Antimicrobial Activity of Neutrophils Against Mycobacteria.

The mycobacterium genus contains a broad range of species, including the human pathogens M. tuberculosis and M. leprae. These bacteria are best known for their residence inside host cells. Neutrophils are frequently observed at sites of mycobacterial infection, but their role in clearance is not well understood. In this review, we discuss how neutrophils attempt to control mycobacterial infections, either through the ingestion of bacteria into intracellular phagosomes, or the release of neutrophil extracellular traps (NETs). Despite their powerful antimicrobial activity, including the production of reactive oxidants such as hypochlorous acid, neutrophils appear ineffective in killing pathogenic mycobacteria. We explore mycobacterial resistance mechanisms, and how thwarting neutrophil action exacerbates disease pathology. A better understanding of how mycobacteria protect themselves from neutrophils will aid the development of novel strategies that facilitate bacterial clearance and limit host tissue damage.

Oral administration of Debaryomyces hansenii CBS8339-ß-glucan induces trained immunity in newborn goats.

Beta-glucans from yeast can induce trained immunity in in vitro and in vivo models. Intraperitoneal doses of ß-glucans in mammals have shown to induce trained immunity, but the training effects of orally administering ß-glucans are unknown. Newborn goats are susceptible to infections in the neonatal stage, so the induction of trained immunity could improve animal survival. This study aimed to describe the in vitro effects of immunological training by ß-glucan from Debaryomyces hansenii (ß-Dh) on caprine monocytes, as well as its in vivo effects using oral doses on newborn goats upon challenge with lipopolysaccharide (LPS). Hence in vitro, goat monocytes trained with ß-Dh up-regulated the gene expression of macrophage surface markers (CD11b and F4/80) whereas enhanced cell survival and high phagocytic ability was found upon LPS challenge. In the in vivo experiment, newborn goats stimulated with two doses (day -7 and - 4) of ß-Dh (50 mg/kg) and challenged (day 0) with LPS showed an increase in respiratory burst activity, IL-1ß, IL-6, and TNFα production in plasma, and transcription of the macrophage surface markers. This study has demonstrated for the first time that trained immunity was induced with oral doses of ß-glucan upon LPS challenge in mammals using newborn goats.

M. leprae interacts with the human epidermal keratinocytes, neonatal (HEKn) via the binding of laminin-5 with α-dystroglycan, integrin-ß1, or -ß4.

Although Mycobacterium leprae (M.leprae) is usually found in macrophages and nerves of the dermis of patients with multibacillary leprosy, it is also present in all layers of the epidermis, basal, suprabasal, prickle cells, and keratin layers. However, the mechanism by which M.leprae invades the dermis remains unknown, whereas the underlying mechanism by which M.leprae invades peripheral nerves, especially Schwann cells, is well defined. M. leprae binds to the α-dystroglycan (DG) of Schwann cells via the interaction of α-DG and laminin (LN) -α2 in the basal lamina, thus permitting it to become attached to and invade peripheral nerves. In the current study, we investigated the issue of how M.leprae is phagocytosed by human epidermal keratinocytes, neonatal (HEKn). LN-5 is the predominant form of laminin in the epidermis and allows the epidermis to be stably attached to the dermis via its interaction with α/ß-DG as well as integrins that are produced by keratinocytes. We therefore focused on the role of LN-5 when M. leprae is internalized by HEKn cells. Our results show that M.leprae preferentially binds to LN-5-coated slides and this binding to LN-5 enhances its binding to HEKn cells. The findings also show that pre-treatment with an antibody against α-DG, integrin-ß1, or -ß4 inhibited the binding of LN-5-coated M.leprae to HEKn cells. These results suggest that M. leprae binds to keratinocytes by taking advantage of the interaction of LN-5 in the basal lamina of the epidermis and a surface receptor of keratinocytes, such as α-DG, integrin-ß1, or -ß4.

CR3 Engaged by PGL-I Triggers Syk-Calcineurin-NFATc to Rewire the Innate Immune Response in Leprosy.

