Universiẗat, Diss., 2005--Würzburg. / Erscheinungsjahr an der Haupttitelstelle: 2005.

In der vorliegenden Arbeit wurde die zelluläre Regulation des monocyten-/ macrophagenspezifischen Oberflächenproteins CD163 untersucht und klinische Aspekte der löslichen Form des CD163 (sCD163) diskutiert. sCD163 wird in vivo durch einen inflammatorischen Reiz von der Zelloberfläche abgespalten. Bislang waren jedoch noch keine Mediatoren charakterisiert worden, die immer unter Entzündungsbedingungen vorhanden sind. In den eigenen Untersuchungen des Shedding von CD163 konnten für die Generierung des sCD163 neue endogene Aktivatoren identifiziert werden. Sowohl reaktive Sauerstoffspezies, wie Wasserstoffperoxid oder Stickstoffmonoxid, als auch das Produkt von endogenen Oxidationsreaktionen mit reaktiven Sauerstoffspezies 8-iso Prostaglandin F2a erwiesen sich als potente Aktivatoren des Shedding von CD163. Neben den bekannten physiologischen Funktionen des 8-iso Prostaglandin F2a konnte erstmals eine neue Funktion bei Entzündungen definiert werden. Dieser Effekt wurde spezifisch durch 8-iso Prostaglandin F2a hervorgerufen, da das isomere Prostaglandin F2a unter gleichen Bedingungen keinen Einfluss auf das Shedding von CD163 ausübte. Das Immunsuppressivum Cyclosporin A konnte ebenfalls als Induktor des Shedding von CD163 ermittelt werden. Damit konnte zusätzlich zur bekannten immunmodulatorischen Wirkung des Cyclosporin A eine weitere antiinflammatorische Wirkung über Monocyten/Macrophagen aufgezeigt werden. Durch Untersuchungen der Inhibierung des Shedding von CD163 konnten Gemeinsamkeiten bezüglich der in die sCD163-Generierung involvierten Mediatoren dargestellt werden. Obwohl die untersuchten Verbindungen wahrscheinlich über unterschiedliche Signalwege das Shedding von CD163 induzieren, waren die Anwesenheit von reaktiven Sauerstoffspezies und intrazellulärem Calcium und die Beteiligung einer TIMP-3-sensitiven Metalloproteinase an diesem Prozess essentiell. Bei einem Vergleich zwischen dem Shedding von CD163 und Tumor necrosis factor-a (TNF-a) ergaben sich Gemeinsamkeiten durch die Induktion des Shedding beider Verbindungen durch 8-iso Prostaglandin F2a und in sehr viel geringerem Maße durch Wasserstoffperoxid. Im Gegensatz dazu waren deutliche Unterschiede in dem Ausmaß der induzierten Stimulation des Shedding durch Stickstoffmonoxid, Prostaglandin F2a und Cyclosporin A zu erkennen. Im Hinblick auf die entgegengesetzten Wirkungen von sCD163 als antiinflammatorisch wirkende Verbindung und TNF-a als proinflammatorisches Cytokin, konnte dargestellt werden, dass die Freisetzung durch unterschiedliche Aktivatoren erfolgt. Nach Bestimmung der Konzentrationen von sCD163 und 8-iso PGF2a in bronchoalveolärer Lavage-Flüssigkeit von Patienten mit Cystischer Fibrose konnten keine abschließende Aussagen über die Eignung beider Parameter als biologische Marker für chronische Entzündungen der Lunge bei diesen Patienten getroffen werden. Weiterführend zu der Kenntnis, dass sCD163 die antiinflammatorische Wirkung über eine Interaktion des Proteins mit humanen T-Lymphocyten und nachfolgender Hemmung der Proliferation dieser Zellen ausübt, wurde diese Wechselwirkung genauer untersucht. In quantitativen Bestimmungen des sCD163 in isolierten T-Lymphocyten verschiedener Spender konnte erstmals gezeigt werden, dass sCD163 zu einem Teil konstitutiv in die T-Lymphocyten aufgenommen wird und dass diese Aufnahme durch proinflammatorische Aktivierung der T-Lymphocyten stark gesteigert werden kann. Durch fluoreszenzmikroskopische Aufnahmen unstimulierter T-Lymphocyten konnte die intrazelluläre Lokalisation des sCD163 visualisiert werden. Nach Aktivierung der Zellen mit einem proinflammatorischen Reiz fand innerhalb der Zellen eine Translokalisation des sCD163 aus dem cytoplasmatischen Bereich zur Zellmembran statt. Damit konnte erstmals gezeigt werden, dass abhängig vom Aktivierungsstatus der T-Lymphocyten eine Umverteilung des sCD163 innerhalb der Zellen erfolgt. Eine quantitative Bestimmung des sCD163 und seines Bindungspartners in T-Lymphocyten nichtmuskuläres Myosin Typ IIA gelang mittels eines neu entwickeltem ELISA, der spezifisch sCD163 und Myosin ausschließlich im Komplex