Makrophagen sind professionelle phagozytische Zellen, welche körpereigene gealterte oder toten Zellen und in den Körper eingedrungene Krankheitserreger aufnehmen. Die phagozytierten Partikel werden von lysosomalen Hydrolasen abgebaut und daraus hervorgehende Antigene an Zellen des spezifischen Immunsystems präsentiert. Aufgabe der lysosomalen Phospholipase A1 (PLA1) ist der Abbau von Phospholipiden. Sie spielt damit nicht nur eine elementare Rolle bei dem Abbau von Phospholipidmembranen nach Phago- und Autophagozytose, sondern kann auch an der Generation von Lipidantigenen beteiligt sein. Die vorliegende Arbeit bietet zum ersten Mal Hinweise auf die Sequenz der lysosomalen PLA1. Mittels proteinbiochemischer Reinigung und nachfolgender massenspektrometrischer Sequenzanalyse wurden zwei Proteinkandidaten identifiziert, welche der lysosomalen PLA1-Aktivität zugrunde liegen können. Des Weiteren werden ausführliche Untersuchungen zu den Katalyseeigenschaften des Enzyms an Liposomen präsentiert. Die Lipidzusammensetzung der Membran beeinflusst maßgeblich die Aktivität der lysosomalen PLA1. So haben in die Membran integrierte anionische Phospholipide eine stark enzymaktivierende Wirkung. Eine Erhöhung der Ionenstärke oder des pH-Wertes vermindern die Bindungsfähigkeit der lysosomalen PLA1 an die Membran und damit deren Aktivität. Dies lässt vermuten, dass elektrostatische Wechselwirkungen eine Rolle bei der Membranbindung spielen. Das Enzym besitzt pIs / Macrophages are professional phagocytes which engulf and degrade senescent and dead cells as well as pathogens. Phagocytosed particles are subsequently degraded by lysosomal enzymes. The lysosomal phospholipase A1 (PLA1) degrades phospholipids, the major components of biological membranes and, hence, plays a mandatory role in decomposition of phagocytosed and autophagocytosed membranes. Furthermore the enzyme might play a role in the processing of lipid antigens for immune presentation. Nevertheless, the gene encoding this important enzyme activity is as yet unknown. Here, we used proteinbiochemical methods to isolate the lysosomal PLA1 activity from RAW B cells and identified resulting sequences by tandem mass spectrometry. This analysis revealed for the first time two putative protein candidates responsible for lysosomal PLA1 activity. Using native enzyme fractions and liposome-embedded substrate, we show that PLA1 activity depends on the presence of anionic phospholipids, low pH and low ionic strength. Lysosomal PLA1 only attaches to membranes with anionic but not zwitterionic charges. High ionic strength impairs binding demonstrating that electrostatic attraction is responsible for membrane partitioning. Upon binding the enzyme remains on membranes for numerous catalytic cycles. The enzyme’s pIs at

HIV remains an incurable viral infection and a significant global health concern. Despite the advent of antiretroviral therapy, there are 36.9 million recorded cases of HIV worldwide, with an additional 1.8 million new infections recorded in 2017 alone. An HIV cure is therefore one of several priorities within the field, and will require HIV “reservoir” cells—comprised of latently-HIV infected CD4+ T cells and productively-infected, tissue resident macrophages—to be selectively killed in vivo. HIV reservoir cells are rarely found within the peripheral circulation, residing instead within inaccessible tissue sanctuaries. Consequently, their characterization has been limited to in vitro laboratory models. To complicate matters further, a definitive cellular surface marker of HIV infected cells has yet to be identified. Impairment of the type I interferon (IFN1) response has been observed during HIV infection, however, making it a unique intracellular maker of HIV-infected cells. The recent development of oncolytic viruses (OV) designed to selectively kill IFN-defective cancer cells also suggests that these IFN1 defects possess therapeutic value. It was therefore hypothesized that the impairment of the IFN1 response in HIV-infected CD4+ cells and macrophages could serve as a target for oncolytic virus-mediated killing. The induction of several antiviral IFN-stimulated proteins, including PKR and ISG15, was inhibited in HIV-infected monocyte-derived macrophages (MDM) following stimulation with IFNα or a synthetic RNA. Consequently, HIV-infected MDM were more susceptible to infection and killing by the oncolytic Maraba virus, MG1. Importantly, MG1-mediated killing required the presence of replication-competent OV, and could not be potentiated by UV-inactivated MG1 or supernatants from MG1-infected cells. The ability of MG1 to target the HIV reservoir was further confirmed using alveolar macrophages collected from the lungs of cART-suppressed individuals living with HIV. These findings indicate that IFN1 defects are a feature of HIV infected cells, which can be exploited for selective killing by OV. This project is therefore unique in that it demonstrates that HIV reservoir cells can be eradicated in a targeted manner by exploiting an intracellular marker of HIV infection. As MG1-based cancer therapies are currently being explored in Phase I/II clinical trials, there is potential for this approach to be adapted for use within the HIV cure field.

