M-DC8+leukocytes - A novel human dendritic cell population

Dendritic cells (DC) constitute a heterogeneous leukocyte population having in common a unique capacity to induce primary T cell responses and are therefore most attractive candidates for immunomodulatory strategies. Two populations of blood DC (CD11c+ CD123(dim) and CD11c- CD123(high)) have been defined so far. However, their direct isolation for experimental purposes is hampered by their low frequency and by the lack of selective markers allowing large scale purification from blood. Here we describe the monoclonal antibody (mAb) M-DC8, which was generated by immunizing mice with highly enriched blood DC. This mAb specifically reacts with 0.2-1% of blood leukocytes and enables their direct isolation by a one-step immunomagnetic procedure from fresh mononuclear cells. These cells can be differentiated from T cells, B cells, NK cells and monocytes using lineage-specific antibodies. M-DC8+ cells express HLA class It molecules, CD33 and low levers of the costimulatory molecules CD86 and CD40. Upon in vitro culture M-DC8+ cells spontaneously mature into cells with the phenotype of highly stimulatory cells as documented by the upregulation of HLA-DR, CD86 and CD40; in parallel CD80 expression is induced. M-DC8+ cells display an outstanding capacity to present antigen. In particular, they proved to be excellent stimulators of autologous mixed leukocyte reaction and to activate T cells against primary antigens such as keyhole limpet hemocyanin. Furthermore, they induce differentiation of purified allogeneic cytotoxic T cells into alloantigen-specific cytotoxic effector cells. While the phenotypical analysis reveals similarities with the two known blood DC populations, the characteristic expression of Fc gamma RIII (CD16) and the M-DC8 antigen clearly defines them as a novel population of blood DC. The mAb M-DC8 might thus be a valuable tool to determine circulating DC for diagnostic purposes and to isolate these cells for studies of antigen-specific T cell priming. Copyright (C) 2000 S. Karger AG, Basel

Nature as a 'Lifeline': The Power of Photography when Exploring the Experiences of Older Adults living with Memory Loss and Memory Concerns

The visual is an underutilised modality through which to investigate experiences of memory loss in older people. We describe a visual ethnography with older adults experiencing subjective or objective memory loss, receiving a cognitive wellbeing group intervention designed to prevent cognitive decline and dementia (APPLE-Tree programme). We aimed to explore lived experiences of people with memory concerns, how participants engaged with this photography and co-design project, and how collaboration with an artist/photographer enhanced this process. Nineteen participants shared photographs reflecting what they valued in their daily lives, their experiences of memory concerns, and the intervention. Fourteen participated in qualitative photo-elicitation interviews, and thirteen collaborated with a professional artist/photographer to co-create an exhibition, in individual meetings and workshops, during which a researcher took ethnographic field notes. Eight participants were re-interviewed after the exhibition launch. We contextualise images produced by participants in relation to discourses around the visual and ageing and highlight their relationship with themes developed through thematic analysis that interconnects photographic, observational and interview data. We present themes around the use of photographs to: (1) celebrate connections to nature as a lifeline; (2) anchor lives within the context of relationships with family; (3) reflect on self and identity, enduring through ageing, memory concerns, pandemic, and ageing stereotypes. We explore visual research as a powerful tool for eliciting meaningful accounts from older adults experiencing cognitive change and to connect the arts and social sciences within ageing studies

Measurement of Th1/Th2 serum cytokines by flow cytometry in classical Hodgkin lymphoma