Mycobacterium leprae, the causative agent of leprosy, is unique amongst human pathogens in its capacity to produce the virulence factor phenolic glycolipid (PGL)-I. In addition to mediating bacterial tropism for neurons, PGL-I interacts with Complement Receptor (CR)3 on macrophages (MPs) to promote infection. We demonstrate here that PGL-I binding to CR3 also enhances bacterial invasion of both polymorphonuclear neutrophils (PMNs) and dendritic cells (DCs). Moreover, in all cell types CR3 engagement by PGL-I activates the Syk tyrosine kinase, inducing calcineurin-dependent nuclear translocation of the transcription factor NFATc. This selectively augments the production of IL-2 by DCs, IL-10 by PMNs and IL-1ß by MPs. In intranasally-infected mice PGL-I binding to CR3 heightens mycobacterial phagocytosis by lung PMNs and MPs, and stimulates NFATc-controlled production of Syk-dependent cytokines. Our study thus identifies the CR3-Syk-NFATc axis as a novel signaling pathway activated by PGL-I in innate immune cells, rewiring host cytokine responses to M. leprae.

Debaryomyces hansenii CBS 8339 ß-glucan enhances immune responses and down-stream gene signaling pathways in goat peripheral blood leukocytes.

Debaryomyces hansenii-derived ß-glucan has shown immunostimulant effect on aquaculture species and recently on goat peripheral blood leukocytes. Moreover, the marine yeast D. hansenii CBS 8339 has demonstrated to enhance fish immune response. Nonetheless, the associated immune signaling pathways induced by ß-glucan from this marine yeast have not been characterized yet. This study described the effects of ß-glucan from D. hansenii CBS 8339 against challenge with Escherichia coli and activation of possible mechanisms on goat peripheral blood leukocytes. The proton nuclear magnetic resonance spectra showed that D. hansenii had ß-(1,3)(1,6)-glucan. The phagocytic ability enhanced after E. coli challenge, and nitric oxide production increased before and after challenge in leukocytes stimulated with D. hansenii ß-glucan. In addition, an early gene expression stimulation was found related to ß-glucan recognition by TLR2 and Dectin-1 receptors, intracellular regulation by Syk, TRAF6, MyD88 and transcription factor NFκB, and effector functions of pro-inflammatory cytokine, such as IL-1ß and TNF-α. Interestingly, simulation with D. hansenii-derived ß-glucan increased leukocyte viability after E. coli challenge. In conclusion, ß-glucan from D. hansenii CBS 8339 reduced cytotoxic effects of E. coli and modulated signaling pathways and innate immune response in goat peripheral blood leukocytes.

Genome-Wide Analysis of Protein-Coding Variants in Leprosy.

Although genome-wide association studies have greatly advanced our understanding of the contribution of common noncoding variants to leprosy susceptibility, protein-coding variants have not been systematically investigated. We carried out a three-stage genome-wide association study of protein-coding variants in Han Chinese, of whom were 7,048 leprosy patients and 14,398 were healthy control subjects. Seven coding variants of exome-wide significance were discovered, including two rare variants: rs145562243 in NCKIPSD (P = 1.71 × 10-9, odds ratio [OR] = 4.35) and rs149308743 in CARD9 (P = 2.09 × 10-8, OR = 4.75); three low-frequency variants: rs76418789 in IL23R (P = 1.03 × 10-10, OR = 1.36), rs146466242 in FLG (P = 3.39 × 10-12, OR = 1.45), and rs55882956 in TYK2 (P = 1.04 × 10-6, OR = 1.30); and two common variants: rs780668 in SLC29A3 (P = 2.17 × 10-9, OR = 1.14) and rs181206 in IL27 (P = 1.08 × 10-7, OR = 0.83). Discovered protein-coding variants, particularly low-frequency and rare ones, showed involvement of skin barrier and endocytosis/phagocytosis/autophagy, in addition to known innate and adaptive immunity, in the pathogenesis of leprosy, highlighting the merits of protein-coding variant studies for complex diseases.

The formation of lipid droplets favors intracellular Mycobacterium leprae survival in SW-10, non-myelinating Schwann cells.

