Development Of Lymphoid Organs Research Articles

Not just for lymphoid cells: The role of the noncanonical NF-κB signaling pathway in early and late myelopoiesis with a focus on hypereosinophilic disorders.

The noncanonical NF-κB pathway is involved in lymphoid organ development, B cell maturation, and cytokine production. However, new research has demonstrated that this pathway is also key for the orderly and sequential maturation of myeloid cells, including neutrophils and eosinophils. When this pathway is disrupted or constitutively activated, aberrations in hematopoietic stem and progenitor cell (HSPC) survival and proliferation, as well as subsequent granulopoiesis and eosinophilopoiesis are affected. Disturbance of such a coordinated and delicate process can manifest in devastating clinical disease including acute and chronic myeloid leukemias (AML and CML, respectively), pre-leukemic processes such as myelodysplastic syndrome (MDS) or hyperinflammatory conditions like Hypereosinophilic Syndrome (HES). In this review, we will discuss the molecular machinery within the noncanonical NF-κB pathway, crosstalk with the canonical NF-κB pathway, murine models of noncanonical signaling, as well as how aberrations in this pathway manifest in leukemic or hyperinflammatory disease with a focus on HES. Potential and promising drug therapies will also be discussed, emphasizing the noncanonical NF-κB pathway as a potential target for improved treatment for patients suffering from leukemia or idiopathic HES. The hope is that review of such mechanisms and treatments may eventually result in findings that aid physicians in rapidly diagnosing and more accurately classifying patients suffering from such complex and overlapping hematopoietic diseases.

Genetic Knockin Approaches to Reconstructing DLBCL-Subtypes from Human Germinal Center B-Cells

Background and Significance: The clinical and genetic heterogeneity of DLBCL has led to the recent classification of the disease into five genetically distinct subtypes. The MCD/C5 subtype of DLBCL is defined by genetic alterations of CD79B at the first tyrosine residue of its ITAM motif (CD79B Y196H), crucial for signaling downstream of the BCR, and MYD88 (MYD88 L265P), a signaling adaptor protein essential for Toll Like receptor signaling. CD79B Y196H and MYD88 L265P mutations together define the MCD/C5 subtype and were found to be co-dependent for proliferation and oncogenic BCR-signaling in vitro. However, the mechanism of epistatic interaction between CD79B Y196H and MYD88 L265P is unknown. The MCD/C5 DLBCL subtype is associated with particularly poor clinical outcomes, however only a few human cell lines and a genetic mouse model are available for preclinical testing. Here, we developed a novel genetic approach to engineer the MCD/C5 subtype of DLBCL in primary human tonsillar germinal center B cells. To this end, we introduced the CD79B Y196H and MYD88 L265P mutations as genetic knockin alleles utilizing CRISPR-Cas9 homology directed repair templates (HDRT). In addition, we developed humanized NBSGW mice carrying a mouse Tslp-transgene to enable development of lymph nodes and secondary lymphoid organs to propagate genetically engineered GC B-cells carrying CD79B Y196H and MYD88 L265P knockin mutations for lymphoma development ( Figure 1). Results: Primary human germinal center B-cells were isolated from tonsils and grown on follicular dendritic feeder cells expressing CD40L and IL21. Single strand DNA templates were designed for CD79B, with and without Y196H mutation. Constructs were linked to fluorescent reporters by T2A sites and flanked by homology arms of ~280 bp targeting the guide RNA cut site. The target site was selected to disrupt the endogenous gene, while placing the construct under transcriptional control of endogenous promoter/enhancer elements. We achieved a knock-in efficiency of 5-8%, prior to single-cell cloning and genotyping to confirm the presence of heterozygous CD79B Y196H mutant CD79B Y196 wild-type mutations ( Figure 1). Preliminary biochemical studies show a delayed response and reduced intensity of signaling downstream of the BCR upon stimulation in CD79B Y196H mutant, compared to CD79B Y196 wild-type knockin germinal center B-cells. A similar approach was taken to design temples encoding Exon 5 of MYD88, with and without the L265P mutation, utilizing two guide RNAs to excise and replace Exon 5, linked to a mCherry reporter by a T2A site. Upon successfully introducing the MYD88 HDR templates into our CD79B wild-type and mutant clones to generate four distinct genotypes based on wildtype and CD79B Y196H and MYD88 L265P mutations alone or in combination. In addition, traditional NSG mice lack lymph nodes and secondary lymphoid organs. Lack of lymphoid follicles in NSG mice, owing to defective IL7Rα-signaling, contributed to lack of lymphoma formation observed in the previous model. In our model, we substituted the missing IL7Rα-signal with transgenic expression of murine Tslp, which enables development of embryonic lymphoid tissue inducer (LTi) cells that are required for the development of secondary lymphoid organs and germinal center follicles supporting initiation of human DLBCL development. Conclusion: Previous approaches to model DLBCL starting from germinal center B-cells were based on lentiviral overexpression of mutant oncogenes. Thereby, tonsillar germinal center B-cells with lentiviral BCL2 and BCL6 overexpression did not form lymphomas in NSG mice, however, were permissive to malignant transformation by MYC. Building on this previous work, we propose an improved model for preclinical testing for genetically defined DLBCL subtypes. Our two main improvements involve (i) genetic modeling based on knockin-strategies targeting endogenous loci and transcriptional control elements to phenocopy physiological gene expression levels and transcriptional regulation, and (ii) substitution of defective IL7Rα-signaling by transgenic expression of murine Tslp to rescue development of LTi cells. Transgenic expression of mTslp enables LTi-dependent formation of lymph nodes and secondary lymphoid organs supporting lymphoma development.

