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To evaluate the levels of YKL40, IL-6(interleukin-6), IL-8(interleukin-8), IL-10(interleukin-10), TNF-α (tumor necrosis factor-α) in OSAS (obstructive sleep apnea syndrome)children and explore the mechanism of YKL40 promoting inflammatory factors overexpression in tonsils. qPCR and ELISA were used to identify the expression of YKL40, IL-6, IL-8, IL-10, and TNF-α in the tonsils of OSAS children. Primary tonsil lymphocytes (PTLCs) were cultured and recombinant human YKL40(rhYKL40)was used to stimulate PTLCs in different concentrations and at different time points. The activation of NF-κB in PTLCs was screened by western blotting. Relative mRNA of YKL40, IL-6, IL-8, TNF-α was over expressed in OSAS-derived tonsil tissue and the levels of YKL40, IL-6, IL-8, and TNF-α was increased in OSAS-derived protein supernatant of tonsil tissue.The relative mRNA expression of IL-6, IL-8 and TNF-α increased under the treatment of YKL40 (100 ng/mmol for 24 h). The phosphorylation of p65 in NF-κB pathway was stimulated in the process. The levels of YKL40, IL-6, IL-8, and TNF-α increases in OSAS children, and YKL40 promotes the overexpression of IL-6, IL-8 and TNF-α in PTLCs via NF-κB pathway. The result implements that inflammation may play an important role in the pathogenesis of OSAS in children., (© 2024. The Author(s).)

Background: Obstructive sleep-disordered breathing (oSDB) is a heterogeneous phenotype that is increasing in prevalence worldwide and has many potential comorbidities that could severely affect quality of life. There is a need to identify biomarkers for oSDB and its comorbidities to improve clinical management, particularly in children. Methods: We performed bulk mRNA-sequencing, differential expression analysis, and qPCR replication of selected differentially expressed genes (DEGs) using RNA samples extracted from tonsils of children with oSDB. Two variables were used as classifier, namely, detection of Epstein-Barr virus (EBV) in tonsils and need for continuous positive airway pressure (CPAP) treatment. Standard statistical tests were used to determine associations across clinical, EBV, and DEG variables. Results: Nineteen genes were dysregulated in tonsils that are EBV+ or from children needing CPAP. Of these genes, APOBR was downregulated in both EBV+ and CPAP+ tonsils, and this downregulation was replicated by qPCR in an independent set of pediatric samples. In the tonsils of adult patients with oSDB, APOBR was positively correlated with age, and potentially with diastolic blood pressure. Conclusion s: Taken together, APOBR and DEGs in tonsillar tissues may be useful as potential biomarkers of oSDB severity and comorbidity across the lifespan, with APOBR levels being dependent on latent EBV infection.

B cells play a key role in humoral immune responses by producing antibodies. Although there are numerous research on memory B cells definition markers and cytokines on B cell development, different studies have yielded contradictory conclusions due to species studied, the different cells and stimulating agents used. In the current study, we conducted a detailed characterization of B cells in human CBMCs, PBMCs and tonsil, including expression of Igs, activation and memory markers. Furthermore, we found that considerable amounts of IgA and IgG were expressed by CD27 - B cells. These "Atypical" memory B cells corresponded to approximately 50% of IgG + and IgA + B cells in blood, this proportion even reached 90% in tonsil. In addition, we investigated the effect of IL-21 and TGF-β1 on the membrane-bound form and secreted form of Igs using PBMCs and purified blood B cells. There were actual differences between the effect of cytokines on Igs secretion and surface expression. Our study will be helpful to advance the knowledge and understanding of humoral memory., (© 2024. The Author(s).)

