Functional Interactions Between Helicobacter-activated b (hact-b) Cells And Cd4+ T Cells

Tez (Yüksek Lisans) -- İstanbul Teknik Üniversitesi, Fen Bilimleri Enstitüsü, 2015Thesis (M.Sc.) -- İstanbul Technical University, Institute of Science and Technology, 2015Helicobacter pylori, dünya popülasyonunun yarısından fazlasını enfekte etmiş bulunan, gram-negatif, spiral yapıda, mikroaerofilik bir bakteri olmakla beraber gastritten mide kanserine kadar uzanan gastrik patolojilerin temel risk faktörü olarak tanımlanmıştır. Enfeksiyonlar genelde çocukluk çağlarında meydana geldiğinden, dünya genelinde enfekte olan kişi sayısı oldukça fazla olmasına karşın, enfekte bireylerin büyük bir kısmı herhangi bir semptom göstermezken, popülasyonun yalnızca % 20’sinde gastrite, gastrik ve duodenal ülserlere ve ilerleyen zamanda gastrik adenokarsinoma sebep olmaktadır. Helicobacter’in etkisini incelemek için fare modelleri kullanılarak yapılan çalışmalarda, Helicobacter pylori’ye yakınlığıyla bilinen ve CagA virülans faktörü bulundurmamasına rağmen enfeksiyonlarda pylori’ye oldukça benzer sonuçlar veren Helicobacter felis kullanılmaktadır. Helicobacter felis de gram negatif, helikal şekilli ve mikroaerofilik bir bakteridir. İmmün sistem hücreleri, mikroçevrelerinde bulunan patojenleri tanımak için hücre yüzeylerinde bazı reseptörlere ihtiyaç duymaktadır.  Toll benzeri reseptörler (Toll-like receptors -TLR), birçok patojenin varlığında sinyal üreterek doğal immün cevabın oluşmasını sağlayan bir grup tip 1 transmembran proteinidir. Hayvan ve hücre kültürü deneyleri, H.pylori kaynaklı ligandların B hücrelerinde aktivasyonu TLR2 yolu ile yaptığı ortaya konmuştur. Antijen sunucu hücreler (APC- antiger presenting cell) olan B hücrelerinin ve T hücrelerinin IL-10 üreten regülatör alt tipleri olduğu bilinmektedir. Güncel araştırmalar, Helicobacter felis (H.felis) enfeksiyonlu fare modellerinde TLR-2 yoluyla  IL-10 üreten B hücrelerinin immün cevabı baskılayıcı ve düzenleyici rolü olduğunu ortaya koymuştur.  Yardımcı T (Th) hücrelerinin bir alt kümesi olan Th17 (T helper 17) hücrelerinin ve salgıladıkları interlökin 17 (IL-17) sitokininin, Helicobacter gibi bakterilerin temizlenmesinde kritik rolü vardır. Th17 dönüşümü için transforme edici büyüme faktörü-β (TGF-β), IL-21, IL-6 gibi sitokinleri etkili olduğu önerilmiştir. Th17 için oldukça önemli bir transkripsiyon faktörü olan RORγt, IL-17 gen transkripsiyonunu tetikler. Fakat kronik gastrik enflamasyon sırasında, üretilen IL-17 seviyesinin enfeksiyonu temizlemeye yeterli olmadığı görülmektedir. Buna sebep olarak, bakterinin kendi devamlılığını sağlamak amacıyla diğer immün hücreleri aracılığıyla, T hücrelerini, Th17 etkisini bastıran ve düzenleyici etkiler gösteren regülatör T hücrelerine dönüştürdüğü düşünülmektedir. Doğal regülatör T hücreleri olarak bilinen nTreg’ler (CD4+CD25+FoxP3+) timustaki gelişim sırasında fonksiyon kazanırken, indüklenebilir Treg (iTreg) periferde naif CD4+ T hücrelerin farklılaşması sırasında gelişir. Fare ve insan Tr1 hücreleri ile yapılan çalışmalarda, hücre yüzeyinde bulunan CD49b ve lenfosit aktivasyon geni 3 (LAG-3)’ün her ikisinin de aynı anda yüzeyde eksprese edilmesinin IL-10 salgılayan Tr1 hücrelerini Th1, Th2 ve Th17 hücrelerinden ayırdığı ortaya atılmıştır.  Helicobacter felis ile yapılan fare enfeksiyon modeli çalışmalarında, Helicobacter ile aktive edilen B hücrelerinin in vivo ve in vitro koşullarda T hücrelerini IL-10 üreten CD4+CD25+ Tr1-benzeri hücrelere dönüştürdükleri gösterilmiştir. Bu çalışmalarda, B hücreleri tarafından indüklenen Tr1 hücrelerinin in vitro koşullarda baskılayıcı aktivite gösterdiği ve in vivo koşullarda Helicobacter’e bağlı immünopatolojiyi baskıladığı ortaya konmuştur. Ayrıca, daha önce laboratuvarımızda yapılan çalışmalarda, Helicobacter-aktive B hücrelerinin TGF-β ürettiği gösterilmiştir. Ancak Helicobacter-aktive B hücrelerinden, IL-10 üreten ya da üretmeyen gruplardan hangisinin T hücrelerini IL-10 üreten Tr1-benzeri hücrelere dönüştürmede rolü olduğu bilinmemektedir. Bu nedenle, bu çalışmada Helicobacter-aktive B hücre alt gruplarının CD4+ T hücre farklılaşmaları (Tr1 veya