Bursal Cells Research Articles

Muscle weakness and mitochondrial stress occur before severe metastasis in a novel mouse model of ovarian cancer cachexia

ObjectivesA high proportion of women with advanced epithelial ovarian cancer (EOC) experience weakness and cachexia. This relationship is associated with increased morbidity and mortality. EOC is the most lethal gynecological cancer, yet no preclinical cachexia model has demonstrated the combined hallmark features of metastasis, ascites development, muscle loss and weakness in adult immunocompetent mice. MethodsHere, we evaluated a new model of ovarian cancer-induced cachexia with the advantages of inducing cancer in adult immunocompetent C57BL/6J mice through orthotopic injections of EOC cells in the ovarian bursa. We characterized the development of metastasis, ascites, muscle atrophy, muscle weakness, markers of inflammation, and mitochondrial stress in the tibialis anterior (TA) and diaphragm ∼45, ∼75 and ∼90 days after EOC injection. ResultsPrimary ovarian tumour sizes were progressively larger at each time point while severe metastasis, ascites development, and reductions in body, fat and muscle weights occurred by 90 Days. There were no changes in certain inflammatory (TNFα), atrogene (MURF1 and Atrogin) or GDF15 markers within both muscles whereas IL-6 was increased at 45 and 90 Day groups in the diaphragm. TA weakness in 45 Day preceded atrophy and metastasis that were observed later (75 and 90 Day, respectively). The diaphragm demonstrated both weakness and atrophy in 45 Day. In both muscles, this pre-severe-metastatic muscle weakness corresponded with considerable reprogramming of gene pathways related to mitochondrial bioenergetics as well as reduced functional measures of mitochondrial pyruvate oxidation and creatine-dependent ADP/ATP cycling as well as increased reactive oxygen species emission (hydrogen peroxide). Remarkably, muscle force per unit mass at 90 days was partially restored in the TA despite the presence of atrophy and severe metastasis. In contrast, the diaphragm demonstrated progressive weakness. At this advanced stage, mitochondrial pyruvate oxidation in both muscles exceeded control mice suggesting an apparent metabolic super-compensation corresponding with restored indices of creatine-dependent adenylate cycling. ConclusionsThis mouse model demonstrates the concurrent development of cachexia and metastasis that occurs in women with EOC. The model provides physiologically relevant advantages of inducing tumour development within the ovarian bursa in immunocompetent adult mice. Moreover, the model reveals that muscle weakness in both TA and diaphragm precedes severe metastasis while weakness also precedes atrophy in the TA. An underlying mitochondrial bioenergetic stress corresponded with this early weakness. Collectively, these discoveries can direct new research towards the development of therapies that target pre-atrophy and pre-severe-metastatic weakness during EOC in addition to therapies targeting cachexia.

Deciphering infected cell types, hub gene networks and cell-cell communication in infectious bronchitis virus via single-cell RNA sequencing.

Infectious bronchitis virus (IBV) is a coronavirus that infects chickens, which exhibits a broad tropism for epithelial cells, infecting the tracheal mucosal epithelium, intestinal mucosal epithelium, and renal tubular epithelial cells. Utilizing single-cell RNA sequencing (scRNA-seq), we systematically examined cells in renal, bursal, and tracheal tissues following IBV infection and identified tissue-specific molecular markers expressed in distinct cell types. We evaluated the expression of viral RNA in diverse cellular populations and subsequently ascertained that distal tubules and collecting ducts within the kidney, bursal mucosal epithelial cells, and follicle-associated epithelial cells exhibit susceptibility to IBV infection through immunofluorescence. Furthermore, our findings revealed an upregulation in the transcription of proinflammatory cytokines IL18 and IL1B in renal macrophages as well as increased expression of apoptosis-related gene STAT in distal tubules and collecting duct cells upon IBV infection leading to renal damage. Cell-to-cell communication unveiled potential interactions between diverse cell types, as well as upregulated signaling pathways and key sender-receiver cell populations after IBV infection. Integrating single-cell data from all tissues, we applied weighted gene co-expression network analysis (WGCNA) to identify gene modules that are specifically expressed in different cell populations. Based on the WGCNA results, we identified seven immune-related gene modules and determined the differential expression pattern of module genes, as well as the hub genes within these modules. Our comprehensive data provides valuable insights into the pathogenesis of IBV as well as avian antiviral immunology.