Composition and dynamics of intestinal fungi during the postnatal 2 months of very low birth weight infants

It has been found that intestinal fungi play a role in the composition of the intestinal microecology and in the formation and development of the immunity during childhood. We investigated the gut fungi composition of preterm infants to analysis composition and dynamics of intestinal fungi during the postnatal 2 months of very low birth weight infants. We collected feces from 34 very low birth weight infants (VLBWI) and 28 preterm infants with birth weight >1500 g. We extracted total fungal DNA from feces and analyzed the composition of gut fungus through ITS sequencing. The fungal detectable rate in the experimental group peaked on day 3 (85.19%), then gradually decreased and started to show an increasing trend again by day 28. There were significant differences in the alpha diversity of intestinal fungus between VLBWI and controls, and the VLBWI had its own characteristics at different time points in richness and diversity. A total of 10 phylums and 342 genera were identified in all VLBWI samples. The dominant fungal phylum of the VLBWI group is Ascomycota (50.3%)and Basidiomycota (48.8%). The functional metabolic activity of the experimental group was lower than that of the control group. Conclusion: The composition and abundance of VLBWI intestinal fungal showed several alterations during the first 2 months of life. The prediction of gut microbiota function suggests that intestinal metabolic function may be altered in VLBWI.What is Known:• A limited number of studies has been found that symbiont fungi may be able to calibrate host immunological responses, promote development of peripheral lymphoid organs, promote T cell responses, and even may be associated with the development of certain diseases, such as inflammatory bowel disease (IBD), NEC, and allergic diseases. However, previous studies on intestinal microecology have mainly focused on adults while neglecting the role of fungi in the gut of children due to the much lower abundance of intestinal fungi than bacteria, limitations of techniques for detecting fungi, the difficulty of obtaining samples, and the absence of largescale reference databases.What is New:• In recent years, the discovery and development of fungal detection technologies such as 18s rDNA sequencing technology, Internal Transcribed Spacer(ITS), and DNA fingerprinting technology have further broadened the perspective on the impact of intestinal fungal exposure in early life.

Effects of dietary supplementation with garlic powder (Allium sativum L.) on broiler performance, carcass traits, lymphoid organ development and intestinal biometrics

Aim of study: To assess the effects of dietary supplementation with garlic (Allium sativum L.) powder as a growth-promoting additive on the productive performance, carcass traits, intestinal biometrics, and lymphoid organ development of broilers.
 Area of study: Machado, Minas Gerais, southeast region of Brazil.
 Material and methods: A total of 660 one-day-old chicks were used in a completely randomized design with 5 treatments (0, 2.5, 5.0, 7.5, and 10.0 g/kg garlic powder), 6 replications, and 22 animals per experimental unit during 42 days of production, divided into phases: initial (1 to 7 days), growth (8 to 22 days), and finishing (23 to 42 days).
 Main results: In the total production period, the birds that were fed rations containing 5, 7.5 and 10 g/kg garlic powder showed increased weight gain and feed conversion ratio (p<0.05). Among the carcass yield variables, live weight and carcass yield showed a positive linear effect (p<0.05). Birds supplemented with garlic powder showed increased weight (p<0.05) and relative length of the small intestine (p<0.05). The absolute weight of the lymphoid organs, thymus and bursa of Fabricius, and the relative weight of the thymus, bursa of Fabricius and spleen also increased linearly with the level of dietary supplementation with garlic powder (p<0.05).
 Research highlights: Dietary supplementation with 5 g/kg and higher concentrations of garlic powder is recommended as a growth promoter for broilers because this feed additive enhances lymphoid organ development, improves intestinal biometric variables and consequently maximises the productive performance and carcass yield of broilers from 1 to 42 days of age.