Spatial omics technologies decipher functional components of complex organs at cellular and subcellular resolutions. We introduce Spatial Graph Fourier Transform (SpaGFT) and apply graph signal processing to a wide range of spatial omics profiling platforms to generate their interpretable representations. This representation supports spatially variable gene identification and improves gene expression imputation, outperforming existing tools in analyzing human and mouse spatial transcriptomics data. SpaGFT can identify immunological regions for B cell maturation in human lymph nodes Visium data and characterize variations in secondary follicles using in-house human tonsil CODEX data. Furthermore, it can be integrated seamlessly into other machine learning frameworks, enhancing accuracy in spatial domain identification, cell type annotation, and subcellular feature inference by up to 40%. Notably, SpaGFT detects rare subcellular organelles, such as Cajal bodies and Set1/COMPASS complexes, in high-resolution spatial proteomics data. This approach provides an explainable graph representation method for exploring tissue biology and function., (© 2024. The Author(s).)

In clinical situations, peripheral blood accessible CD3 + CD4 + CXCR5 + T-follicular helper (T FH ) cells may have to serve as a surrogate indicator for dysregulated germinal center responses in tissues. To determine the heterogeneity of T FH cells in peripheral blood versus tonsils, CD3 + CD4 + CD45RA - CXCR5 + cells of both origins were sorted. Transcriptomes, TCR repertoires and cell-surface protein expression were analysed by single-cell RNA sequencing, flow cytometry and immunohistochemistry. Reassuringly, all blood-circulating CD3 + CD4 + CXCR5 + T-cell subpopulations also appear in tonsils, there with some supplementary T FH characteristics, while peripheral blood-derived T FH cells display markers of proliferation and migration. Three further subsets of T FH cells, however, with bona fide T-follicular gene expression patterns, are exclusively found in tonsils. One additional, distinct and oligoclonal CD4 + CXCR5 + subpopulation presents pronounced cytotoxic properties. Those 'killer T FH (T FK ) cells' can be discovered in peripheral blood as well as among tonsillar cells but are located predominantly outside of germinal centers. They appear terminally differentiated and can be distinguished from all other T FH subsets by expression of NKG7 (TIA-1), granzymes, perforin, CCL5, CCR5, EOMES, CRTAM and CX3CR1. All in all, this study provides data for detailed CD4 + CXCR5 + T-cell assessment of clinically available blood samples and extrapolation possibilities to their tonsil counterparts., (© 2024. The Author(s).)

Mesenchymal stem cells (MSCs) are promising biological therapeutic candidates in cancer treatment. As a source of MSCs, palatine tonsil tissue is one of the secondary lymphoid organs that form an essential part of the immune system, and the relation between the secondary lymphoid organs and cancer progression leads us to investigate the effect of tonsil-derived MSCs (T-MSC) on cancer treatment. We aimed to determine the anti-tumoral effects of T-MSCs cultured at the febrile temperature (40 °C) on hematological cancer cell lines. The co-culture of cancer cells with T-MSCs was carried out under fever and normal culture conditions, and then the cell viability was determined by cell counting. In addition, apoptosis rate and cell cycle arrest were determined by flow cytometry. We confirmed the apoptotic effect of T-MSC co-culture at the transcriptional level by using real-time polymerase chain reaction (RT-PCR). We found that co-culture of cancer cells with T-MSCs significantly decreased the viable cell number under the febrile and normal culture conditions. Besides, the T-MSC co-culture induced apoptosis on K562 and MOLT-4 cells and induced the cell cycle arrest at the G2/M phase on MOLT-4 cells. The apoptotic effect of T-MSC co-culture under febrile stimulation was confirmed at the transcriptional level. Our study has highlighted the anti-tumoral effect of the cellular interaction between the T-MSCs and human hematological cancer cells during in vitro co-culture under hyperthermia., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Mesenchymal stem cells (MSCs) are widely used in the development of therapeutic tools in regenerative medicine. However, their quality decreases during in vitro expansion because of heterogeneity and acquired cellular senescence. We investigated the potential role of podoplanin (PDPN) in minimizing cellular senescence and maintaining the stemness of tonsil-derived MSCs (TMSCs)., Methods: TMSCs were isolated from human tonsil tissues using an enzymatic method, expanded, and divided into two groups: early-passaged TMSCs, which were cultured for 3-7 passages, and late-passaged TMSCs, which were passaged more than 15 times. The TMSCs were evaluated for cellular senescence and MSC characteristics, and PDPN-positive and -negative cells were identified by fluorescence-activated cell sorting. In addition, MSC features were assessed in siRNA-mediated PDPN-depleted TMSCs., Results: TMSCs, when passaged more than 15 times and becoming senescent, exhibited reduced proliferative rates, telomere length, pluripotency marker (NANOG, OCT4, and SOX2) expression, and tri-lineage differentiation potential (adipogenesis, chondrogenesis, or osteogenesis) compared to cells passaged less than five times. Furthermore, PDPN protein levels significantly decreased in a passage-dependent manner. PDPN-positive cells maintained their stemness characteristics, such as MSC-specific surface antigen (CD14, CD34, CD45, CD73, CD90, and CD105) and pluripotency marker expression, and exhibited higher tri-lineage differentiation potential than PDPN-negative cells. SiRNA-mediated silencing of PDPN led to decreased cell-cycle progression, proliferation, and migration, indicating the significance of PDPN as a preliminary senescence-related factor. These reductions directly contributed to the induction of cellular senescence via p16 Ink4a /Rb pathway activation., Conclusion: PDPN may serve as a novel biomarker to mitigate cellular senescence in the clinical application of MSCs., (© 2024. The Author(s).)

