T cells are an essential part of the immune system. They determine the specificity of the immune response to foreign substances and, thus, help to protect the body from infections and cancer. Recently, T cells have gained much attention as promising tools in adoptive T cell transfer for cancer treatment. However, it is crucial not only for medical purposes but also for research to obtain T cells in large quantities, of high purity and functionality. To fulfill these criteria, efficient and robust isolation methods are needed. We used three different isolation methods to separate CD3-specific T cells from leukocyte concentrates (buffy coats) and Ficoll purified PBMCs. To catch the target cells, the Traceless Affinity Cell Selection (TACS®) method, based on immune affinity chromatography, uses CD-specific low affinity Fab-fragments; while the classical Magnetic Activated Cell Sorting (MACS®) method relies on magnetic beads coated with specific high affinity monoclonal antibodies. The REAlease® system also works with magnetic beads but, in contrast to MACS®, low-affinity antibody fragments are used. The target cells separated by TACS® and REAlease® are “label-free”, while cells isolated by MACS® still carry the cell specific label. The time required to isolate T cells from buffy coat by TACS® and MACS® amounted to 90 min and 50 min, respectively, while it took 150 min to isolate T cells from PBMCs by TACS® and 110 min by REAlease®. All methods used are well suited to obtain T cells in large quantities of high viability (>92%) and purity (>98%). Only the median CD4:CD8 ratio of approximately 6.8 after REAlease® separation differed greatly from the physiological conditions. MACS® separation was found to induce proliferation and cytokine secretion. However, independent of the isolation methods used, stimulation of T cells by anti CD3/CD28 resulted in similar rates of proliferation and cytokine production, verifying the functional activity of the isolated cells.

No description available.

