Bone marrow maintains isotype switched memory B cells in stromal niches

The adaptive immune system has the unique ability to remember and rapidly mount protective response against previously encountered pathogen. This feature of the immune system is termed immunological memory and the functional duty is carried out by differentiated T and B cells. The maintenance of memory cells is important to confer long-lasting protection for the organism. Also, strategic positioning of these memory cells throughout the organism in crucial to ensure timely response against recurrent antigenic stimulation. While the tissue distribution and maintenance of memory T and plasma cells has been described, the lifestyle of memory B cells (Bmem) has not been well studied so far. In my doctoral thesis I investigated the tissue organization and lifestyle of memory B cells in mice, which would serve as the starting point for further translational studies in humans. To determine the tissue distribution, isotype-switched Bmem of spleen, bone marrow (BM), peripheral blood, and lymph nodes were enumerated under different immunization and infection protocols. The majority of isotype-switched Bmem were localized in the spleen, but a significant population was also contained within the BM. Comparison of the repertoire of B cell receptor (BCR), a unique identifier of each individual B cell, the repertoires of isotype-switched Bmem of spleen and BM revealed limited overlap of B cells with same BCR (clonotypes) generated during a specific immune response. The majority of Bmem clonotypes are expressed exclusively in either organ, demonstrating that isotype-switched Bmem of the two organs represent distinct resident populations with minimal exchange between them via blood circulation. Phenotypically, isotype-switched Bmem of the two organs differ in surface protein expression of CD21 (complement receptor) and CD62L (L-selectin) with subsets of CD21low and CD21high populations in the BM but not in spleen, also, isotype-switched Bmem of BM express higher levels of CD62L compared to those in spleen. Isotype-switched Bmem of BM and spleen are resting in the G0 of cell cycle as determined by the expression of the proliferative marker Ki67, and are refractory to in vivo treatment with cyclophosphamide (a DNA alkylating agent which kills proliferating cells). In the BM, isotype-switched Bmem are located in close proximity to reticular stromal cells expressing VCAM-1. To further understand the role of BM stromal cells in organization of survival niches for memory cells, the biology and functional properties of VCAM-1+ stromal cells were analyzed. Next generation sequencing single cell mRNA transcriptomes profiling of directly ex vivo isolated BM VCAM-1+ stromal cells revealed distinct subpopulation of stromal cells defined by the expression of cytokines and chemokines which have been described to be important for the maintenance and survival of subsets of hematopoietic and immune cells subsets. Altogether, the findings of this thesis demonstrate that murine Bmem are residing in the BM as distinct population of B cell memory and that distinct subsets of BM stromal cells organize survival niches for different hematopoietic cells, including memory cells.Das adaptive Immunsystem hat die einzigartige Fähigkeit sich an zuvor vorgefundene Krankheitserreger zu erinnern und schnell gegen diese schützend zu reagieren. Diese Fähigkeit des Immunsystems heißt immunologisches Gedächtnis und wird von differenzierten T- und B-Zellen ausgeübt. Die Aufrechterhaltung der Gedächtniszellen ist wichtig um dem Organismus einen dauerhaften Schutz zu verleihen. Weiterhin ist eine strategische Positionierung dieser Gedächtniszellen im gesamten Organismus entscheidend um eine zeitnahe Reaktion gegen wiederkehrende antigenische Stimulierungen sicherzustellen. Während die Gewebeverteilung und -erhaltung von Gedächtnis T-Zellen und Plasma Zellen bereits gut beschrieben wurde, wurde der Lebensstil von Gedächtnis-B-Zellen (Bmem) bislang nicht weiter untersucht. In meiner Doktorarbeit habe ich die Gewebeorganisation und den Lebensstil von Gedächtnis B-Zellen in Mäusen untersucht, welche als Ausgangspunkt für weitere translationale Studien am Menschen dient. Um die Gewebeverteilung zu untersuchen, wurden Isotyp-veränderte Bmem der Milz, des Knochenmarks, von peripherem Blut und von Lymphknoten unter verschiedenen Immunisierungs- und Infektionsprotokollen ausgezählt. Die Mehrheit der Isotyp-veränderten Bmem wurden in der Milz lokalisiert, es ist aber auch eine signifikante Population im Knochenmark enthalten. Der Vergleich des Repertoires des B-Zell-Rezeptors (BCR), einer einzigartigen Bezeichnung jeder individuellen B-Zelle, des Repertoires der Isotyp-veränderten Bmem der Milz und des Knochenmarks hat gezeigt, dass B-Zellen mit derselben BCR (Klonotypen), welche während einer spezifischen Immunreaktion generiert wurden, sich kaum überlappen. Die Mehrheit der Bmem Klonotypen wird ausschließlich in einem der beiden Organe exprimiert, was beweist, dass Isotyp-veränderte Bmem zweier Organe verschiedene Populationen mit nur minimalen Austausch über den Blutkreislauf repräsentieren. Phänotypisch, Isotyp-veränderte Bmem der beiden Organe unterscheiden sich in der Oberflächenproteinexpression von CD21 (Komplementrezeptor) und CD62L (L-Selektin) mit Untergruppen von CD21low und CD21high Populationen im Knochenmark, aber nicht in der Milz. Weiterhin beinhalten Isotyp-veränderte Bmem des Knochenmarks einen höheren Spiegel von CD62L im Vergleich zur Milz. Isotyp-veränderte Bmem des Knochenmarks und der Milz ruhen im G0 des Zellzyklus, wie durch die Expression des proliferativen Markers Ki67 bestimmt wurde, und sind widerstandsfähig gegen in vivo Behandlungen mit Cyclophosphamid (einem DNA alkylierenden Wirkstoff, der wuchernde Zellen tötet). Im Knochenmark befinden sich Isotyp-veränderte Bmem in unmittelbarer Nähe zu retikulären Stromazellen, die VCAM-1 exprimieren. Um die Rolle der Knochenmark Stromazellen bei der Organisation von Überlebensnischen für Gedächtniszellen genauer zu verstehen, wurden die Biologie und die funktionellen Eigenschaften von VCAM-1+ Stromazellen analysiert. Die Sequenzierung von Einzelzell-mRNA-Transkriptomen von direkt ex vivo isolierten Knochenmark VCAM-1+ Stromazellen ergab eindeutige Subpopulationen von Stromazellen, die durch die Expression von Cytokinen und Chemokinen definiert wurden, welche als wichtig für die Aufrechterhaltung und das Überleben von Subpopulationen von hämatopoetischen Zellen und Immunzellen beschrieben wurden. Zusammenfassend beschreiben die Ergebnisse der Dissertation, dass murine Bmem im Knochenmark als ausgeprägte ansässige Population des B-Zell-Gedächtnisses vorkommen und das Subpopulationen von Knochenmark Stromazellen Überlebensnischen für verschiedene hämatopoetische Zellen, inklusive den Gedächtniszellen des Immunsystems, organisieren