Single-cell spatial proteomic analysis holds great promise to advance our understanding of the composition, organization, interaction, and function of the various cell types in complex biological systems. However, the current multiplexed protein imaging technologies suffer from low detection sensitivity, limited multiplexing capacity, or are technically demanding. To tackle these issues, here, we report the development of a highly sensitive and multiplexed in situ protein profiling method using off-the-shelf antibodies. In this approach, the protein targets are stained with horseradish peroxidase (HRP) conjugated antibodies and cleavable fluorophores via click chemistry. Through repeated cycles of target staining, fluorescence imaging, and fluorophore cleavage, many proteins can be profiled in single cells in situ. Applying this approach, we successfully quantified 28 different proteins in human formalin-fixed paraffin-embedded (FFPE) tonsil tissue, which represents the highest multiplexing capacity among the tyramide signal amplification (TSA) methods. Based on their unique protein expression patterns and their microenvironment, ∼820,000 cells in the tissue are classified into distinct cell clusters. We also explored the cell-cell interactions between these varied cell clusters and observed that different subregions of the tissue are composed of cells from specific clusters.

Spatial tissue proteomics integrating whole-slide imaging, laser microdissection, and ultrasensitive mass spectrometry is a powerful approach to link cellular phenotypes to functional proteome states in (patho)physiology. To be applicable to large patient cohorts and low sample input amounts, including single-cell applications, loss-minimized and streamlined end-to-end workflows are key. We here introduce an automated sample preparation protocol for laser microdissected samples utilizing the cellenONE robotic system, which has the capacity to process 192 samples in 3 h. Following laser microdissection collection directly into the proteoCHIP LF 48 or EVO 96 chip, our optimized protocol facilitates lysis, formalin de-crosslinking, and tryptic digest of low-input archival tissue samples. The seamless integration with the Evosep ONE LC system by centrifugation allows 'on-the-fly' sample clean-up, particularly pertinent for laser microdissection workflows. We validate our method in human tonsil archival tissue, where we profile proteomes of spatially-defined B-cell, T-cell, and epithelial microregions of 4000 μm 2 to a depth of ∼2000 proteins and with high cell type specificity. We finally provide detailed equipment templates and experimental guidelines for broad accessibility., Competing Interests: Conflict of interest D. H. and A. S. are employees at Cellenion. All other authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Recent technological innovations have enabled the high-throughput quantification of gene expression and epigenetic regulation within individual cells, transforming our understanding of how complex tissues are constructed 1-6 . However, missing from these measurements is the ability to routinely and easily spatially localize these profiled cells. We developed a strategy, Slide-tags, in which single nuclei within an intact tissue section are tagged with spatial barcode oligonucleotides derived from DNA-barcoded beads with known positions. These tagged nuclei can then be used as an input into a wide variety of single-nucleus profiling assays. Application of Slide-tags to the mouse hippocampus positioned nuclei at less than 10 μm spatial resolution and delivered whole-transcriptome data that are indistinguishable in quality from ordinary single-nucleus RNA-sequencing data. To demonstrate that Slide-tags can be applied to a wide variety of human tissues, we performed the assay on brain, tonsil and melanoma. We revealed cell-type-specific spatially varying gene expression across cortical layers and spatially contextualized receptor-ligand interactions driving B cell maturation in lymphoid tissue. A major benefit of Slide-tags is that it is easily adaptable to almost any single-cell measurement technology. As a proof of principle, we performed multiomic measurements of open chromatin, RNA and T cell receptor (TCR) sequences in the same cells from metastatic melanoma, identifying transcription factor motifs driving cancer cell state transitions in spatially distinct microenvironments. Slide-tags offers a universal platform for importing the compendium of established single-cell measurements into the spatial genomics repertoire., (© 2023. The Author(s).)