Die Polarisierung von Makrophagen resultiert in einer Vielzahl von Subtypen, z.B. M1 oder M2. In dieser Studie lege ich die Makrophagen-Polarisierung im allergischen Asthma mit Fokus auf microRNA (miRNA) dar. Nach Etablierung von Subtyp–charakteristischen mRNA und miRNA Expressionsprofilen in vitro polarisierter, humaner blutstämmiger Makrophagen wurden diese Profile genutzt, um den Polarisierungsstatus isolierter Lungenmakrophagen in einem Mausmodell des Asthmas zu ermitteln. Es wurden humane blutstämmige Makrophagen in vitro durch IFNg/LPS (M1) bzw. IL4/IL13 (M2) polarisiert. M1-assoziierte Gene waren TNFa, IL6 und IL1b, während in M2 Makrophagen eine Induktion von CD209 und PPARg beobachtet wurde. Herauf-regulierte miRNAs waren z.B. hsa-miR-187-3p, hsa-miR-155-5p und hsa-miR-146a-5p (M1) bzw. hsa-miR-193b-3p und hsa-miR-511-5p (M2). Eine in silico Vorhersage von mRNA/miRNA Interaktionspartnern wurde in einem Luciferase Reportermodell überprüft, das u.a. hsa-miR-187-3p als Regulator von SH2B2 und hsa-miR-187-3p sowie hsa-miR-155-5p als kooperative Regulatoren von LAMP2 bestätigte. Unter physiologischen Bedingungen regulierte hsa-miR-187-3p die SH2B2 mRNA herunter, aber es lag kein Einfluß von hsa-miR-187-3p oder hsa-miR-155-5p auf LAMP2 mRNA oder Protein vor. Weiterhin wurden die miRNA Profile von murinen Makrophagen erhoben, die aus der bronchoalveolären Lavage und aus Lungengewebe gewonnen wurden. Diese Profile wurden in einer vergleichenden Analyse von gesunden Mäusen und Mäusen mit akuter Ovalbumin-induzierter eosinophiler Atemwegsentzündung eingesetzt. In Reaktion auf Ovalbumin regulierte miRNAs waren z.B. mmu-miR-21a-5p und mmu-miR-155-5p (heraufreguliert), sowie mmu-miR-126-3p und mmu-miR-146a-5p (herunterreguliert). Sowohl M1 als auch M2 assoziierte Muster zeigten sich vor allem in der reziproken Regulation von mmu-miR-155-5p (heraufreguliert) und mmu-miR-146a-5p (herunterreguliert). / Macrophage polarization gives rise to a plethora of macrophage subtypes, e.g. M1 or M2. In this thesis, I aim to point out some features of macrophage polarization in allergic asthma with a focus on microRNA (miRNA). The chosen approach started with the establishment of subtype-characteristic mRNA and miRNA profiles of in vitro polarized human macrophages. In a second step, the miRNA patterns were used to interpret the polarization status of isolated lung macrophages from a murine model of asthma. In vitro polarization of human blood-derived macrophages was performed by IFNgLPS (M1) and IL4/IL13 (M2), and global mRNA and miRNA profiling ensued. M1-induced genes included TNFa, IL6 and IL1b, whereas in M2 macrophages, CD209 and PPARg were induced. M1-associated miRNAs were hsa-miR-187-3p, hsa-miR-155-5p and hsa-miR-146a-5p, while hsa-miR-193b-3p and hsa-miR-511-5p were induced in M2 macrophages. In silico prediction of mRNA/miRNA interaction partners was experimentally validated in a luciferase-based reporter assay that confirmed hsa-miR-187-3p as a regulator of SH2B2 and the pair of hsa-miR-187-3p and hsa-miR-155-5p as cooperative regulators of LAMP2. Under physiologic conditions, hsa-miR-187-3p was able to down-regulate SH2B2 transcript, but there was no impact of either hsa-miR-187-3p or hsa-miR-155-5p on LAMP2 mRNA or protein. Furthermore, the miRNA profiles of murine macrophages from the bronchoalveolar lavage fluid and from lung tissue were established and compared between healthy mice and mice with acute Ovalbumin-induced eosinophilic airway inflammation. Individual miRNAs responding to Ovalbumin were e.g. mmu-miR-21a-5p and mmu-miR-155-5p (up-regulated), and mmu-miR-126-3p and mmu-miR-146a-5p (down-regulated). Characteristics of both M1- and M2-associated miRNA patterns were most evident in the concomitant reciprocal expression of mmu-miR-155-5p (up-regulated) and mmu-miR-146a-5p (down-regulated).