Leprosy is a chronic infectious disease that is caused by the obligate intracellular pathogen Mycobacterium leprae (M.leprae), which is the leading cause of all non-traumatic peripheral neuropathies worldwide. Although both myelinating and non-myelinating Schwann cells are infected by M.leprae in patients with lepromatous leprosy, M.leprae preferentially invades the non-myelinating Schwann cells. However, the effect of M.leprae infection on non-myelinating Schwann cells has not been elucidated. Lipid droplets (LDs) are found in M.leprae-infected Schwann cells in the nerve biopsies of lepromatous leprosy patients. M.leprae-induced LD formation favors intracellular M.leprae survival in primary Schwann cells and in a myelinating Schwann cell line referred to as ST88-14. In the current study, we initially characterized SW-10 cells and investigated the effects of LDs on M.leprae-infected SW-10 cells, which are non-myelinating Schwann cells. SW-10 cells express S100, a marker for cells from the neural crest, and NGFR p75, a marker for immature or non-myelinating Schwann cells. SW-10 cells, however, do not express myelin basic protein (MBP), a marker for myelinating Schwann cells, and myelin protein zero (MPZ), a marker for precursor, immature, or myelinating Schwann cells, all of which suggests that SW-10 cells are non-myelinating Schwann cells. In addition, SW-10 cells have phagocytic activity and can be infected with M. leprae. Infection with M. leprae induces the formation of LDs. Furthermore, inhibiting the formation of M. leprae-induced LD enhances the maturation of phagosomes containing live M.leprae and decreases the ATP content in the M. leprae found in SW-10 cells. These facts suggest that LD formation by M. leprae favors intracellular M. leprae survival in SW-10 cells, which leads to the logical conclusion that M.leprae-infected SW-10 cells can be a new model for investigating the interaction of M.leprae with non-myelinating Schwann cells.

Debaryomyces hansenii up regulates superoxide dismutase gene expression and enhances the immune response and survival in Pacific red snapper (Lutjanus peru) leukocytes after Vibrio parahaemolyticus infection.

Application of yeast is increasing to improve welfare and promotes growth in aquaculture. The halotolerant yeast Debaryomyces hansenii is normally a non-pathogenic yeast with probiotic properties and potential source of antioxidant enzymes as superoxide dismutase. Here, first, we characterized the sequence features of MnSOD and icCu/ZnSOD from Pacific red snapper, and second, we evaluated the potential antioxidant immune responses of the marine yeast Debaryomyces hansenii strain CBS004 in leukocytes which were then subjected to Vibrio parahaemolyticus infection. In silico analysis revealed that LpMnSOD consisted of 1186 bp, with an ORF of 678 bp encoding a 225 amino acid protein and LpicCu/ZnSOD consisted of 1090 bp in length with an ORF of 465 bp encoding a 154 amino acid protein. Multiple alignment analyzes revealed many conserved regions and active sites among its orthologs. In vitro assays using head-kidney and spleen leukocytes immunostimulated with D. hansenii and zymosan in response to V. parahaemolyticus infection reveled that D. hansenii strain CBS004 significantly increased transcriptions of MnSOD and icCu/ZnSOD genes. Flow cytometry assay showed that D. hansenii was able to inhibit apoptosis caused by V. parahaemolyticus in the Pacific red snapper leukocytes and enhanced the phagocytic capacity in head-kidney leukocytes. Immunological assays reveled an increased in superoxide dismutase and peroxidase activities, as well as, in nitric oxide production and reactive oxygen species production (respiratory burst) in fish stimulated with D. hansenii. Finally, our results. These results strongly support the idea that marine yeast Debaryomyces hansenii strain CBS004 can stimulate the antioxidant immune mechanism in head-kidney and spleen leukocytes.

Trisaccharides of Phenolic Glycolipids Confer Advantages to Pathogenic Mycobacteria through Manipulation of Host-Cell Pattern-Recognition Receptors.