erfasst. Damit konnte durch die in dieser Arbeit beschriebenen Untersuchungen ein grundlegender Beitrag zur Charakterisierung der Regulation der Proteinexpression und des Shedding von CD163 in humanen Monocyten sowie der Interaktion des sCD163 mit T-Lymphocyten geleistet werden. / In the present thesis, cellular regulation of the monocyte/macrophage specific membrane protein CD163 was investigated and clinical aspects of the soluble form of CD163 (sCD163) were discussed. sCD163 is shed from the cell surface in vivo upon an inflammatory stimulus. So far no mediators had been characterized that are persistently present under inflammatory conditions. In the present investigation of the shedding of CD163, new endogenous activators for the generation of sCD163 were successfully identified. Both reactive oxygen species, as hydrogen peroxide or nitric monoxide, and the product of endogenous oxidative reactions 8-iso prostaglandin F2a turned out to be potent activators of the shedding of CD163. In addition to the known physiological functions of 8-iso prostaglandin F2a a novel function of this compound in inflammation was defined. This effect was specifically generated by 8-iso prostaglandin F2a as prostaglandin F2a did not influence the shedding of CD163 under the same conditions. The immunosuppressive drug cyclosporine A was also established as an inductor of the shedding of CD163. Accordingly, another anti-inflammatory effect via monocytes/macrophages was pointed out in addition to the well known immunomodulatory effects of cyclosporine A. Investigations of the inhibition of shedding of CD163 led to the identification of key mediators involved in the generation of sCD163. Even though the tested compounds may induce shedding via different signal transduction pathways, the presence of reactive oxygen species and intracellular calcium and the involvement of a TIMP-3 sensitive metalloproteinase were essential in this process. The tested activators revealed different abilities for inducing the formation of reactive oxygen species. Comparing shedding of CD163 and tumor necrosis factor-a (TNF-a), similarities in induced shedding by 8-iso prostaglandin F2a and to a lower extend by hydrogen peroxide were seen. In contrast, significant differences were recognized in the extent of shedding induced via stimulation by nitric oxide, prostaglandin F2a, and cyclosporine A. With regard to the opposite effects of sCD163 as an anti-inflammatory compound and TNF-a as a pro-inflammatory cytokine it was demonstrated that shedding occurred via induction by different activators. The determination of concentrations of sCD163 and 8-iso prostaglandin F2a in bronchoalveolar lavage fluid of patients with cystic fibrosis allowed no conclusive statement about the suitability of both parameters as marker for chronic inflammation. In extension to earlier insights in the anti-inflammatory effects of sCD163 via interaction of the protein with human T-lymphocytes and subsequent inhibition of the proliferation of these cells these interactions were analyzed in detail. Quantifications of sCD163 in isolated T-lymphocytes from different donors revealed for the first time that sCD163 is constitutively taken up into T-lymphocytes and that this process can significantly be enhanced by pro-inflammatory activation of T-lymphocytes. The intracellular localization of sCD163 was visualized by fluorescence microscopy of T-lymphocytes. Upon activation of the cells by a pro-inflammatory stimulus a translocalization of sCD163 was observed within the cells from the cytoplasmatic region to the cell membrane. Thus, it was shown for the first time that an activation dependent translocalization of sCD163 occurs within the T-lymphocytes. The quantitative determination of sCD163 and non-muscular myosin type IIA as its intracellular binding partner in T-lymphocytes was successful using a newly developed ELISA that detects specifically sCD163 and myosin as a complex. To conclude, the investigations described in this thesis contributed substantially to the understanding of the regulation of the protein expression and shedding of CD163 in human monocytes and of the interaction of sCD163 with T-lymphocytes.