Bursal Tissue Harvested During Rotator Cuff Repair Contains Viable Mesenchymal Stem Cells

PurposeThe purpose of this study was to evaluate the effect of intra operative ablation on the viability, distribution, phenotype, and potential for culture expansion of bursal cells harvested during arthroscopic rotator cuff surgery. MethodsTissue was collected during primary arthroscopic rotator cuff repair on six healthy, randomly selected patients from a fellowship-trained surgeon’s practice between September 2020 and January 2021. There were 3 females (age 60 ± 8 years) and 3 males (age 61 ± 10 years). At the time of surgery, subacromial bursal tissue was subjected to no ablation, one second, or three seconds of ablation. Tissues were collected by an autograft harvesting system connected to an arthroscopic shaver and a pituitary grasper. Tissue fragments from each condition were sampled for viability testing or cell isolation. A viability kit with confocal microscopy was used to assess live and dead cells. Cell isolation consisted of collagenase digestion or placing tissue fragments onto tissue culture treated plates that induced migration of cells out of the tissue. Cell proliferation rates were monitored and surface markers for mesenchymal stem cells (MSC) and pericytes were analyzed via multi-color flow cytometry. ResultsIncreased ablation time significantly reduced cell viability. The mean percentage of live cells was 55.2 ± 27.2% (range 26-90% live) in the control group, 46.8 ± 23.8% (range 9.6-69.6% p = 0.045) in the short, and 35.5 ± 19% (range 11-54% p = 0.03) in the long ablation group. No significant differences in population doubling level (1.6 ± 0.5 days) and population doubling time (6.7 ± 2.4 days) were observed in cells from any treatment. The surface marker profile indicated an MSC phenotype with absence of a pericyte population. Ablation or cell isolation procedure had no significant effect on the surface marker profile of isolated cells. ConclusionRadiofrequency ablation significantly reduced the overall tissue viability, but had no significant effect on cell proliferation, or expression of surface markers on isolated subacromial bursal cells harvested arthroscopically. Clinical RelevanceAnalysis of the viability and performance of cells harvested after the use of ablation devices using mechanical surgical collection during RCR surgery could further our understanding of subacromial bursal tissue and its potential role in augmenting rotator cuff repair healing.

Antioxidant Properties of Brassica Rapa L. Seeds and Immunostimulant Effect on Immunity Broilers Cells (Gallus Gallus Domesticus)

Background: In the past few decades, researchers have focused on finding the benefits of natural substances derived from plants. Objective: This study aimed to evaluate the use of Brassica rapa seeds in poultry feed as an antioxidant and immunostimulant of host defenses. Methods: We prepared three extracts using ethanol, Ethyl Acetate, and water. Spectrophotometric methods determined the total phenolic and flavonoid content in the three extracts. Antioxidant activity was assessed using 1,1-diphenyl-2-picrylhydrazyl (DPPH), ferric reducing/antioxidant power (FRAP), 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) radicals and β-Carotene bleaching methods. An assessment of their immunostimulant activities in vitro was performed on chicken immunity cell proliferation (splenocytes, thymocytes, and Bursa cells) and IgY production. method: We prepared three different extracts using ethanol, Ethyl Acetate, and water. The total phenolic and flavonoid content in the three extracts was determined by spectrophotometric methods.Antioxidant activity has been assessed using 1,1-diphenyl-2-picrylhydrazyl (DPPH), ferric reducing/antioxidant power (FRAP), 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) radicals and β-Carotene bleaching methods. Assessment of their immunostimulant activities in vitro was tested on chicken immunity cell proliferation (splenocytes, thymocytes, and Bursa cells) and IgY production Results: The total phenolic contents ranged from 462.5 to 794.8 mg /g of extract, the order of TPC was as follows: EtOAc > EtOH >Water. Flavonoid contents were, 63.7 mg in EtOH extract, 81.2 mg in EtOAc extract, and 108.7 mg/g Extract in aqueous extract. By DPPH, the IC50s of EtOH, EtOAc, and water, were 1.8μg/ml, 2.4μg/ml, and 1.5μg/ml, respectively. Using the Ferric- Reducing Antioxidant Power (FRAP), we remarked that the EtOH and EtOAc extracts have important antioxidant powers. The ABTS assay indicated that EtOH and water had the highest activities with IC50s of 0.19 and 0.07, respectively. Finally, by the β-carotene bleaching test, we observed that the IC50 of the EtOH, EtOAc, and water were 62.1 μg/mL; 72.7 μg/mL, and 45.8 μg/mL, respectively. The results obtained indicate that Brassica aqueous extract stimulates humoral immunity by stimulating splenocyte (B and T-lymphocytes) and Bursa cell (B-lymphocyte) proliferation by more than 200% of response vs control. In addition, the aqueous extract highly stimulated the function of bursa cells by 208% of the reaction. In the same conditions, we recorded a stimulation of cellular immunity mediated by thymocytes by an increase in cell proliferation (352.7% of response) implicated in virus protection. These extracts also possessed an antimicrobial effect against diverse microorganisms such as coliforms and Staphylococcus. The FT-IR spectrum indicated that the hydroxyl group (phenol), hydroxybenzoic acid family, carbohydrate molecules such as glucopyranose, a carbonyl ester group, hydrocarbon chains (alkyls groups)) were present. result: The total phenolic contents ranged from 462.5 to 794.8 mg /g of Extract, the order of TPC was as follows: EtOAc > EtOH >Water. Flavonoid contents were, 63.7 mg in EtOH extract, 81.2 mg in EtOAc extract, and 108.7 mg/g Extract in aqueous extract.By DPPH, the IC50s of EtOH, EtOAc, and water, were respectively 1.8µg/ml, 2.4µg/ml, and 1.5µg/ml. Using the ferric-reducing antioxidant power (FRAP) we remarked that the EtOH and EtOAc extracts have important antioxidant powers. The ABTS assay indicated that EtOH and water had the highest activities with IC50s of 0.19 and 0.07 respectively. Finally, by the β-carotene bleaching test, we observed that the IC50 of the EtOH, EtOAc, and water were 62.1 µg/mL; 72.7 µg/mL, and 45.8 µg/mL respectively. The results obtained indicate that Brassica aqueous extract stimulates humoral immunity by stimulating splenocyte (B and T-lymphocytes) and Bursa cell (B-lymphocyte) proliferation by more than 200% of response vs control. In addition, the aqueous extract stimulated highly the function of bursa cells by 208% of the response. In the same conditions, we recorded a stimulation of cellular immunity mediated by thymocytes by an increase in cell proliferation (352.7% of response) implicated in virus protection. These extracts possessed also an antimicrobial effect against diverse microorganisms such as coliforms and Staphylococcus. The FT-IR spectrum indicated that the hydroxyl group (phenol), hydroxybenzoic acid family, carbohydrate molecules such as glucopyranose, a carbonyl ester group, hydrocarbon chains (alkyls groups)) were present. Conclusion: This result could have interesting applications in the poultry feed industry to enhance the performance of animal development.