Th17) üzerine olan etkilerinin incelenmesi amaçlanmıştır. Manyetik izolasyon teknikleri kullanılarak, CD19+ B hücreleri ve CD4+ T hücreleri, C57BL/6 farelerin dalaklarından %90’dan yüksek saflıklarla izole edilmiştir. B hücre izolasyonunu takiben hücreler Helicobacter felis sonikatı ile 24 saat muamele edilmiştir. Optimum bir IL-10 üretimi için inkübasyonun son 5 saatinde PMA ve iyonomisin hücrelere eklenmiştir. B hücrelerinin Helicobacter felis sonikatı ile in vivo tetiklenmesinin ardından IL-10 üreten hücreler manyetik olarak etiketlenerek bu sitokini üretmeyen IL-10 negatif B hücrelerinden manyetik ayrım yöntemi ile ayrılmıştır. Helicobacter-aktive B hücre alt grupları olan IL-10+ HAKT-B hücreleri ve IL-10- HAKT-B hücrelerinin CD4+ T hücreleri üzerindeki etkilerinin incelenmesi için bu hücreler 24 saat boyunca ko-kültüre konmuştur. Helicobacter-aktive B hücre alt gruplarının CD4+ T hücre farklılaşmaları (Tr1 veya Th17) üzerine olan etkileri RNA (kantitatif PZR) ve protein düzeyinde (ELIZA, hücre içi akan hücre ölçer analizi) incelenmiştir.  Hücre içinde özgün antikorlar ile boyanan IL-10 analiz sonuçları göstermektedir ki, hem IL-10+ HAKT-B hücreleri hem de IL-10- HAKT-B hücreleri CD4+ T hücrelerinde IL-10 üretimine yol açarak bu hücrelerin Tr1-benzeri hücrelere dönüşmesinde rol almaktadır. 24 saat ko-kültür boyunca hücre dışına salgılanan IL-10’un ölçümünde kullanılan IL-10 ELISA sonuçları da akan hücre ölçer sonuçlarını desteklemektedir. Aktive olmuş ve regülatör T hücrelerinin yüzeyinde bulunan CD25’in ve T hücrelerinde aktive olduktan sonra ekspresyonu azalan CD62L’nin özgün antikorlarla etiketlenerek akan hücre ölçerde analizlerinin sonucunda IL-10- HAKT-B hücreleri ile ko-kültürde tutulan CD4+ T hücrelerinin daha fazla aktive olduğu ya da farklılaştığı önerilmektedir. Literatürde regülatör T hücre belirteci olarak kullanılan CD49b ve LAG-3 yüzey belirteçlerinin birlikte eksprese edilmesinin incelenmesi sonucunda, IL-10+ HAKT-B hücreleri ile birlikte tutulan T hücrelerinin yaklaşık %20’si bu belirteçleri bir arada gösterirken, IL-10- HAKT-B hücreleri ile ko-kültürde tutulan T hücrelerinin ancak %10’u bu belirteci aynı anda eksprese etmektedir. İkincil olarak, Helicobacter-aktive B hücreleri ile etkileşime giren T hücrelerindeki IL-17 üretimi incelenmiştir. Hücre dışına salınımı engellenen IL-17 sitokininin hücre içi boyama sonuçlarına göre IL-10- HAKT-B hücreleri ile ko-kültürde tutulan T hücrelerinin %15’i IL-17 üretimi gerçekleştirirken, IL-10+ HAKT-B hücreleri T hücrelerinin yaklaşık %8’ini IL-17 üretmesi için tetiklemiştir. IL-17 ELISA sonuçlarının yanı sıra IL-17 üretiminde önemli bir transkripsiyon faktörü olan Ror gamma T ve IL-17’nin gen düzeyinde kantitatif PZR ile incelenmesinin sonuçları da akan hücre ölçer sonuçları ile paralellik göstermiştir. Bütün bu sonuçlar bir araya getirildiğinde, Helicobacter-aktive B hücre alt grupları her ikisinin de CD4+ T hücrelerinin IL-10 ürettiğini göstermiştir. Bu sonuçlara dayanarak, IL-10+ HAKT-B ve IL-10- HAKT-B hücrelerinin T hücrelerinin Tr-1 benzeri hücrelere dönüşmesinde etkisinin olduğu ortaya atılmıştır. Regülatör T hücre belirteci olarak kabul edilen CD49b-LAG3 yüzey belirteçlerinin ko-ekspresyonunun, IL-10+ HAKT-B hücreleri ile birlikte tutulan CD4+ T hücrelerinde daha yüksek olması bu hücrelerin regülatör profile daha yakın olduğunu önerirken, CD25 ve CD62L sonuçları IL-10- HAKT-B hücreleri ile ko-kültürde tutulan T hücrelerinin daha aktive olmuş/farklılaşmış olduğunu desteklemektedir. Fakat bu aktivasyonun ya da farklılaşmanın özgün olup olmadığı belli değildir. Bununla beraber, IL-17’nin hem protein düzeyinde hem de gen düzeyindeki incelemeleri CD4+ T hücrelerini Th17 benzeri hücrelere farklılaşmasında IL-10- HAKT-B hücrelerinin IL-10+ HAKT-B hücrelerine kıyasla daha fazla rolü olduğunu göstermiştir. Th17 farklılaşmasında TGF-betanın önemi göz önünde bulundurulduğunda, bu sonuçlar, daha önceden IL-10- HAKT-B hücrelerinin, IL-10 pozitif gruba göre daha fazla TGF-beta ve IL-6 ürettiğinin gösterilmesi ile tutarlıdır. Bu çalışma literatüre, Helicobacter-aktive B hücre alt grupları, IL10+ HAKT B ve IL10- HAKT