José Daniel Lopes

Photodynamic therapy for acne vulgaris: A critical review from basics to clinical practice: Part I. Acne vulgaris: When and why consider photodynamic therapy?

UNLABELLED The first-line treatments for acne vulgaris are conventional topical and/or oral medications. However, many patients have contraindications, only partial response, significant adverse effects, or recurrence. Light-based treatments and photodynamic therapy (PDT) using topical precursors of porphyrins are off-label alternative treatments for acne vulgaris, with their own advantages and disadvantages. To date, there is no consensus on PDT methodology and parameters. An understanding of pathophysiology of acne, light-tissue interactions, and PDT mechanisms of action is helpful whenever PDT is considered as an alternative treatment. In general, blue light alone improves acne because of its antiinflammatory effects. PDT using 5-aminolevulenic acid (ALA) or ALA derivatives induces transient antimicrobial and antiinflammatory effects. At high doses, red light PDT may induce inhibition or destruction of sebaceous glands, resulting in clinical improvement. LEARNING OBJECTIVES After completing this learning activity, participants should be able to compare different treatments of acne, recognize when photodynamic therapy may be a useful off-label treatment for acne vulgaris, and identify variables that may affect the efficacy of photodynamic therapy.

GP43 from Paracoccidioides brasiliensis inhibits macrophage functions. An evasion mechanism of the fungus.

Macrophages constitute one of the primary cellular mechanisms that impairs parasite invasion of host tissues. The phagocytic and microbicidal properties of these cells can be modulated by specific membrane receptors involved in cell-microorganism interactions. Gp43, the main antigen secreted by Paracoccidiodes brasiliensis (Pb), the causative agent of Paracoccidioidomycosis, is a high mannose glycoprotein. The role played by gp43 in the pathogenesis of the disease is not completely known. Here, we describe the influence of this molecule on the interaction between peritoneal murine macrophages and Pb. Phagocytosis of Pb, live or heat-killed, by adherent peritoneal cells from both, B10.A (susceptible) and A/Sn (resistant) mice, was evaluated. Addition of different concentrations of gp43 to the culture medium inhibited, in a dose-dependent pattern, phagocytosis of live or heat-killed Pb by peritoneal macrophages from both B10.A and A/Sn mice. Gp43 also inhibits phagocytosis of zymosan particles but did not interfere with the uptake of opsonized sheep red blood cells. It was also shown that both gp43 and heat-killed Pb have an inhibitory effect on the release of NO by zymosan stimulated macrophages. Finally, we demonstrated that gp43 inhibits the fungicidal ability of macrophages from both lineages. Based on these data, it is suggested that gp43 can be considered one of the evasion mechanisms for the installation of primary infection in susceptible hosts.

Interleukin-10 secreted by B-1 cells modulates the phagocytic activity of murine macrophages in vitro

As demonstrated previously in our laboratory, B‐1 cells migrate from the peritoneal cavity of mice and home to a distant site of inflammation to become macrophage‐like cells. However, the influence that these cells might have on the kinetics and fate of the inflammatory process is not known. Considering that macrophages are pivotal in the inflammatory reaction, we decided to investigate the possible influence B‐1 cells could have on macrophage activities in vitro. Our results show that peritoneal macrophages from Xid mice, a mouse strain deprived of B‐1 cells, have higher phagocytic indexes for zymozan particles when compared with macrophages from wild‐type mice. Moreover, macrophages from wild‐type mice have a lower ability to release nitric oxide and hydrogen peroxide when compared with macrophages from Xid mice. Experiments using cocultures of B‐1 cells and macrophages from Xid mice in transwell plates demonstrated that B‐1 cells down‐regulate macrophage activities. These observations also indicate that this phenomenon is not due to a physical interaction between these two cell populations. As B‐1 cells are one of the main sources of interleukin (IL)‐10, we demonstrate in this study that adherent peritoneal cells from Xid mice produce significantly less amounts of this cytokine in culture when compared with IL‐10 production by cells from wild‐type mice. When B‐1 cells from IL‐10 knock‐out mice and macrophages from wild‐type mice were cocultured in transwell plates, the phagocytic index of macrophages was not altered demonstrating that B‐1 cells can influence the effector functions of macrophages in vitro via IL‐10 secretion.