Pro-Resolving Mediators in Rotator Cuff Disease: How Is the Bursa Involved?

So far, tendon regeneration has mainly been analyzed independent from its adjacent tissues. However, the subacromial bursa in particular appears to influence the local inflammatory milieu in the shoulder. The resolution of local inflammation in the shoulder tissues is essential for tendon regeneration, and specialized pro-resolving mediators (SPMs) play a key role in regulating the resolution of inflammation. Here, we aimed to understand the influence of the bursa on disease-associated processes in neighboring tendon healing. Bursa tissue and bursa-derived cells from patients with intact, moderate and severe rotator cuff disease were investigated for the presence of pro-resolving and inflammatory mediators, as well as their effect on tenocytes and sensitivity to mechanical loading by altering SPM signaling mediators in bursa cells. SPM signal mediators were present in the bursae and altered depending on the severity of rotator cuff disease. SPMs were particularly released from the bursal tissue of patients with rotator cuff disease, and the addition of bursa-released factors to IL-1β-challenged tenocytes improved tenocyte characteristics. In addition, mechanical loading modulated pro-resolving processes in bursa cells. In particular, pathological high loading (8% strain) increased the expression and secretion of SPM signaling mediators. Overall, this study confirms the importance of bursae in regulating inflammatory processes in adjacent rotator cuff tendons.

In ovo feeding of probiotic lactobacilli differentially alters expression of genes involved in the development and immunological maturation of bursa of Fabricius in pre-hatched chicks

Compelling evidence indicates that immunological maturation of the gut-associated lymphoid tissues, including the bursa of Fabricius, is dependent upon antigenic stimulation post-hatch. In view of these data, the present study investigated the impact of exposing the immune system of chick embryos to antigenic stimuli, via in ovo delivery of poultry-specific lactobacilli, on the expression of genes associated with early bursal development and maturation. Broiler line embryonated eggs were inoculated with 106 and 107 colony-forming units (CFUs) of an individual or a mixture of Lactobacillus species, including L. crispatus (C25), L. animalis (P38), L. acidophilus (P42), and L. reuteri (P43), at embryonic day 18 (ED18). The bursa of Fabricius was collected from pre-hatched chicks (ED20) to measure the expression levels of various immune system genes. The results revealed that L. acidophilus and the mixture of Lactobacillus species at the dose of 106 CFU consistently elicited higher expression of genes responsible for B cell development, differentiation, and survival (B cell activating factor (BAFF), BAFF-receptor (BAFF-R)), and antibody production (interleukin (IL)-10) and diversification (TGF-β). Similar expression patterns were also noted in T helper (Th) cell-associated cytokine genes, including Th1-type cytokines (interferon (IFN)-γ and IL-12p40), Th2-type cytokines (IL-4 and IL-13) and Th17 cytokine (IL-17). Overall, these results suggest that the supplementation of poultry-specific lactobacilli to chick embryos might be beneficial for accelerating the development and immunological maturation of the bursa of Fabricius. However, further studies are required to determine if the changes in gene expression are associated with the developmental trajectory and phenotypes of bursal cells.