Impact of Cassava Leaf Meal as a Rice Bran Substitute and Enzyme Supplementation on Lymphoid Organ Weight and Digestibility in Broiler Chickens

Cassava leaves are a potential alternative feed ingredient due to their high levels of crude protein and energy. However, their utilization in poultry feed is constrained by factors such as cyanic acid, fiber content, and nutrient digestibility. This study aimed to investigate the impact of including cassava leaf meal (CLM) as a rice bran substitute and enzyme supplementation on lymphoid organ development and metabolizable energy parameters in broiler chickens. A 2x3 completely randomized factorial design was conducted using 48 Cobb-strain broilers unsexing at 35 days old. The treatments included different levels of CLM and enzyme (NSP and protease) supplementation at a dose of 250 g/ton of feed. The treatments consisted of R0E0: 0% CLM without enzyme, R0E1: 0% CLM with enzyme, R1E0: 1.5% CLM without enzyme, R1E1: 1.5% CLM with enzyme, R2E0: 3% CLM without enzyme, and R2E1: 3% CLM with enzyme. The variables assessed were lymphoid organ development (thymus, bursa Fabricius, and spleen) and metabolizable energy parameters (Apparent Metabolizable Energy (AME), True Metabolizable Energy (TME), Apparent Metabolizable Energy Corrected to Nitrogen (AMEn), and True Metabolizable Energy Corrected to Nitrogen (TMEn)). The data were subjected to analysis of variance (ANOVA) with post-hoc tests conducted for significant differences. Results indicated no interaction between CLM and enzymes in lymphoid organ development and energy metabolizable. The inclusion of CLM led to a reduction in AME and TME (p<0.05). However, enzyme supplementation significantly increased the relative weight of lymphoid organs (thymus, bursa Fabricius, spleen) and metabolizable energy parameters (AME, TME, AMEn, and TMEn) (p<0.05). Importantly, the inclusion of CLM up to a level of 3.0% did not negatively impact the health of broiler chickens. Furthermore, the addition of enzymes effectively mitigated the negative effects associated with CLM inclusion in the feed, suggesting their potential as a strategy to improve feed utilization in broiler production systems.

BMSC-exosomes miR-25-3p Regulates the p53 Signaling Pathway Through PTEN to Inhibit Cell Apoptosis and Ameliorate Liver Ischemia‒reperfusion Injury.

Hepatic ischemia‒reperfusion injury (HIRI) is a pathological phenomenon during liver surgery, and bone marrow-mesenchymal stem cell (BMSC) exosomes (BMSC-Exos) regulate cell apoptosis and reduce ischemia‒reperfusion injury. We aimed to investigate the roles of BMSC-Exos and miR-25b-3p (enriched in BMSC-Exos) in HIRI and elucidate the underlying mechanisms. An HIRI mouse model was constructed and preinjected with BMSC-Exos, agomir-miR-25, agomir-miR-NC, or PBS via the tail vein. Compared with mice with HIRI, mice with HIRI preinjected with BMSC-Exos had significantly decreased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels and alleviated liver necrosis (P < 0.05). Quantitative hepatic transcriptomics showed that mice with HIRI preinjected with BMSC-Exos exhibited increased cell division, hematopoietic or lymphoid organ development and metabolic processes. miRNA sequencing of BMSC-Exos revealed that miR-25, which is related to I/R injury, was enriched in the exosomes. Compared with HIRI + NC mice, HIRI + miR-25b-3p mice had significantly increased miR-25b-3p expression, decreased ALT/AST levels and apoptosis-related protein expression (P < 0.05), and alleviated liver necrosis. The proliferation of AML-12 cells transfected with miR-25b-3p was significantly higher than that in the mimic NC group (P < 0.01) after hypoxia induction, and the apoptosis rate of cells was significantly lower than that in the NC group (P < 0.01). PTEN was identified as a miR-25b-3p target gene. PTEN expression was significantly diminished in miR-25b-3p-transfected AML12 cells (P < 0.05). HIRI + agomir-miR-25 mice displayed reduced PTEN expression and decreased p53 and cleaved caspase 3 levels compared to HIRI + NC mice. We revealed the roles and underlying mechanisms of BMSC-Exos and miR-25 in HIRI, contributing to the prevention and treatment of HIRI.

Growth and response to Escherichia coli lipopolysaccharide challenge in Lohmann LSL-Lite pullets when fed a source of omega-3 fatty acids and yeast bioactives from hatch through to 16 wk of age