Detection of circulating gp43 antigen in serum, cerebrospinal fluid, and bronchoalveolar lavage fluid of patients with paracoccidioidomycosis

ABSTRACT Paracoccidioidomycosis (PCM) is an important systemic fungal disease, particularly among individuals living and working in rural areas of endemicity in Latin America, who, without antifungal therapy, may develop fatal acute or chronic infection. For such patients, the detection of antibody responses by immunodiffusion is of limited value due to false-negative results. In contrast, the detection of Paracoccidioides brasiliensis gp43 circulating antigen may represent a more practical approach to the rapid diagnosis of the disease. Accordingly, an inhibition enzyme-linked immunosorbent assay (inh-ELISA) was developed for the detection of a 43-kDa P. brasiliensis-specific epitope incorporating a species-specific murine monoclonal antibody. With sera from patients with acute and chronic forms of the disease (n = 81), the overall sensitivity of the test was found to be 95.1%, while specificity was found to be 97.5% compared to that with normal human sera from blood donors (n = 93) and sera from patients with other chronic fungal infections (histoplasmosis [n = 33] and cryptococcosis [n = 20]). The inh-ELISA detected circulating antigen in 100% of patients with the acute form of PCM and in 95.31 and 100% of patients with the chronic multifocal and unifocal forms of PCM according to the patients clinical presentation. Cerebrospinal fluid from 14 patients with neuroparacoccidioidomycosis and 13 samples of bronchoalveolar lavage fluid from patients with pulmonary unifocal PCM were also tested for gp43 detection, with the test showing 100% sensitivity and specificity. This novel, highly specific inh-ELISA represents a significant addition to the existing tests for the diagnosis of PCM.

A Subset of Host B Lymphocytes Controls Melanoma Metastasis through a Melanoma Cell Adhesion Molecule/MUC18-Dependent Interaction: Evidence from Mice and Humans

Host immunity affects tumor metastasis but the corresponding cellular and molecular mechanisms are not entirely clear. Here, we show that a subset of B lymphocytes (termed B-1 population), but not other lymphocytes, has prometastatic effects on melanoma cells in vivo through a direct heterotypic cell-cell interaction. In the classic B16 mouse melanoma model, one mechanism underlying this phenomenon is a specific up-regulation and subsequent homophilic interaction mediated by the cell surface glycoprotein MUC18 (also known as melanoma cell adhesion molecule). Presence of B-1 lymphocytes in a panel of tumor samples from melanoma patients directly correlates with MUC18 expression in melanoma cells, indicating that the same protein interaction exists in humans. These results suggest a new but as yet unrecognized functional role for host B-1 lymphocytes in tumor metastasis and establish a biochemical basis for such observations. Our findings support the counterintuitive central hypothesis in which a primitive layer of the immune system actually contributes to tumor progression and metastasis in a mouse model and in melanoma patients. Given that monoclonal antibodies against MUC18 are in preclinical development but the reason for their antitumor activity is not well understood, these translational results are relevant in the setting of human melanoma and perhaps of other cancers.

Immune alterations after selective rapid eye movement or total sleep deprivation in healthy male volunteers.

We investigated the impact of two nights of total sleep deprivation (SD) or four nights of rapid eye movement (REM) SD on immunological parameters in healthy men. Thirty-two volunteers were randomly assigned to three protocols (control, total SD or REM SD). Both SD protocols were followed by three nights of sleep recovery. The control and REM SD groups had regular nights of sleep monitored by polysomnography. Circulating white blood cells (WBCs), T- (CD4/CD8) and B-lymphocytes, Ig classes, complement and cytokine levels were assessed daily. Two nights of total SD increased the numbers of leukocytes and neutrophils compared with baseline levels, and these levels returned to baseline after 24 h of sleep recovery. The CD4+ T-cells increased during the total SD period (one and two nights) and IgA levels decreased during the entire period of REM SD. These levels did not return to baseline after three nights of sleep recovery. Levels of monocytes, eosinophils, basophils and cytokines (IL-1β, IL-2, IL-4, IL-6, IL-10, TNF-α and IFN-γ) remained unchanged by both protocols of SD. Our findings suggest that both protocols affected the human immune profile, although in different parameters, and that CD4+ T-cells and IgA levels were not re-established after sleep recovery.

Characterization of gp70 and Anti-gp70 Monoclonal Antibodies in Paracoccidioides brasiliensis Pathogenesis

ABSTRACT Paracoccidioidomycosis (PCM) is a systemic granulomatous mycosis whose agent is Paracoccidioides brasiliensis. In the culture supernatant, the fungus expresses glycoproteins of from 13 to 148 kDa. A cell surface glycoprotein of 43 kDa is the major antigenic component of P. brasiliensis. Another expressed glycoprotein, gp70, is recognized by 96% of sera from PCM patients and is able to induce lymphoproliferation. Since, little is known about this glycoprotein, we produced monoclonal antibodies (MAbs) against gp70 to isolate the molecule from total fungus extracts and to investigate its possible role in the pathogenesis of PCM. Using these MAbs, it was observed by confocal microscopy that gp70 is located mainly in the intracellular compartment of the fungus, although it was also detected in the culture supernatant. Based on observations showing that gp43 has a down-regulatory effect on mouse peritoneal macrophages, we tested the effects of gp70 on their phagocytic ability. Purified gp70 was able to inhibit the activity of macrophages through the mannose receptors and also through the Fc receptors; the latter effect was not observed with gp43. gp70 inhibits NO and H2O2 liberation by peritoneal macrophages in vitro, as does gp43. Results obtained with gp43 led us to hypothesize that gp70 could act as an escape mechanism for fungal establishment in primary infections. To corroborate this hypothesis, we analyzed the effect of passive immunization of mice during infection with P. brasiliensis using anti-gp70 MAbs. This treatment almost completely abolished granuloma formation in the lungs, suggesting that the protein facilitates fungal establishment and progression of lesions in primary infection.