BACKGROUND: Subarachnoid Hemorrhage (SAH) accounts for 3-5% of total stroke patients annually. Despite its rare incidence, SAH carries a 50% mortality rate. Survivors are often left with varying degrees of disability, many will never return to their previous jobs and require long-term care. One of the leading causes for this high mortality and morbidity rate in SAH is Delay Cerebral Ischemia (DCI). Researchers are now beginning to investigate neuroinflammation as the underlying cause for DCI. Studies have shown the activation of the innate immune system in the central nervous system is initiated by excess hemoglobin in the subarachnoid space. This process is mediated by the Toll-Like Receptor 4 expressed on the tissue-resident macrophages. Activated macrophages release pro-inflammatory cytokines and cause neuronal apoptosis in the surrounding tissue. However, macrophages may also mediate neuroprotection in SAH. A macrophage surface receptor called CD163 is responsible for the recognition and endocytosis of excess hemoglobin. The thesis provides a closer assessment of the neuroprotective role of macrophages in SAH patients. METHODS: Cerebrospinal fluid (CSF) was obtained from twenty three patients diagnosed with SAH (on day 1 and day 7 post-admission) or unruptured aneurysms. Immune cells were separated from CSF and analyzed by flow cytometry. The following antibody panel was used in this study: PE-anti-CD163, PeCy7-anti-CD15, and APC-anti-CD14. Macrophage expression of CD14 and CD163 was quantified using FlowJo. SAH patients were graded by the Hunt and Hess scale for the clinical states upon admission; modified Fisher scale for the size of the hemorrhage; and modified Rankin scale for clinical outcome at the time of discharge. RESULTS: Significant increase in macrophage CD14 and CD163 expression is observed in day 1 SAH patients (p<0.05) as compared to the control group. Male SAH patients have equivocal macrophages CD163 expression on day 1 as compared to the control group (p>0.05), and significantly higher expression on day 7 as compared to day 1(p<0.05). Female SAH patients have significantly higher macrophages CD163 expression on day 1 as compared to control patients (p<0.05), but slightly decreased expression on day 7 as compared to day 1(p>0.05). Lower macrophages CD163 expression is observed in SAH patients with more severe hemorrhage (marked by higher modified Fisher score), but not in patients with more severe clinical states at admission (marked by higher Hunt and Hess score). Furthermore, SAH patients with low day 1 macrophage CD163 expression and low expression on day 7 may be correlated with better clinical outcome (marked by lower modified Rankin score). However, more patients are required before correlation can be established. CONCLUSION: The data further support our previous findings in mouse SAH model that macrophages in the central nervous system may mediate inflammation via the increased expression TLR4, measured by increased expression of its co-receptor CD14. Macrophages also may be neuroprotective, mediated by increased expression of CD163 in SAH patients. The macrophage CD163 expression may be the key in determining clinical outcome in SAH patients, but additional patients are required to establish statistical significance. / 2017-06-16T00:00:00Z

INTRODUÇÃO: A Leishmaniose Tegumentar Americana (LTA) é uma doença infecciosa endêmica no Brasil, causada por protozoários do gênero Leishmania e possui duas formas clínicas principais, a leishmaniose cutânea (LC) e a leishmaniose mucocutânea (LMC). Os macrófagos têm papel crucial na patogênese da Leishmaniose como hospedeiros do parasita e atuam como células apresentadoras de antígenos, promovendo uma resposta imune de perfil Th1 contra a Leishmania. Os macrófagos polarizados M2, relacionados com resposta anti-inflamatória (Th2), são envolvidos no controle da inflamação e dano tecidual em várias doenças e estão associados à cronicidade nas doenças infecciosas. O objetivo deste trabalho foi verificar a participação dos macrófagos M2 nos sítios de lesão cutânea da LTA MÉTODOS: Sessenta e três biópsias de pele de lesões de LTA, obtidas de 48 doentes com LC e 15 de LMC, foram submetidas ao método imuno-histoquímico com os anticorpos anti-CD163 e anti-CD206, marcadores de macrófagos M2. Dez espécimes foram também submetidos à técnica imuno-histoquímica de dupla marcação com a utilização dos marcadores pSTAT-1 e CD68 para identificação de macrófagos M1 e CMAF e CD163 para identificação de macrófagos M2. RESULTADOS: Os espécimes de pele com reação inflamatória difusa (n = 26) exibiram maior número de células imunomarcadas com CD163, quando comparadas ao grupo com reação granulomatosa bem organizada (n = 37) (p= 0,01). Os macrófagos CD163+ também foram mais numerosos nas lesões de pele com a presença de formas amastigotas de Leishmania (n = 39) ao exame histopatológico (p=0,02), quando comparadas às lesões onde os parasitas não foram observados (n = 24). O mesmo ocorreu quando comparados somente os espécimes do grupo de doentes com LC com a presença de parasitas (n = 31) e ausência dos mesmos (n = 17) (p = 0,04). As lesões de pele de doentes com LMC mostraram maior número de células CD206+ (n = 15) que a de doentes com LC (n = 48) (p=0,008). Houve correlação positiva entre o número de células imunomarcadas com os anticorpos CD163 e CD206 (r de