL) patients have the characteristic inability to generate T helper 1 (Th1) responses against the bacterium. In this study, we investigated the unresponsiveness to M. leprae in peripheral blood mononuclear cells (PBMC) of LL patients by analysis of IFN-γ responses to M. leprae before and after depletion of CD25+ cells, by cell subsets analysis of PBMC and by immunohistochemistry of patients' skin lesions. Depletion of CD25+ cells from total PBMC identified two groups of LL patients: 7/18 (38.8%) gained in vitro responsiveness towards M. leprae after depletion of CD25+ cells, which was reversed to M. leprae-specific T-cell unresponsiveness by addition of autologous CD25+ cells. In contrast, 11/18 (61.1%) remained anergic in the absence of CD25+ T-cells. For both groups mitogen-induced IFN-γ was, however, not affected by depletion of CD25+ cells. In M. leprae responding healthy controls, treated lepromatous leprosy (LL) and borderline tuberculoid leprosy (BT) patients, depletion of CD25+ cells only slightly increased the IFN-γ response. Furthermore, cell subset analysis showed significantly higher (p = 0.02) numbers of FoxP3+ CD8+CD25+ T-cells in LL compared to BT patients, whereas confocal microscopy of skin biopsies revealed increased numbers of CD68+CD163+ as well as FoxP3+ cells in lesions of LL compared to tuberculoid and borderline tuberculoid leprosy (TT/BT) lesions. Thus, these data show that CD25+ Treg cells play a role in M. leprae-Th1 unresponsiveness in LL.

Number and brightness analysis of LRRK2 oligomerization in live cells.

Leucine-rich repeat kinase 2 (LRRK2) is a large multidomain protein that contains enzymatically functional GTPase and kinase domains. Several noncoding LRRK2 gene polymorphisms have been associated with susceptibility to Parkinson's disease (PD), Crohn's disease, and leprosy. Many LRRK2 coding polymorphisms have been associated with or causally linked to PD. The G2019S point mutation within the LRRK2 kinase domain is the most common cause of familial PD. The G2019S mutation appears to alter LRRK2 kinase activity. Some but not all studies have reported that LRRK2 kinase activity is dependent upon LRRK2 dimerization and membrane localization. It is important to define the oligomeric state(s) of LRRK2 in living cells, which to date have only been characterized in vitro. Here we use confocal and total internal reflection microscopy coupled with number and brightness analysis to study the oligomeric states of LRRK2 within the cytosol and on the plasma membrane of live CHO-K1 cells. Our results show, for the first time to our knowledge, that LRRK2 is predominantly monomeric throughout the cytosol of living cells, but attains predominately higher oligomeric states in the plasma membrane.

TLR6-driven lipid droplets in Mycobacterium leprae-infected Schwann cells: immunoinflammatory platforms associated with bacterial persistence.

The mechanisms responsible for nerve injury in leprosy need further elucidation. We recently demonstrated that the foamy phenotype of Mycobacterium leprae-infected Schwann cells (SCs) observed in nerves of multibacillary patients results from the capacity of M. leprae to induce and recruit lipid droplets (LDs; also known as lipid bodies) to bacterial-containing phagosomes. In this study, we analyzed the parameters that govern LD biogenesis by M. leprae in SCs and how this contributes to the innate immune response elicited by M. leprae. Our observations indicated that LD formation requires the uptake of live bacteria and depends on host cell cytoskeleton rearrangement and vesicular trafficking. TLR6 deletion, but not TLR2, completely abolished the induction of LDs by M. leprae, as well as inhibited the bacterial uptake in SCs. M. leprae-induced LD biogenesis correlated with increased PGE(2) and IL-10 secretion, as well as reduced IL-12 and NO production in M. leprae-infected SCs. Analysis of nerves from lepromatous leprosy patients showed colocalization of M. leprae, LDs, and cyclooxygenase-2 in SCs, indicating that LDs are sites for PGE(2) synthesis in vivo. LD biogenesis Inhibition by the fatty acid synthase inhibitor C-75 abolished the effect of M. leprae on SC production of immunoinflammatory mediators and enhanced the mycobacterial-killing ability of SCs. Altogether, our data indicated a critical role for TLR6-dependent signaling in M. leprae-SC interactions, favoring phagocytosis and subsequent signaling for induction of LD biogenesis in infected cells. Moreover, our observations reinforced the role of LDs favoring mycobacterial survival and persistence in the nerve. These findings give further support to a critical role for LDs in M. leprae pathogenesis in the nerve.

Sidestream dark field imaging: the evolution of real-time visualization of cutaneous microcirculation and its potential application in dermatology.