Effect of Intermediate Plus Vaccine and vvIBDV on Bursa Secretory Cells and Their Glycoprotein Production.

There are two types of secretory cells in the chicken bursa of Fabricius (BF): (a) interfollicular epithelial cells (IFE), and (b) bursal secretory dendritic cells (BSDC) in the medulla of bursal follicles. Both cells produce secretory granules, and the cells are highly susceptible to IBDV vaccination and infection. Before and during embryonic follicular bud formation, an electron-dense, scarlet-acid fuchsin positive substance emerges in the bursal lumen, the role of which is unknown. In IFE cells, IBDV infection may induce rapid granular discharge, and in several cells, peculiar granule formation, which suggests that the glycosylation of protein is injured in the Golgi complex. In control birds, the discharged BSDC granules appear in membrane-bound and subsequently solubilized, fine-flocculated forms. The solubilized, fine-flocculated substance is Movat-positive and can be a component of the medullary microenvironment, which prevents the medullary B lymphocytes from nascent apoptosis. Vaccination interferes with the solubilization of the membrane-bound substance, resulting in: (i) aggregation of a secreted substance around the BSDC, and (ii) solid lumps in the depleted medulla. The non-solubilized substance is possibly not "available" for B lymphocytes, resulting in apoptosis and immunosuppression. In IBDV infection, one part of the Movat-positive Mals fuse together to form a medullary, gp-containing "cyst". The other part of Mals migrate into the cortex, recruiting granulocytes and initiating inflammation. During recovery the Movat-positive substance appears as solid, extracellular lumps between the cells of FAE and Mals. Possibly the Mals and Movat-positive extracellular lumps glide into the bursal lumen via FAE to eliminate cell detritus from the medulla.

Characterization and pathogenicity of a novel variant infectious bursal disease virus in China.

Infectious bursal disease (IBD) is a highly epidemic and immunosuppressive disease of 3- to 6-week-old chicks caused by infectious bursal disease virus (IBDV). Since 2017, there has been a notable increase in the isolation rates of novel variant IBDV strains in China, of which characteristic amino acid residues were different from those of early antigen variants. In this study, one IBDV strain was isolated from a farm with suspected IBD outbreak in Shandong Province, China, which was designated LY21/2. The strain LY21/2 could replicate in MC38 cells with previous culture adaption in SPF chick embryos. Phylogenetic analysis revealed that LY21/2 formed one branch with novel variant IBDVs and shared 96.8-98.6% nucleotide sequence identity with them. Moreover, LY21/2 serving as the major parent underwent the recombination event of a variant strain (19D69), while the minor parent was a very virulent strain (Harbin-1). SPF chicks inoculated with LY21/2 showed no gross clinic symptom, whereas bursal atrophy was exhibited and apoptosis was occurred in 55.21% of bursal cells. The results of histopathology and immunohistochemical staining showed that lymphocyte depletion and connective tissue hyperplasia and IBDV antigen-positive cells were observed in the bursa of LY21/2-infected chicks. Besides, DNA fragmentation was detected in the LY21/2-infected bursal tissue section by TUNEL assay. Collectivtely, these data presented analysis and evaluation of the genetic characteristics and pathogenicity of a novel variant IBDV strain. This study may help in the development of biosafety strategies for the prevention and control of IBDV in poultry.

Glycoprotein Production by Bursal Secretory Dendritic Cells in Normal, Vaccinated, and Infectious Bursal Disease Virus (IBDV)-Infected Chickens.

The aim of this study is to follow the gp production in IBDV-vaccinated and challenged birds. The progress of IBDV infection was monitored using anti-VP2 immunocytochemistry, light and transmission electron microscopy. In the medulla of the bursal follicle, the Movat pentachrome staining discovered an extracellular glycoprotein (gp) produced by bursal secretory dendritic cells (BSDCs). The secretory granules of BSDCs either discharge resulting in extracellular gp or fuse together forming intracellular corpuscles. The double fate of granules suggests a dual function of BSDCs: (a.) For the discharged granules, gp contributes to the medullary microenvironment (ME). (b.) The intracellular corpuscles may be the sign of BSDC transformation to a macrophage-like cell (Mal). Infectious bursal disease virus (IBDV) infection accelerates the BSDC transformation to Mal. The decreased number of BSDCs is feedback for the precursor cells of BSDCs lodging in the cortico-medullary epithelial arches (CMEA), where they proliferate. Opening the CMEA, the precursor cells enter the medulla, and differentiate to immature BSDCs. The virus uptake in the corpuscles prevents the granular discharge resulting in the absence of gp and alteration in ME. In vaccine-take birds, the mitotic rate of BSDC precursor cells cannot restore the precursor pool; therefore, in the case of IBDV challenge, the number of newly formed BSDCs is too low for outbreak of clinical disease. The BSDCs, as a primary target of IBDV, may contribute to the long-lasting immunosuppressive status of IBDV-infected chickens.