Spearman= 0,369). Os macrófagos +pSTAT1/CD68+ (M1) mostraram-se mais numerosos que os macrófagos CMAF+/CD163+ (M2) nos sítios de lesão cutânea da LTA. CONCLUSÕES: Os resultados obtidos sugerem que os macrófagos M2 têm um papel na resposta imune \"in situ\" da LTA. Essas células devem atuar como células alvo, facilitando a entrada dos parasitas, a progressão da doença e parecem estar envolvidos nos mecanismos de cronicidade e nos processos de remodelamento tecidual dessa doença. As moléculas CD163 e CD206 podem ser consideradas bons marcadores teciduais de macrófagos M2, uma vez que mostraram correlação positiva em sua expressão. A técnica de dupla marcação confirmou a presença dos macrófagos M2 nas lesões cutâneas da LTA. Embora ocorra a presença de macrófagos M2, os macrófagos M1 são a população macrofágica predominante da resposta imune \"in situ\" da LTA / INTRODUCTION: American tegumentary leishmaniasis (ATL) is an endemic infectious disease in Brazil, caused by Leishmania parasites and it has two main clinical forms, cutaneous leishmaniasis (CL) and mucocutaneous leishmaniasis (MCL). Macrophages play a key role in leishmaniasis pathogenesis. They are definite parasite host and act as antigen presenting cells, eliciting a Th1 pattern of immune response (M1) against Leishmania. M2 polarized macrophages, related to an anti-inflammatory response (Th2), are involved in controlling inflammation and tissue damage in several diseases and are related to chronicity in infectious diseases. The aim of this study was verifying M2 macrophages \"in situ\" participation in ATL. METHODS: Sixty-three skin biopsies from LTA lesions, obtained from patients with CL (n=48) and MCL (n=15), were subjected to imunohistochemical technique with M2 macrophages markers CD163 and CD206 antibodies. Ten samples were selected for double staining technique with pSTAT1 and CD68 markers for M1 identification, and CMAF and CD163 markers for M2 macrophages identification. RESULTS: Skin samples displaying diffuse inflammatory reaction (n = 26) exhibited higher number of CD163 immune stained macrophages when compared to the well-organized granulomatous reaction group (n = 37) (p=0.01). CD163+ macrophages were higher in samples displaying Leishmania parasites (n = 39) at histopathology exam than the samples without parasites (n = 24) (p=0.02). Same result was observed when comparing the lesions of CL lesions with (n = 31) and without (n = 17) parasites (p = 0.04). MCL skin lesions (n = 15) showed higher number of CD206+ cells (p = 0.008) in comparison with skin lesions taken from CL patients (n = 48). Spearman test demonstrated a positive correlation between the number of CD163+ and CD206+ immune stained cells (Spearman r = 0,369). M1 macrophages (pSTAT1+/CD68+) were present in higher number when compared to M2 macrophages (CMAF+/CD163+) in the samples studied. CONCLUSIONS: The results suggest that M2 macrophages play a role in the in situ immune response of ATL. M2 polarized macrophages may act as host target cells facilitating parasites entry and promoting disease progression, but also seem to be involved with chronicity and tissue remodeling of ATL lesions. CD163 and CD206 molecules may be considered as good markers for M2 macrophages. Double staining technique confirmed the presence of M2 macrophages, although M1 macrophages are the predominant macrophagic component in skin lesions of ATL

INTRODUÇÃO: A Leishmaniose Tegumentar Americana (LTA) é uma doença infecciosa endêmica no Brasil, causada por protozoários do gênero Leishmania e possui duas formas clínicas principais, a leishmaniose cutânea (LC) e a leishmaniose mucocutânea (LMC). Os macrófagos têm papel crucial na patogênese da Leishmaniose como hospedeiros do parasita e atuam como células apresentadoras de antígenos, promovendo uma resposta imune de perfil Th1 contra a Leishmania. Os macrófagos polarizados M2, relacionados com resposta anti-inflamatória (Th2), são envolvidos no controle da inflamação e dano tecidual em várias doenças e estão associados à cronicidade nas doenças infecciosas. O objetivo deste trabalho foi verificar a participação dos macrófagos M2 nos sítios de lesão cutânea da LTA MÉTODOS: Sessenta e três biópsias de pele de lesões de LTA, obtidas de 48 doentes com LC e 15 de LMC, foram submetidas ao método imuno-histoquímico com os anticorpos anti-CD163 e anti-CD206, marcadores de macrófagos M2. Dez espécimes foram também submetidos à técnica imuno-histoquímica de dupla marcação com a utilização dos marcadores pSTAT-1 e CD68 para identificação de macrófagos M1 e CMAF e CD163 para identificação de macrófagos M2. RESULTADOS: Os espécimes de pele com reação inflamatória difusa (n = 26) exibiram maior número de células imunomarcadas com CD163, quando comparadas ao grupo com reação granulomatosa bem organizada (n = 37) (p= 0,01). Os macrófagos CD163+ também foram mais numerosos nas lesões de pele com a presença de formas amastigotas de Leishmania (n = 39) ao exame histopatológico (p=0,02), quando comparadas às lesões onde os parasitas não foram observados (n = 24). O mesmo ocorreu quando comparados somente os espécimes do grupo de doentes com LC com a presença de parasitas (n = 31) e ausência dos mesmos (n = 17) (p = 0,04). As lesões de pele de doentes com LMC mostraram maior número de células CD206+ (n = 15) que a de doentes com LC (n = 48) (p=0,008). Houve correlação