Growth and response to Escherichia coli lipopolysaccharide (LPS) challenge in Lohmann LSL-Lite pullets when fed a source of omega-3 fatty acids (n-3 FA) and yeast bioactives (YB) from hatch through to 16 wk of age (woa) were investigated. Co-extruded full fat flaxseed and pulse mixture (FFF; 1:1 wt/wt) supplied n-3 FA and YB were yeast cell walls processed with β-1,3-glucan hydrolase. A total of 1,064-day-old pullets were placed in cages (19 birds/cage) and allocated to 7 diets (n=8). The iso-caloric and iso-nitrogenous diets were control, control+1, 3, or 5% FFF and+0.025, 0.05, or 0.1% YB. The birds had ad libitum access to feed and water. Body weight (BW), feed intake, and lymphoid organs weight were recorded. At 15 woa, 2 pairs of pullets/cage received intravenous injection of either 1 mL of sterile saline without or with 8 mg LPS/kg BW. Injected pullets were bled, monitored for BW and cloaca temperature at time points within 168 h. Birds fed 1% FFF were heavier (P < 0.04) than birds fed other diets at 16 woa with FFF and YB exhibiting non-linear responses. Control birds had heavier (P=0.02) thymus at 4 woa. Spleen weight increased quadratically (P < 0.05) in response to FFF at 8 and YB at 16 woa. The LPS increased cloaca temperature and altered concentration of several plasma metabolites (P < 0.05). Theinteraction (P < 0.05) between LPS and diet was such thatcontrol birds exhibited lower creatine kinase (CK) upon challenge with LPS relative to birds fed other diets. The LPS birds fed 1% FFF and 0.05% YB showed higher plasmaalbumin than non-LPS cohorts. Non-LPS birds fedcontrol, 1 and 3% FFF had higher plasma K than LPS cohorts. In general, FFF and YB exhibited linear and quadratic effects (P < 0.05) onselect plasma metabolites. In conclusion, dietary provision of n-3 FA and YB influenced pullet BW at sexual maturity, development of lymphoid organs and modulated some plasma metabolites in response to LPS.

New insights into triazole fungicide-caused hematopoietic abnormality in zebrafish based on GRα screening developmental toxicity

Triazole fungicides (TFs) are known to be common environmental contaminants that can be toxic to aquatic animals, but their developmental toxicity is not fully understood. To address this gap, we first used a glucocorticoid receptor α (GRα)-mediated dual luciferase reporter gene system to explore the possible development toxicity of ten TFs and found that flusilazole (FLU) exhibited stronger agonistic activity against GRα. Subsequent transcriptome sequencing showed that FLU exposure affected GRα activation and hematopoiesis associated with a variety of biological processes, including responses to corticosteroid release, embryonic hematopoiesis, erythroid differentiation, and the development of hematopoietic or lymphoid organs. Furthermore, based on in situ hybridization and staining techniques, we clarified that FLU decreased the expression of the primitive hematopoietic marker genes gata1 and pu.1. and caused the defects in the posterior blood island (PBI), thereby impacting intermediate hematopoietic processes. Also, FLU significantly reduced the expression of the crucial hematopoietic gene cmyb and disrupted the production of erythrocytes and bone marrow cells during definitive hematopoiesis. Consistently, we found that FLU induced lesions in the kidney, a hematopoietic organ, including the infiltration of inflammatory cells, tubular collapse, reduced tubular filtration area, and interstitial hydronephrosis. We also found that FLU increased aberrant red blood cells in the peripheral blood of zebrafish. These findings provide new insights into the developmental toxicity and ecotoxicological risk of TFs.

Effect of two brooding systems and four stocking densities on immune response and stress indicators of broiler chicks during the brooding period

This experiment investigated the effects of the brooding system (BS) and stocking density (SD) on the immune response of 1-14-day-old broiler chicks. A 3672 one-day-old Cobb 500 chicks were raised in two BSs (conventional floor brooding system (CFBS) and the newly developed multi-tier closed brooding system (NDBS)) at four different SDs (30, 60, 90, and 120 chicks/m2), resulting in a 2 × 4 factorial arrangement. Immunity data on lymphoid organ weights, maternal antibody titres to IBV, IBDV, and NDV, and stress indicators were collected on days 6 and 12. The results showed that CFBS and NDBS had similar effects on the relative development of chick lymphoid organs (Bursa, spleen, and thymus), the durability of the maternal humoral immune response against IBDV, and the maintenance of normal levels of stress indicators. Birds in CFBS outperformed NDBS in terms of the durability of maternal immunity to IBV and NDV. Birds in NDBS had a higher liver weight percentage than CFBS. Maternal immunity, lymphoid organ weights %, glucose and cholesterol concentrations were not altered by SD. In contrast to relative weights, differences in absolute lymphoid weights (g) were attributed to differences in live weights. An increase in SD per area unit resulted in an increase in heterophils and a decrease in lymphocyte percentage resulted in an increase in the ratio of heterophils to lymphocytes(H:L). In conclusion, birds in the higher SD had lower live weights and higher H:L ratios. Smarter brooding systems are needed to usher in a new era for the poultry industry. Highlights The brooding system and/or stocking density accounted for the stress factors of the birds. Both brooding systems increased the relative development of chick lymphoid organs and the durability of the maternal humoral immune response to IBDV. Smarter brooding systems are needed to apply the critical control point standard and usher in a new era for the poultry industry. As stocking density per unit area increased, live and liver weights decreased, and the lymphocyte ratio increased.