B hücrelerinin, CD4+ T hücreleri ile ex vivo ilişkisini gösteren ilk çalışma olarak katkıda bulunmaktadır.Helicobacter pylori is a spiral-shaped, gram-negative bacterium that infects the gastric mucosa of more than half of the world’s population. The infection initially occurs in childhood, becomes persistent and the chronic infection leads to gastric inflammation. A major virulence factor of H. pylori is the cytotoxin-associated gene A (CagA) protein and this CagA protein interacts with several intracellular components of signal transduction and activates some crucial signaling pathways. Helicobacter has developed a variety of mechanisms to persist in the gastric mucosa. Gastric epithelial cells (GECs) are primary target for H. pylori infection, therefore they are the first point of contact for H. pylori and activate an innate immune response through TLRs. Rather than being a strong TLR4 ligand, H. pylori LPS is thought to activate TLR2 on gastric epithelial cells. Animal and cell culture experiments suggested that ligands in Helicobacter species can bind to TLR2 and activate NF-κB in epithelial cells. In mouse studies, Helicobacter felis (H. felis) is mainly used because it is more immunogenic than H.pylori in mice. B cells and their cytokines have important roles in Helicobacter infections by balancing between the infection and T cell driven gastric immunopathology. The ability of B cells to interact with pathogenic T cells and to produce anti-inflammatory cytokines such as IL-10 is crucial to dampen harmful immune responses. It has been found that B-cells exposed to Helicobacter sonicate produced large amounts of the regulatory cytokine IL-10. Using mouse models of Helicobacter-induced gastric premalignant pathology, it is shown that IL-10 secretion by purified B cells absolutely required MyD88 signaling and TLR-2.  The TLR-2- dependent Helicobacter activation of B cells differentiates them into IL-10 and TGF-β producing regulatory B cells. Both IL-10 and TGF-β have crucial effects on T cell differentiation. When co-cultured with Helicobacter-activated B cells, naive CD4+ T-cells are shown to produce IL-10 and differentiate into T regulatory 1 (Tr1)-like cells. In addition to that, it is suggested that Breg cells contribute to regulatory T-cell induction by producing TGF-β. At the same time, studies have shown convincingly that TGF-β is required for Th17 differentiation in vitro and in vivo. In previous studies in our laboratory, Helicobacter-activated total B cells were separated into two subgroups: IL-10+ B cells and IL-10- B cells. The experimental results showed that Helicobacter-activated IL-10+ B cells are the source of the IL-10 production while Helicobacter-activated IL-10-B cells are mostly TGF-β positive. However, it was not clear if the Helicobacter-acitvated-IL-10+ B cells or the Helicobacter-activated-IL-10- B were specifically causing the Tr1 differentiation. Taking account that Bregs are known for producing IL-10 and TGF-β which are key cytokines in T cell differentiation, the interaction between Helicobacter-activated B cell subgroups and CD4+ T cell differentiation was investigated. By magnetic isolation techniques, CD19+B cells and CD4+T cells were isolated from the spleens of C57BL/6 mice with high purities (with an average of 90% and 93%, respectively). Following the B cell isolation, cells were treated with Helicobacter felis sonicate (10 µg/ml) for 24 hours. For the last 5 hours of incubation, to induce an optimal IL-10 production and secretion, PMA (50 ng/ml) and ionomycin (500 ng/ml) were added. After the in vitro stimulation of B cells, IL-10 producing B cells were labeled and the IL-10+B and IL-10-B cells were separated. To observe the interaction between the Helicobacter-activated-B cell subgroups and CD4+ T cells, isolated CD4+ T cells were put on co-culture in 1:1 ratio with the Helicobacter-activated-IL-10+ B cells and Helicobacter-activated-IL-10- B cells, for 24 hours. The cell surface markers and intracellular cytokine productions were examined by flow cytometry. While the supernatants of the co-culture groups were subjected to ELISA