The mononuclear phagocyte system revisited

The mononuclear phagocyte system (MPS) was defined as a family of cells comprising bone marrow progenitors, blood monocytes, and tissue macrophages. In this review, we briefly consider markers for cells of this lineage in the mouse, especially the F4/80 surface antigen and the receptor for macrophage colony-stimulating factor. The concept of the MPS is challenged by evidence that there is a separate embryonic phagocyte lineage, the blurring of the boundaries between macrophages and other cells types arising from phenotypic plasticity and transdifferentiation, and evidence of local renewal of tissue macrophage populations as opposed to monocyte recruitment. Nevertheless, there is a unity to cells of the MPS suggested by their location, morphology, and shared markers. We discuss the origins of macrophage heterogeneity and argue that macrophages and antigen-representing dendritic cells are closely related and part of the MPS.

Single-nucleotide polymorphism in the CD14 promoter and periodontal disease expression in a Japanese population

It has been reported that there is a relationship between a single-nucleotide polymorphism (SNP) in the promoter region of the CD 14 gene at position -159 (C-->T) and infectious diseases. The aim of the present study was to test the hypthesis that expression of this SNP correlates with periodontal disease in a Japanese population. The CD14 genotype was determined in 163 subjects with periodontitis and in 104 age- and gender-matched control subjects without periodontitis. The genotype distribution and allele frequency within the periodontitis patients were not significantly different from those of control subjects. There was, however, a significant difference in the genotype distribution between young patients (< 35 yrs) and older patients (greater than or equal to 35 yrs). These findings suggest that CD14-159C/T polymorphism is not related to the development of periodontitis in a Japanese population, but that, within the periodontitis subjects, expression of the SNP may be related to early disease activity.

CD40 and dendritic cell function

CD40 has emerged as a key signaling pathway for the function of B cells, monocytes, and dendritic cells (DC) in the immune system, and plays a major role in inflammatory pathways of nonhemopoletic cells. CD40 is expressed by monocytes and DC and is up-regulated when DC migrate from the periphery to draining lymph nodes (DLN) in response to microbial challenge. CD154 signaling by MHC-restricted, activated CD4* T cells induces differentiation of DC, as defined by an increased surface expression of MHC, costimulatory, and adhesion molecules. Thus, CD40 functions in the adaptive immune response as a trigger for the expression of costimulatory molecules for efficient T-cell activation. CD40 ligation of DC also has the capacity to induce high levels of the cytokine IL-12, which polarizes CD4(+) T cells toward a T helper 1 (Th1) type, enhances proliferation of CD8(+) T cells, and activates NK cells. CD40 may also play an important role in the decision between tolerance and immunity and the generation of regulatory CD4(+) T cells that are thought to maintain peripheral self-tolerance in vivo.

Recent advances on the role of CD40 and dendritic cells in immunity and tolerance

CD40 is a key signaling pathway for the function of B cells, monocytes, and dendritic cells in the immune system, and plays an important role in inflammatory pathways of nonhemopoietic cells. The NFkappaB family of transcription factors is a critical mediator in inflammation. NFkappaB is involved both in the regulation of CD40 expression and in cell signaling after CD40 ligation. This positive feedback loop linking NFkappaB and CD40 plays an important role in the control of the adaptive immune response, with fundamental implications for immunity and tolerance in vivo.

Expression of circulating leucocytes before, during and after myiasis by Dermatobia hominis in experimentally infected rats

Expression of circulating white blood cells was investigated in rats (Rattus norvegicus) experimentally infected with larvae of Dermatobia hominis, the human bot fly. Leucocytes were counted prior to infection (control group) as well as at 6, 10, 15, 20 and 28 days post-infection (dpi) and at 7, 15, 30 and 60 days post-larval emergence (dple). Total leucocyte numbers did not differ markedly among the groups. Significant differences were registered when values from control and animals harboring each larval stage of D. hominis were compared; with crescent rank: L1-, L2-, control and L3-infected groups. Leucocyte numbers were significantly higher in the control, 15, 20 or 28 dpi groups than in the 6 dpi animals. Higher counts were observed in control, L2- or L3-infected rats than L1-infected animals. Neutrophils, eosinophils and both large and small lymphocytes were also counted and analyzed. Basophils and monocytes were insufficient in number to permit statistical studies. These results stimulate the continuity of the studies about the host-parasite relationship in the dermatobiosis.