Multiplex Recombinase Polymerase Amplification Assay for Simultaneous Detection of Treponema pallidum and Haemophilus ducreyi in Yaws-Like Lesions

Yaws is a skin debilitating disease caused by Treponema pallidum subspecies pertenue with most cases reported in children. World Health Organization (WHO) aims at total eradication of this disease through mass treatment of suspected cases followed by an intensive follow-up program. However, effective diagnosis is pivotal in the successful implementation of this control program. Recombinase polymerase amplification (RPA), an isothermal nucleic acid amplification technique offers a wider range of differentiation of pathogens including those isolated from chronic skin ulcers with similar characteristics such as Haemophilus ducreyi (H. ducreyi). We have developed a RPA assay for the simultaneous detection of Treponema pallidum (T. pallidum) and H. ducreyi (TPHD-RPA). The assay demonstrated no cross-reaction with other pathogens and enable detection of T. pallidum and H. ducreyi within 15 min at 42 °C. The RPA assay was validated with 49 clinical samples from individuals confirmed to have yaws by serological tests. Comparing the developed assay with commercial multiplex real-time PCR, the assay demonstrated 94% and 95% sensitivity for T. pallidum and H. ducreyi, respectively and 100% specificity. This simple novel TPHD-RPA assay enables the rapid detection of both T. pallidum and H. ducreyi in yaws-like lesions. This test could support the yaws eradication efforts by ensuring reliable diagnosis, to enable monitoring of program success and planning of follow-up interventions at the community level