tests, the cell pellets were used for gene expression analyses. The intracellular staining of IL-10 of T cells co-cultured with Helicobacter-activated B cell subgroups showed that about 15% of T cells co-cultured with HACT-IL-10+ B cells produced IL-10 while almost 20% of the T cell population was IL-10 positive when T cells were co-cultured with HACT-IL-10- B cells. In addition, according to IL-10 ELISA results, CD4+T cells co-cultured with IL-10+B cells and IL-10-B cells secreted twice IL-10 when compared to only T cells. For IL-10+B cell and T cell co-culture, a part of secreted IL-10 came from B cells while most of the IL-10 secreted from IL-10-B cell and T cell co-culture originated is suggested to be from T cells.  CD25 has been used as a marker to identify activated T cells as well as  some regulatory T cell subsets in mice; while CD62L has been known to rapidly shed from lymphocytes upon cellular activation. Both CD25 and CD62L levels in co-culture groups showed significant differences compared to T only groups. Furthermore, the differences in T cell CD25 and CD62L levels together might indicate that T cells co-cultured with Helicobacter-activated IL-10-B cells are more activated/differentiated compared to the T cells interacting with IL-10+B cells. In literature, it has been shown that B cells which were activated by Helicobacter induce IL-10–producing CD4+CD25+ Tr1–like cells in vitro. In addition, it has been identified that the co-expression of CD49b and LAG-3 distinguishes Tr1 cells from Th1, Th2 and Th17 cells. Flow cytometry results of CD4-CD49b-LAG3 stainings showed that  almost 20% of  T cells co-cultured with IL-10+B cells express CD4-CD49b-LAG3 surface markers while only around 10% of CD4+ T cells co-cultured with IL-10-B cells express CD49b and LAG3 together.  CD4+ T cells obtain distinct functional properties in response to signals sent by commensal and pathogenic microbe-activated cells of the innate immune system. Th17 cells secrete interleukin-17 (IL-17), IL-17F, and IL-22 and have significant roles in protecting the host from bacterial and fungal infections, particularly at mucosal surfaces. Data of four independent experiments showed that the approximately 15%  of CD4+ T cells co-cultured with IL10-B cells turned into IL17 producing CD4+ T cells while surprisingly about 8% of T cells also produced IL-17 when co-cultured with IL10+B cells. IL-17 ELISA results were parallel with the IL-17 intracellular cytokine staining analyses. Ror gamma T  and IL-17 relative gene expression levels in co-culture groups showed similar results with flow cytometry and ELISA results. Main research focus of this study was to investigate the interaction between HACT-B cell subgroups and CD4+ T cells and understand the effects of these B cell subgroups on T cell differentiation ex vivo. The results revealed that both Helicobacter-activated IL-10+B and IL-10-B cells induce IL-10 production from CD4+T cells and it might suggest that T cells differentiate into Tr1-like cells in both co-culture conditions. Although the CD25 and CD62L expression on T cell surfaces show significantly higher activation/differentiation in Helicobacter-activated IL-10-B and T cell co-cultures, regulatory type indicator CD4-CD49b-LAG3 surface marker co-expressions were higher on T cells in the Helicobacter-activated IL-10+B and T cell co-cultures. Furthermore, intracellular IL-17A  levels and relative gene expression experiments of IL-17A  and RorgammaT showed significantly higher results  in IL-10- B and T cell co-culture groups, as expected. On the other hand, interestingly IL-17 production was also observed in T cells co-cultured with IL-10- B cells. The reason behind the IL-17 and RorgammaT expressions in IL-10+ B–T cell co-culture groups was suggested to be the IL-6 and TGF- β produced by IL-10 negative B cell population in the IL-10 positive co-culture group. This study has contributed to the literature through providing a first step to show the ex vivo interactions of Helicobacter-activated B cells subgroups, IL10+ HACT B cells and IL-10- HACT B cells, with CD4+ T cells.Yüksek LisansM.Sc