INTRODUÇÃO: Os macrófagos associados aos tumores (MAT) sólidos estão relacionados à progressão ou à involução das neoplasias, dependendo da diferenciação em M1 ou M2. No carcinoma basocelular (CBC), as formas mais agressivas apresentam aumento de macrófagos às custas do fenótipo M2, se comparadas às formas não invasivas. O tratamento do CBC sólido pela Cirurgia Micrográfica de Mohs (CMM) proporciona elevados índices de cura, porém recidivas podem ocorrer. OBJETIVOS: Comparar a população total de macrófagos e as subpopulações M1 e M2 nos casos de CBC sólidos recidivados e não recidivados após exérese pela CMM. METODOLOGIA: Cortes histológicos obtidos a partir dos blocos de parafina de nove casos de CBC sólidos recidivados após CMM e de 18 casos de CBC sólido operados pela CMM não recidivados foram marcados imunoistoquimicamente para iNOS, CD204, CD163 e CD68. A expressão desses marcadores foi analisada pelo método de análise de imagens. RESULTADOS: Não foram encontradas diferenças significativas entre os grupos em relação à porcentagem média de células M1 (INOS), células M2 (CD163 e CD204) e total de células (CD68). CONCLUSÃO: A recidiva dos tumores estudados não ocorreu por influência do MAT, mas pode ser decorrente da falha técnica na realização da CMM ou de algum outro mecanismo imunológico desconhecido / INTRODUCTION: The macrophages associated with solid tumors (MAT) are related to the progression or regression of tumors, depending on the differentiation in M1 or M2. In basal cell carcinoma (BCC), the most aggressive forms show an increase in macrophages at the expense of M2 phenotype compared to non-invasive forms. The treatment of BCC solid by Mohs micrographic surgery (MMS) provides high cure rates, but relapses can occur. OBJECTIVES: To compare the total population of macrophages and subpopulations M1 and M2 in cases of recurrent BCC solid and not recurrent after excision by MMS. METHODS: Histological sections obtained from paraffin blocks of 9 cases of recurrent solid CBC after MMS and 18 cases of solid CBC operated by MMS not relapsed were labeled immunohistochemically for iNOS, CD204, CD163 and CD68. The expression of these markers was analyzed by image analysis. RESULTS: No significant differences were found between the groups in relation to the average percentage of M1 cells (INOS), M2 cells (CD163 and CD204) and total cells (CD68). CONCLUSION: The recurrence of the tumors studied did not occur under the influence of MAT, but may be due to technical failure in achieving MMS or some other unknown immune mechanism

Os venenos de serpentes contêm concentrações elevadas de fosfolipases A2 secretadas (sFLA2), que apresentam homologia com as FLA2s de mamíferos, cujos níveis estão aumentados em doenças inflamatórias. Neste estudo, investigou-se a ativação e a expressão de fatores envolvidos na formação de corpúsculos lipídicos (CLs) em células fagociticas e o papel desses fatores na resposta imune inata, induzida pela MT-III, uma sFLA2s de veneno. A MT-III induziu aumento dos níveis de triacilglicerol, colesterol e lisofosfolipideos e a ativação e expressão dos fatores PPAR-g, PPAR-d/b, SREBP2 e do CD36. Sob estimulo da MT-III, o receptor PPAR-b/d, as enzimas DGAT, ACAT e FAS foram relevantes para a formação de CLs e para a expressão da PLIN2. O CD36 participa da expressão da COX-2, sem modificar a liberação de PGE2. O TLR2 e a MyD88 foram essenciais para a formação de CLs