EINLEITUNG: Eine Trachealrekonstruktion bedingt Komplikationen wie z.B. Infektionen und Stenosierungen durch Granulationsgewebe. Diese werden durch ein differenziertes respiratorisches Epithel, das eine mukoziliäre Clearance ermöglicht, deutlich reduziert. Die Basalzellen gelten als die Vorläuferzellen des humanen respiratorischen Epithels (HRE), d.h. sie können sich teilen und besitzen das Potenzial zur Differenzierung. Durch die magnetische Zellseparation (MACS) sollen Vorläuferzellen aus dem HRE angereichert und anschließend kultiviert werden. METHODEN: Die Conchae nasales inferiores von 80 Patienten (mittleres Alter 40 ± 14 Jahre) dienen als Zellquelle für HRE-Zellen, die mittels enzymatischen Verdaus mit Dispase II (2,4 U/ml) aus dem Gewebeverband isoliert werden. Die Kultivierung der HRE-Zellen erfolgt auf Kollagen-A-beschichteten Kulturgefäßen in serumfreiem AECG-Medium. Die Bindungsspezifität von verschiedenen extrazellulären Zellmarkern wie GSA I B4, CD44S und CD44v6 wird immunhistochemisch überprüft. Dabei erweist sich nur CD44v6 als spezifisch für Basalzellen. Die Vorläuferzellen aus dem HRE-Zellgemisch werden durch monoklonale Antikörper gegen CD44v6 und Goat-Anti-Mouse-Microbeads magnetisch konjugiert. Anschließend werden sie mittels MACS positiv selektiert. ERGEBNISSE: Die Präparation der Nasenmuscheln ergibt Einzelzellsuspensionen aus vitalen HRE-Zellen (Vitalität > 80%, n = 30). Eine Beschichtung der Kulturgefäße mit Kollagen A steigert die Adhärenz der HRE-Zellen signifikant (p < 0,0145, n = 5). Die Proliferationskinetik der HRE-Zellkulturen lässt sich durch die Populationsverdoppelungszeit charakterisieren (tPD = 23 ± 3h, n = 3). Während eines Monats wird die Proliferationskapazität der HRE-Zellen durch Zellvermehrung (383fach, n = 6) ermittelt. Die magnetische Separation ergibt eine Zellfraktion (20 ± 2%, n = 5), die sich positiv zu CD44v6 verhält. Anschließend werden die separierten Zellen eine Woche auf Kollagen A kultiviert, wobei sie alle ein adäquates Proliferationsverhalten aufweisen. SCHLUSSFOLGERUNG: Die Ergebnisse zeigen, dass CD44v6 ein spezifischer Marker für Basalzellen ist, der sich für die Anreicherung einer positiven Zellfraktion mittels MACS eignet. In weiteren Studien muss überprüft werden, inwiefern eine solche magnetisch separierte Population aus Basalzellen die Besiedelung eines Trachealersatzes mit einem differenzierten respiratorischen Epithel ermöglicht. / OBJECTIVE: Common problems affecting patients with tracheal replacement are infections and stenosis caused by granulation tissue. These complications can be minimized by establishing a differentiated respiratory epithelium, which facilitates mucocilliary clearance. The basal cells are regarded as the progenitor cells of the human respiratory epithelium (HRE). They are known to divide and possess the ability to differentiate. These cells can be enriched by means of magnetic cell sorting (MACS) for the purpose of cultivation. METHODS: The inferior nasal turbinates of 80 patients (mean age 40 ± 14 years) are used as cell source. The HRE-cells are isolated by a standard preparation using an enzymatic digestion with Dispase II (2,4 U/ml). The HRE-cells are plated on culture dishes coated with Collagen A in serum-free AECG-Medium. Several extracellular cell markers including GSA I B4, CD44S and CD44v6 are tested by immunohistochemistry. Only CD44v6 shows specific staining of basal cells. The progenitor cells of mixed single cell suspensions of HRE-cells are marked with monoclonal antibodies against CD44v6 and are conjugated with Goat-Anti-Mouse-Microbeads. Enrichment of progenitor cells is achieved by MACS using a positive selection protocol. RESULTS: The preparation of the nasal turbinates yields viable single cell suspensions of HRE-cells (viability > 80%, n = 30). Adhesion of HRE-cells is enhanced significantly (p < 0,0145, n = 5) by coating the culture dishes with Collagen A. The kinetics of proliferation of HRE-cell-cultures can be characterized by the population doubling time (tPD = 23 ± 3h, n = 3). In the course of one month the capacity of proliferation is approximated by cell expansion (383fold, n = 6). Magnetic cell sorting results in a cell fraction (20 ± 2%, n = 5) positive for CD44v6. The separated cells are cultured on Collagen A for one week, where they all show adequate proliferation. CONCLUSIONS: The results indicate that CD44v6 is a specific marker for basal cells and enables the enrichment of a positive cell fraction via application of MACS. Further studies will be required to investigate the potential of such a magnetically separated population of basal cells to generate a differentiated respiratory epithelium on a tracheal prosthesis.

