BACKGROUND: Immune cells promote periodontal bone loss through an unresolved inflammatory response to bacterial pathogens. The limited availability of ex vivo gingival immune cells severely impedes identification of cell types and cell-specific functions that drive human periodontitis and thus impedes the development of effective pharmacotherapeutics. Previous studies have largely relied on mRNA analysis and confocal microscopy to imprecisely estimate gingival immune cell function. The aim of the study was to develop a cell type-specific technique to quantitate function of resident gingival immune cells. METHODS: Diseased tissues from chronic periodontitis in non-diabetes or type 2 diabetes subjects or relatively healthy gingival tissues were removed during standard-of-care surgery for pocket reduction surgery or crown lengthening, respectively. Gingiva was dissociated with collagenase to generate single cell suspensions, then 9-color flow cytometry was used quantitate and/or isolate myeloid cells (CD11b+), B cells (CD20+), T cells (CD4+ or CD8+) and natural killer (NK) cells (CD56+). We stimulated the sorted cells with lineage-appropriate activators for 36 hrs and measured cytokine production by ELISPOT, an assay that identifies individual cytokine-producing cells by fixed “spots” on a solid support. RESULTS: A higher proportion of gingival CD4+ T helper cells and not CD8+cytoxic T cells from subjects with periodontal disease with or without type 2 diabetes produce pro-inflammatory cytokines compared to CD4+ T cells from crown lengthening subjects. CD4+ T cells were the dominant cell population in gingiva from all three groups, and all groups contained similar proportions of cytotoxic (CD8+) T cells, myeloid cells (CD11b+), B cells (CD20+) and natural killer cells (CD56+). CONCLUSION: The combination of flow cytometry, cell sorting and ELISPOT identified CD4+ T cells as dominant immune cells in human periodontal lesions, and identified T cell cytokines that may uniquely promote periodontitis in type 2 diabetes.

Multiple myeloma is an agressive hemato-oncological disease the diagnosis and treatment possibilities of which have been developing for the last 15 years. The diagnostic methods include flow cytometry which uses antigen detection to distinguish between pathological and physiological plasmocytes. One of the monitored markers is marker CD45 which could be, according to our monitoring, of a negative prognostic value. 71 patients in our group were divided according to the intensity of CD45 antigen expression and the group with a lower expression showed a statistically higher risk of relapse within 12 months 62% vs. 25 % (p=0,0011). We did not find any connection with the influences of induction therapy or cytogenetics which are otherwise considered the most important prognostic markers. Multiple myeloma treatment involves a lot of combined protocols; and the repeated autologous transplantation is still considered to be the most efficient. We observed more positive results in planning the second autologous transplant early in patients who reached just partial remission after the 1st autologous transplant - significantly better TFS (treatment free survival) and overall survival (OS) were in the group of patients with early tandem transplantation: 18 months vs. 10 months (p=0.04) and the OS median was not reached...

Multiple myeloma is an agressive hemato-oncological disease the diagnosis and treatment possibilities of which have been developing for the last 15 years. The diagnostic methods include flow cytometry which uses antigen detection to distinguish between pathological and physiological plasmocytes. One of the monitored markers is marker CD45 which could be, according to our monitoring, of a negative prognostic value. 71 patients in our group were divided according to the intensity of CD45 antigen expression and the group with a lower expression showed a statistically higher risk of relapse within 12 months 62% vs. 25 % (p=0,0011). We did not find any connection with the influences of induction therapy or cytogenetics which are otherwise considered the most important prognostic markers. Multiple myeloma treatment involves a lot of combined protocols; and the repeated autologous transplantation is still considered to be the most efficient. We observed more positive results in planning the second autologous transplant early in patients who reached just partial remission after the 1st autologous transplant - significantly better TFS (treatment free survival) and overall survival (OS) were in the group of patients with early tandem transplantation: 18 months vs. 10 months (p=0.04) and the OS median was not reached...