Activated Serum Increases In Vitro Cellular Proliferation and Growth Factor Expression of Musculoskeletal Cells.

The purpose of this study was to investigate proteomic alteration that occurs to whole blood when converted to activated serum (AS) using an autologous thrombin system. This study further sought to evaluate the functional in vitro effect of AS on tenocytes, chondrocytes, subacromial bursal cells, and osteoblasts. The peptide/protein composition of AS was analyzed by liquid chromatography–mass spectrophotometry (LC-MS). The cell lines were treated with AS, and cellular proliferation was quantified 48 h after treatment. Platelet-derived growth factor (PDGF), insulin-like growth factor 1 (IGF-1), vascular endothelial growth factor (VEGF), interleukin-1 beta (IL-1β), and interleukin-1 receptor antagonist (IL-1Ra) were quantified utilizing enzyme-linked immunosorbent assays (ELISAs). LC-MS identified 357 proteins across the AS and whole blood. Fifty-four of the proteins identified had significant differences between the relative protein abundance of the AS samples compared to whole blood. Treatment with AS in all cell lines significantly increased proliferation compared to control cells at 48 h. Increased PDGF, VEGF, and IGF-1 in all cell lines exposed to AS compared to the control (p < 0.05) were observed. These findings suggest that treatment with AS increases in vitro cellular proliferation and the release of growth factors that may play a role in tissue repair.

Blood B Cell Depletion Reflects Immunosuppression Induced by Live-Attenuated Infectious Bursal Disease Vaccines.

Immunosuppression in poultry production is a recurrent problem worldwide, and one of the major viral immunosuppressive agents is Infectious Bursal Disease Virus (IBDV). IBDV infections are mostly controlled by using live-attenuated vaccines. Live-attenuated Infectious Bursal Disease (IBD) vaccine candidates are classified as “mild,” “intermediate,” “intermediate-plus” or “hot” based on their residual immunosuppressive properties. The immunosuppression protocol described by the European Pharmacopoeia (Ph. Eur.) uses a lethal Newcastle Disease Virus (NDV) infectious challenge to measure the interference of a given IBDV vaccine candidate on NDV vaccine immune response. A Ph. Eur.-derived protocol was thus implemented to quantify immunosuppression induced by one mild, two intermediate, and four intermediate-plus live-attenuated IBD vaccines as well as a pathogenic viral strain. This protocol confirmed the respective immunosuppressive properties of those vaccines and virus. In the search for a more ethical alternative to Ph. Eur.-based protocols, two strategies were explored. First, ex vivo viral replication of those vaccines and the pathogenic strain in stimulated chicken primary bursal cells was assessed. Replication levels were not strictly correlated to immunosuppression observed in vivo. Second, changes in blood leukocyte counts in chicks were monitored using a Ph. Eur. - type protocol prior to lethal NDV challenge. In case of intermediate-plus vaccines, the drop in B cells counts was more severe. Counting blood B cells may thus represent a highly quantitative, faster, and ethical strategy than NDV challenge to assess the immunosuppression induced in chickens by live-attenuated IBD vaccines.

Evaluation of cellular and humoral immune response in chickens immunized with flagellin-adjuvanted inactivated newcastle disease virus

The aim of the present study was to evaluate the potential effect of flagellin as adjuvant in Newcastle disease virus (NDV) vaccine on the cellular and humoral immunity in chickens. Fifty-six specific pathogen-free chickens were assigned to seven groups of eight chickens and immunized twice with a two-week interval, intramuscularly. Group 1, received phosphate buffered saline as control (C), groups 2, 3, 4, 5, 6 and 7 were immunized with inactivated NDV [Ag], Ag + full FliC protein [AgF], Ag + truncated Flic protein [AgT], Ag + native Flic protein [AgN], commercial NDV vaccine [Vac] and Vac + N [VacN], respectively. After 45 days, spleen and bursa of Fabricius samples were collected and analyzed by flow cytometry and responses in control/vaccinated chickens were studied by immunophenotyping. Humoral response was also, evaluated by ELISA during the experiment. Results showed that immunized chickens with Ag + flagellin proteins had significantly higher frequency of circulating CD3+, CD4+ and CD8+ T cells in bursa of Fabricius in AgF, AgT and AgN, respectively, compared with other groups. Similar results were observed for spleen; however, the highest frequency of circulating CD3+, CD4+ and CD8+ T cells belonged to AgT and AgF, respectively. ELISA results showed that all flagellin-adjuvanted groups had higher antibody titers than other groups with the highest antibody response in VacN. It can be concluded that flagellin may induce both humoral and cellular immune responses against ND and is suggested for use as an efficient adjuvant.