Prover segurança às informações trafegadas nos mais diversos tipos de redes é algo essencial. Entretanto, redes altamente dependentes de dispositivos com recursos limitados (como sensores, tokens e smart cards) apresentam um desafio importante: a reduzida disponibilidade de memória e energia destes dispositivos, bem como sua baixa capacidade de processamento, dificultam a utilização de diversos algoritmos criptográficos considerados seguros atualmente. Este é o caso não apenas de cifras simétricas, que proveem confidencialidade aos dados, mas também de MACs (Message Authentication Code, ou Código de Autenticação de Mensagem), que garantem sua integridade e autenticidade. De fato, algumas propostas recentes de cifras de bloco dedicadas a plataformas limitadas (e.g., o Curupira-2) proveem segurança e desempenho em um nível mais adequado a este tipo de cenário do que soluções tradicionais. Seguindo uma linha semelhante, o presente trabalho concentra-se no projeto e análise MACs leves e seguros voltados a cenários com recursos limitados, com foco especial em redes de sensores sem fio (RSSF). Marvin é o nome do algoritmo de MAC proposto neste trabalho. Marvin adota a estrutura Alred, que reutiliza porções de código de uma cifra de bloco subjacente e, assim, introduz um reduzido impacto em termos de ocupação de memória. Este algoritmo apresenta uma estrutura bastante flexível e é altamente paralelizável, permitindo diversas otimizações em função dos recursos disponíveis na plataforma alvo. Como vantagem adicional, Marvin pode ser usado tanto em cenários que necessitam apenas da autenticação de mensagens quanto em esquemas de AEAD (Authenticated- Encryption with Associated Data, ou Encriptação Autenticada com Dados Associados), que aliam encriptação e autenticação. O esquema de AEAD proposto neste trabalho, denominado LetterSoup, explora as características da estrutura do Mar vin e adota uma cifra de bloco operando no modo LFSRC (Linear Feedback Shift Register Counter, ou Contador-Registrador de Deslocamento Linear com Retroalimentação). Além da especificação de ambos os algoritmos, este documento apresenta uma análise detalhada da segurança e desempenho dos mesmos em alguns cenários representativos. / Security is an important concern in any modern network. However, networks that are highly dependent on constrained devices (such as sensors, tokens and smart cards) impose a difficult challenge: their reduced availability of memory, processing power and (specially) energy hinders the deployment of many modern cryptographic algorithms known to be secure. This inconvenience affects not only the deployment of symmetric ciphers, which provide data confidentiality, but also Message Authentication Codes (MACs), used to attest the messages integrity and authenticity. Due to the existence of dedicated block ciphers whose performance and security are adequate for use in resource-constrained scenarios (e.g., the Curupira-2), the focus of this document is on the design and analysis of message authentication algorithms. Our goal is to develop a secure and lightweight solution for deployment on resource constrained scenarios, with especial focus on Wireless Sensor Networks (WSNs). Marvin is the name of the MAC algorithm proposed in this document. Marvin adopts the Alred structure, allowing it to reuse parts of an underlying block cipher machinery; as a result, Marvins implementation builds on the ciphers efficiency and introduces little impact in terms of memory occupation. Moreover, this algorithm presents a flexible and highly parallelizable structure, allowing many implementation optimizations depending on the resources available on the target platform. Marvin can be used not only as an authentication-only function, but also in an Authenticated- Encryption with Associated Data (AEAD) scheme, combining authentication and encryption. In this document, we define a new AEAD proposal called LetterSoup, which is based on the LFSRC (Linear Feedback Shift Register Counter) mode of operation and builds on Marvin. Together with the specification of both algorithms, we provide a detailed security analysis and evaluate their performance in some representative scenarios.