O linfoma de Hodgkin clássico (LHC) é uma neoplasia com distúrbio na produção de citocinas. Estudos demonstram que o padrão anormal das citocinas no linfonodo acometido pela lesão contribui não somente com a proliferação das células malignas H-RS, como também com o característico infiltrado hiper-reativo que compõe o tecido no LHC. Esta disfunção pode ser observada tanto no quadro clínico dos pacientes, como nas características histopatológicas: sintomas B, deficiência na resposta imune celular, bandas de colágeno e eosinofilia. As concentrações séricas das citocinas Th1 (IL-2, TNF, INF-&#947;) e Th2 (IL-4, IL-5, IL-10) foram estudadas em 45 pacientes com LHC, ao diagnóstico, e em 34 doadores saudáveis, por citometria de fluxo (CBA - cytometric beads array). Houve aumento das concentrações das citocinas TNF (p<0,01), INF-&#947; (p<0,01), IL-4 (p=0,01), IL-5 (p<0,01) e IL-10 (p<0,01) dos pacientes quando comparados com o grupo controle. Não foi evidenciada diferença em relação a IL-2. Ao correlacionarmos as concentrações das citocinas Th1/Th2 com as variáveis clínico-laboratoriais dos pacientes, observou-se que níveis elevados da IL-10 (Th2) estão correlacionados com as variáveis que implicam em pior prognóstico: estádios III/IV (p=0,01), presença de sintomas B (p=0,04), hemoglobina < 10,5g/dL (p=0+,01), linfócitos <600 mm³ (p=0,01) e, de acordo com o IPI, os pacientes de alto risco (p=0,01). Por outro lado, níveis séricos elevados da IL-2 (Th1) foram encontrados em estádio I/II, quando comparados com III/IV (p=0,03), o que indica que a IL-2 diminui com a progressão da doença. Os resultados sugerem que a IL-10 possa estar regulando negativamente a resposta imune citotóxica (Th1) pela inibição da IL-2. Há uma possível associação entre progressão da doença e níveis elevados da IL-10. Esse estudo evidenciou que a utilização do CBA é factível na detecção das citocinas, e que as alterações encontradas podem estar envolvidas na biologia do LHC.Classical Hodgkin lymphoma (CHL) is a malignancy with an abnormal or unbalanced secretion/production of cytokines, which might support the growth of H-RS cells, their surrounding reactive bystander cells and may be responsible for the typical clinical and histopathologic features of CHL: systemic B symptoms, an apparent defect in cell-mediated immune response, tumor fibrosis and eosinophilic infiltrate. Serum concentrations of IL-2, IL-4, IL5, IL-10, TNF and IFN-&#947; (Th1/Th2) were measured in 45 patients at diagnosis of classical Hodgkin lymphoma and in 34 healthy controls by cytometric beads array (CBA). Levels of TNF (p<0.01), INF-&#947;(p<0.01), IL-4 (p=0.01), IL-5 (p<0.01) e IL-10 (p<0.01) were significantly higher in patients compared to the control group. No difference was observed for IL-2 between the two groups. On correlating Th1/Th2 cytokine concentrations with clinical risk factors, elevated IL-10 (Th2) levels are associated with variables that suggest worse prognoses including III/IV stage (p=0.01), B-symptoms (p=0.04), hemoglobin < 10.5g/dL (p=0.01), lymphocytes < 600/mm³ (p=0.01) and according to the seven-factored international prognostic score (IPI), a subset of patients with a particularly high risk of failure (p=0.01). Furthermore, the serum levels of IL-2 (Th1) were significantly higher in a group of I/II stage patients compared to III/IV patients (p=0.03) which implies that, the levels of IL-2 might decrease with disease progression. The elevated IL-10 levels in a subset of patients with poor clinical risk factors might down regulate a Th1 immune response by inhibiting IL-2 production causing survival disadvantage by suppression of the cytotoxic immune response against the tumor. This suggests an association between progression of CHL and higher levels of the IL-10 cytokine. This study showed that measurement of serum cytokines using the CBA methodology is highly reproducible, and that changes in concentrations seem to be involved in the biology of this diseas

Helicobacter pylori Type IV Secretion Apparatus Exploits β1 Integrin in a Novel RGD-Independent Manner