Influence of Immune Parameters after Enterococcus faecium AL41 Administration and Salmonella Infection in Chickens

Immune response of day-old chicks infected with Salmonella Enteritidis PT4 and preventive administration of Enterococcus faecium AL41 were studied using hematology and flow cytometry of immunocompetent cells in blood, cecum, bursa and spleen for 11 days, and included 220 animals divided into four groups (n = 55). E. faecium AL41 was administered for 7 days to EF and EFSE groups and on day 4 SE and EFSE groups were infected with Salmonella Enteritidis. Values of monocytes at 4 dpi significantly increased in EFSE and lymphocytes at 7 dpi in EF groups. Blood CD3, CD4, CD8 and IgM lymphocytes improved in EF and EFSE groups and IgA in EF group at 4 dpi. Phagocytic activity of probiotic groups was improved in both samples. Cecal IEL and LPL lymphocytes showed at 7 dpi stimulation of CD3, CD4 and CD8 subpopulations in probiotic groups, especially in EFSE group, IgA IEL and IgA with IgM LPL in EF groups. Bursa Fabricii at 7 dpi presented overstimulation of IgG subpopulation in SE group, spleen CD3 and CD8 in EF and EFSE groups. E. faecium AL41 revealed the protective effect and positive influence on the local and systemic immune response in Salmonella Enteritidis PT4 infected chickens.

Characterization and functional properties of a novel monoclonal antibody which identifies a B cell subpopulation in bursa of Fabricius

The bursa of Fabricius (BF) plays a central role in the development of B lymphocytes in birds. During embryonic development the BF primordium is colonized by myeloid and lymphoid prebursal stem cells to form the follicle buds, which ultimately develop into lymphoid follicles with a central medullary and an outer cortical region. Lympho-myeloid differentiation within the medulla is fundamental to normal B cell development. In contrast, the complexity of the cellular composition of the follicular cortex and its role in B cell differentiation has only recently begun to be studied. As an effort to characterize the different bursal cells we have produced a large panel of monoclonal antibodies (mAbs) by immunizing mice with a BF cell suspension of guinea fowl (Numida meleagris). One of these antibodies (clone: 7H3) was found to recognize a 80 kDa cell surface antigen expressed first in the yolk sac blood island of 2-day-old guinea fowl and chicken embryos, and later detected in the embryonic circulation and primary lymphoid organs. Double immunofluorescence revealed that chB6+ (Bu-1+) B cells of embryonic BF co-express the 7H3 antigen. 7H3 immunoreactivity of the bursal follicles gradually diminished after hatching and only a subpopulation of cortical B cells expressed the 7H3 antigen. In addition, in post-hatched birds 7H3 mAb recognizes all T lymphocytes of the thymus, peripheral lymphoid organs and blood. Embryonic BF injected with the 7H3 mAb showed a near complete block of lymphoid follicle formationIn conclusion, 7H3 mAb labels a new differentiation antigen specific for avian hematopoietic cells, which migrate through the embryonic mesenchyme, colonize the developing BF lymphoid follicles, and differentiate into a subpopulation of cortical B cells. The staining pattern of the 7H3 mAb and the correlation of expression with cell migration suggest that the antigen will serve as valuable immunological marker for studying the ontogeny of avian B cells.

Xenograft Models of Ovarian Cancer for Therapy Evaluation.

The evaluation of novel treatment regimes in ovarian cancer, ranging from cytotoxic agents and targeted therapy to surgery, demands clinically relevant mouse models to mimic human disease. These more advanced preclinical models provide a tool to obtain robust data on the mechanism of action, cytotoxicity and therapeutic efficacy of newly emerging antitumor therapies.In this chapter, we describe how to generate ovariancancer xenograft models through injection of human tumor cell lines in immunocompromised mice. Detailed methodological descriptions are provided for both the commonly applied subcutaneous model and the more technically challenging orthotopic tumor model that involves inoculation of cancer cells in the ovarian bursa. We demonstrate how to monitor tumor growth and metastases in orthotopic ovarian models through noninvasive optical imaging and the procedures for treatment strategy, including administration of test compounds and debulking surgery. We comment on the strengths, limitations, and procedural challenges associated with each of the models.