Co‐ordinated expression of lymphoid and myeloid specific transcription factors during B‐1b cell differentiation into mononuclear phagocytes in vitro

We previously demonstrated that B‐1b cells can undergo differentiation to acquire a mononuclear phagocyte phenotype upon attachment to substrate in vitro. Here we followed the expression of surface markers and transcription factors during this differentiation. B‐1b cells spontaneously express both myeloid and lymphoid restricted transcription factors. When induced to differentiate into a phagocyte, the lymphoid genes E box protein (E2A), early B‐cell factor (EBF), paired box 5 (Pax5) are down‐modulated, while expression of genes related to myeloid commitment is sustained. Furthermore, B‐1b cell‐derived phagocytes (B‐1CDPs) decrease immunoglobulin M (IgM) expression but retain the expression of the heavy chain variable gene VH11 or VH12, an immunoglobulin gene rearrangement predominantly expressed by B‐1 cells. The maintenance of lymphoid characteristics in B‐1CDPs characterizes a unique type of phagocyte, not related to monocyte‐derived macrophages.

Detection of Paracoccidioides brasiliensis gp70 Circulating Antigen and Follow-Up of Patients Undergoing Antimycotic Therapy

ABSTRACT Paracoccidioidomycosis (PCM), one of the most important systemic mycoses in Central and South America, is caused by the dimorphic fungus Paracoccidioides brasiliensis and has a high prevalence in Brazil. Glycoproteins of 43 and 70 kDa are the main antigenic compounds of P. brasiliensis and are recognized by Western blotting by 100 and 96% of PCM patient sera, respectively. In the present study, an inhibition enzyme-linked immunosorbent assay (ELISA) was used to detect gp70 in different biological samples from patients with PCM. gp70 was detected in 98.76% of 81 serum samples, with an average concentration of 8.19 μg/ml. The test was positive for 100% of the patients with the acute and chronic unifocal forms of PCM and 98.43% of the patients with the multifocal chronic form, with average concentrations of 11.86, 4.83, and 7.87 μg/ml, respectively. Bronchoalveolar lavage fluid from 23 patients with pulmonary unifocal PCM and 14 samples of cerebrospinal fluid from patients with neurological PCM were also tested for gp70 detection, with the test showing 100% sensitivity and 100% specificity, with mean gp70 concentrations of 7.5 and 6.78 μg/ml, respectively. To investigate the potential of gp70 detection by inhibition ELISA for the follow-up of PCM patients during antimycotic therapy with itraconazole (ITZ), the sera of 23 patients presenting with the chronic multifocal form of PCM were monitored at regular intervals of 1 month for 12 months. The results showed a decrease in circulating gp70 levels during treatment which paralleled the reduction in anti-P. brasiliensis antibody levels. The detection of P. brasiliensis gp70 from the biological fluids of patients suspected of having PCM proved to be a promising method for diagnosing infection and evaluating the efficacy of ITZ treatment.

B-1 cells facilitate Paracoccidioides brasiliensis infection in mice via IL-10 secretion.

Protective immunity in paracoccidioidomycosis is mainly mediated by cellular immunity. The role of B cells in this disease, in particular B-1 cells, is poorly understood. The aim of this study was to characterize the participation of B-1 cells in resistance or susceptibility of BALB/c and BALB/Xid mice to P. brasiliensis (Pb) pulmonary infection. BALB/Xid, which lacks B-1 cells, exhibited higher resistance to infection when compared with BALB/c mice. However, adoptive transfer of B-1 cells to BALB/Xid mice drastically increased the susceptibility of these animals to Pb infection. The fungal burden in BALB/c and B-1-reconstituted BALB/Xid was significantly higher as compared to BALB/Xid strain. Compact, well-organized granulomas were observed in the lungs of BALB/Xid mice, whereas large lesions with necrotic center with a plethora of fungi developed in BALB/c mice. It was also shown that B-1 cells impair phagocytosis of Pb by macrophages in vitro via secretion of IL-10, which was increased upon stimulation with a purified Pb antigen, gp43. Finally, in vivo blockade of IL-10 led to a better control of infection by the highly susceptible B10.A mouse. These findings suggest that B-1 cells play a major role in resistance/susceptibility to Pb infection in murine models, most likely via production of IL-10.