Single-cell transcriptomics reveals striking heterogeneity and functional organization of dendritic and monocytic cells in the bovine mesenteric lymph node.

Dendritic and monocytic cells co-operate to initiate and shape adaptive immune responses in secondary lymphoid tissue. The complexity of this system is poorly understood, also because of the high phenotypic and functional plasticity of monocytic cells. We have sequenced mononuclear phagocytes in mesenteric lymph nodes (LN) of three adult cows at the single-cell level, revealing ten dendritic-cell (DC) clusters and seven monocyte/macrophage clusters with clearly distinct transcriptomic profiles. Among DC, we defined LN-resident subsets and their progenitors, as well as subsets of highly activated migratory DC differing in transcript levels for T-cell attracting chemokines. Our analyses also revealed a potential differentiation path for cDC2, resulting in a cluster of inflammatory cDC2 with close transcriptional similarity to putative DC3 and monocyte-derived DC. Monocytes and macrophages displayed sub-clustering mainly driven by pro- or anti-inflammatory expression signatures, including a small cluster of cycling, presumably self-renewing, macrophages. With this transcriptomic snapshot of LN-derived mononuclear phagocytes, we reveal functional properties and differentiation trajectories in a "command center of immunity", and identify elements that are conserved across species

Single-cell transcriptomics reveals striking heterogeneity and functional organization of dendritic and monocytic cells in the bovine mesenteric lymph node