From nasal to basal: single-cell sequencing of the bursa of Fabricius highlights the IBDV infection mechanism in chickens

BackgroundChickens, important food animals and model organisms, are susceptible to many RNA viruses that invade via the nasal cavity. To determine the nasal entry site of the virus and clarify why avians are susceptible to RNA viruses, infectious bursal disease virus (IBDV) was selected because it is a typical avian RNA virus that infects chickens mainly via the nasal route.ResultsFirst, we found that IBDV infected the posterior part of the nasal cavity in chickens, which is rich in lymphoid tissue and allows the virus to be easily transferred to the blood. Via the blood circulation, IBDV infected peripheral blood mononuclear cells (PBMCs) and was transferred to the bursa of Fabricius to damage the IgM + B lymphocyte population. Subsequently, the single-cell RNA sequencing (scRNA-seq) results suggested the more detailed response of different bursal cell populations (B cells, epithelial cells, dendritic cells, and fibroblasts) to IBDV. Regarding B cells, IBDV infection greatly decreased the IgM + B cell population but increased the IgA + B cell population in the bursal follicles. In contrast to B cells, bursal epithelial cells, especially basal cells, accumulated a large number of IBDV particles. Furthermore, we found that both innate RNA sensors and interferon-stimulated genes (ISGs) were highly expressed in the IBDV-infected groups, while dicer and ago2 expression was largely blocked by IBDV infection. This result suggests that dicer-related RNA interference (RNAi) might be an effective antiviral strategy for IBDV infection in avian.ConclusionOur study not only comprehensively elaborates on the transmission of airborne IBDV via the intranasal route and establishes the main target cell types for productive IBDV infection but also provides sufficient evidence to explain the cellular antiviral mechanism against IBDV infection.Graphical

Trochanteric Bursa Is a Source of Connective Tissue Progenitor Cells

PurposeTo investigate the presence of connective tissue progenitor cells (CTPs) in the trochanteric bursa harvested over the gluteus medius muscle belly and tendon during open hip procedures.MethodsTrochanteric bursa samples from nine patients (63.1 ± 8.6 years) undergoing total hip arthroplasty for primary osteoarthritis were obtained from 2 sites: over the gluteus medius tendon at the greater trochanter and over the muscle belly. Bursal tissue was digested with collagenase and grown in culture. The nucleated cell count, cellular concentration, cellular proliferation, fluorescence-activated cell sorting (FACS) analysis, and differentiation using immunostaining and quantitative polymerase chain reaction (PCR) were used to determine and quantify the presence of CTPs.ResultsBursa-derived CTPs were identified in all harvested samples. At t = 0, there was no difference in nucleated cell count over muscle and tendon (1.69 ± 1.26 × 108 and 1.41 ± 1.12 × 108 cells/g, respectively; P = .162). Similarly, the cellular concentration at 3 weeks was not significantly different between bursa harvested over muscle and tendon (6.61 ± 5.14 × 106 and 5.58 ± 4.70 × 106 cells/g, respectively; P = .532). High cellular proliferation was identified for both bursal tissue overlying muscle and tendon (2.28 ± .95 and 1.66 ± 1.05, respectively; P = .194). FACS analysis revealed high positivity rates (>95%) of CTP-specific surface epitopes (CD105, CD90, and CD73) and low positivity rates (<1.3%) of negative markers (CD45, CD31). Osteogenic, adipogenic, and chondrogenic differentiation potential was demonstrated with immunostaining and quantitative PCR for gene expression.ConclusionsConnective tissue progenitor cells are found in the trochanteric bursa overlying the muscle and tendon of the hip abductors.Clinical RelevanceDuring open hip procedures, the trochanteric bursa is often partially excised to identify muscular boundaries and tissue planes for surgical exposure. The function of the trochanteric bursa remains largely unknown. However, this tissue is a source of connective tissue progenitor cells, which may be important in the healing response of surgically repaired abductor tendons.

Analysis of Patient Factors Affecting In Vitro Characteristics of Subacromial Bursal Connective Tissue Progenitor Cells during Rotator Cuff Repair.

Unsatisfactory failure rates following rotator cuff (RC) repair have led orthopaedic surgeons to explore biological augmentation of the healing enthesis. The subacromial bursa (SB) contains abundant connective tissue progenitor cells (CTPs) that may aid in this process. The purpose of the study was to investigate the influence of patient demographics and tear characteristics on the number of colony-forming units (CFUs) and nucleated cell count (NCC) of SB-derived CTPs. In this study, we harvested SB tissue over the supraspinatus tendon and muscle in 19 patients during arthroscopic RC repair. NCC of each sample was analyzed on the day of the procedure. After 14 days, CFUs were evaluated under a microscope. Spearman’s rank correlation coefficient was then used to determine the relationship between CFUs or NCC and patient demographics or tear characteristics. The study found no significant correlation between patient demographics and the number of CFUs or NCC of CTPs derived from the SB (p > 0.05). The study did significantly observe that increased tear size was negatively correlated with the number of CFUs (p < 0.05). These results indicated that increased tear size, but not patient demographics, may influence the viability of CTPs and should be considered when augmenting RCrepairs with SB.