Technological advances during the last years have enhanced the image quality of the microcirculation. Intravital microscopy (IM) has been considered the "gold standard" for many years, but it can be used mostly in anesthetized animals which is a disadvantage. The nailfold videocapillaroscopy, a non-invasive examination that includes a microscope with an epiillumination system, came afterward, but its major disadvantage is the restricted area available for investigation namely the nailfold capillary bed. The orthogonal polarization spectral (OPS) imaging technique, where reflected light allows the visualization of the microcirculation, was the next non-invasive exam, but it still presents some drawbacks such as suboptimal capillary visualization and image blurring due to red blood cell movements. Excessive probe pressure modifies red blood cell velocity. There is suboptimal imaging of capillaries due to motion-induced image blurring by movements of OPS device, tissue and/or flowing red blood cells. Sidestream dark field (SDF) imaging is the newest tool for microcirculatory research. Illumination is provided by concentrically placed light-emitting diodes to avoid image blurring and to enhance image contrast. It represents a simple and non-invasive imaging technique, with low cost, good portability and high sensitivity that provides fine, well-defined images. In addition, the microcirculation can be studied through laser Doppler flowmetry (LDF) or reflectance-mode confocal-laser-scanning microscopy (RCLM). However, LDF cannot show microcirculatory vessels and high cost of RCLM can be an inconvenience. New applications of SDF technique could include skin microcirculatory evaluation and allow dermatological studies on psoriasis, skin tumors and leprosy.

High matrix metalloproteinase production correlates with immune activation and leukocyte migration in leprosy reactional lesions.

Gelatinases A and B (matrix metalloproteinase 2 [MMP-2] and MMP-9, respectively) can induce basal membrane breakdown and leukocyte migration, but their role in leprosy skin inflammation remains unclear. In this study, we analyzed clinical specimens from leprosy patients taken from stable, untreated skin lesions and during reactional episodes (reversal reaction [RR] and erythema nodosum leprosum [ENL]). The participation of MMPs in disease was suggested by (i) increased MMP mRNA expression levels in skin biopsy specimens correlating with the expression of gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha), (ii) the detection of the MMP protein and enzymatic activity within the inflammatory infiltrate, (iii) increased MMP levels in patient sera, and (iv) the in vitro induction of MMP-9 by Mycobacterium leprae and/or TNF-alpha. It was observed that IFN-gamma, TNF-alpha, MMP-2, and MMP-9 mRNA levels were higher in tuberculoid than lepromatous lesions. In contrast, interleukin-10 and tissue inhibitor of MMP (TIMP-1) message were not differentially modulated. These data correlated with the detection of the MMP protein evidenced by immunohistochemistry and confocal microscopy. When RR and ENL lesions were analyzed, an increase in TNF-alpha, MMP-2, and MMP-9, but not TIMP-1, mRNA levels was observed together with stronger MMP activity (zymography/in situ zymography). Moreover, following in vitro stimulation of peripheral blood cells, M. leprae induced the expression of MMP-9 (mRNA and protein) in cultured cells. Overall, the present data demonstrate an enhanced MMP/TIMP-1 ratio in the inflammatory states of leprosy and point to potential mechanisms for tissue damage. These results pave the way toward the application of new therapeutic interventions for leprosy reactions.

Metabolon formation in dhurrin biosynthesis.

Synthesis of the tyrosine derived cyanogenic glucoside dhurrin in Sorghum bicolor is catalyzed by two multifunctional, membrane bound cytochromes P450, CYP79A1 and CYP71E1, and a soluble UDPG-glucosyltransferase, UGT85B1 (Tattersall, D.B., Bak, S., Jones, P.R., Olsen, C.E., Nielsen, J.K., Hansen, M.L., Høj, P.B., Møller, B.L., 2001. Resistance to an herbivore through engineered cyanogenic glucoside synthesis. Science 293, 1826-1828). All three enzymes retained enzymatic activity when expressed as fluorescent fusion proteins in planta. Transgenic Arabidopsis thaliana plants that produced dhurrin were obtained by co-expression of CYP79A1/CYP71E1-CFP/UGT85B1-YFP and of CYP79A1/CYP71E1/UGT85B1-YFP but not by co-expression of CYP79A1-YFP/CYP71E-CFP/UGT85B1. The lack of dhurrin formation upon co-expression of the two cytochromes P450 as fusion proteins indicated that tight interaction was necessary for efficient substrate channelling. Transient expression in S. bicolor epidermal cells as monitored by confocal laser scanning microscopy showed that UGT85B1-YFP accumulated in the cytoplasm in the absence of CYP79A1 or CYP71E1. In the presence of CYP79A1 and CYP71E1, the localization of UGT85B1 shifted towards the surface of the ER membrane in the periphery of biosynthetic active cells, demonstrating in planta dhurrin metabolon formation.