Dendritic and monocytic cells co-operate to initiate and shape adaptive immune responses in secondary lymphoid tissue. The complexity of this system is poorly understood, also because of the high phenotypic and functional plasticity of monocytic cells. We have sequenced mononuclear phagocytes in mesenteric lymph nodes (LN) of three adult cows at the single-cell level, revealing ten dendritic-cell (DC) clusters and seven monocyte/macrophage clusters with clearly distinct transcriptomic profiles. Among DC, we defined LN-resident subsets and their progenitors, as well as subsets of highly activated migratory DC differing in transcript levels for T-cell attracting chemokines. Our analyses also revealed a potential differentiation path for cDC2, resulting in a cluster of inflammatory cDC2 with close transcriptional similarity to putative DC3 and monocyte-derived DC. Monocytes and macrophages displayed sub-clustering mainly driven by pro- or anti-inflammatory expression signatures, including a small cluster of cycling, presumably self-renewing, macrophages. With this transcriptomic snapshot of LN-derived mononuclear phagocytes, we reveal functional properties and differentiation trajectories in a “command center of immunity”, and identify elements that are conserved across species

Precise Delineation and Transcriptional Characterization of Bovine Blood Dendritic-Cell and Monocyte Subsets

A clear-cut delineation of bovine bona fide dendritic cells (DC) from monocytes has proved challenging, given the high phenotypic and functional plasticity of these innate immune cells and the marked phenotypic differences between species. Here, we demonstrate that, based on expression of Flt3, CD172a, CD13, and CD4, a precise identification of bovine blood conventional DC type 1 and 2 (cDC1, cDC2), plasmacytoid DC (pDC), and monocytes is possible with cDC1 being Flt3+CD172adimCD13+CD4−, cDC2 being Flt3+CD172a+CD13−CD4−, pDC being Flt3+CD172adimCD13−CD4+, and monocytes being Flt3−CD172ahighCD13−CD4−. The phenotype of these subsets was characterized in further detail, and a subset-specific differential expression of CD2, CD5, CD11b, CD11c, CD14, CD16, CD26, CD62L, CD71, CD163, and CD205 was found. Subset identity was confirmed by transcriptomic analysis and subset-specific transcription of conserved key genes. We also sorted monocyte subsets based on their differential expression of CD14 and CD16. Classical monocytes (CD14+CD16−) clustered clearly apart from the two CD16+ monocyte subsets probably representing intermediate and non-classical monocytes described in human. The transcriptomic data also revealed differential gene transcription for molecules involved in antigen presentation, pathogen sensing, and migration, and therefore gives insights into functional differences between bovine DC and monocyte subsets. The identification of cell-type- and subset-specific gene transcription will assist in the quest for “marker molecules” that—when targeted by flow cytometry—will greatly facilitate research on bovine DC and monocytes. Overall, species comparisons will elucidate basic principles of DC and monocyte biology and will help to translate experimental findings from one species to another

Monocyte biology conserved across species: Functional insights from cattle

Similar to human monocytes, bovine monocytes can be split into CD14highCD16- classical, CD14highCD16high intermediate and CD14-/dimCD16high nonclassical monocytes (cM, intM, and ncM, respectively). Here, we present an in-depth analysis of their steady-state bulk- and single-cell transcriptomes, highlighting both pronounced functional specializations and transcriptomic relatedness. Bulk gene transcription indicates pro-inflammatory and antibacterial roles of cM, while ncM and intM appear to be specialized in regulatory/anti-inflammatory functions and tissue repair, as well as antiviral responses and T-cell immunomodulation. Notably, intM stood out by high expression of several genes associated with antigen presentation. Anti-inflammatory and antiviral functions of ncM are further supported by dominant oxidative phosphorylation and selective strong responses to TLR7/8 ligands, respectively. Moreover, single-cell RNA-seq revealed previously unappreciated heterogeneity within cM and proposes intM as a transient differentiation intermediate between cM and ncM

Monocyte biology conserved across species: Functional insights from cattle.