Translocation of the Helicobacter pylori (Hp) cytotoxin-associated gene A (CagA) effector protein via the cag-Type IV Secretion System (T4SS) into host cells is a major risk factor for severe gastric diseases, including gastric cancer. However, the mechanism of translocation and the requirements from the host cell for that event are not well understood. The T4SS consists of inner- and outer membrane-spanning Cag protein complexes and a surface-located pilus. Previously an arginine-glycine-aspartate (RGD)-dependent typical integrin/ligand type interaction of CagL with α5β1 integrin was reported to be essential for CagA translocation. Here we report a specific binding of the T4SS-pilus-associated components CagY and the effector protein CagA to the host cell β1 Integrin receptor. Surface plasmon resonance measurements revealed that CagA binding to α5β1 integrin is rather strong (dissociation constant, KD of 0.15 nM), in comparison to the reported RGD-dependent integrin/fibronectin interaction (KD of 15 nM). For CagA translocation the extracellular part of the β1 integrin subunit is necessary, but not its cytoplasmic domain, nor downstream signalling via integrin-linked kinase. A set of β1 integrin-specific monoclonal antibodies directed against various defined β1 integrin epitopes, such as the PSI, the I-like, the EGF or the β-tail domain, were unable to interfere with CagA translocation. However, a specific antibody (9EG7), which stabilises the open active conformation of β1 integrin heterodimers, efficiently blocked CagA translocation. Our data support a novel model in which the cag-T4SS exploits the β1 integrin receptor by an RGD-independent interaction that involves a conformational switch from the open (extended) to the closed (bent) conformation, to initiate effector protein translocation

Twist exome capture allows for lower average sequence coverage in clinical exome sequencing

Background Exome and genome sequencing are the predominant techniques in the diagnosis and research of genetic disorders. Sufficient, uniform and reproducible/consistent sequence coverage is a main determinant for the sensitivity to detect single-nucleotide (SNVs) and copy number variants (CNVs). Here we compared the ability to obtain comprehensive exome coverage for recent exome capture kits and genome sequencing techniques. Results We compared three different widely used enrichment kits (Agilent SureSelect Human All Exon V5, Agilent SureSelect Human All Exon V7 and Twist Bioscience) as well as short-read and long-read WGS. We show that the Twist exome capture significantly improves complete coverage and coverage uniformity across coding regions compared to other exome capture kits. Twist performance is comparable to that of both short- and long-read whole genome sequencing. Additionally, we show that even at a reduced average coverage of 70× there is only minimal loss in sensitivity for SNV and CNV detection. Conclusion We conclude that exome sequencing with Twist represents a significant improvement and could be performed at lower sequence coverage compared to other exome capture techniques

A Solve-RD ClinVar-based reanalysis of 1522 index cases from ERN-ITHACA reveals common pitfalls and misinterpretations in exome sequencing

Purpose Within the Solve-RD project (https://solve-rd.eu/), the European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies aimed to investigate whether a reanalysis of exomes from unsolved cases based on ClinVar annotations could establish additional diagnoses. We present the results of the “ClinVar low-hanging fruit” reanalysis, reasons for the failure of previous analyses, and lessons learned. Methods Data from the first 3576 exomes (1522 probands and 2054 relatives) collected from European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies was reanalyzed by the Solve-RD consortium by evaluating for the presence of single-nucleotide variant, and small insertions and deletions already reported as (likely) pathogenic in ClinVar. Variants were filtered according to frequency, genotype, and mode of inheritance and reinterpreted. Results We identified causal variants in 59 cases (3.9%), 50 of them also raised by other approaches and 9 leading to new diagnoses, highlighting interpretation challenges: variants in genes not known to be involved in human disease at the time of the first analysis, misleading genotypes, or variants undetected by local pipelines (variants in off-target regions, low quality filters, low allelic balance, or high frequency). Conclusion The “ClinVar low-hanging fruit” analysis represents an effective, fast, and easy approach to recover causal variants from exome sequencing data, herewith contributing to the reduction of the diagnostic deadlock