positiva entre o número de células imunomarcadas com os anticorpos CD163 e CD206 (r de Spearman= 0,369). Os macrófagos +pSTAT1/CD68+ (M1) mostraram-se mais numerosos que os macrófagos CMAF+/CD163+ (M2) nos sítios de lesão cutânea da LTA. CONCLUSÕES: Os resultados obtidos sugerem que os macrófagos M2 têm um papel na resposta imune \"in situ\" da LTA. Essas células devem atuar como células alvo, facilitando a entrada dos parasitas, a progressão da doença e parecem estar envolvidos nos mecanismos de cronicidade e nos processos de remodelamento tecidual dessa doença. As moléculas CD163 e CD206 podem ser consideradas bons marcadores teciduais de macrófagos M2, uma vez que mostraram correlação positiva em sua expressão. A técnica de dupla marcação confirmou a presença dos macrófagos M2 nas lesões cutâneas da LTA. Embora ocorra a presença de macrófagos M2, os macrófagos M1 são a população macrofágica predominante da resposta imune \"in situ\" da LTA / INTRODUCTION: American tegumentary leishmaniasis (ATL) is an endemic infectious disease in Brazil, caused by Leishmania parasites and it has two main clinical forms, cutaneous leishmaniasis (CL) and mucocutaneous leishmaniasis (MCL). Macrophages play a key role in leishmaniasis pathogenesis. They are definite parasite host and act as antigen presenting cells, eliciting a Th1 pattern of immune response (M1) against Leishmania. M2 polarized macrophages, related to an anti-inflammatory response (Th2), are involved in controlling inflammation and tissue damage in several diseases and are related to chronicity in infectious diseases. The aim of this study was verifying M2 macrophages \"in situ\" participation in ATL. METHODS: Sixty-three skin biopsies from LTA lesions, obtained from patients with CL (n=48) and MCL (n=15), were subjected to imunohistochemical technique with M2 macrophages markers CD163 and CD206 antibodies. Ten samples were selected for double staining technique with pSTAT1 and CD68 markers for M1 identification, and CMAF and CD163 markers for M2 macrophages identification. RESULTS: Skin samples displaying diffuse inflammatory reaction (n = 26) exhibited higher number of CD163 immune stained macrophages when compared to the well-organized granulomatous reaction group (n = 37) (p=0.01). CD163+ macrophages were higher in samples displaying Leishmania parasites (n = 39) at histopathology exam than the samples without parasites (n = 24) (p=0.02). Same result was observed when comparing the lesions of CL lesions with (n = 31) and without (n = 17) parasites (p = 0.04). MCL skin lesions (n = 15) showed higher number of CD206+ cells (p = 0.008) in comparison with skin lesions taken from CL patients (n = 48). Spearman test demonstrated a positive correlation between the number of CD163+ and CD206+ immune stained cells (Spearman r = 0,369). M1 macrophages (pSTAT1+/CD68+) were present in higher number when compared to M2 macrophages (CMAF+/CD163+) in the samples studied. CONCLUSIONS: The results suggest that M2 macrophages play a role in the in situ immune response of ATL. M2 polarized macrophages may act as host target cells facilitating parasites entry and promoting disease progression, but also seem to be involved with chronicity and tissue remodeling of ATL lesions. CD163 and CD206 molecules may be considered as good markers for M2 macrophages. Double staining technique confirmed the presence of M2 macrophages, although M1 macrophages are the predominant macrophagic component in skin lesions of ATL

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Kyeong-Ok Chang / Positive sense RNA viruses include a diverse group of pathogens that cause a wide array of diseases that can range from sub-clinical to lethal. These viruses infect humans and mammals as well as a variety of other hosts. For their successful replication, viruses interact closely with host cells from the binding to the receptor to the exit as complete viral progenies. During the events, viruses are dependent on host factors for receptor bindings, genome synthesis, and trafficking of viral genome and proteins. Thus there have been major efforts on the studies of understanding the virus-host interactions in the field of virology. In my PhD program, I have studied the host factors that regulate the replication of viruses using porcine reproductive and respiratory syndrome virus (PRRSV) and hepatitis C virus (HCV). I found that modulation of either the viral receptor or cellular signaling pathways had pronounced effects in the replication of PRRSV or HCV respectively. Using PRRSV, I found that the modulation of the level of the putative receptor CD163 on cells with cytokines significantly influence virus replication, suggesting the importance of cytokine presence in environments to determine the replication and pathogenicity of PRRSV via receptor expression in vivo. With HCV, I found that the enhancement of the virus replication occurs through the activation of the epidermal growth factor receptor/extracellular signal-regulated kinase pathway by bile acids which are abundant in the liver where the virus targets in vivo. Furthermore, I found that the bile acid-mediated signaling pathway significantly inhibited the antiviral activities against HCV. These results indicate the importance of environmental factors such as bile acids and signaling pathways in the replication and pathogenicity of HCV in vivo.