Despite mycobacterial pathogens continue to be a threat to public health, the mechanisms that allow them to persist by modulating the host immune response are poorly understood. Among the factors suspected to play a role are phenolic glycolipids (PGLs), produced notably by the major pathogenic species such as Mycobacterium tuberculosis and Mycobacterium leprae. Here, we report an original strategy combining genetic reprogramming of the PGL pathway in Mycobacterium bovis BCG and chemical synthesis to examine whether sugar variations in the species-specific PGLs have an impact on pattern recognition receptors (PRRs) and the overall response of infected cells. We identified two distinct properties associated with the trisaccharide domains found in the PGLs from M. leprae and M. tuberculosis. First, the sugar moiety of PGL-1 from M. leprae is unique in its capacity to bind the lectin domain of complement receptor 3 (CR3) for efficient invasion of human macrophages. Second, the trisaccharide domain of the PGLs from M. tuberculosis and M. leprae share the capacity to inhibit Toll-like receptor 2 (TLR2)-triggered NF-κB activation, and thus the production of inflammatory cytokines. Consistently, PGL-1 was found to also bind isolated TLR2. By contrast, the simpler sugar domains of PGLs from M. bovis and Mycobacterium ulcerans did not exhibit such activities. In conclusion, the production of extended saccharide domains on PGLs dictates their recognition by host PRRs to enhance mycobacterial infectivity and subvert the host immune response.

Elastophagocytosis and Elastolysis in Leprosy.

Elastophagocytosis is the engulfment of the elastic fibres by the histiocytes, multinucleated giant cells, or both. The cutaneous lesions showing elastophagocytosis are annular elastolytic giant cell granuloma, actinic keratoses, persistent insect-bite reactions, elastosis perforans serpiginosa, foreign body granuloma. Occasionally, it may occur in infectious diseases like leprosy, granulomatous syphilis, North-American blastomycosis, bacterial folliculitis, and cutaneous leishmaniasis. We report a case of lepromatous leprosy with necrotic erythema nodosum leprosum with secondary anetoderma. Histopathology from the atrophic macule of anetoderma revealed periappendageal, perineural infiltration, elastophagocytosis and reduction in elastic fibres.

Interaction of Mycobacterium leprae with the HaCaT human keratinocyte cell line: new frontiers in the cellular immunology of leprosy.

Leprosy is a chronic granulomatous disease caused by Mycobacterium leprae affecting the skin and peripheral nerves. Despite M. leprae invasion of the skin and keratinocytes importance in innate immunity, the interaction of these cells in vitro during M. leprae infection is poorly understood. Conventional and fluorescence optical microscopy, transmission electronic microscopy, flow cytometry and ELISA were used to study the in vitro interaction of M. leprae with the HaCaT human keratinocyte cell line. Keratinocytes uptake of M. leprae is described, and modulation of the surface expression of CD80 and CD209, cathelicidin expression and TNF-α and IL-1ß production of human keratinocytes are compared with dendritic cells and macrophages during M. leprae interaction. This study demonstrated that M. leprae interaction with human keratinocytes enhanced expression of cathelicidin and greatly increased TNF-α production. The highest spontaneous expression of cathelicidin was by dendritic cells which are less susceptible to M. leprae infection. In contrast, keratinocytes displayed low spontaneous cathelicidin expression and were more susceptible to M. leprae infection than dendritic cells. The results show, for the first time, an active role for keratinocytes during infection by irradiated whole cells of M. leprae and the effect of vitamin D on this process. They also suggest that therapies which target cathelicidin modulation may provide novel approaches for treatment of leprosy.

Phagocytosed Clofazimine Biocrystals Can Modulate Innate Immune Signaling by Inhibiting TNFα and Boosting IL-1RA Secretion.

Clofazimine (CFZ) is an FDA-approved leprostatic and anti-inflammatory drug that massively accumulates in macrophages, forming insoluble, intracellular crystal-like drug inclusions (CLDIs) during long-term oral dosing. Interestingly, when added to cells in vitro, soluble CFZ is cytotoxic because it depolarizes mitochondria and induces apoptosis. Accordingly, we hypothesized that, in vivo, macrophages detoxify CFZ by sequestering it in CLDIs. To test this hypothesis, CLDIs of CFZ-treated mice were biochemically isolated and then incubated with macrophages in vitro. The cell biological effects of phagocytosed CLDIs were compared to those of soluble CFZ. Unlike soluble CFZ, phagocytosis of CLDIs did not lead to mitochondrial destabilization or apoptosis. Rather, CLDIs altered immune signaling response pathways downstream of Toll-like receptor (TLR) ligation, leading to enhanced interleukin-1 receptor antagonist (IL-1RA) production, dampened NF-κB activation and tissue necrosis factor alpha (TNFα) production, and ultimately decreased TLR expression levels. In aggregate, our results constitute evidence that macrophages detoxify soluble CFZ by sequestering it in a biocompatible, insoluble form. The altered cellular response to TLR ligation suggests that CLDI formation may also underlie CFZ's anti-inflammatory activity.