Tubarões e Raias pertencem à classe dos Chondrichthyes, por serem animais que possuem esqueleto cartilaginoso. O Tubarão-azul (Prionace glauca), popularmente conhecido como \"cação-azul\" dentre todas as espécies de tubarão é a mais abundante no ambiente marinho, podendo ser encontrado em toda a parte do mundo. O estudo teve como objetivo estudar a morfologia das nadadeiras do tubarão-azul (Prionace glauca) e os efeitos sobre células tumorais \"in vitro . Os resultados foram obtidos através da microscopia de luz e Citometria de fluxo. Com os caracteres encontrados na analise macroscópica foi possível identificar as nadadeiras obtidas como sendo do Tubarão-azul (Prionace glauca). A histologia da cartilagem da nadadeira do Tubarãoazul, demonstrou que é formada de cartilagem hialina, apresentando três regiões distintas, sendo no seu interior formado por condrócitos, na periferia de cartilagem calcificada e nas bordas formada por pericôndrio com a presença de colágeno tipo I,II e III. Os resultados obtidos das amostras do Elemento Radial não evidenciam alterações funcionais, quanto ao armazenamento, transporte e obtenção do suprimento celular, estes são viáveis e satisfatórios. O composto da cartilagem de tubarão para o tratamento de tumor \"in vitro\" neste estudo sugeriu que o mesmo apresenta uma atividade anti-tumoral significativa. Mostrou efeito tóxico sobre os tumores de mama murina (TAE) e tumor de mama canino (TMC) em baixas concentrações, não apresentando efeito tóxico nas células de fibroblasto nas mesmas concentrações. / Sharks and rays belong to the Chondrichthyes Class, once they have cartilaginous skeleton. The blue shark (Prionaceglauca), popularly known as \"blue-cation\", among all shark species is the most abundant in the marine environment and can be found everywhere in the world. This study aimed to study the fin morphology in the blue shark (Prionaceglauca) and its effects on \"in vitro\" tumor cells. The results were obtained using light microscopy and flow cytometry. Using the gross morphology we confirm that the fins belonged to the blue shark (Prionaceglauca). The fin cartilage of the blue shark was formed of hyaline cartilage. It showed three distinct regions with chondrocytes inside, calcified cartilage in the periphery, and perichondrium with collagen type I, II and III in the margins. The results obtained from the Radial Element not showed functional changes as storage, transport and cellular supplies obtaining, they were feasible and satisfactory. The use of shark cartilage compound for the treatment of \"in vitro\" tumor cells suggested that it showed a significant anti-tumor activity. It showed a toxic effect on murine breast tumors (MBT) and canine breast tumor (CBT) at low concentrations, with no significant toxic effect on fibroblast cells using the same concentrations.

<p>Flowcytometric technology has been widely used for measurement of the absolute numbers of T-lymphocytes subsets in Human Immunodeficiency virus (HIV), defining the disease state, monitoring antiviral treatment, and identifying any risk for opportunistic infections. A manual preparing of the samples is required. More recently an automated and enclosed blood cell counting, Celldyn Sapphire has been introduced. In this study the performance of the Flow cytometer EPICS XL as a reference method for analysis of CD3+, CD4+ and CD8 T-lymphocytes was evaluated with blood from 40 individual’s samples. EPICS XL was also compared with Celldyn Sapphire in the analysis of T-lymphocyte subsets in 39 blood samples from patients with low, high and normal lymphocyte counts. The result showed that the precision was high for both EPICS XL (2.5%) and Celldyn (10%). The method was linear over a wide range. Comparisons of CD3+, CD4+, and CD8+ T-lymphocytes analysis showed high coefficients of correlation (r0.9) and agreement (y>0.9x) between two instruments. A lower degree of agreement was observed at low concentration of CD3+ and CD4+ T-lymphocytes (0.757, 0.739). This means that cell counts obtained by Celldyn were 30% lower than those obtained with EPICS XL. This study shows that both EPICS XL and Celldyn Sapphire were suitable for CD4+ and CD8+ T cell counts. It is however preferable to use Flowcytometry for counting of low concentration of CD4+ T-lymphocytes (<200 cells/µL).</p>