MicroRNA-31 Reduces the Motility of Proinflammatory T Helper 1 Lymphocytes

Proinflammatory type 1 T helper (Th1) cells are enriched in inflamed tissues and contribute to the maintenance of chronic inflammation in rheumatic diseases. Here we show that the microRNA- (miR-) 31 is upregulated in murine Th1 cells with a history of repeated reactivation and in memory Th cells isolated from the synovial fluid of patients with rheumatic joint disease. Knock-down of miR-31 resulted in the upregulation of genes associated with cytoskeletal rearrangement and motility and induced the expression of target genes involved in T cell activation, chemokine receptor– and integrin-signaling. Accordingly, inhibition of miR-31 resulted in increased migratory activity of repeatedly activated Th1 cells. The transcription factors T-bet and FOXO1 act as positive and negative regulators of T cell receptor (TCR)–mediated miR-31 expression, respectively. Taken together, our data show that a gene regulatory network involving miR-31, T-bet, and FOXO1 controls the migratory behavior of proinflammatory Th1 cells

Diarrhoeagenic E. coli occurrence and antimicrobial resistance of Extended Spectrum Beta-Lactamases isolated from diarrhoea patients attending health facilities in Accra, Ghana.

IntroductionDiarrhoea accounts for high morbidity and mortality in children and adults worldwide. Extended Spectrum Beta-Lactamase-Producing Enterobacteriaceae (ESBL-PE) and Diarrhoeagenic Escherichia coli (DEC) contribute to prolonged hospitalization because of their resistance and virulence properties aiding in the spread of diarrhoeal disease and delayed treatment.AimTo determine DEC and the antimicrobial resistance of ESBL-PE isolated among diarrhoea patients attending two health facilities in Ghana.MethodsStool samples were collected from 122 diarrhoeal patients who attended Maamobi General Hospital and Kaneshie Polyclinic between January 2019 and March 2020. Identification of bacteria was performed by using the Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Using disk diffusion, antimicrobial susceptibility testing (AST) was conducted and interpreted according to the 2018 CLSI guidelines. Detection of ESBL and DEC genes was performed using Polymerase chain reaction (PCR).ResultsA total of 80.3% (98/122) Enterobacteriaceae was recovered from the patients in the study with an overall ESBL occurrence of 20.4% (20/98), predominantly among E. coli showed 13.2% (10/76), Klebsiella pneumoniae,35.7%(5/14) and Proteus mirabilis, 57.1%(4/7). Among the ESBL genes detected, blaTEM (n = 14) was common, followed by blaCTX-M (n = 13) and blaSHV (n = 4). Thirty-four E. coli isolates possessed the heat labile (Lt) gene of Enterotoxigenic E. coli (ETEC).ConclusionOur findings confirm the existence of DEC and the antimicrobial resistance patterns of ESBL-PE among stool isolates, limiting the options of commonly used drugs for diarrhoeal treatment in Ghana. Routine laboratory testing in health care facilities and strengthened surveillance systems among hospital networks are encouraged for a better understanding of their epidemiology and clinical implications

Multiplex Recombinase Polymerase Amplification Assay for Simultaneous Detection of Treponema pallidum and Haemophilus ducreyi in Yaws-Like Lesions

Yaws is a skin debilitating disease caused by Treponema pallidum subspecies pertenue with most cases reported in children. World Health Organization (WHO) aims at total eradication of this disease through mass treatment of suspected cases followed by an intensive follow-up program. However, effective diagnosis is pivotal in the successful implementation of this control program. Recombinase polymerase amplification (RPA), an isothermal nucleic acid amplification technique offers a wider range of differentiation of pathogens including those isolated from chronic skin ulcers with similar characteristics such as Haemophilus ducreyi (H. ducreyi). We have developed a RPA assay for the simultaneous detection of Treponema pallidum (T. pallidum) and H. ducreyi (TPHD-RPA). The assay demonstrated no cross-reaction with other pathogens and enable detection of T. pallidum and H. ducreyi within 15 min at 42 °C. The RPA assay was validated with 49 clinical samples from individuals confirmed to have yaws by serological tests. Comparing the developed assay with commercial multiplex real-time PCR, the assay demonstrated 94% and 95% sensitivity for T. pallidum and H. ducreyi, respectively and 100% specificity. This simple novel TPHD-RPA assay enables the rapid detection of both T. pallidum and H. ducreyi in yaws-like lesions. This test could support the yaws eradication efforts by ensuring reliable diagnosis, to enable monitoring of program success and planning of follow-up interventions at the community level

Multiplex Recombinase Polymerase Amplification Assay for Simultaneous Detection of Treponema pallidum and Haemophilus ducreyi in Yaws-Like Lesions