CD163, the receptor for haptoglobin–hemoglobin complex, is expressed on monocytes/macrophages and neutrophils. A soluble form (sCD163) has been associated with the M2 macrophage phenotype. M2 macrophages down-modulate the inflammatory response and has previously been described in the most severe, lepromatous (LL) clinical presentation of leprosy as well as tuberculosis. We hypothesized that sCD163 would correlate with severity of diseases caused by intracellular pathogens. Our general objective was to verify the presence of sCD163 in sera of Leprosy and Visceral leishmaniasis (VL) patients. Sera of patients with diferent clinical forms of leprosy (n = 47), and VL (n = 65) were tested for the presence of sCD163. The levels of sCD163 were compared in the diferent clinical forms of leprosy (Indeterminate - IL, Tuberculoid-TL, Boderline-BL and Lepromatous leprosy-LL). The levels of sCD163 were also compared in sera of VL patients before treatment (D0) (n = 46), and at D30 post treatment (n = 19), and also compared at D0, between patients with classical VL (n = 33) and severe VL (n = 13). High levels of sCD163 were detected in sera from leprosy patients as compared to contact controls, and confirmed the association of sCD163 with LL leprosy, finding it at elevated levels in this clinical form. ROC curves showed high sensitivity and specificity for the association between higher levels of this molecule with the most severe LL clinical form. VL patients also presented with high levels of sCD163 in sera at D0 as compared with healthy individuals, and even higher in sera from severe VL patients. Further stratification on infection and disease status revealed a clear association with clinical parameters of disease severity and clinical cure, with a direct correlation sCD163 concentration and liver and spleen sizes. ROC curves also demonstrated high sensitivity and specificity of this molecule to associate this molecule with disease severity of VL. Additionally, sCD163 concentrations reduces after treatment (D30). In vitro cultures indicated that Leishmania infection induced CD163 expression on the surface of monocyte/macrophages and neutrophils, suggesting these cells as possible sources of sCD163 in the sera. Interestingly, the association of sCD163 levels with disease status was not mirrored by levels of haptoglobin, heme-oxygenase, or arginase. Taken together, our data support the use of sCD163 as a biomarker of disease and severity in both leprosy and VL, and indicate a role of Leishmania infection in induce the expression of CD163 in macrophages and neutrophils. This data also suggest CD163 as a marker for M2 and N2, which down modulate inflammatory responses and interfere in the outcome of intracellular infections and the severity of their associated diseases. / O marcador CD163, receptor para o complexo Haptoglobina-Hemoglobina, é expresso em monócitos/macrófagos e neutrófilos. Sua forma solúvel (sCD163) tem sido associada com o fenótipo de macrófagos M2, que modulam negativamente a resposta inflamatória e descritos como relacionados à apresentação Virchowiana da Hanseníase bem como à Tuberculose. Este trabalho se baseou na hipótese de que sCD163 poderia se correlacionar com a gravidade de doenças provocadas por patógenos intracelulares. O objetivo geral foi verificar a presença e caracterizar a associação do marcador CD163 com apresentação clínica da Hanseníase e Leishmaniose Visceral (LV). Soros de pacientes com diferentes formas clínicas de hanseníase (n = 47), e de pacientes com LV foram testados para a presença do sCD163. Foram comparadas as concentracões do sCD163 entre as formas clínicas da hanseníase (Indeterminada - HI, Tuberculóide - HT, Dimorfa - HD e Virchowiana - HV). A presença do sCD163 foi também avaliada nos soros de pacientes com LV (n = 46) no D0 (antes do tratamento) (n = 33), D30 (pós-tratamento) (n = 19) e comparada entre os pacientes no D0 com LV grave (n = 13). Foram observados altos níveis do sCD163 nos soros dos pacientes com hanseníase em comparação com controles contactantes, e confirmou-se a associação desta molécula com a forma HV. Curvas ROC demontraram alta sensibilidade e especificidade da dosagem desta molécula para a associação com a forma mais grave HV da doença. Na LV, foram observadas concentrações elevadas do sCD163 no D0, sendo estas ainda mais elevadas nos pacientes com LV grave. Além disso, observou-se redução do sCD163 após o tratamento (D30). Análises de correlação confirmaram inter-relação entre o biomarcador e parâmetros de gravidade da LV, sendo observadas correlacões diretas entre as concentrações de sCD163 e as medidas do fígado e do baço. Curva ROC, também confirmou a associação entre o nível sérico alto desta molécula com a forma mais grave da LV. Adicionalmente, há uma redução significativa do sCD163 com cura clínica pós-tratamento. Experimentos in vitro indicaram indução da expressão de CD163 na superfície de macrófagos e neutrófilos por Leishmania amazonensis, sugerindo que estas células sejam possíveis fontes para a molécula sérica encontrada nos pacientes com LV. Não houve diferenças nos níveis de haptoglobina, heme-oxigenase-1 e arginase-1 entre os pacientes com LV clássico e LV grave. Em conjunto, esses dados um possível papel da molécula de sCD163 como biomarcador de doença e de gravidade na hanseníase e LV, e sugere um papel da leishmania na indução da expressão dessa molécula. Além disso, os achados sugerem que CD163 pode serum marcador de macrófagos e neutrófilos M2 e N2, os quais podem modular a resposta imune e contribuir para a apresentação clínica de infecções por patógenos intracelulares.