In ovo given sunset yellow adversely affects embryonic development of chick thymus and bursa Fabricii as evidenced by histological and enzyme histochemical findings

BackgroundSunset yellow (E110) has largely been used as food coloring agent. Complaints accumulated on E110 for possible detrimental effects on the immune functions such as allergenicity in the children. In this study, the effects of different doses of E110 on embryonic development of chicken primary lymphoid organs, thymus and bursa of Fabricius were determined by means of histological, histomorphometrical and histochemical methods. A total of 250 fertilized eggs from the Ross 508 line were used in the study. The eggs were divided into 5 groups as non-treated, sham-exposed and 100 ng/egg, 500 ng/egg, 1000 ng/egg E110-injected groups, each having 50 eggs.ResultsIn the 1.000 ng/egg E110-administered group, embryonic development of chicken thymus and bursa of Fabricius were retarded.ConclusionsE110 given in ovo before incubation retarded the embryonic development of the avian thymus and bursa of Fabricii. The effect is more pronounced in the 1.000 ng/egg group.

Single-cell and spatial transcriptomics reveal aberrant lymphoid developmental programs driving granuloma formation

Granulomas are lumps of immune cells that can form in various organs. Most granulomas appear unstructured, yet they have some resemblance to lymphoid organs. To better understand granuloma formation, we performed single-cell sequencing and spatial transcriptomics on granulomas from patients with sarcoidosis and bioinformatically reconstructed the underlying gene regulatory networks. We discovered an immune stimulatory environment in granulomas that repurposes transcriptional programs associated with lymphoid organ development. Granuloma formation followed characteristic spatial patterns and involved genes linked to immunometabolism, cytokine and chemokine signaling, and extracellular matrix remodeling. Three cell types emerged as key players in granuloma formation: metabolically reprogrammed macrophages, cytokine-producing Th17.1 cells, and fibroblasts with inflammatory and tissue-remodeling phenotypes. Pharmacological inhibition of one of the identified processes attenuated granuloma formation in a sarcoidosis mouse model. We show that human granulomas adopt characteristic aspects of normal lymphoid organ development in aberrant combinations, indicating that granulomas constitute aberrant lymphoid organs.

Deciphering the Somatic and Germline Structural Variation Landscape in Pediatric B-Cell Precursor Acute Lymphoblastic Leukemia By Whole Genome Optical Mapping

Background: B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is the most frequent pediatric cancer. The most common numerical and structural chromosomal alterations are high hyperdiploidy (HHD, 51-67 chromosomes) and t(12;21)(p13;q22) leading to ETV6::RUNX1 fusion. However, for a full-blown leukemia, secondary alterations are required. Furthermore, it is increasingly recognized that germline variants may enhance ALL susceptibility in ~4% of the patients (Bloom et al. 2019, Klco & Mullighan 2020). In large-scale genomic studies, the mutational landscape of somatic and germline single nucleotide variants and small indels has been well described in BCP-ALL. However, a comprehensive analysis of somatic and germline structural variants (SVs) in ETV6::RUNX1 and HHD tumors is still lacking. Recent feasibility studies showed that whole genome optical mapping (WGOM) is a novel method for reliable SV detection which could abrogate conventional cytogenetics and array based methods in the future (Neveling et al. 2021, Mantere et al. 2021). The aim of the study was to analyze the structural variation landscape in pediatric BCP-ALL patients by employing WGOM to gain significant insights on leukemia predisposition and development. Methods: We studied 60 pediatric patients diagnosed with ETV6::RUNX1 (n=30) or HHD (n=30) BCP-ALL with a median age at diagnosis of 3.9 (range 1.7-17) and a male to female ratio of 1.14:1. We collected tumor material at initial diagnosis and included fibroblast or remission material as matched non-tumor control of each patient. Ultra-high molecular weight DNA was extracted from 120 samples and labeled by a sequence specific enzyme. Fluorescently labeled DNA was analyzed using a Saphyr instrument (Bionano Genomics). We integrated comprehensive datasets of 60 matched tumor/non-tumor genomes to identify somatically acquired SVs >500bp. Corresponding data of standard methods as cyto- (karyotype, fluorescence in situ hybridization) and molecular genetics (SNP-array) of the leukemia samples were compared to WGOM data. In addition, the prevalence of rare germline SVs (<1% of 160 healthy control samples [Bionano] and minor allele frequency <0.01 in dbVar) occurring in known cancer predisposition genes was analyzed in the non-tumor samples. Results: WGOM was able to detect 94% (438/464) of previously observed events by conventional methods, including ETV6::RUNX1 and high hyperdiploid hallmark alterations in all BCP-ALL cases. Beyond that, we discovered 895 additional SVs with WGOM not detected by standard cyto- and molecular genetics. WGOM detected a median of 25 (range 0-70) somatic SVs secondary to the hallmark alterations in ETV6::RUNX1 tumors compared to 13 (range 0-28) SVs in HHD cases. We identified 60 recurrently altered regions occurring in ≥3 BCP-ALL cases including 12p13.2 (ETV6, 19/60), 9p13.2 (PAX5, 13/60), 9p21.3 (CDKN2A/B, 12/60), 11p12 (RAG2, 10/60), 20p12.2 (MKKS/SLXIP, 10/60) or 3q13.2 (CD200/BTLA, 8/60). We detected ETV6::RUNX1 subtype specific recurrent focal deletions in ATF7IP (10/30), BTG1 (10/30), NSD2 (5/30) and TBL1XR1 (4/30). Interestingly, we discovered recurrent SVs affecting putative novel genes involved in BCP-ALL leukemogenesis such as STAG2, GPN3 and UBA2 which are affected in ~10% of the BCP-ALL patients. Strikingly, WGOM revealed in total 9 novel potential in-frame fusion transcripts in both tumor subtypes, including a potential fusion of the cohesin complex members PDS5B::STAG2, arising through a t(X;13)(q25;q13.1) in an ETV6::RUNX1 patient (Figure 1). In addition, we detected 28 rare germline SVs targeting 22 known cancer predisposing genes in 35% (21/60) of the pediatric patients. 40% (9/22) of the affected genes were involved in hematopoietic or lymphoid organ development as ETV6, FANCA, IKZF1, ITCH or PTPRC. In 18% (11/60) of the children, the identified germline alteration potentially altered a coding region of a known cancer predisposing gene including a duplication of CIITA in an ETV6::RUNX1 patient. Conclusions: By applying the novel technology WGOM, we could detect a significant amount of previously hidden SVs in our BCP-ALL cohort in addition to conventional methods. We further generated a comprehensive landscape of somatic and germline SVs in childhood BCP-ALL, revealing recurrently altered regions and novel potential players in ALL leukemogenesis and predisposition. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Characterization of SHARPIN knockout Syrian hamsters developed using CRISPR/Cas9 system.