Herausforderungen und erste Erfahrungen bei der Implementierung des Qualitätsgedankens in die betriebliche Ausbildung – Erfahrungen und Ansätze aus dem Verbundprojekt von ZWH und Handwerkskammer Hannover

Diskussionen zur Qualitätsentwicklung im Berufsbildungssystem fokussierten lange die Implementierung von Qualitätsmanagementsystemen der Wirtschaft an berufsbildenden Schulen. Aktuell erstreckt sich die Qualitätsdebatte zunehmend auch auf die Ausbildungsprozesse auf betrieblicher Seite. Systematisches Qualitätsmanagement betrieblicher Erstausbildung ist aktuell im Handwerk nicht erkennbar. Ebenso wenig haben sich einheitlichen Qualitätskriterien etabliert. Hinzu kommt, dass die Notwendigkeit attraktiver Ausbildung als Mittel zur langfristigen Fachkräftesicherung noch zu wenig gesehen wird. Vor dem Hintergrund eines zunehmenden Wettbewerbs der Branchen, um talentierte Ausbildungsplatzbewerber(innen) und einer Ausbildungsabbruchsquote im Handwerkskammerbezirk Hannover von 20 Prozent, erscheint eine Sensibilisierung der Ausbildungsbetriebe für diese Qualitätsfragen unabdingbar. Das 2010 initiierte Verbundprojekt „Qualitätsentwicklung in der Ausbildung in Handwerksbetrieben: Entwicklungsinstrumente und Qualifizierungskonzepte“ der Handwerkskammer Hannover und der Zentralstelle für Weiterbildung im Handwerk (ZWH) fokussiert diese Thematik. Projektziele sind die Identifizierung des vorherrschenden Qualitätsverständnisses sowie handwerksspezifische Ausbildungsprobleme. Erkenntnisse dazu werden durch Befragung von Ausbildungsverantwortlichen und Auszubildenden gewonnen und der Entwicklung praktikabler Qualitäts- und Beratungskonzepte zugrunde gelegt. Der Beitrag stellt die Ausgangssituation und die Eckdaten des oben genannten Verbundprojektes vor und fasst bisherige zentrale Ergebnisse und Erkenntnisse zusammen. Darüber hinaus werden die Frage nach praktikablen Qualitätsbegriffen und Verstetigungsansätze des Qualitätsgedankens im Handwerk herausgearbeitet sowie die Chancen und Grenzen einer Kooperation zwischen HWK und ZWH mit den Ausbildungsbetrieben diskutiert. (Orig.)Discussions on quality development in the vocational education and training system have long focused the implementation of quality management systems from industry at vocational schools. Currently the quality debate also extends increasingly to the training processes on the in-companyside. Systematic quality management of in-company initial training is not currently discernible in the craft trades. Uniform quality criteria have also hardly been established. In addition, the necessity of attractive training as a means for long-term securing of skilled staff is still given too little importance. Against the background of increasing competition in the sectors over talented applicants for training places, and a rate of attrition amongst trainees in the Hanover region Chamber of Trade of 20% it would seem that a sensibilisation of the training companies for these questions of quality is indispensable. The joint project that was initiated in 2010: “Quality development in training in craft businesses: Development instruments and qualification concepts” at the Chamber of Trade in Hanover and the central office for further education and training in craft trades (ZWH) focuses these themes. The aims of the project are the identification of the prevailing understanding of quality as well as training problems that are specific to the craft trades. Insights in this regard are gained through surveys of those responsible for training and trainees and these provide the foundation for the development of practicable quality and advisory concepts. This paper presents the current situation and the parameters of the above mentioned joint project, and summarises the key results and insights that have been gained thus far. In addition the question of practicable quality concepts and approaches for permanent adoption of quality thinking in trade crafts is developed, and the opportunities and limits of a cooperation between the HWK and the ZWH with the training companies are also discussed. (Orig.