Long-term survival and virulence of Mycobacterium leprae in amoebal cysts.

Leprosy is a curable neglected disease of humans caused by Mycobacterium leprae that affects the skin and peripheral nerves and manifests clinically in various forms ranging from self-resolving, tuberculoid leprosy to lepromatous leprosy having significant pathology with ensuing disfiguration disability and social stigma. Despite the global success of multi-drug therapy (MDT), incidences of clinical leprosy have been observed in individuals with no apparent exposure to other cases, suggestive of possible non-human sources of the bacteria. In this study we show that common free-living amoebae (FLA) can phagocytose M. leprae, and allow the bacillus to remain viable for up to 8 months within amoebic cysts. Viable bacilli were extracted from separate encysted cocultures comprising three common Acanthamoeba spp.: A. lenticulata, A. castellanii, and A. polyphaga and two strains of Hartmannella vermiformis. Trophozoites of these common FLA take up M. leprae by phagocytosis. M. leprae from infected trophozoites induced to encyst for long-term storage of the bacilli emerged viable by assessment of membrane integrity. The majority (80%) of mice that were injected with bacilli extracted from 35 day cocultures of encysted/excysted A. castellanii and A. polyphaga showed lesion development that was similar to mice challenged with fresh M. leprae from passage mice albeit at a slower initial rate. Mice challenged with coculture-extracted bacilli showed evidence of acid-fast bacteria and positive PCR signal for M. leprae. These data support the conclusion that M. leprae can remain viable long-term in environmentally ubiquitous FLA and retain virulence as assessed in the nu/nu mouse model. Additionally, this work supports the idea that M. leprae might be sustained in the environment between hosts in FLA and such residence in FLA may provide a macrophage-like niche contributing to the higher-than-expected rate of leprosy transmission despite a significant decrease in human reservoirs due to MDT.

Effect of apoptotic cell recognition on macrophage polarization and mycobacterial persistence.

Intracellular Mycobacterium leprae infection modifies host macrophage programming, creating a protective niche for bacterial survival. The milieu regulating cellular apoptosis in the tissue plays an important role in defining susceptible and/or resistant phenotypes. A higher density of apoptotic cells has been demonstrated in paucibacillary leprosy lesions than in multibacillary ones. However, the effect of apoptotic cell removal on M. leprae-stimulated cells has yet to be fully elucidated. In this study, we investigated whether apoptotic cell removal (efferocytosis) induces different phenotypes in proinflammatory (Mϕ1) and anti-inflammatory (Mϕ2) macrophages in the presence of M. leprae. We stimulated Mϕ1 and Mϕ2 cells with M. leprae in the presence or absence of apoptotic cells and subsequently evaluated the M. leprae uptake, cell phenotype, and cytokine pattern in the supernatants. In the presence of M. leprae and apoptotic cells, Mϕ1 macrophages changed their phenotype to resemble the Mϕ2 phenotype, displaying increased CD163 and SRA-I expression as well as higher phagocytic capacity. Efferocytosis increased M. leprae survival in Mϕ1 cells, accompanied by reduced interleukin-15 (IL-15) and IL-6 levels and increased transforming growth factor beta (TGF-ß) and IL-10 secretion. Mϕ1 cells primed with M. leprae in the presence of apoptotic cells induced the secretion of Th2 cytokines IL-4 and IL-13 in autologous T cells compared with cultures stimulated with M. leprae or apoptotic cells alone. Efferocytosis did not alter the Mϕ2 cell phenotype or cytokine secretion profile, except for TGF-ß. Based on these data, we suggest that, in paucibacillary leprosy patients, efferocytosis contributes to mycobacterial persistence by increasing the Mϕ2 population and sustaining the infection.

Interaction of Mycobacterium leprae with human airway epithelial cells: adherence, entry, survival, and identification of potential adhesins by surface proteome analysis.