Flowcytometric technology has been widely used for measurement of the absolute numbers of T-lymphocytes subsets in Human Immunodeficiency virus (HIV), defining the disease state, monitoring antiviral treatment, and identifying any risk for opportunistic infections. A manual preparing of the samples is required. More recently an automated and enclosed blood cell counting, Celldyn Sapphire has been introduced. In this study the performance of the Flow cytometer EPICS XL as a reference method for analysis of CD3+, CD4+ and CD8 T-lymphocytes was evaluated with blood from 40 individual’s samples. EPICS XL was also compared with Celldyn Sapphire in the analysis of T-lymphocyte subsets in 39 blood samples from patients with low, high and normal lymphocyte counts. The result showed that the precision was high for both EPICS XL (2.5%) and Celldyn (10%). The method was linear over a wide range. Comparisons of CD3+, CD4+, and CD8+ T-lymphocytes analysis showed high coefficients of correlation (r0.9) and agreement (y&gt;0.9x) between two instruments. A lower degree of agreement was observed at low concentration of CD3+ and CD4+ T-lymphocytes (0.757, 0.739). This means that cell counts obtained by Celldyn were 30% lower than those obtained with EPICS XL. This study shows that both EPICS XL and Celldyn Sapphire were suitable for CD4+ and CD8+ T cell counts. It is however preferable to use Flowcytometry for counting of low concentration of CD4+ T-lymphocytes (&lt;200 cells/µL).

Tubarões e Raias pertencem à classe dos Chondrichthyes, por serem animais que possuem esqueleto cartilaginoso. O Tubarão-azul (Prionace glauca), popularmente conhecido como \"cação-azul\" dentre todas as espécies de tubarão é a mais abundante no ambiente marinho, podendo ser encontrado em toda a parte do mundo. O estudo teve como objetivo estudar a morfologia das nadadeiras do tubarão-azul (Prionace glauca) e os efeitos sobre células tumorais \"in vitro . Os resultados foram obtidos através da microscopia de luz e Citometria de fluxo. Com os caracteres encontrados na analise macroscópica foi possível identificar as nadadeiras obtidas como sendo do Tubarão-azul (Prionace glauca). A histologia da cartilagem da nadadeira do Tubarãoazul, demonstrou que é formada de cartilagem hialina, apresentando três regiões distintas, sendo no seu interior formado por condrócitos, na periferia de cartilagem calcificada e nas bordas formada por pericôndrio com a presença de colágeno tipo I,II e III. Os resultados obtidos das amostras do Elemento Radial não evidenciam alterações funcionais, quanto ao armazenamento, transporte e obtenção do suprimento celular, estes são viáveis e satisfatórios. O composto da cartilagem de tubarão para o tratamento de tumor \"in vitro\" neste estudo sugeriu que o mesmo apresenta uma atividade anti-tumoral significativa. Mostrou efeito tóxico sobre os tumores de mama murina (TAE) e tumor de mama canino (TMC) em baixas concentrações, não apresentando efeito tóxico nas células de fibroblasto nas mesmas concentrações. / Sharks and rays belong to the Chondrichthyes Class, once they have cartilaginous skeleton. The blue shark (Prionaceglauca), popularly known as \"blue-cation\", among all shark species is the most abundant in the marine environment and can be found everywhere in the world. This study aimed to study the fin morphology in the blue shark (Prionaceglauca) and its effects on \"in vitro\" tumor cells. The results were obtained using light microscopy and flow cytometry. Using the gross morphology we confirm that the fins belonged to the blue shark (Prionaceglauca). The fin cartilage of the blue shark was formed of hyaline cartilage. It showed three distinct regions with chondrocytes inside, calcified cartilage in the periphery, and perichondrium with collagen type I, II and III in the margins. The results obtained from the Radial Element not showed functional changes as storage, transport and cellular supplies obtaining, they were feasible and satisfactory. The use of shark cartilage compound for the treatment of \"in vitro\" tumor cells suggested that it showed a significant anti-tumor activity. It showed a toxic effect on murine breast tumors (MBT) and canine breast tumor (CBT) at low concentrations, with no significant toxic effect on fibroblast cells using the same concentrations.