e síntese da IL-1b e IL-10. Ainda, o TLR2 foi relevante para a liberação de PGE2, PGD2 e LTB4, enquanto MyD88 foi fundamental somente para a liberação de PGE2 e expressão da PLIN2, induzidas pela MT-III. / Snake venoms contain high concentrations of secreted phospholipase A2 (sPLA2) with homology to mammalian PLA2s, whose levels are elevated in inflammatory diseases. In this study, we investigated activation and expression of factors involved in lipid droplets formation (LDs) and participation that factors in the innate immune response induced by MT-III, sPLA2s from snake venom, in phagocytic cells. MT-III induced increase of triacylglycerol, cholesterol and lysophospholipids levels and activation and expression of factors PPAR-g, PPAR-d/b, SREBP2 and CD36. PPAR-b/d receptor, DGAT, ACAT and FAS enzymes were relevant to LDs formation and critical to PLIN2 expression induced by MT-III. CD36 participates in COX-2 expression without modifying PGE2 release stimulated by MT-III. TLR2 and MyD88 were essential to LDs formation and IL-1b and IL-10 synthesis stimulated by MT-III. Moreover, TLR2 was relevant to PGE2, PGD2 and LTB4 biosynthesis, while MyD88 is essential only for PGE2 release and PLIN2 expression induced by MT-III.

O Histoplasma capsulatum é um fungo dimórfico, patogênico e responsável por graves lesões pulmonares. A infecção é adquirida pela inalação de conídios e posterior conversão para leveduras nos alvéolos e bronquíolos, onde são fagocitadas por macrófagos alveolares residentes e leucócitos que migram para o local da infecção. Recentemente, demonstramos que animais infectados com H. capsulatum e tratados com inibidor da síntese de prostaglandinas apresentaram diminuição de carga fúngica nos pulmões e baço, aumento da produção de nitrito e da fagocitose de leveduras por macrófagos alveolares, e maior sobrevivência, quando comparados com os animais somente infectados. Porém, neste estudo não foram determinados quais subtipos de prostaglandinas participam na patogênese da histoplasmose. Vários grupos de pesquisa têm demonstrado que PGD2 e PGE2 podem ter ações biológicas distintas quanto à remoção de microrganismos no hospedeiro. Desta maneira, é fundamental o entendimento do papel da PGD2 e da PGE2 nos mecanismos efetores dos macrófagos na defesa do hospedeiro, especialmente na histoplasmose. Portanto, o objetivo deste estudo foi investigar a participação da PGD2 e PGE2 na infecção experimental por H. capsulatum. Assim, demonstramos que a PGD2 aumentou a fagocitose e mecanismos microbicidas de macrófagos alveolares infectados in vitro com H. capsulatum. Observamos ainda que a 15dPGJ2, metabólito da PGD2, aumentou somente a fagocitose, e PGE2 inibiu os mecanismos efetores do macrófago. Mostramos ainda o aumento de BLT1 em macrófagos alveolares após adição de PGD2, e a possível ligação desta ao BLT1, e de LTB4 em DP2. Além disso, caracterizamos micropartículas de PLGA contendo PGD2 (MS-PGD2), e investigamos seus efeitos. O tamanho, carga elétrica e morfologia das micropartículas foram adequados para um tratamento intranasal e para fagocitose por macrófagos alveolares. As MS-PGD2 foram fagocitadas e capazes de ativar NF-B, e consequentemente, influenciar na produção de nitrito, IL-1, TNF-, IL-6 e TGF-. Com base nestes dados, avaliamos os efeitos do tratamento da MS-PGD2 ou da MS-PGE2 em animais infectados com H. capsulatum. Estas foram administradas via intranasal em animais infectados e tratados ou não com celecoxibe. Verificamos a diminuição da carga fúngica nos pulmões e baço, diminuição do infiltrador celular no espaço broncoalveolar e de citocinas inflamatórias no pulmão após