Cytokine and nitric oxide production by mouse macrophages infected with brazilian flaviviruses

The Flaviviridae family, Flavivirus genus includes viruses that are transmitted to vertebrates by infected mosquitoes or ticks. The genus Flavivirus includes a variety of viruses that cause diseases such as acute febrile illness, encephalitis, and hemorrhagic fever. Flaviviruses primarily infect blood monocytes and tissue macrophages, which have been shown to be permissive, supporting viral replication and serving as virus reservoirs. On the other hand, these cells may have an important antiviral activity related to modulation by cytokine production and by the capacity of these cells to synthesize reactive free radicals such as nitric oxide (NO) which can have a microbicidal effect. The present study was performed in order to determine the production of cytokines interleukin-1beta (IL-1β), tumor necrosis factor -alpha (TNF-α), transforming growth factor- beta (TGF-β) and interferon -alpha (IFN-α) and NO by macrophages infected with one of four Brazilian flaviviruses, Bussuquara virus (BUSV), Yellow Fever virus (YFV), Rocio virus (ROCV) and Encephalitis Saint Louis virus (SLEV), and to verify the possible antiviral effect of NO during macrophage infection with ROCV. Moreover, we asked if the different viruses were able to regulate bacterial lipopolysaccharide (LPS) induced cytokine production. Our results showed that YFV and SLEV reduced the production of IL-1β and TGF-β by LPS-stimulated macrophages, while ROCV only diminished LPS-stimulated TGF-β synthesis. On the other hand, BUSV more likely favored an enhancement of the LPS-induced production of IL-1β by macrophages. Additionally, while most of the viruses stimulated the production of IFN-α, none of them altered the production of TNF-α by murine macrophages. Interestingly, all viruses induced synthesis of NO that was not correlated with antiviral activity for ROCV.

FATAL DISSEMINATED CRYPTOCOCCOSIS WITH RENAL INVOLVEMENT IN AN HIV-INFECTED PATIENT

SUMMARY Introduction: We present a fatal case of disseminated cryptococcosis in a young man whose diagnosis of HIV infection was made at the time of admission to the emergency room. Case report: The patient was a twenty-three-year-old man, with a history of daily fever during one month associated with diarrhea, weight loss, headache, vomiting and generalized seizures. He also had a history of diabetes mellitus, alcoholism and drug addiction. Upon physical examination the patient was pale, disoriented and had periods of agitation. White blood cells count was 3,440/mm3 (5% lymphocytes), hemoglobin was 10g/dL, platelets were 83,000/ mm3. Creatinine was 0.7 mg/dL; urea 19 mg/dL; Na, K, and liver enzymes were within normal limits. Lactic dehydrogenase was 494 IU/L. Cerebrospinal fluid (CSF) analysis revealed 10 white blood cells/mm3 (58% neutrophils, 31% lymphocytes, 11% monocytes) and 2 red blood cells/mm3. India ink test revealed six Cryptococcus yeasts/mm3. CSF glucose was 122 mg/dL and protein was 36 mg/ dL. VDRL test was negative and anti-HIV test was positive. Intravenous hydration, insulin, phenytoin, fluconazole, pyrimethamine, sulfadiazine, folinic acid, and amphotericin B were started. The patient did not improve and became obtunded and hypotensive. He was intubated and put on mechanical respiration. He received vasoactive drugs and died less than 24 hours after admission. A postmortem examination was performed and revealed disseminated cryptococcosis, with severe involvement of the kidneys. Conclusion: Cryptococcosis, as a rule, is a systemic disease that affects mostly immunocompromised individuals, especially patients with AIDS. When diagnosed late in its course it has a very high mortality.

Effects of lethal and non-lethal malaria on the mononuclear phagocyte system

The effects ofone non-lethal species ofmalarialparasite, Plasmodium yoelii, and one lethal species, P. berghei, on the mononuclear phagocyte system (MPS) of BALB/c mice were studied. P. yoelii caused a greater and more sustained expansion and activation of the MPS, and the two major populations of spleen phagocytic cells-red pulp and marginal zone macrophages - exhibited a greater increase in numbers in this infection. During the course of P. berghei mataria, the spleen was progressively occupied by haematopoietic tissue and, at the terminal stage of infection, an extensive depletion of lymphocytes and macrophages was apparent. The possibility was suggested that the outcome of mataria may be inftuenced by the particular way the parasite interacts with the MPS.