Prover segurança às informações trafegadas nos mais diversos tipos de redes é algo essencial. Entretanto, redes altamente dependentes de dispositivos com recursos limitados (como sensores, tokens e smart cards) apresentam um desafio importante: a reduzida disponibilidade de memória e energia destes dispositivos, bem como sua baixa capacidade de processamento, dificultam a utilização de diversos algoritmos criptográficos considerados seguros atualmente. Este é o caso não apenas de cifras simétricas, que proveem confidencialidade aos dados, mas também de MACs (Message Authentication Code, ou Código de Autenticação de Mensagem), que garantem sua integridade e autenticidade. De fato, algumas propostas recentes de cifras de bloco dedicadas a plataformas limitadas (e.g., o Curupira-2) proveem segurança e desempenho em um nível mais adequado a este tipo de cenário do que soluções tradicionais. Seguindo uma linha semelhante, o presente trabalho concentra-se no projeto e análise MACs leves e seguros voltados a cenários com recursos limitados, com foco especial em redes de sensores sem fio (RSSF). Marvin é o nome do algoritmo de MAC proposto neste trabalho. Marvin adota a estrutura Alred, que reutiliza porções de código de uma cifra de bloco subjacente e, assim, introduz um reduzido impacto em termos de ocupação de memória. Este algoritmo apresenta uma estrutura bastante flexível e é altamente paralelizável, permitindo diversas otimizações em função dos recursos disponíveis na plataforma alvo. Como vantagem adicional, Marvin pode ser usado tanto em cenários que necessitam apenas da autenticação de mensagens quanto em esquemas de AEAD (Authenticated- Encryption with Associated Data, ou Encriptação Autenticada com Dados Associados), que aliam encriptação e autenticação. O esquema de AEAD proposto neste trabalho, denominado LetterSoup, explora as características da estrutura do Mar vin e adota uma cifra de bloco operando no modo LFSRC (Linear Feedback Shift Register Counter, ou Contador-Registrador de Deslocamento Linear com Retroalimentação). Além da especificação de ambos os algoritmos, este documento apresenta uma análise detalhada da segurança e desempenho dos mesmos em alguns cenários representativos. / Security is an important concern in any modern network. However, networks that are highly dependent on constrained devices (such as sensors, tokens and smart cards) impose a difficult challenge: their reduced availability of memory, processing power and (specially) energy hinders the deployment of many modern cryptographic algorithms known to be secure. This inconvenience affects not only the deployment of symmetric ciphers, which provide data confidentiality, but also Message Authentication Codes (MACs), used to attest the messages integrity and authenticity. Due to the existence of dedicated block ciphers whose performance and security are adequate for use in resource-constrained scenarios (e.g., the Curupira-2), the focus of this document is on the design and analysis of message authentication algorithms. Our goal is to develop a secure and lightweight solution for deployment on resource constrained scenarios, with especial focus on Wireless Sensor Networks (WSNs). Marvin is the name of the MAC algorithm proposed in this document. Marvin adopts the Alred structure, allowing it to reuse parts of an underlying block cipher machinery; as a result, Marvins implementation builds on the ciphers efficiency and introduces little impact in terms of memory occupation. Moreover, this algorithm presents a flexible and highly parallelizable structure, allowing many implementation optimizations depending on the resources available on the target platform. Marvin can be used not only as an authentication-only function, but also in an Authenticated- Encryption with Associated Data (AEAD) scheme, combining authentication and encryption. In this document, we define a new AEAD proposal called LetterSoup, which is based on the LFSRC (Linear Feedback Shift Register Counter) mode of operation and builds on Marvin. Together with the specification of both algorithms, we provide a detailed security analysis and evaluate their performance in some representative scenarios.

We have acquired Hubble Space Telescope (HST) and Very Large Telescope near-infrared spectra and images of supernova (SN) Refsdal after its discovery as an Einstein cross in fall 2014. The HST light curve of SN Refsdal has a shape consistent with the distinctive, slowly rising light curves of SN. 1987A-like SNe, and we find strong evidence for a broad H alpha P-Cygni profile and Na I D absorption in the HST grism spectrum at the redshift (z = 1.49) of the spiral host galaxy. SNe. IIn, largely powered by circumstellar interaction, could provide a good match to the light curve of SN Refsdal, but the spectrum of a SN IIn would not show broad and strong H alpha and Na I D absorption. From the grism spectrum, we measure an H alpha expansion velocity consistent with those of SN. 1987A-like SNe at a similar phase. The luminosity, evolution, and Gaussian profile of the H alpha emission of the WFC3 and X-shooter spectra, separated by similar to 2.5 months in the rest frame, provide additional evidence that supports the SN. 1987A-like classification. In comparison with other examples of SN. 1987A-like SNe, photometry of SN Refsdal favors bluer B - V and V - R colors and one of the largest luminosities for the assumed range of potential magnifications. The evolution of the light curve at late times will provide additional evidence about the potential existence of any substantial circumstellar material. Using MOSFIRE and X-shooter spectra, we estimate a subsolar host-galaxy metallicity (8.3 +/- 0.1 dex and <8.4 dex, respectively) near the explosion site.