La leucémie lymphoïde chronique (LLC) est l'hémopathie maligne la plus fréquente des pays occidentaux. Elle se caractérise par une accumulation de cellules leucémiques dans le sang, la moelle osseuse et les organes lymphoïdes secondaires. Malgré les nouvelles thérapies, la LLC reste incurable. Dans la LLC, comme dans tout cancer, le microenvironnement tumoral, un milieu complexe contenant des cellules immunitaires pouvant être pro-tumorales, prend une part croissante aussi bien dans la physiopathologie que le pronostic. La recherche de nouvelles thérapies ciblant les interactions des cellules leucémiques avec ce microenvironnement représente aujourd'hui une piste prometteuse pour traiter cette pathologie. Les " nurse-like " cells (NLC) semblaient être les macrophages associés aux tumeurs (TAM) de la LLC. Ces cellules, retrouvées dans les ganglions lymphatiques de patients, favorisent in vitro la survie des cellules leucémiques. Elles partagent avec les TAM de nombreuses caractéristiques telles que des capacités immunosuppressives, un profil transcriptomique proche et une implication dans la résistance aux traitements. Nous l'avons confirmé par une analyse fonctionnelle et phénotypique. Nos travaux indiquent clairement que les NLC sont les TAM de la LLC avec une forte expression de CD68 et de CD163 et qu'elles favorisent la survie des cellules leucémiques. Suite à ce travail, nous avons déterminé l'impact pronostique des NLC dans la LLC, en émettant l'hypothèse que, comme dans d'autres cancers, une forte infiltration par les TAM pouvait être de mauvais pronostic. Nous avons alors montré que le taux de NLC présentes au sein du ganglion lymphatique chez le patient est corrélé à la progression de la LLC. Les NLC relarguent également un facteur soluble le CD163 soluble (sCD163) dans le sang des patients, dont le taux est corrélé à des marqueurs de mauvais pronostic dans la LLC tels que le statut IgHV non muté, le caryotype complexe et les mutations de TP53. Enfin, le taux de sCD163 est un marqueur pronostique indépendant dans la LLC : un fort taux étant associé à un raccourcissement du temps avant retraitement, de la survie sans progression et de la survie globale des patients. Ensuite nous avons étudié l'impact d'une thérapie innovante, l'ibrutinib, un inhibiteur de tyrosine kinase ciblant spécifiquement la Bruton Tyrosine Kinase, sur ces NLC. In vitro, les monocytes se différencient toujours en NLC et leur phénotype n'est pas modifié. De plus, ces cellules sont toujours capables de protéger les cellules leucémiques de l'apoptose. Finalement, nous avons mis en évidence qu'elles pouvaient participer à la chimiorésistance, en protégeant in vitro les cellules leucémiques de l'ibrutinib mais pas du dasatinib, de l'idélalisib, du vénétoclax, de la bendamustine et du rituximab. L'ensemble de nos résultats indique que cibler les NLC est une piste prometteuse dans le traitement de la LLC. Les interactions cellulaires entre NLC et cellules leucémiques pourraient représenter une cible thérapeutique intéressante. Nous avons montré qu'in vitro le contact entre NLC et cellules leucémiques est nécessaire à la survie de ces dernières. Suite à une étude transcriptomique, nous avons mis en évidence plusieurs couples moléculaires potentiellement impliqués dans ces interactions. Seul le couple LFA3/CD2 s'est révélé être nécessaire dans ce contact via un mécanisme dépendant d'Akt et son blocage inhibe totalement les effets pro-survie des NLC sur les cellules leucémiques. En conclusion, les NLC via des interactions dépendantes du couple LFA-3/CD2 protègent les cellules leucémiques de l'apoptose in vitro. De plus, elles participent à la chimiorésistance en protégeant aussi les cellules leucémiques de l'ibrutinib. Elles relarguent également du sCD163, dosable dans le sérum, un facteur pronostique indépendant de la LLC. La recherche de nouvelles thérapies plus spécifiques, ciblant les NLC constitue donc une piste thérapeutique intéressante dans la LLC. / Chronic lymphocytic leukemia (CLL) is the most common hemopathy in western countries. This pathology is characterized by an accumulation of leukemic cells in blood, bone marrow and secondary lymphoid organs. Despite new therapies, CLL is still incurable. In CLL, the microenvironment, a complex media containing immune cells witch can favor the tumor, takes now a large place in the physiopathology and the prognosis. The research of new therapies targeting interactions between CLL cells and microenvironment is a promising runway to find new drugs in CLL. "Nurse-like" cells (NLC) should be the tumor associated macrophages (TAM) of CLL. This cells, found in the lymph node of CLL patients, protect CLL cells against in vitro apoptosis. They share with TAM many characteristics including immunosubversive proprieties chemoresistance induction and a close gene expression profiling. We confirmed that by functional