SHARPIN (SHANK-associated RH domain interactor) is a component of the linear ubiquitination complex that regulates the NF-κB signaling pathway. To better understand the function of SHARPIN, we sought to establish a novel genetically engineered Syrian hamster with SHARPIN disruption using the CRISPR/Cas9 system. A single-guide ribonucleic acid targeting exon 1 of SHARPIN gene was designed and constructed. The zygotes generated by cytoplasmic injection of the Cas9/gRNA ribonucleoprotein were transferred into pseudopregnant hamsters. Neonatal mutants were identified by genotyping. SHARPIN protein expression was detected using Western blotting assay. Splenic, mesenteric lymph nodes (MLNs), and thymic weights were measured, and organ coefficients were calculated. Histopathological examination of the spleen, liver, lung, small intestine, and esophagus was performed independently by a pathologist. The expression of lymphocytic markers and cytokines was evaluated using reverse transcriptase-quantitative polymerase chain reaction. All the offspring harbored germline-transmitted SHARPIN mutations. Compared with wild-type hamsters, SHARPIN protein was undetectable in SHARPIN-/- hamsters. Spleen enlargement and splenic coefficient elevation were spotted in SHARPIN-/- hamsters, with the descent of MLNs and thymuses. Further, eosinophil infiltration and structural alteration in spleens, livers, lungs, small intestines, and esophagi were obvious after the deletion of SHARPIN. Notably, the expression of CD94 and CD22 was downregulated in the spleens of knockout (KO) animals. Nonetheless, the expression of CCR3, CCL11, Il4, and Il13 was upregulated in the esophagi. The expression of NF-κB and phosphorylation of NF-κB and IκB protein significantly diminished in SHARPIN-/- animals. A novel SHARPIN KO hamster was successfully established using the CRISPR/Cas9 system. Abnormal development of secondary lymphoid organs and eosinophil infiltration in multiple organs reveal its potential in delineating SHARPIN function and chronic inflammation.

Lymphotoxin-alpha expression in the meninges causes lymphoid tissue formation and neurodegeneration.

Organized meningeal immune cell infiltrates are suggested to play an important role in cortical grey matter pathology in the multiple sclerosis brain, but the mechanisms involved are as yet unresolved. Lymphotoxin-alpha plays a key role in lymphoid organ development and cellular cytotoxicity in the immune system and its expression is increased in the CSF of naïve and progressive multiple sclerosis patients and post-mortem meningeal tissue. Here we show that persistently increased levels of lymphotoxin-alpha in the cerebral meninges can give rise to lymphoid-like structures and underlying multiple sclerosis-like cortical pathology. Stereotaxic injections of recombinant lymphotoxin-alpha into the rat meninges led to acute meningeal inflammation and subpial demyelination that resolved after 28 days, with demyelination being dependent on prior subclinical immunization with myelin oligodendrocyte glycoprotein. Injection of a lymphotoxin-alpha lentiviral vector into the cortical meningeal space, to produce chronic localized overexpression of the cytokine, induced extensive lymphoid-like immune cell aggregates, maintained over 3 months, including T-cell rich zones containing podoplanin + fibroblastic reticular stromal cells and B-cell rich zones with a network of follicular dendritic cells, together with expression of lymphoid chemokines and their receptors. Extensive microglial and astroglial activation, subpial demyelination and marked neuronal loss occurred in the underlying cortical parenchyma. Whereas subpial demyelination was partially dependent on previous myelin oligodendrocyte glycoprotein immunization, the neuronal loss was present irrespective of immunization. Conditioned medium from LTα treated microglia was able to induce a reactive phenotype in astrocytes. Our results show that chronic lymphotoxin-alpha overexpression alone is sufficient to induce formation of meningeal lymphoid-like structures and subsequent neurodegeneration, similar to that seen in the progressive multiple sclerosis brain.