This study examined the in vitro interaction between Mycobacterium leprae, the causative agent of leprosy, and human alveolar and nasal epithelial cells, demonstrating that M. leprae can enter both cell types and that both are capable of sustaining bacterial survival. Moreover, delivery of M. leprae to the nasal septum of mice resulted in macrophage and epithelial cell infection in the lung tissue, sustaining the idea that the airways constitute an important M. leprae entry route into the human body. Since critical aspects in understanding the mechanisms of infection are the identification and characterization of the adhesins involved in pathogen-host cell interaction, the nude mouse-derived M. leprae cell surface-exposed proteome was studied to uncover potentially relevant adhesin candidates. A total of 279 cell surface-exposed proteins were identified based on selective biotinylation, streptavidin-affinity purification, and shotgun mass spectrometry; 11 of those proteins have been previously described as potential adhesins. In vitro assays with the recombinant forms of the histone-like protein (Hlp) and the heparin-binding hemagglutinin (HBHA), considered to be major mycobacterial adhesins, confirmed their capacity to promote bacterial attachment to epithelial cells. Taking our data together, they suggest that the airway epithelium may act as a reservoir and/or portal of entry for M. leprae in humans. Moreover, our report sheds light on the potentially critical adhesins involved in M. leprae-epithelial cell interaction that may be useful in designing more effective tools for leprosy control.

[Phagocytosis of Mycobacterium leprae down-regulates anti-microbial activity of murine macrophages against Mycobacterium intracellulare].

Patients with highly bacillated lepromatous leprosy (LL) essentially lack T cell-mediated immune responses specific to Mycobacterium leprae (ML) antigens, resulting in severely impaired host resistance to leprosy bacilli. Such type of immune unresponsiveness characteristic of LL patients is mainly attributable to markedly depressed T cell ability to activate/expand in response to ML antigens. In this study, we examined profiles of antimycobacterial activity of macrophages, which phagocytized leprosy bacilli, because there is another possibility that, in LL patients, host macrophages in the leprosy lesions are impaired in their antimicrobial activity due to their interaction with infected leprosy bacilli, particularly cellular events through binding with and/or internalization of the pathogens, thereby causing the reduction in host resistance to ML pathogens. The present study indicated the following. First, the anti-M. avium complex activity of murine peritoneal macrophages was significantly reduced when they had phagocytosed heat-killed leprosy bacilli. Second, infection of macrophages with leprosy bacilli did not affect macrophage-mediated suppressor activity against T cell proliferative response to Concanavalin A. These findings indicate that macrophage's intracellular signaling pathways that are up-regulated in response to phagocytosis of leprosy bacilli are linked to the signaling cascades participating in macrophage antimicrobial functions, but not cross-talk with those allowing the expression of macrophage's suppressor activity against T cell functions.

Identification of a novel gene product that promotes survival of Mycobacterium smegmatis in macrophages.

BACKGROUND: Bacteria of the suborder Corynebacterineae include significant human pathogens such as Mycobacterium tuberculosis and M. leprae. Drug resistance in mycobacteria is increasingly common making identification of new antimicrobials a priority. Mycobacteria replicate intracellularly, most commonly within the phagosomes of macrophages, and bacterial proteins essential for intracellular survival and persistence are particularly attractive targets for intervention with new generations of anti-mycobacterial drugs. METHODOLOGY/PRINCIPAL FINDINGS: We have identified a novel gene that, when inactivated, leads to accelerated death of M. smegmatis within a macrophage cell line in the first eight hours following infection. Complementation of the mutant with an intact copy of the gene restored survival to near wild type levels. Gene disruption did not affect growth compared to wild type M. smegmatis in axenic culture or in the presence of low pH or reactive oxygen intermediates, suggesting the growth defect is not related to increased susceptibility to these stresses. The disrupted gene, MSMEG_5817, is conserved in all mycobacteria for which genome sequence information is available, and designated Rv0807 in M. tuberculosis. Although homology searches suggest that MSMEG_5817 is similar to the serine:pyruvate aminotransferase of Brevibacterium linens suggesting a possible role in glyoxylate metabolism, enzymatic assays comparing activity in wild type and mutant strains demonstrated no differences in the capacity to metabolize glyoxylate. CONCLUSIONS/SIGNIFICANCE: MSMEG_5817 is a previously uncharacterized gene that facilitates intracellular survival of mycobacteria. Interference with the function of MSMEG_5817 may provide a novel therapeutic approach for control of mycobacterial pathogens by assisting the host immune system in clearance of persistent intracellular bacteria.