Transcriptomic Analysis of Inbred Chicken Lines Reveals Infectious Bursal Disease Severity Is Associated with Greater Bursal Inflammation In Vivo and More Rapid Induction of Pro-Inflammatory Responses in Primary Bursal Cells Stimulated Ex Vivo.

In order to better understand differences in the outcome of infectious bursal disease virus (IBDV) infection, we inoculated a very virulent (vv) strain into White Leghorn chickens of inbred line W that was previously reported to experience over 24% flock mortality, and three inbred lines (15I, C.B4 and 0) that were previously reported to display no mortality. Within each experimental group, some individuals experienced more severe disease than others but line 15I birds experienced milder disease based on average clinical scores, percentage of birds with gross pathology, average bursal lesion scores and average peak bursal virus titre. RNA-Seq analysis revealed that more severe disease in line W was associated with significant up-regulation of pathways involved in inflammation, cytoskeletal regulation by Rho GTPases, nicotinic acetylcholine receptor signaling, and Wnt signaling in the bursa compared to line 15I. Primary bursal cell populations isolated from uninfected line W birds contained a significantly greater percentage of KUL01+ macrophages than cells isolated from line 15I birds (p < 0.01) and, when stimulated ex vivo with LPS, showed more rapid up-regulation of pro-inflammatory gene expression than those from line 15I birds. We hypothesize that a more rapid induction of pro-inflammatory cytokine responses in bursal cells following IBDV infection leads to more severe disease in line W birds than in line 15I.

Marine algal polysaccharides alleviate aflatoxin B1-induced bursa of Fabricius injury by regulating redox and apoptotic signaling pathway in broilers

Aflatoxin B1 (AFB1) causes toxic effect and leads to organ damage in broilers. Marine algal polysaccharides (MAP) of Enteromorpha prolifera exert multiple biological activities, maybe have a potential detoxification effect on AFB1, but the related research in broilers is extremely rare. Therefore, the purpose of this study was to investigate whether MAPs can alleviate AFB1-induced oxidative damage and apoptosis of bursa of Fabricius in broilers. A total of 216 five-week-old male indigenous yellow-feathered broilers (with average initial body weight 397.35 ± 6.32 g) were randomly allocated to one of three treatments (6 replicates with 12 broilers per replicate), and the trial lasted 4 wk. Experimental groups were followed as basal diet (control group); basal diet mixed with 100 μg/kg AFB1 (AFB1 group, the AFB1 is purified form); basal diet with 100 μg/kg AFB1 + 2,500 mg/kg MAPs (AFB1 + MAPs group). The results showed that the diet with AFB1 significantly decreased the relative weight of bursa of Fabricius (P < 0.05), antioxidant enzymes activities of total superoxide dismutase (T-SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione S-transferase (GST), and total antioxidation capacity (T-AOC), while increased malondialdehyde (MDA) content (P < 0.05). Besides, compared with AFB1 group, dietary MAPs improved the relative weight of bursa of Fabricius and activities of antioxidant enzymes (T-SOD, GSH-Px, CAT, GST) with decreased MDA contents (P < 0.05). Moreover, the consumption of AFB1 downregulated the mRNA expression of SOD1, SOD2, GSTA3, CAT1, GPX1, GPx3, GSTT1, Nrf2, HO-1, and p38MAPK (P < 0.05). Dietary MAPs upregulated the mRNA expression of SOD2, GSTA3, CAT1, GPX1, GSTT1, p38MAPK, Nrf2, and HO-1 in comparison with AFB1 group (P < 0.05). The histological analysis confirmed restoration of apoptotic cells of bursa of Fabricius (P < 0.01), which seen with MAPs supplemented broilers. Besides, dietary MAPs down-regulated the mRNA expression of caspase-3 and Bax (P < 0.05), while up-regulated the mRNA expression of Bcl-2 (P < 0.05) compared with AFB1 group. In addition, according to protein expression results, dietary MAPs up-regulated the protein expression level of antioxidant and apoptosis-associated proteins (Nrf2, HO-1, p38MAPK, Bcl-2) (P < 0.01), but down-regulated the protein expression level of caspase-3 and Bax (P < 0.01). In conclusion, dietary MAPs alleviated AFB1-induced bursa of Fabricius injury through regulating Nrf2-mediated redox and mitochondrial apoptotic signaling pathway in broilers.