Establishing a cell biology platform: isolation and preservation of human blood products

Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina

Osteoclast activity in ankylosing spondylitis patients before and after TNF-blocking therapy

Ankylosing spondylitis (AS) is a chronic inflammatory rheumatic disease of the axial skeleton. The major outcome of this disease is defined by new bone formation, commonly observed in the ligaments of the intervertebral joints, that can lead to the formation of bony spurs, known as syndesmophytes. Previous studies have shown that serum levels of TNF, IL-6 and IL-17 are increased in AS patients and may be implicated in the development of secondary osteoporosis, since these cytokines are able to induce osteoclast (OC) differentiation and, therefore, bone resorption. In this work we aimed to assess the effects of TNF-blocking therapy in the systemic inflammatory environment of AS patients with active disease as well as in OC differentiation and activity. To accomplish this objective, we cultured circulating monocytes from AS patients, before and after therapy, under osteoclastogenic conditions and we performed two functional assays (TRAP staining and resorption pit assay) and analyzed the expression of osteoclast specific genes. We have shown that AS patients with active disease have increased levels of pro-inflammatory cytokines when compared with healthy subjects. We also found that IL-17, TGF-β and osteoprotegerin are decreased after TNF-blocking therapy. Interestingly, we also observed that after TNF-blocking therapy the expression of some genes is favoring osteoclastogenesis and that differentiated OCs have increased resorption activity. These results suggest that in active AS there may be an uncoupling between inflammation and OC activity that is reset by TNF-blocking therapy.

Contribuição para o estudo da anexina V na apoptose celular em concentrados de eritrócitos

A hemoterapia moderna baseia-se na utilização correcta dos diversos componentes sanguíneos, associados a um maior controle de qualidade do sangue, o que a torna mais segura e, actualmente, muitos doentes sao beneficiados pois, a transfusão de componentes sanguineos, em situaçoes várias, está na linha da frente na manutenção da vida e em casos extremos, o último recurso que salva vidas. A qualidade e a segurança nas transfusões de sangue são grandes preocupações da área médica, autoridades de saúde e doente1. O sangue obtido pelos Centros de Sangue provem de dadores voluntários, dotados de uma enorme sensibilidade social, que periodicamente assumem uma postura benevola e altruista e consequentemente mantêm os bancos de sangue providos de um produto imprescindivel no tratamento de diversas patologias. O produto final disponível – concentrado de eritrócitos (CE´s), plasma e concentrado plaquetário – tem de assumir um carácter seguro e viável de modo a que os riscos para o doente sejam diminutos2. O controlo de qualidade aplicado a todo o sangue doado realiza provas de conformidade nas unidades com especificações previamente definidas, sendo a hémolise um dos parâmetros importantes na avaliação da qualidade dos concentrados de eritrócitos, pois, pode ocasionar implicações clinicas para o receptor. Para além disso a avaliação da concentração de hemoglobina (Hg) no sangue doado mostra-se um controlo imprescindivel que salvaguarda a qualidade e segurança do componente a transfundir3;4.Até se obter um CE há todo um processo moroso e de responsabilidade vital. Todo o sangue obtido passa por várias etapas fundamentais até à obtenção do componente pretendido (analise, produção e armazenamento). Os CE’s obtidos quando armazenados, num ambiente de refrigeração, têm uma vida útil de 42 dias. Após este período, o sangue deve ser inutilizado por se verificar alterações bioquímicas, biomecânicas, e imunológicas nos CE’s e por consequência a sua instabilidade vital no que ao tratamento de patologias, para as quais este componente está indicado, diz respeito5. Foi realizado um estudo experimental com o objetivo de avaliar a contribuição da Anexina V na apoptose celular nos concentrados de eritrócitos, constatando a degradação dos mesmos ao longo de todo o período de armazenamento e validar o paradigma que a ciência preconiza: “Os CE’s após os 42 dias armazenados, em condições específicas (2 a 6º centígrados), são inviaveis para transfundir”6;7. A avaliação dos níveis de apoptose por citometria de fluxo é geralmente realizada por métodos que utilizam Anexina V como marcador vital, que se associa aos resíduos de fosfatidilserina, externalizados no início do processo apoptótico. A Anexina V é uma proteína humana endógena dependente do ião Ca+2, amplamente distribuída intracelularmente em altas concentrações na placenta e em concentrações mais baixas nos eritrócitos, plaquetas e monócitos. Apresenta como principal característica a capacidade de se ligar à fosfatidilserina, um fosfolipído presente na camada interna da bicamada lipídica, que durante a apoptose celular é translocada para a camada externa da membrana celular. A determinação da Anexina V é normalmente utilizada para verificar se as células são viáveis, apoptóticas ou necróticas por meio de diferenças na integridade da membrana plasmática. Assim, ao conjugar a Anexina V ao FITC (Isotiocianato de fluoresceína) é possível identificar e quantificar as células apoptóticas por citometria de fluxo7. Numa amostra de 15 CE’s, a qual foi induzida a hemólise, verificou-se, por citometria de fluxo, que a viabilidade deste componente se desvanesce ao longo do tempo, confirmando assim que o tratamento, manuseamento e armazenamento do sangue compromete a vitalidade terapeutica deste insubstituivel produto vital.