Yaws is a skin debilitating disease caused by Treponema pallidum subspecies pertenue with most cases reported in children. World Health Organization (WHO) aims at total eradication of this disease through mass treatment of suspected cases followed by an intensive follow-up program. However, effective diagnosis is pivotal in the successful implementation of this control program. Recombinase polymerase amplification (RPA), an isothermal nucleic acid amplification technique offers a wider range of differentiation of pathogens including those isolated from chronic skin ulcers with similar characteristics such as Haemophilus ducreyi (H. ducreyi). We have developed a RPA assay for the simultaneous detection of Treponema pallidum (T. pallidum) and H. ducreyi (TPHD-RPA). The assay demonstrated no cross-reaction with other pathogens and enable detection of T. pallidum and H. ducreyi within 15 min at 42 °C. The RPA assay was validated with 49 clinical samples from individuals confirmed to have yaws by serological tests. Comparing the developed assay with commercial multiplex real-time PCR, the assay demonstrated 94% and 95% sensitivity for T. pallidum and H. ducreyi, respectively and 100% specificity. This simple novel TPHD-RPA assay enables the rapid detection of both T. pallidum and H. ducreyi in yaws-like lesions. This test could support the yaws eradication efforts by ensuring reliable diagnosis, to enable monitoring of program success and planning of follow-up interventions at the community level

Multiplex Recombinase Polymerase Amplification Assay for Simultaneous Detection of Treponema pallidum and Haemophilus ducreyi in Yaws-Like Lesions

Yaws is a skin debilitating disease caused by Treponema pallidum subspecies pertenue with most cases reported in children. World Health Organization (WHO) aims at total eradication of this disease through mass treatment of suspected cases followed by an intensive follow-up program. However, effective diagnosis is pivotal in the successful implementation of this control program. Recombinase polymerase amplification (RPA), an isothermal nucleic acid amplification technique offers a wider range of differentiation of pathogens including those isolated from chronic skin ulcers with similar characteristics such as Haemophilus ducreyi (H. ducreyi). We have developed a RPA assay for the simultaneous detection of Treponema pallidum (T. pallidum) and H. ducreyi (TPHD-RPA). The assay demonstrated no cross-reaction with other pathogens and enable detection of T. pallidum and H. ducreyi within 15 min at 42 °C. The RPA assay was validated with 49 clinical samples from individuals confirmed to have yaws by serological tests. Comparing the developed assay with commercial multiplex real-time PCR, the assay demonstrated 94% and 95% sensitivity for T. pallidum and H. ducreyi, respectively and 100% specificity. This simple novel TPHD-RPA assay enables the rapid detection of both T. pallidum and H. ducreyi in yaws-like lesions. This test could support the yaws eradication efforts by ensuring reliable diagnosis, to enable monitoring of program success and planning of follow-up interventions at the community level

Using drones to transport suspected COVID-19 samples; experiences from the second largest testing centre in Ghana, West Africa.

BackgroundThe declaration of COVID-19 as a pandemic on March 11 2020, by the World Health Organisation prompted the need for a sustained and a rapid international response. In a swift response, the Government of Ghana, in partnership with Zipline company, launched the use of Unmanned Automated Vehicles (UAV) to transport suspected samples from selected districts to two foremost testing centres in the country. Here, we present the experiences of employing this technology and its impact on the transport time to the second largest testing centre, the Kumasi Centre for Collaborative Research in Tropical Medicine (KCCR) in Kumasi, Ghana.MethodsSwab samples collected from suspected COVID-19 patients were transported to the Zipline office by health workers. Information on the samples were sent to laboratory personnel located at KCCR through a WhatsApp platform to get them ready to receive the suspected COVID-19 samples while Zipline repackaged samples and transported them via drone. Time of take-off was reported as well as time of drop-off.ResultsA total of 2537 COVID-19 suspected samples were received via drone transport from 10 districts between April 2020 to June 2021 in 440 deliveries. Ejura-Sekyedumase District Health Directorate delivered the highest number of samples (765; 30%). The farthest district to use the drone was Pru East, located 270 km away from KCCR in Kumasi and 173 km to the Zipline office in Mampong. Here, significantly, it took on the average 39 minutes for drones to deliver samples compared to 117 minutes spent in transporting samples by road (pConclusionThe use of drones for sample transport during the COVID-19 pandemic significantly reduced the travel time taken for samples to be transported by road to the testing site. This has enhanced innovative measures to fight the pandemic using technology