The mesenchymal stromal cell (MSC) is of great interest for its immunomodulatory and regenerative properties. However, to research and use these MSCs it is essential to identify and characterize them as such. They need to fulfill the MSCs' minimal criteria which assess the differentiation potential, cell surface markers, and adherence. In this study, cells donated from human bone marrow were identified as MSC according to the minimal criteria. Methods used were flow cytometry, immunofluorescent staining, and ELISA. Furthermore, the population was cultured in three different media (DMEM-LG with either 10% FBS, 2% FBS, or 10% FBS supplemented with 10% conditioned media from human urinary bladder carcinoma cells (T24)) for 21 days whereupon tested for the mesenchymal characteristics, cells were counted and size measured at every passage. All cultures maintained their mesenchymal character, however, cells grown in 2% FBS became a considerably more heterogenous population regarding cell size and granularity, perhaps because of senescence. Additionally, these cells somewhat decreased in proliferation and resulted in 1 x 106 cells after 21 days, however, this was not a significant decrease when compared to the 10% FBS culture which had 2.16 x 106 cells after 21 days (p=0.061). On the contrary, the culture supplemented with T24 conditioned media resulted in a significantly higher cell count with 4.75 x 106 cells (p=0.008). Further studies could investigate which components in the conditioned media contributed to the proliferation. Moreover, the cell population in this study could not be characterized as MSC with certainty as additional cell surface markers should be tested.