tratamento com MS-PGD2. Contrariamente, após tratamento da MS-PGE2 observamos maior carga fúngica nos pulmões e baço, e aumento da inflamação no tecido e maior produção de IL-10. Além disso, demonstramos que no 21° dia após infecção, referente ao 7° dia após o término do tratamento com MS-PGD2, a carga fúngica manteve-se reduzida nos pulmões, comprovando assim a eficácia deste tratamento. Posteriormente, utilizando inibidores específicos, HQL-79 e CAY10526, mostramos respectivamente o papel protetor da PGD2 e o deletério da PGE2 na histoplasmose. Em conjunto, nossos dados contribuíram para o entendimento das funções antagônicas da PGD2 e PGE2 nesta micose. / Histoplasma capsulatum is a pathogenic dimorphic fungus and responsible for severe pulmonary lesions. Infection is acquired by inhalation of conidia and posterior conversion to yeasts in the alveoli and bronchioles, in which they are phagocyted by resident alveolar macrophages and leukocytes that migrate to the local infection. Recently, we demonstrate that mice infected by H. capsulatum and treated with inhibitor of prostaglandins synthesis presented a decrease in fungal burden in lungs and spleen, increase in nitrite production and uptake of yeasts by alveolar macrophages, and more survival, when compared with animals only infected. However, in this study, it was not determined what subtypes of prostaglandins participate in pathogenesis of histoplasmosis. Many research groups have demonstrated that PGD2 and PGE2 can have different biological effects regarding to microorganisms elimination in the host. Thus, it is primordial the understanding about the role of PGD2 and PGE2 on effector mechanisms of macrophages in host defense, especially in histoplasmosis. Therefore, the aim of this study was to investigate the role of PGD2 and PGE2 on experimental infection by H. capsulatum. So, we verify that PGD2 increased the uptake and microbicidal mechanisms of alveolar macrophages infected in vitro by H. capsulatum. 15dPGJ2, a PGD2 metabolite, increased only the phagocytosis, and PGE2 inhibited the effector mechanisms of macrophages. Among these results, we showed an increase of BLT1 expression on alveolar macrophages after addition of PGD2, and a possible binding of this mediator to BLT1, and of LTB4 to DP2. Later, as tool of therapeutic investigation, we used PGD2 encapsulation in biodegradable polymer, PLGA, in order to preserve its stability. Size, zeta potential and morphology were adequate for a possible intranasal treatment and uptake by alveolar macrophages. MS-PGD2 were phagocyted and able to activate NF-B, and consequently, to modulate nitrite, IL-1, TNF-, IL-6 and TGF- production. In this context, we purpose a treatment of the infection with MS-PGD2, in comparison to treatment with PGE2. MS-PGD2 were administrated via intranasal in infected mice, treated or not with celecoxib. We verify a decrease of fungal burden in lungs and spleen, less cellular infiltrate and decrease of some inflammatory cytokines. In contrast, after treatment of MS-PGE2, we observed greater fungal burden in the lungs and spleen, and an increase of the tissue inflammation and production of IL-10. Furthermore, we show that on day 21 after infection, referring to the 7th day after the treatment with MS-PGD2, fungal burden remained reduced in the lungs, thus proving the effectiveness of the treatment. Subsequently, using specific inhibitors, HQL-79 and CAY10526, respectively show the protective role of PGD2 and in deleterious to PGE2 in histoplasmosis. Together, our data contribute to the understanding of the antagonistic functions of PGD2 and PGE2 in this mycosis.