Abnormal spontaneous interleukin 8 receptor expression: a brief report of two cases

Interleukin 8 (CXCL8) is an autocrine chemokine specific for the chemoattraction and activation of granulocytes, NKT cells and T lymphocytes. Patients with tuberculosis and latent Mycobacterium tuberculosis infection were assessed for the spontaneous expression of CXCR1 (CD128) and CXCR2 on lymphocytes and monocytes. Compared with ex vivo profiles, increased spontaneous CXCR2 expression and normal CXCR1 expression were found on lymphocytes in two out of 59 individuals. Monocytes showed normal ex vivo profiles for both receptors. After stimulation with purified protein derivative, the in vitro levels of CXCL8 were below the median levels of all patients with prior tuberculosis. Spontaneous CXCR2 modulation did not cause notable variation in the in vitro levels of CXCL8.

Interaction of an opportunistic fungus Purpureocillium lilacinum with human macrophages and dendritic cells

IntroductionPurpureocillium lilacinum is emerging as a causal agent of hyalohyphomycosis that is refractory to antifungal drugs; however, the pathogenic mechanisms underlying P. lilacinum infection are not understood. In this study, we investigated the interaction of P. lilacinum conidia with human macrophages and dendritic cells in vitro.MethodsSpores of a P. lilacinum clinical isolate were obtained by chill-heat shock. Mononuclear cells were isolated from eight healthy individuals. Monocytes were separated by cold aggregation and differentiated into macrophages by incubation for 7 to 10 days at 37°C or into dendritic cells by the addition of the cytokines human granulocyte-macrophage colony stimulating factor and interleukin-4. Conidial suspension was added to the human cells at 1:1, 2:1, and 5:1 (conidia:cells) ratios for 1h, 6h, and 24h, and the infection was evaluated by Giemsa staining and light microscopy.ResultsAfter 1h interaction, P. lilacinum conidia were internalized by human cells and after 6h contact, some conidia became inflated. After 24h interaction, the conidia produced germ tubes and hyphae, leading to the disruption of macrophage and dendritic cell membranes. The infection rate analyzed after 6h incubation of P. lilacinumconidia with cells at 2:1 and 1:1 ratios was 76.5% and 25.5%, respectively, for macrophages and 54.3% and 19.5%, respectively, for cultured dendritic cells.ConclusionsP. lilacinum conidia are capable of infecting and destroying both macrophages and dendritic cells, clearly demonstrating the ability of this pathogenic fungus to invade human phagocytic cells.

Osteoclastogenesis, inflammatory cytokines and biomarkers of bone metabolism in psoriatic arthritis