Background: Estrogen receptor (ER) testing performed using immunohistochemistry (IHC) is a critical predictive tool for breast cancer treatment. This study aimed to investigate the use of tonsil control for monitoring ER staining and hypothesize that optimal staining would reduce interlaboratory variations., Methods: A proficiency test for ER IHC was conducted using 21 tissue cores. The staining quality was centrally reviewed based on tonsil ER staining., Results: We found that 64.9% of participant samples demonstrated optimal or good staining quality. Poor staining quality was significantly associated with the use of Ventana autostainers and concentrated antibodies. Although the concordance rate did not show significant differences across staining quality levels, interparticipant agreement declined as staining quality deteriorated. Among the 19 discordant responses, 63.2% could be attributed to staining problems, whereas 36.8% could be due to misinterpretation. Poor staining quality due to inadequate staining was the primary reason for undercalls, which can lead to false-negative results. Misinterpretations of nonspecific faint staining that was weaker than the staining of the tonsil control were the cause of most overcalls., Conclusion: Tonsil tissue is an ideal control for monitoring ER staining and can serve as a reference for determining the lower bound for ER positivity. Optimal ER staining and appropriate references for ER positivity can further improve ER IHC quality., Competing Interests: Conflicts of Interest: The authors declare that they have no conflicts of interest related to the subject matter or materials discussed in this article., (Copyright © 2023, the Chinese Medical Association.)

Background: Mesenchymal stem cells (MSCs) are widely used in regenerative medicine and cell-based transplantations. However, an in-depth comparison of the different MSC origins is lacking. This study aimed to compare the expression of adipose-derived (AMSCs), bone marrow-derived (BMSCs), and tonsil-derived (TMSCs) and evaluate whether TMSCs are good alternatives for AMSCs or BMSCs., Methods: We analyzed the expression levels of 47,000 transcripts in AMSCs (n = 4), BMSCs (n = 4), and TMSCs (n = 4) using GeneChip. Microarray data were analyzed using the LIMMA package to compare the TMSCs, AMSCs, and BMSCs. Hub genes were analyzed using STRING and Cytoscape. To ascertain the functional roles of AURKA and AURKB, small interfering RNA (siRNA) molecules specifically targeting AURKA and AURKB mRNA were synthesized and employed to induce knockdown of AURKA and AURKB in TMSC and AMSC. We analyzed the expression level of OCT4, SOX-2, and NANOG genes in TMSC and AMSCs by cell culture and real-time PCR., Results: We identified commonly increased 256 and decreased 160 genes in TMSCs from the differentially expressed genes (DEGs) between the TMSCs, AMSCs, and BMSCs. In the DEG-based protein-protein interaction and gene set enrichment analysis, hub genes (AURKA, AURKB, CDC20, and BUB1) highly expressed in TMSCs were enriched for development- and progression-related oocyte meiosis, the cell cycle, and ubiquitin-mediated proteolysis. In vitro analysis demonstrated that cells with downregulated expression of AURKA and AURKB exhibited a significant reduction in proliferation compared to control cells. However, silencing of the genes did not affect the differentiation capacity in TMSCs and AMSCs., Conclusion: Our study compared MSCs of different origins to better understand the similarities and differences among these cell types., (© 2023. Korean Tissue Engineering and Regenerative Medicine Society.)