Macrophages are leukocytes of the innate immune system that display great phenotypic plasticity to mediate diverse functions. The ontogeny of tissue resident macrophages has been debated in recent decades. It is now recognised that tissue macrophages can be replenished from embryonically-derived precursors, and/or monocyte intermediates in a tissue specific manner. Interferon Regulatory Factor 5 (IRF5) is a transcription factor that promotes a pro-inflammatory phenotype in macrophages in vitro and in vivo. Indeed, IRF5 contributes to the pathogenesis of experimental inflammatory arthritis, lupus, and obesity via recruitment and activation of effector cells. Research described here as part of this thesis, involves the profiling of the intestinal Mononuclear Phagocyte system to investigate the role of IRF5 in the development of monocyte-derived macrophages in the Colonic Lamina Propria (cLP) which are exclusively replenished by adult Ly6C^hi monocytes. Using Mixed Bone Marrow Chimaeras (MBMCs) we showed that in shared environment Wild-Type (WT) cLP macrophages dominated IRF5-deficient (Irf5^-/-) cLP macrophages in both steady state and inflammation. The development of in vitro bone marrow derived macrophages, and the reconstitution of the haematopoietic compartment in bone marrow of MBMCs were not significantly affected by IRF5 deficiency. IRF5 promoted the accumulation of WT monocytes in the cLP of MBMCs in a process possibly dependent on the CCL2/CCR2 axis. Furthermore, IRF5 expression committed Ly6C^hi monocytes to a pro-inflammatory macrophage fate in the inflamed cLP, characterised by protein expression of the cytokines IL1β, and TNFα, and the expression of Ccl4 and Ccl8 transcripts, whilst loss of IRF5 favoured accumulation of CD11b⁺ IRF4-dependent Dendritic Cells. Of significance, IRF5 expression might have prevented further differentiation of inflammatory macrophages into tissue-resident macrophages, thus supporting an inflammatory state. Irf5-/- mice were protected from Helicobacter hepaticus + αIL10R colitis. Intriguingly, protection from colitis may also be conferred by the presence of Irf5-/- haematopoietic cells, evidenced by WT:Irf5-/- MBMCs . Modulation of IRF5 activity may therefore be a viable therapeutic strategy. RNA sequencing identified that C1q, Cd81, and Ccl8 were upregulated in WT macrophages from MBMC, which may prove therapeutic targets.

The diagnosis of cancer in early stages is an unmet clinical need, especially in view that current treatments for cancer cannot address metastatic disease. Cancer aberration processes are associated to an increase in the production of reactive oxygen species (ROS). Chemical probes that can specifically detect these species are potentially useful as medical diagnostics and research tools for cancer imaging. One of the aims of my thesis was the design and synthesis of the activatable fluorescent probes based on small molecule fluorophores modified with chemically reactive moieties. The activation of these moieties by defined targets (e.g. ROS) results in the activation of the fluorophore and subsequent emission of a fluorescent signal. Two libraries of fluorescence probes for the detection of ROS have been designed and synthesised: 1) hydrocyanine-based probes as silent fluorophores that can be activated with superoxide ions, 2) coumarin-based hydrogen peroxide probes with red-shifted fluorescent properties and different boronate activatable groups for hydrogen peroxide sensing. We have performed in vitro assays to evaluate the fluorescence response of our probes as well as experiments in relevant live cells to assess their application for detection of ROS in live cells with molecular resolution. Moreover, cancer cells also overexpress Epidermal Growth Factor Receptors (EGFR). Surface-enhanced Raman scattering (SERS) nanotags that can recognize specifically EGFR receptors in cells are promising tools for the enhanced diagnosis of cancer. Two near-infrared cyanine Raman reporters were synthesized with a carboxylic group that was conjugated to cysteamine for derivatization of gold nanoparticles (AuNPs). This work was performed in the CSIR-NIIST (Kerala, India), where I did a 3-month PhD placement. I conjugated the cyanine reporters to spherical AuNPs of 40 nm diameter, and measured their Raman intensity and stability. The best SERS nanotags were selected for encapsulation with PEG and subsequently derivatization with anti-EGFR-EP22 antibodies. In vitro characterization of the SERS nanotags was performed: SERS and absorbance spectra, electron microscopy images as well as SERS imaging experiments in A549 lung cancer cells.