Similar to human monocytes, bovine monocytes can be split into CD14highCD16- classical, CD14highCD16high intermediate and CD14-/dimCD16high nonclassical monocytes (cM, intM, and ncM, respectively). Here, we present an in-depth analysis of their steady-state bulk- and single-cell transcriptomes, highlighting both pronounced functional specializations and transcriptomic relatedness. Bulk gene transcription indicates pro-inflammatory and antibacterial roles of cM, while ncM and intM appear to be specialized in regulatory/anti-inflammatory functions and tissue repair, as well as antiviral responses and T-cell immunomodulation. Notably, intM stood out by high expression of several genes associated with antigen presentation. Anti-inflammatory and antiviral functions of ncM are further supported by dominant oxidative phosphorylation and selective strong responses to TLR7/8 ligands, respectively. Moreover, single-cell RNA-seq revealed previously unappreciated heterogeneity within cM and proposes intM as a transient differentiation intermediate between cM and ncM

Efficacy of three innovative bacterin vaccines against experimental infection with Mycoplasma hyopneumoniae

International audienceAbstractNew vaccine formulations that include novel strains of Mycoplasma hyopneumoniae and innovative adjuvants designed to induce cellular immunity could improve vaccine efficacy against this pathogen. The aim of this experimental study was to assess the efficacy of three experimental bacterin formulations based on M. hyopneumoniae field strain F7.2C which were able to induce cellular immunity. The formulations included a cationic liposome formulation with the Mincle receptor ligand trehalose 6,6-dibehenate (Lipo_DDA:TDB), a squalene-in-water emulsion with Toll-like receptor (TLR) ligands targeting TLR1/2, TLR7/8 and TLR9 (SWE_TLR), and a poly(lactic-co-glycolic acid) micro-particle formulation with the same TLR ligands (PLGA_TLR). Four groups of 12 M. hyopneumoniae-free piglets were primo- (day (D) 0; 39 days of age) and booster vaccinated (D14) intramuscularly with either one of the three experimental bacterin formulations or PBS. The pigs were endotracheally inoculated with a highly and low virulent M. hyopneumoniae strain on D28 and D29, respectively, and euthanized on D56. The main efficacy parameters were: respiratory disease score (RDS; daily), macroscopic lung lesion score (D56) and log copies M. hyopneumoniae DNA determined with qPCR on bronchoalveolar lavage (BAL) fluid (D42, D56). All formulations were able to reduce clinical symptoms, lung lesions and the M. hyopneumoniae DNA load in the lung, with formulation SWE_TLR being the most effective (RDSD28–D56 −61.90%, macroscopic lung lesions −88.38%, M. hyopneumoniae DNA load in BAL fluid (D42) −67.28%). Further experiments raised under field conditions are needed to confirm these results and to assess the effect of the vaccines on performance parameters

The spike gene is a major determinant for the SARS-CoV-2 Omicron-BA.1 phenotype.

Variant of concern (VOC) Omicron-BA.1 has achieved global predominance in early 2022. Therefore, surveillance and comprehensive characterization of Omicron-BA.1 in advanced primary cell culture systems and animal models are urgently needed. Here, we characterize Omicron-BA.1 and recombinant Omicron-BA.1 spike gene mutants in comparison with VOC Delta in well-differentiated primary human nasal and bronchial epithelial cells in vitro, followed by in vivo fitness characterization in hamsters, ferrets and hACE2-expressing mice, and immunized hACE2-mice. We demonstrate a spike-mediated enhancement of early replication of Omicron-BA.1 in nasal epithelial cultures, but limited replication in bronchial epithelial cultures. In hamsters, Delta shows dominance over Omicron-BA.1, and in ferrets Omicron-BA.1 infection is abortive. In hACE2-knock-in mice, Delta and a Delta spike clone also show dominance over Omicron-BA.1 and an Omicron-BA.1 spike clone, respectively. Interestingly, in naïve K18-hACE2 mice, we observe Delta spike-mediated increased replication and pathogenicity and Omicron-BA.1 spike-mediated reduced replication and pathogenicity, suggesting that the spike gene is a major determinant of replication and pathogenicity. Finally, the Omicron-BA.1 spike clone is less well-controlled by mRNA-vaccination in K18-hACE2-mice and becomes more competitive compared to the progenitor and Delta spike clones, suggesting that spike gene-mediated immune evasion is another important factor that led to Omicron-BA.1 dominance