Cecal tonsils are the main organs which generate an immune response and also the part of the GALT, thus they are in the close proximity of the intestinal microbiota and continuously exposed to microbe-associated molecular patterns. GALT developed regulatory and anti-inflammatory mechanisms which eliminate or tolerate microbiota. Bioactive substances in ovo administration ensures an early contact between the GALT and beneficial bacteria, which greatly promotes the development of tolerance. Our previous studies have shown that the administration of bioactive substances in ovo silences gene expression in the cecal tonsils. The research hypothesis assumes that negative silencing of expression is correlated with the level of methylation in the tonsils. Therefore the current study aimed to analyze the global and gene-specific DNA methylation profiles in the cecal tonsils of two distinct chicken genotypes administered in ovo with bioactive substances. Eggs of Ross 308 and Green-legged Partridgelike were stimulated on day 12 of incubation. The injected compounds were: probiotic-Lactococcus lactis subsp. cremoris, prebiotic-galactooligosaccharides, and synbiotic-combination of both. Chickens were sacrificed on d 42 post-hatching. Cecal tonsils was collected, RNA and DNA were isolated and intended to gene expression, gene methylation and global methylation analysis. Cecal tonsils changes were observed in the methylation of 6 genes: SYK, ANGPTL4, TNFRSF14, IKZF1, CYR61, SERPING. Analyzes showed that the suppression of gene expression is related to the level of methylation of individual genes. Based on the results obtained in the cecal tonsils, it can be concluded that the silencing of gene expression is of an epigenetic nature. This is another study aimed at analyzing the relationship between the host, its intestinal microbiota and the possibilities of its programming., (© 2023. The Author(s).)

Innate immune cells represent the first line of cellular immunity, comprised of both circulating and tissue-resident natural killer cells and innate lymphoid cells. These innate lymphocytes arise from a common CD34+ progenitor that differentiates into mature natural killer cells and innate lymphoid cells. The successive stages in natural killer cell maturation are characterized by increased lineage restriction and changes to phenotype and function. Mechanisms of human natural killer cell development have not been fully elucidated, especially the role of signals that drive the spatial localization and maturation of natural killer cells. Cytokines, extracellular matrix components, and chemokines provide maturation signals and influence the trafficking of natural killer cell progenitors to peripheral sites of differentiation. Here we present the latest advances in our understanding of natural killer and innate lymphoid cell development in peripheral sites, including secondary lymphoid tissues (i.e. tonsil). Recent work in the field has provided a model for the spatial distribution of natural killer cell and innate lymphoid cell developmental intermediates in tissue and generated further insights into the developmental niche. In support of this model, future studies using multifaceted approaches seek to fully map the developmental trajectory of human natural killer cells and innate lymphoid cells in secondary lymphoid tissues., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Leukocyte Biology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