Eine wichtige Eigenschaft der Plasmamembran eukaryotischer Zellen ist die stabile transversale Asymmetrie der Phospholipide. Sie wird energieabhängig durch die Aktivität einer Aminophospholipidtranslokase aufrechterhalten und gilt als wichtige Voraussetzung für die Homöostasis der Zellen. Die Plasmamembran einiger Säugerzellen weist zudem laterale Lipiddomänen auf, denen eine wesentliche Bedeutung bei der Signaltransduktion zugeschrieben wird. Während der Genese durchlaufen die Membranen der Säugerspermien intensive Veränderungen. Um die Bedeutung der Phospholipidasymmetrie für die Funktion der Spermien zu untersuchen, wurde die Lokalisation und Dynamik von Phosphatidylserin in der Zell- und Akrosommembran von Eberspermien im Verlauf von Kapazitation und Akrosomreaktion betrachtet. Unter Ausnutzung der selektiven, kalziumabhängigen Bindung von AnnexinV an endogenes Phosphatidylserin konnte dessen Lokalisation an morphologisch differenzierten Zellen verfolgt werden. Eine Markierung der Zellen mit NBD-markierten Phospholipidanaloga lieferte zudem Informationen zur Dynamik der Phospholipide in der Plasmamembran. Die Differenzierung der Zellen erfolgte entweder am Durchflusszytometer oder fluoreszenz- bzw. elektronenmikroskopisch. Die Ergebnisse der vorliegenden Arbeit weisen sowohl auf eine transversale als auch laterale Ungleichverteilung der Lipide in der Zell- und Akrosommembran während der Genese der Spermien hin. Neben der stabilen transversalen Phospholipidasymmetrie der Plasmamembran konnte erstmals eine zytoplasmatische Lokalisation von Phosphatidylserin auf der äußeren Akrosommembran nachgewiesen werden. Somit akkumulieren die beiden einander zugewandten zytoplasmatischen Monolayer von Plasmamembran und äußerer Akrosommembran Phosphatidylserin. Kapazitationsbedingt kommt es zu einer engen Wechselwirkung zwischen Plasmamembran und äußerer Akrosommembran. Die Ausbildung lateraler Membrandomänen, in denen Phosphatidylserin zytoplasmatisch akkumuliert, wird als Voraussetzung für diese enge Assoziation diskutiert. Weitere Hinweise auf eine funktionelle Bedeutung lateraler Membrandomänen lieferten die Arbeiten zur Isolation Triton-unlöslicher Lipiddomänen aus der Plasmamembran von Forellenspermien. / One of the essential qualities of cell membranes in Eucaryotae is a stable transverse phospholipid asymmetry. It is regulated and maintained by ATP-dependent action of an aminophospholipid translocase and is a major prerequisite for cell homeostasis. The plasma membranes of several mammalian cells show moreover lateral lipid domains, which are imputed to play a significant role in signal transduction. The membranes of mammalian spermatozoa undergo significant changes during genesis. The localisation and dynamics of phosphatidylserine in the cell as well as acrosome membranes of boar sperm cells was studied during capacitation and acrosome reaction to assess the relevance of lipid asymmetry for sperm function. The localisation of endogenous phosphatidylserine in morphologically differentiated cells was followed using the selective calcium depending binding of annexinV. Information on the transverse dynamics of phospholipids in the plasma membrane was obtained by labelling the cells with a NBD-phospholipid analogues. The morphological status of the cells was assessed by flow cytometry, fluorescence and electron microscopy. The results of this study indicate both a transversal and lateral inhomogenous distribution of lipids in the cell membrane as well as in the outer acrosome membrane during sperm genesis. The plasma membrane of boar sperm shows a stable transversal lipid asymmetry characterised by an accumulation of phosphatidylserine in the cytoplasmic monolayer. Moreover a cytoplasmic localisation of phosphatidylserine on the outer acrosome membrane could be detected for the first time. Therefore the two facing cytoplasmic leaflets of the outer acrosome and cell membrane contain phosphatidylserine. Applying microscopy substantiated the hypothesis that there are close interactions between the cell membrane and the outer layer of the acrosome membrane because of capacitation. The cytoplasmic accumulation of phosphatidylserine in lateral lipid domains is probably essential for the strong association of plasma and outer acrosome membrane finally leading to local fusions of both membranes. An indication for the functional meaning of lateral membrane domains in sperm cells was futher deduced from the isolation of Triton-insoluble lipid domains from membranes of trout sperm cells.

This thesis describes studies in patients with inflammatory bowel disease (IBD) and collagenous colitis (CC). We investigated mucosal eosinophil and neutrophil granulocytes and T-cells involved in the inflammatory processes and aimed at determining whether these processes are reflected in the faecal (F) contents of specific proteins secreted by cells in the intestinal mucosa. Thus, we measured eosinophil cationic protein (ECP) and eosinophil protein X (EPX) and the neutrophil derived myeloperoxidase (MPO) and calprotectin (C); and in addition, chromogranin A (CgA), Chromogranin B (CgB) and secretoneurin (SN), derived from EEC cells and cells in the enteric nervous system. We found that a normalised FC level can serve as a surrogate marker for successful treatment in patients with IBD, but persistently high FC levels need further evaluation (study I). Furthermore, FC and F-MPO appear to relate better than F-EPX to treatment outcome in IBD. We evaluated F-ECP, F-EPX, F-MPO and FC as markers of disease activity and treatment outcome in patients with CC (study III) and concluded that F-ECP was the best discriminator of detecting active CC. Normalised F-ECP and F-EPX could serve as markers of successful treatment. We showed that the inflammation in CC is characterised by activated eosinophils, but that there is no neutrophil activity (study II). T-cells have a lower grade of activity in active CC than in control subjects. During budesonide treatment the normal activation of eosinophils and T-cells is restored, with concomitant clinical remission. The findings in studies II and III indicate that the eosinophils have an essential role in the pathophysiology of CC. Markedly higher values of F-CgA, F-CgB and F-SN were found in patients with CC than in those with IBD and controls (study IV) indicating a crucial role for the intestinal neuro-endocrine system in the pathogenesis of collagenous colitis.