INTRODUÇÃO: Os macrófagos associados aos tumores (MAT) sólidos estão relacionados à progressão ou à involução das neoplasias, dependendo da diferenciação em M1 ou M2. No carcinoma basocelular (CBC), as formas mais agressivas apresentam aumento de macrófagos às custas do fenótipo M2, se comparadas às formas não invasivas. O tratamento do CBC sólido pela Cirurgia Micrográfica de Mohs (CMM) proporciona elevados índices de cura, porém recidivas podem ocorrer. OBJETIVOS: Comparar a população total de macrófagos e as subpopulações M1 e M2 nos casos de CBC sólidos recidivados e não recidivados após exérese pela CMM. METODOLOGIA: Cortes histológicos obtidos a partir dos blocos de parafina de nove casos de CBC sólidos recidivados após CMM e de 18 casos de CBC sólido operados pela CMM não recidivados foram marcados imunoistoquimicamente para iNOS, CD204, CD163 e CD68. A expressão desses marcadores foi analisada pelo método de análise de imagens. RESULTADOS: Não foram encontradas diferenças significativas entre os grupos em relação à porcentagem média de células M1 (INOS), células M2 (CD163 e CD204) e total de células (CD68). CONCLUSÃO: A recidiva dos tumores estudados não ocorreu por influência do MAT, mas pode ser decorrente da falha técnica na realização da CMM ou de algum outro mecanismo imunológico desconhecido / INTRODUCTION: The macrophages associated with solid tumors (MAT) are related to the progression or regression of tumors, depending on the differentiation in M1 or M2. In basal cell carcinoma (BCC), the most aggressive forms show an increase in macrophages at the expense of M2 phenotype compared to non-invasive forms. The treatment of BCC solid by Mohs micrographic surgery (MMS) provides high cure rates, but relapses can occur. OBJECTIVES: To compare the total population of macrophages and subpopulations M1 and M2 in cases of recurrent BCC solid and not recurrent after excision by MMS. METHODS: Histological sections obtained from paraffin blocks of 9 cases of recurrent solid CBC after MMS and 18 cases of solid CBC operated by MMS not relapsed were labeled immunohistochemically for iNOS, CD204, CD163 and CD68. The expression of these markers was analyzed by image analysis. RESULTS: No significant differences were found between the groups in relation to the average percentage of M1 cells (INOS), M2 cells (CD163 and CD204) and total cells (CD68). CONCLUSION: The recurrence of the tumors studied did not occur under the influence of MAT, but may be due to technical failure in achieving MMS or some other unknown immune mechanism

Benzodiazepínicos (BDZ) são fármacos amplamente utilizados devido às suas propriedades ansiolíticas e sedativas, mediadas pelo complexo GABAA no Sistema Nervoso Central (SNC). Além destes receptores centrais, os BDZ possuem afinidade por receptores do tipo periféricos (PBR) os quais estão presentes em células do sistema imune, como linfócitos e macrófagos, em células tumorais e em glândulas adrenais. O presente trabalho avaliou os efeitos do diazepam, um BDZ freqüentemente utilizado, sobre o crescimento tumoral e a imunidade de animais portadores do Tumor Ascítico de Ehrlich (TAE). Mais especificamente, este trabalho avaliou os seguintes parâmetros da resposta imune: atividade de macrófagos, populações de linfócitos B, T helper e citotóxicos esplênicos, citotoxicidade de células Natural Killer (NK). Além disso, a marcação para PBR em células do TAE e avaliação de possíveis efeitos do diazepam ou Ro5-4864 in vitro sobre o ciclo celular do TAE foram avaliadas. Os resultados mostraram os seguintes efeitos do tratamento com diazepam in vivo (3,0 mg/kg): (1) aumento do crescimento do TAE; sem modificação das fases do ciclo celular do tumor, após 7 dias, (2) diminuição do número de leucócitos da cavidade peritoneal, da produção de NO e do índice de