1. GND 2. RDA 3. CrissCross 4. MACS 5. Petrus

Therapeutic Assessment (TA) is a hybrid of assessment and therapy techniques in which assessors actively collaborate with clients during an individualized assessment. TA is centered around client assessment questions and provides a safe environment where clients can create shifts in their ‘story’ of self. More specifically, TA with children and their parents has demonstrated more confident parenting and parents’ better understanding of their child’s difficulties, while children have shown decreased problem behaviors and improved social/emotional functioning. The theoretical framework behind TA emphasizes the importance of the interpersonal interactions between the assessor and client, such as the development of a strong assessor client relationship and collaboration. These interpersonal processes are conceptualized as catalysts for greater depth of parent investment in the assessment and deeper levels of feedback results. The need for greater parent involvement and partnership in child mental health services is increasingly recognized in the client/parent satisfaction literature. Parent feedback to child mental health services is most often acquired through satisfaction questionnaires. However, the satisfaction literature has well known limitations, specifically a lack of unifying theory and methodological issues in scale development. Parent satisfaction research indicates that interpersonal experiences are more related to satisfaction than outcomes or client characteristics, and that more psychometrically sound measures are needed. Currently, satisfaction surveys do not provide a detailed understanding of parents’ experiences to inform practice and research. The current study outlines the development of the Parent Experience of Assessment Scale (PEAS). The PEAS is anchored in the theoretical orientation of TA and provides a more quantifiable measure of hypothesized underlying TA constructs. Moreover, the development of the PEAS uses advanced statistical techniques, such as Confirmatory Factor Analysis and invariance testing, to provide a higher level of psychometric rigor. The resulting scale consists of 24 items divided among 5 subscales with demonstrated relationships to general satisfaction. Structural equation modeling provides insight via direct and indirect effects among the PEAS subscales and their relationship to general satisfaction. Through the development of the PEAS, this study provides empirical evidence and support for TA theory and a more nuanced understanding of parent experiences related to satisfaction. / text

Despite recent advances in stem cell biology, immunotherapy and transplantation, substantial barriers still exist in the large-scale specific separation of a discrete population of human therapeutic cells from a cell suspension. The ideal purification technique should combine high cell purity, yield and function, with fast processing and affordability. Currently, fluorescence-activated cell sorting with flow cytometry (FACS) and magnetic activated cell sorting (MACS®) are the most used methods for cell separation and purification and have been employed extensively in molecular biology, diagnostic and cell sorting applications, because they are considered to be gentle, fast and scalable. However, these methods have several key disadvantages; they are invariably expensive, yield low log cell reduction (LCR) rates, and suffer from drawbacks when applied to niche cell populations, such as those requiring multiple tandem separation steps and/or involving combined positive and negative cell selection steps. To address this challenge, a new cell affinity selection system was developed. The selectivity is based on the reversible monomeric avidin biotin interaction and it is primary designed for positive selection. The initial studies were performed on flat, nonporous, glass coverslips and the technology was then successfully transferred on high grade smooth non-porous glass beads (with a diameter of 79.12 to 118.59 μm). The multi-step layer-by-layer deposition procedure culminating in dextran-coated supports bearing monomeric avidin was rigorously characterized and subsequently employed in packed bed chromatography experiments with human erythrocytes isolated from cord blood and B lymphocytes from cell lines. The developed affinity selection platform was highly selective, efficient and, most importantly, resulted in high yields, cell purity and viability comparable with MACS® technology. Additionally scale up is possible and could be easily transferred to another chromatographic matrix with the appropriate structure.

B cells have a central role in the pathogenesis of collagen-induced arthritis (CIA), an animal model of the autoimmune disease rheumatoid arthritis. In this report, a specific subset of an innate type of B cells, B-1 B cells, have been studied for the involvement in CIA. The B-1 B cells were shown to produce small amounts of collagen-specific antibodies upon stimulation in vitro, suggesting that they play a minor role in the development of CIA. This report also includes how marginal zone B cells, another innate type of B cells with natural collagen-reactivity, can be identified in the medullary sinuses of lymph nodes of collagen-immunized mice, implying involvement in auto antigen trapping.