The spike gene is a major determinant for the SARS-CoV-2 Omicron-BA. 1 phenotype

Variant of concern (VOC) Omicron-BA.1 has achieved global predominance in early 2022. Therefore, surveillance and comprehensive characterization of Omicron-BA.1 in advanced primary cell culture systems and animal models are urgently needed. Here, we characterize Omicron-BA.1 and recombinant Omicron-BA.1 spike gene mutants in comparison with VOC Delta in well-differentiated primary human nasal and bronchial epithelial cells in vitro, followed by in vivo fitness characterization in hamsters, ferrets and hACE2-expressing mice, and immunized hACE2-mice. We demonstrate a spike-mediated enhancement of early replication of Omicron-BA.1 in nasal epithelial cultures, but limited replication in bronchial epithelial cultures. In hamsters, Delta shows dominance over Omicron-BA.1, and in ferrets Omicron-BA.1 infection is abortive. In hACE2-knock-in mice, Delta and a Delta spike clone also show dominance over Omicron-BA.1 and an Omicron-BA.1 spike clone, respectively. Interestingly, in naïve K18-hACE2 mice, we observe Delta spike-mediated increased replication and pathogenicity and Omicron-BA.1 spike-mediated reduced replication and pathogenicity, suggesting that the spike gene is a major determinant of replication and pathogenicity. Finally, the Omicron-BA.1 spike clone is less well-controlled by mRNA-vaccination in K18-hACE2-mice and becomes more competitive compared to the progenitor and Delta spike clones, suggesting that spike gene-mediated immune evasion is another important factor that led to Omicron-BA.1 dominance

Comparison of pregnant women’s anxiety, depression and birth satisfaction based, on their traumatic childbirth perceptions

This study was conducted to identify and compare pregnant women’s anxiety, depression, and birth satisfaction levels based on their traumatic childbirth perceptions. This study used a cross-sectional design. The data were collected at two stages, namely, the prenatal and postpartum stages. First, the Traumatic Childbirth Perception Scale, the Beck Anxiety Inventory, and the Beck Depression Inventory were applied to the pregnant women. Next, in the postpartum period, the Childbirth Information Form and the Birth Satisfaction Scale-Revised were applied. It was found that the participants with high levels of traumatic childbirth perception had higher mean anxiety and depressive symptom scores (26.13 ± 11.30 and 16.16 ± 9.02, respectively), whereas the participants with low levels of traumatic childbirth perception had a higher mean birth satisfaction score (17.50 ± 4.91). The findings indicated that high levels of traumatic childbirth perception may lead to have anxiety and depression, while low levels of traumatic childbirth perception may enhance their birth satisfaction. IMPACT STATEMENT What is already known on this subject? In the literature, it is stated that the perception of a traumatic birth can lead to permanent or long-term negative consequences in women's lives, negatively affecting their future health, subsequent birth experiences, and family relationships. What do the results of this study add? In this study, 37.7% of the participants were found to have high levels of traumatic childbirth perception. It was determined that the anxiety and depression levels of the participants with high levels of perception of traumatic birth were higher, and the levels of birth satisfaction were higher in the pregnant women with low levels of perception of traumatic birth. The results indicated that severe depressive symptoms, severe anxiety, and low levels of birth satisfaction were likely to raise traumatic childbirth perception levels in pregnant women. What are the implications of these findings for clinical practice and/or further research? The care to be given under the leadership of midwifery professionals is important in terms of reducing pregnant women’s perceptions of a traumatic birth, anxiety, and depression levels and increasing their levels of birth satisfaction