espraiamento; mas sem interferência com a produção de peróxido de hidrogênio e o índice de fagocitose, após 2 dias (3) não modificou o peso relativo do baço e a porcentagem de linfócitos esplênicos, após 2 dias ou 7 dias (4) aumento da citotoxicidade de células NK, após 3 dias (5) diminuição da porcentagem de células do TAE marcadas para PBR, após 7 dias. O tratamento in vitro com diazepam ou Ro5-4864 mostrou um aumento da proliferação de células do TAE. Já o tratamento in vivo com diazepam em doses menores (0,3 mg/kg e 1mg/kg) não modificou o crescimento do TAE, após 7 dias. Desta forma, sugere-se que o diazepam na dose de 3,0 mg/kg tenha aumentado o crescimento do TAE e diminuído a resposta imune inata, observada por meio da diminuição da atividade dos macrófagos peritoneais. Parece-nos plausível excluir de nossos resultados a participação de linfócitos B, T helper e citotóxicos. Por outro lado, não foi possível precisar a relevância das células NK para o desenvolvimento do tumor. Além disso, pode-se afirmar que há expressão de PBRs em células do TAE e que o tratamento in vitro com diazepam ou Ro5-4684 aumentou a proliferação destas células. Desta forma, os resultados dos dois últimos experimentos sugerem que a existência de efeitos do diazepam sobre crescimento tumoral in vivo pode também ser atribuída, ao menos em parte, a uma ação direta deste fármaco sobre células do TAE / Benzodiazepines (BDZ) are drugs widely used due to their anxiolytic and sedative properties, acting on specific sites coupled to GABAA complex in the Central Nervous System (CNS). Besides these central receptors, BDZ have affinity for peripheral-type receptors (PBR), which have been found in immune cells, such as lymphocytes and macrophages, in tumor cells and in the adrenal glands. The present study evaluated the effects of diazepam, a commonly used BDZ, on tumor growth and immunity of mice bearing Ehrlich Ascitic Tumor (EAT). Specifically, this study evaluated the following parameters of the immune system: macrophage activity, populations of B, helper and cytotoxic T lymphocytes, and Natural Killer (NK) cells cytotoxicity. Furthermore, the evaluation of PBR expression in EAT cells and possible in vitro effects of diazepam or Ro5-4864 on EAT cell cycle were performed. Results showed the following diazepam effects in vivo (3.0 mg/kg per day): (1) increased tumor growth without changes in cell cycle, after 7 days; (2) decreased the number of leucocytes in the peritoneal cavity, the production of NO and the spreading index, but did not modify the production of hydrogen peroxide and the phagocytosis index, after 2 days; (3) did not modify the relative spleen weight and the population of lymphocytes after 2 or 7 days, (4) increased NK cytotoxicity after 3 days; (5) reduced the percentage of EAT cells expressing PBR after 7 days. Experiments performed in vitro showed that diazepam or Ro5-4864 increased the proliferation of EAT cells. Diazepam treatment in vivo using lower doses (0.3 mg/kg and 1mg/kg) did not modify tumor growth. Therefore, diazepam in the dose of 3.0 mg/kg increased the growth of EAT and reduced innate immunity, probably through the decrease in the activity of peritoneal macrophages. A role of B and helper or cytotoxic T lymphocytes in our experiments seems unlikely since the population of these cells types remained unchanged. On the other hand, it was not possible to determine the relevance of NK cells cytotoxicity on tumor development. The expression of PBR in EAT cells and the increase of their proliferation induced by in vitro treatment with diazepam or Ro5-4684 were observed. The results of these two last experiments suggest that the increase on tumor growth following diazepam treatment in vivo can be attributed, at least in part, to a direct action of this drug on EAT cells