Protective Effects and Potential Mechanisms of Dietary Resveratrol Supplementation on the Spleen of Broilers Under Heat Stress.

Resveratrol (RSV) is a natural polyphenolic compound with potent antioxidant and anti-inflammatory properties. This study aimed to investigate the protective effects of RSV supplementation on the inflammatory responses of broilers during heat stress. A total of 432 28-d-old white-feathered broilers (817 crossbred chicken) with an average weight of 549 ± 4 g were randomly allotted to 4 equal groups (Half male and half female, 6 replicates/group, 18 chickens/replicate), including normal temperature (NT) group (24 ± 2°C for 24 h/d, basal diet), NT+RSV group (24 ± 2°C for 24 h/d, basal diet + RSV), heat stress (HT) group (37 ± 2°C for 8 h/d, basal diet), and HT+RSV group (37 ± 2°C for 8 h/d, basal diet + RSV). Serum samples were collected on d 7 and 14 of heat stress, and thymus, spleen, jejunum, and bursa of Fabricius samples were collected and analyzed on d14. RSV treatment decreased the feed conversion ratio, partially reversed the negative alternations in body weight, average daily gain, and average daily feed intake caused by heat stress. RSV treatment also decreased the elevated levels of corticosterone on d 14, adrenocorticotropic hormone, and triiodothyronine in serum on d 7 caused by heat stress, and significantly increased the villus height to crypt depth ratio in the jejunum on d 14. Dietary RSV also reduced heat stress-induced splenic pro-inflammatory cytokine concentrations. TUNEL assay showed that RSV significantly reduced heat stress-induced the number of apoptotic cells. Remarkably, RSV down-regulated some splenic related genes for apoptosis genes, including BCL-2, Apaf-1, and MDM2 mRNA levels induced by heat stress. According to GO and KEGG enrichment analyses, the differential genes between HT and HT + RSV groups were mainly associated with immune system process, hematopoietic or lymphoid organ development, and toll-like receptor signaling pathway. The relative mRNA expression of NF-κB, heat shock protein 70 (HSP70), and p38 MAPK were markedly decreased by the combination of RSV and heat stress. These findings showed that RSV might reduce the splenic inflammatory response in heat-stressed white-feather broilers by inhibiting heat stress-induced activation of NF-B, MAPK, and HSP70, as well as inhibiting the activation of mitochondrial apoptotic pathways.

B cells drive tertiary lymphoid organ formation in ileal inflammation

Abstract Crohn’s disease [CD] is one of the two most common forms of inflammatory bowel disease, affecting over half a million Americans. Like many other diseases with chronic inflammation, some patients with CD develop tertiary lymphoid organs [TLO] in areas of the gastrointestinal tract with active disease. TLOs are organized clusters of lymphocytes, similar in structure to secondary lymphoid organs, though they develop after birth and their contribution to pathogenesis in CD, or other diseases, is unclear. We, and others, have also found B cell rich lymphoid aggregates in the mesenteric fat of CD patients along dramatically remodeled lymphatic vessels. TNFΔARE/+ is a murine model of ileal inflammation that recapitulates several key features of ileal CD, including development of mesenteric tertiary lymphoid organs. Use of this model revealed that mesenteric TLOs block cellular and molecular export from the gut, leading us to wonder if mechanisms that interfere with their development might reduce ileitis. TNFΔARE/+ mice that lack B cells revealed that B cells are required for tertiary lymphoid organ formation in this model. Without these TLOs, lymphatic outflow from the intestine was restored. Nonetheless, histological and flow cytometric approaches reveal no difference in local inflammation in the ileum. However, systemic inflammation, as assessed by metabolic cages and changes in body weight over time, increased. This suggests that TLOs may act to trap inflammatory signals locally, preventing systemic dissemination of inflammatory cells or mediators. Work supported by NIH grants DP1-DK109668-04 and T32-DK077653-27 and the Kenneth Rainin Foundation

Expression characteristics and interaction networks of microRNAs in spleen tissues of grass carp (Ctenopharyngodon idella).