and phenotypical analysis. Our results indicate that NLC are the TAM of CLL with a high expression of CD68 and CD163 and that NLC can protect CLL cells against in vitro apoptosis. Thus, we focused on the clinical impact of NLC in CLL. Because in several cancers a high infiltration of TAM is correlated with a poor clinical outcome, we hypothesized that NLC infiltration should be associated with CLL outcome. We showed that infiltration of NLC in the lymph node can be correlated with the progressivity of CLL. Moreover, NLC release a soluble factor, the sCD163 (soluble sCD163), in the blood of CLL patients. High levels of this factor, can be correlated with previously established prognostic makers in CLL such as TP53 mutations, unmutatted IgHV status and complex karyotype. Finally, sCD163 was an independent prognostic marker in CLL and high levels are associated with shorter time to next treatment, progression free survival and overall survival. Next, we studied the impact of ibrutinib, a new therapy in CLL, on NLC. Ibrutinib is a specific tyrosine kinase inhibitor witch targets specifically the Bruton Tyrosine Kinase. In vitro, NLC differentiated from ibrutinib treated patients have the same phenotype as NLC from untreated patients and are still able to protect CLL cells against apoptosis. To end, we tested if in vitro NLC could protect CLL cells against chemotherapy and showed that NLC protect CLL cells against ibrutinib but not against idelalisib, dasatinib, venetoclax, bendamustin and rituximab. All our previous studies reveal that NLC are a good target to find new therapies in CLL. We focused our work on the interaction between NLC and CLL cells. First, we demonstrated that the NLC/CLL cells contact is necessary to prevent CLL cells death in vitro. Next, by gene expression profiling, we screened several couple of molecules potentially implicated in these interactions. Finally, only LFA-3/CD2 couple was necessary for this contact through an Akt pathway dependent and the inhibition of this couple totally inhibited the pro-survival effect of NLC on CLL cells. To conclude, NLC protect CLL cells from in vitro apoptosis through LFA-3/CD2. Moreover, they protect CLL cells against ibrutinib and so facilitate the chemoresistance. They release sCD163, an independent marker in CLL, which can be measured in the serum. The research of new targets, which can be considerate as more specific, targeting NLC is still an interesting way to find new therapies in CLL.

Many research articles describes fish oil as a beneficial for health. Mainly for prevention of cardiovascular diseases or that fish oil reduces cholesterol in blood stream. Aim of this study is to investigate effects of fish oil specifically of his parts EPA+DHA, on a modulation of immune system and phenotypic differentiation. For the experiment were chosen monocytes and macrophages obtained from experimental group of animals which was fed a diet consist of the addition of fish oil in an amount 45mg/kg of live weight. Then inflammatory state was induced by LPS. Cells surface receptors were analyze through flow cytometry specifically CD14, CD163, SLA-DR and theirs combinations after they were statistically processed. Results shows proinflammatory effects of fish oil however they were statistically not significant.

Introduction Breast cancer is the most common cancer among women in Sweden and world-wide. Immunohistochemical (IHC) expression of oestrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) and Ki-67 is routinely performed for diagnostic and prognostic purposes. Based on these biomarkers tumours are divided into subtypes: Luminal A, Luminal B, HER2-enriched and triple negative breast cancer (TNBC). There is evidence that tumour associated macrophages (TAMs) have a crucial role in tumour development. To identify TAMs the antibody CD163 can be used in immunohistochemical staining. Aim The aim of the study was to investigate differences in density of CD163+ macrophages among different subtypes of breast cancer. Material and method Formalin-fixed paraffin-embedded (FFPE) breast cancer tissue from the Biobank of Region Värmland was used. Tissue was de-identified before analysis. Groups were formed based on immunohistochemical expression of ER, PR and HER2, with the exclusion of the Luminal B group. Tissue was stained with CD163. Analysis was performed semi-quantitatively. The border between tumour and healthy tissue was assessed as well as the intra-tumoral tissue. Results At the tumour border, density of CD163+ macrophages was found to be lower in the Luminal group compared to both the HER2-enriched and TNBC.Within intra-tumoral tissue, the density of CD163+ macrophages was found to be lower in the Luminal compared to the HER2-enriched group. Conclusion The density of CD163+ macrophages is higher in HER-enriched and TNBC tumours compared to Luminal A breast cancer tumours.