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Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags
No full textHuman macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to understand the underlying mechanisms of these diseases. We therefore present a straightforward protocol for the isolation of human monocytes from buffy coats, followed by a differentiation procedure which results in high macrophage yields. The technique relies mostly on commonly available lab equipment and thus provides a cost and time effective way to obtain large quantities of human macrophages. Briefly, buffy coats from healthy blood donors are subjected to a double density gradient centrifugation to harvest monocytes from the peripheral blood. These monocytes are then cultured in fluorinated ethylene propylene (FEP) Teflon-coated cell culture bags in the presence of macrophage colony-stimulating factor (M-CSF). The differentiated macrophages can be easily harvested and used for subsequent studies and functional assays. Important methods for quality control and validation of the isolation and differentiation steps will be highlighted within the protocol. In summary, the protocol described here enables scientists to routinely and reproducibly isolate human macrophages without the need for cost intensive tools. Furthermore, disease models can be studied in a syngeneic human system circumventing the use of murine macrophages
Identification and characterisation of novel antimicrobials against Mycobacterium tuberculosis
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Der Verlauf einer experimentellen zerebralen Malaria und der experimentellen Tuberkulose während einer Tuberkulose-Malaria-Koinfektion
Full text linkGoing Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Characterization of macrophage Frizzled1 expression and the role of Wnt3a-induced signaling in experimental M. tuberculosis infection
Full text linkDie Rolle der C-Typ-Lektine DC-SIGN und Makrophagen-Mannose-Rezeptor in der experimentellen Tuberkulose
No full textInnate immune functions, biomarkers of cure and potential roles of cattle for transmission
Full text linkSex differences in vaccine induced immunity and protection against Mycobacterium tuberculosis
No full textTuberkulose (TB), an der jährlich über eine Million Menschen sterben, betrifft in besonders
hohem Maße Männer. Obwohl das häufigere Auftreten von TB bei Männern aus
epidemiologischer Sicht seit langem bekannt ist, ist das mechanistische Verständnis dieser
Unterschiede relativ neu. Der einzige zugelassene TB-Impfstoff, Bacillus Calmette Guérin
(BCG), weist eine unzureichende Wirksamkeit auf, was die Entwicklung effektiverer Impfstoffe
erforderlich macht. Über Geschlechterunterschiede in der Wirksamkeit von BCG ist jedoch nur
wenig bekannt. In der vorliegenden Arbeit wurde erstmals die fehlende Wirksamkeit von BCG
speziell bei männlichen Tieren im C57BL/6-Mausmodell gezeigt. Zwei rekombinante BCG
Derivate (rBCG) - VPM1002 and BCGΔBCG1419c - konnten hingegen das Überleben nach einer
Mycobacterium tuberculosis (Mtb)-Infektion speziell bei männlichen Mäusen gegenüber der
BCG-Impfung signifikant verlängern und zeigten somit eine deutlich verbesserte Wirksamkeit.
Die Unterschiede in der Überlebensrate zwischen rBCG- und BCG-geimpften Männchen waren
dabei nicht auf ihre Fähigkeit zurückzuführen, die Anzahl der koloniebildenden Einheiten (KBE)
in der Lunge zu reduzieren. Darüber hinaus wurde gezeigt, dass BCGΔBCG1419c, das als
Vertreter der beiden rBCGs verwendet wurde, 90 Tage nach der Impfung die CD8-T-Zell
Antworten im Vergleich zu BCG speziell bei männlichen Mäusen signifikant erhöht - eine
Eigenschaft, die eine signifikante positive Korrelation mit dem Überleben nach Mtb-Belastung
aufwies. Desweiteren wurden signifikante positive Korrelationen zwischen der CD4-T
Zellreaktion am Tag 28 nach der Impfung und der CD8-T-Zellreaktion am Tag 90 nach der
Impfung sowie zwischen der CD4-T- und der B-Zellreaktion am Tag 28 nach der Impfung
festgestellt. Die CD4-T-Zell-Antwort am Tag 28 nach der Impfung zeigte ebenfalls eine
signifikante Korrelation mit dem Überleben nach einer Mtb-Infektion. Schließlich zeigten sich
zwischen den Geschlechtern globale Unterschiede in den CD8-T-Zell Populationen in der Milz
28 Tage nach Impfung, wobei sich unabhängig vom Impfstoff spezifische Cluster zwischen
Weibchen und Männchen unterschieden. Zusammenfassend zeigt diese Studie eine
geschlechtsspezifisch fehlende Wirksamkeit von BCG bei männlichen C57BL/6-Mäusen, und
dass neuere rBCG-Derivate die Wirksamkeit bei Männchen signifikant verbessern. Die
zugrundeliegenden Unterschiede in der Immunantwort nach der Impfung, die mit der
Wirksamkeit des Impfstoffs korrelieren, sowie die Unterschiede in der Immunantwort nach der
Impfung zwischen den Geschlechtern wurden ermittelt. Die Aufklärung, wie
geschlechtsspezifische Unterschiede in den CD8-T-Zell-Antworten die Impfstoffwirksamkeit beeinflussen, sowie die mögliche Rolle von CD4-T-Zellen und B-Zellen für die
geschlechtsspezifische Entwicklung von verschiedenen CD8-T-Zell-Populationen, eröffnet
neue Wege für weiterführende Studien.Tuberculosis (TB), a disease killing over a million people per annum, shows a strong male
preponderance in disease development. Although increased male affliction for TB has long been
known from an epidemiological perspective, the mechanistic understanding of those
differences is relatively recent. The only approved vaccine for TB, Bacillus Calmette Guérin
(BCG), shows high variability in its protective efficacy – necessitating the development of
effective vaccine candidates. However, whether the male-biased susceptibility to TB also
applies to the efficacy of the BCG vaccine, has been scarcely explored. In the current study, a
male specific failure of BCG is demonstrated in the C57BL/6 mouse model. However, two
recombinant derivatives of BCG (rBCGs) - VPM1002 and BCGΔBCG1419c - were found to
ameliorate this male specific vulnerability of BCG by significantly improving survival rates in
males upon Mycobacterium tuberculosis (Mtb) challenge. The disparities in survival between
rBCGs and BCG vaccinated males were not attributable to their ability to reduce lung colony
forming units (CFUs). Further analysis revealed that BCGΔBCG1419c, used as a representative
of VPM1002 and BCGΔBCG1419c, significantly enhances CD8 T cell responses 90 days post
vaccination compared to BCG, specifically in males. This enhancement shows a strong positive
correlation with improved survival following Mtb challenge. In addition, significant positive
correlations were identified between the CD4 T cell response on day 28 post-vaccination and
the CD8 T cell response on day 90 post-vaccination, as well as between the CD4 T and B cell
responses on day 28 post-vaccination. The CD4 T cell response at day 28 post-vaccination also
showed a significant direct correlation with survival following Mtb challenge. Lastly, 28 days
post-vaccination, CD8 T cell populations in the spleen showed distinct global differences
between sexes, with specific clusters varying between males and females, independent of
vaccine type. In summary, the current study identified a male specific failure of BCG in the
C57BL/6 mouse model of TB and the ability of rBCGs to significantly improve the protective
efficacy specifically in males. The underlying differences in post-vaccine immune responses
that correlate with vaccine efficacy, as well as, those differences between sexes were
identified. Elucidating how sex-specific differences in CD8 T cell responses influence vaccine
efficacy, as well as the potential role of CD4 T cells and B cells in the sex-specific development
of different CD8 T cell populations open new avenues for future studies
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