[Annular elastolytic giant-cell granuloma]. / Granulome annulaire élastolytique à cellules géantes.

BACKGROUND: Annular elastolytic giant-cell granuloma (AEGCG) is a rare form of granulomatous dermatosis characterized by annular plaques with central atrophy and raised erythematous margins and is usually located on the facial and neck areas. It is characterized histologically by loss of elastic fibre and elastophagocytosis. We report a case of AEGCG. CASE REPORT: A 72-year-old man consulted for annular plaques, some of which were atrophic, and papules that had been present for 2 years. The lesions involved sun-exposed and non-sun-exposed skin. The biopsies showed granulomatous infiltrates and discrete elastophagocytosis. After ruling out various differential clinical and histological diagnoses, the patient was diagnosed with AEGCG. DISCUSSION: We report a case of AEGCG. Diagnosis was not easy. The differential diagnoses of this entity were discussed and we ruled out actinic granuloma, sarcoidosis, leprosy, and granuloma annulare. Our patient presented the classical annular variant combined with a papular variant. We report the first case involving response to isotretinoin.

In vitro and in vivo immunostimulatory activity of Woodfordia fruticosa flowers on non-specific immunity.

CONTEXT: Woodfordia fruticosa Kurz. (Lythraceae), a non-rasayana immunomodulatory Indian medicinal plant, used traditionally as an anthelmintic, in dysentery, leprosy, blood diseases, leucorrhea, and menorrhagia. OBJECTIVE: To investigate the effect of ethanol extract of W. fruticosa flowers on non-specific immune responses in mice. MATERIALS AND METHODS: In vitro immunomodulatory activity of the extract was examined on murine peritoneal macrophage phagocytosis (nitroblue tetrazolium (NBT) dye reduction, lysosomal enzyme activity, nitric oxide and myeloperoxidase) and on proliferation of bone marrow cells by sulforhodamine B (SRB) assay, while the in vivo potential on macrophages and bone marrow cells was evaluated by using carbon clearance test and cyclophosphamide-induced myelosuppression, respectively. RESULTS: Significant increase in the release of myeloperoxidase, nitric oxide lysosomal enzyme and superoxide from macrophages along with significant increase in phagocytic index in carbon clearance test indicate stimulatory activity of the extract on macrophages. The extract also demonstrated 60% increase in bone marrow cell proliferation and offer protection towards cyclophosphamide-induced myelosuppression which represents the stimulation of bone marrow activity. DISCUSSION: Significant increase in mediators released from macrophages and phagocytic index in carbon clearance test suggests the release of cytokines from macrophages and stimulation of reticulo-endothelial system. Proliferation of bone marrow cells indicates the plausible release of colony stimulating factors, which further stimulates the immune system through generation of immune cells. CONCLUSION: The result described here indicates the immunostimulatory activity of ethanol extract of W. fruticosa flowers by stimulating non-specific immune responses, macrophages and bone marrow cells.

Suppression of ROS generation by 4,4-diaminodiphenylsulfone in non-phagocytic human diploid fibroblasts.

The action mode of 4,4-diaminodiphenylsulfone (DDS) is still under debate, although it has long been used in treatment of several dermatologic diseases including Hansens disease. In this study, we tested the effect of DDS as an antioxidant on paraquat-induced oxidative stress in non-phagocytic human diploid fibroblasts (HDFs). Overall, preincubation of HDFs with DDS prevented the oxidative stress and the resulting cytotoxic damages caused by paraquat in these cells. The specific effects of DDS in paraquat-treated HDFs are summarized as follows: a) reducing the expression of NADPH oxidase 4 (NOX4) by inhibiting paraquat-induced activation of PKC; b) inhibiting paraquat-induced decreases in mitochondrial complex protein levels as well as in membrane potentials; c) consequently, inhibiting the generation of cytosolic and mitochondrial superoxide anions. Taken together, these findings suggest that DDS would suppress the radical generation in non-phagocytic HDFs during oxidative stress, and that DDS might have the extended potential to be used further in prevention of other oxidative stress-related pathologies.