The spleen is an important immune organ in fish. MicroRNAs (miRNAs) have been shown to play an important role in the regulation of immune function. However, miRNA expression profiles and their interaction networks associated with the postnatal late development of spleen tissue are still poorly understood in fish. The grass carp (Ctenopharyngodon idella) is an important economic aquaculture species in China. Here, two small RNA libraries were constructed from the spleen tissue of healthy grass carp at one-year-old and three-year-old. A total of 324 known conserved miRNAs and 9 novel miRNAs were identified by using bioinformatic analysis. Family analysis showed that 23 families such as let-7, mir-1, mir-10, mir-124, mir-8, mir-7, mir-9, and mir-153 were highly conserved between vertebrates and invertebrates. In addition, 14 families such as mir-459, mir-430, mir-462, mir-7147, mir-2187, and mir-722 were present only in fish. Expression analysis showed that the expression patterns of miRNAs in the spleen of one-year-old and three-year-old grass carp were highly consistent, and the percentage of miRNAs with TPM > 100 was above 39%. Twenty significant differentially expressed (SDE) miRNAs were identified. Gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that these SDE miRNAs were primarily involved in erythrocyte differentiation, lymphoid organ development, immune response, lipid metabolic process, the B cell receptor signaling pathway, the T cell receptor signaling pathway, and the PPAR signaling pathway. In addition, the following miRNA-mRNA interaction networks were constructed: immune and hematopoietic, cell proliferation and differentiation, and lipid metabolism. This study determined the miRNA transcriptome as well as miRNA-mRNA interaction networks in normal spleen tissue during the late development stages of grass carp. The results expand the number of known miRNAs in grass carp and are a valuable resource for better understanding the molecular biology of the spleen development in grass carp.

Splenic Architecture and Function Requires Tight Control of Transmembrane TNF Expression.

Soluble tumor necrosis factor (sTNF) is an important inflammatory mediator and essential for secondary lymphoid organ (SLO) development and function. However, the role of its transmembrane counterpart (tmTNF) in these processes is less well established. Here, the effects of tmTNF overxpression on SLO architecture and function were investigated using tmTNF-transgenic (tmTNF-tg) mice. tmTNF overexpression resulted in enlarged peripheral lymph nodes (PLNs) and spleen, accompanied by an increase in small splenic lymphoid follicles, with less well-defined primary B cell follicles and T cell zones. In tmTNF-tg mice, the spleen, but not PLNs, contained reduced germinal center (GC) B cell fractions, with low Ki67 expression and reduced dark zone characteristics. In line with this, smaller fractions of T follicular helper (Tfh) and T follicular regulatory (Tfr) cells were observed with a decreased Tfh:Tfr ratio. Moreover, plasma cell (PC) formation in the spleen of tmTNF-tg mice decreased and skewed towards IgA and IgM expression. Genetic deletion of TNFRI or –II resulted in a normalization of follicle morphology in the spleen of tmTNF-tg mice, but GC B cell and PC fractions remained abnormal. These findings demonstrate that tightly regulated tmTNF is important for proper SLO development and function, and that aberrations induced by tmTNF overexpression are site-specific and mediated via TNFRI and/or TNFRII signaling.

Effect of dietary supplementation of oregano and thyme oil on the growth performance, development of lymphoid and digestive organs and carcass quality traits of Turkey poults

Effect of dietary supplementation of oregano and thyme oil on the growth performance, development of lymphoid and digestive organs and carcass quality traits of Turkey poults

Induction and Anti-Tumor Function of Tertiary Lymphoid Organs

To induce the development of tertiary lymphoid organs (TLO) in a mouse model of melanoma and to evaluate TLO's functions in antitumor immunity. Lymphotoxin-beta receptor (LTβR) was overexpressed in NIH3T3 cells through the lentivirus system and the overexpression efficiency of LTβR in LTβR-NIH3T3 cells was examined. Western blot and qPCR were used to examine the non-canonical nuclear factor (NF)-κB signaling pathway in NIH3T3 cells overexpressing LTβR. B16-OVA melanoma mouse model was constructed to explore the induction of TLO and anti-tumor functions of TLO in LTβR-NIH3T3 cells. LTβR was overexpressed in NIH3T3 cells through the lentivirus system, and flow cytometry showed that the proportion of GFP + cells reached 99%. The overexpression of LTβR activated the non-canonical NF-κB signaling pathway in NIH3T3 cells. Findings from the mouse tumor model suggest that the injection of LTβR-NIH3T3 cells successfully induced the development of lymphoid tissue around the tumor and enhanced the tumor infiltration of T cells and MHCⅡ + macrophages, significantly inhibiting tumor growth and prolonging the survival of tumor-bearing mice. LTβR-NIH3T3 cells promoted anti-tumor immunity by inducing TLO development, which may provide new perspectives for tumor immunotherapy.