RESUMO:A artrite psoriática (AP) é uma doença inflamatória crónica caracterizada por várias manifestações nas articulações, nas enteses e na pele. A formação de novo osso após inflamação nas enteses é um dos aspetos mais intrigantes desta doença. Os mecanismos celulares e moleculares deste processo ainda não são completamente conhecidos. Este estudo tem como objetivo compreender melhor os mecanismos subjacentes à formação e reabsorção óssea, bem como o efeito de anti-inflamatórios não esteroides (AINEs) nestes processos. Para atingir este objetivo foram quantificados biomarcadores do metabolismo ósseo e citocinas inflamatórias em doentes AP, antes e após terapêutica com AINEs. Os biomarcadores selecionados foram marcadores de remodelação óssea como CTX-I e P1NP, fatores de diferenciação e ativação de osteoclastos como o RANKL e a OPG, inibidores da via de sinalização Wnt, nomeadamente o DKK-1 e a SOST e ainda citocinas pro-inflamatórias como a IL-22 e a IL-23 e a prostaglandina PGE2. Neste contexto foram também estabelecidas culturas celulares de monócitos, isoladas de doentes AP e de controlos saudáveis. Os monócitos foram cultivados in vitro em condições não estimuladas e estimuladas e realizados dois ensaios funcionais: coloração com TRAP e ensaio de reabsorção. Foi observada uma diminuição nos níveis séricos de CTX-I e OPG em doentes AP em relação aos controlos. De igual forma os níveis séricos de SOST encontram-se significativamente mais baixos, em comparação com os controlos saudáveis. Estes valores de SOST são semelhantes aos dos doentes com espondilite anquilosante (EA), documentados anteriormente. Os ensaios com osteoclastos confirmaram a necessidade da presença de RANKL para estimulação da osteoclastogénese e que o celecoxib parece ter um papel inibitório neste processo. Os resultados obtidos sugerem que a população de doentes com AP analisados têm baixos níveis de reabsorção óssea e alguma atividade na formação óssea. --------------------------- ABSTRACT: Psoriatic arthritis (PsA) is a chronic inflammatory disease characterized by several manifestations involving the joints, enthesis and the skin. New bone formation after inflammation at enthesis site has been one of the most intriguing aspects of the disease. Cellular and molecular mechanisms in this process are still not completely understood. This study aims to understand better the mechanisms underlying bone formation and resorption and the effect of non-steroid anti-inflammatory drugs (NSAIDs) in these processes. To access that, biomarkers of bone metabolism and inflammatory cytokines were measured in PsA patients’ serum before and after NSAID therapy. These selected biomarkers were bone turnover markers such as CTX-I and P1NP, osteoclast differentiation and activation factors RANKL and OPG, Wnt pathway inhibitors DKK-1 and SOST and pro-inflammatory cytokines IL-22, IL-23 and prostaglandin PGE2. In this context monocyte cell culture was also established after PBMC isolation from PsA patients and healthy controls. Monocytes were cultured in vitro under unstimulated and stimulated conditions and two functional assays were performed: TRAP staining and resorption pit assay. It was demonstrated that CTX-I and OPG serum levels in PsA patients were lower than controls. SOST levels were extremely decreased in comparison with controls, resembling the ankylosing spondylitis patients results already documented. Osteoclast assays confirmed the need of RANKL in stimulating osteoclastogenesis and that celecoxib seems to have an inhibitor role in this process. The results obtained suggest that PsA patient population analyzed in this study have low bone resorption levels and some bone formation activity.

Optimization of production of anti-cancer vaccines based on dendritic cells

Cancer remains as one of the top killing diseases in first world countries. It’s not a single, but a set of various diseases for which different treatment approaches have been taken over the years. Cancer immunotherapy comes as a “new” breath on cancer treatment, taking use of the patients’ immune system to induce anti-cancer responses. Dendritic Cell (DC) vaccines use the extraordinary capacity of DCs’ antigen presentation so that specific T cell responses may be generated against cancer. In this work, we report the ex vivo generation of DCs from precursors isolated from clinical-grade cryopreserved umbilical cord blood (UCB) samples. After the thawing protocol for cryopreserved samples was optimized, the generation of DCs from CD14+ monocytes, i.e., moDCs, or CD34+ hematopoietic stem cells (HSCs), i.e, CD34-derived DCs, was followed and their phenotype and function evaluated. Functional testing included the ability to respond to maturation stimuli (including enzymatic removal of surface sialic acids), Ovalbumin-FITC endocytic capacity, cytokine secretion and T cell priming ability. In order to evaluate the feasibility of using DCs derived from UCB precursors to induce immune responses, they were compared to peripheral blood (PB) moDCs. We observed an increased endocytosis capacity after moDCs were differentiated from monocyte precursors, but almost 10-fold lower than that of PB moDCs. Maturation markers were absent, low levels of inflammatory cytokines were seen and T cell stimulatory capacity was reduced. Sialidase enzymatic treatment was able to mature these cells, diminishing endocytosis and promoting higher T cell stimulation. CD34-derived DCs showed higher capacity for both maturation and endocytic capacity than moDCs. Although much more information was acquired from moDCs than from CD34-derived DCs, we conclude the last as probably the best suited for generating an immune response against cancer, but of course much more research has to be performed.