In this study, the effect of photobiomodulation (PBM) treatment using 630 nm light emitting diode (LED) array (continuous wave type, 10 mW power) on tonsil-derived mesenchymal stem cells (TMSCs) and its interaction with RAW 264.7 macrophage cells via co-culture in vitro were investigated. PBM treatment was used as a priming method for TMSCs to improve therapeutic efficacy. TMSCs were subjected to multi-dose PBM treatments before co-culture with M1 activated (1 μg/mL lipopolysaccharide, LPS) macrophage cells with total energy doses of 0, 15, 30, and 60 J. Irradiation set at 15 J (1500 s treatment time) was performed once, twice for 30 J, and four times for 60 J in an incubator kept at 37 °C and 5% CO 2 . No significant anti-inflammatory response was observed for TMSCs co-cultured with macrophage cells without PBM. But PBM treatment of TMSCs with 630 nm LED array at 30 J reduced expression of inducible nitric oxide synthase, iNOS (M1) and increased expression of Arginase-1, Arg-1 (M2) phenotype macrophage markers. Anti-inflammatory cytokine interleukin-1 receptor antagonist (IL-1RA) gene expression also increased significantly. Based on the results, PBM priming of TMSCs supports M2 macrophage polarization. PBM can be used to improve the therapeutic efficacy of TMSCs for potential applications in oral mucositis and wound healing., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Introduction: Human immunodeficiency virus type 1 (HIV-1) causes a chronic, incurable infection leading to immune activation and chronic inflammation in people with HIV-1 (PWH), even with virologic suppression on antiretroviral therapy (ART). The role of lymphoid structures as reservoirs for viral latency and immune activation has been implicated in chronic inflammation mechanisms. Still, the specific transcriptomic changes induced by HIV-1 infection in different cell types within lymphoid tissue remain unexplored., Methods: In this study, we utilized human tonsil explants from healthy human donors and infected them with HIV-1 ex vivo . We performed single-cell RNA sequencing (scRNA-seq) to analyze the cell types represented in the tissue and to investigate the impact of infection on gene expression profiles and inflammatory signaling pathways., Results: Our analysis revealed that infected CD4 + T cells exhibited upregulation of genes associated with oxidative phosphorylation. Furthermore, macrophages exposed to the virus but uninfected showed increased expression of genes associated with the NLRP3 inflammasome pathway., Discussion: These findings provide valuable insights into the specific transcriptomic changes induced by HIV-1 infection in different cell types within lymphoid tissue. The activation of oxidative phosphorylation in infected CD4 + T cells and the proinflammatory response in macrophages may contribute to the chronic inflammation observed in PWH despite ART. Understanding these mechanisms is crucial for developing targeted therapeutic strategies to eradicate HIV-1 infection in PWH., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Freeman, Zhao, Schrode, Fortune, Shroff, Tweel, Beaumont and Swartz.)

Background: Skeletal muscles play many important roles in the human body and any malfunction or disorder of the skeletal muscles can lead to a reduced quality of life. Some skeletal dysfunctions are acquired, such as sarcopenia but others are congenital. Duchenne muscular dystrophy (DMD) is one of the most common forms of hereditary muscular dystrophy and is caused by a deficiency of the protein, Dystrophin. Currently, there is no clear treatment for DMD, there are only methods that can alleviate the symptoms of the disease. Mesenchymal stem cells, including tonsil-derived mesenchymal stem cells (TMSCs) have been shown to differentiate into skeletal muscle cells (TMSC-myocyte) and can be one of the resources for the treatment of DMD. Skeletal muscle cell characteristics of TMSC-myocytes have been confirmed through changes in morphology and expression of skeletal muscle markers such as Myogenin, Myf6, and MYH families after differentiation., Meothds: Based on these characteristics, TMSC-myocytes have been transplanted into mdx mice, a mouse model of DMD, to investigate whether they can help improve the symptoms of DMD. The red fluorescent protein gene was transduced into TMSC (TMSC-R) for tracking transplanted cells., Results: Prior to transplantation (TP), it was confirmed whether TMSC-R-myocytes had the same differentiation potential as TMSC-myocytes. Increased expression of dystrophin and autophagy markers in the TP group compared with the sham group was confirmed in the gastrocnemius muscle 12 weeks after TP., Conclusion: These results demonstrate muscle regeneration and functional recovery of mdx via autophagy activation following TMSC-myocyte TP., (© 2022. Korean Tissue Engineering and Regenerative Medicine Society.)

There are contradictory reports on the use of mesenchymal stromal cells (MSCs) in cancer therapy. Variable outcomes have been associated with several factors including cancer pathology, experimental procedure, MSC source tissue, and individual genetic differences. It is also known that MSCs exert their therapeutic effects with various paracrine factors released from these cells. The profiles of the factors released from MSCs are altered by heat shock, hypoxia, oxidative stress, starvation or various agents such as inflammatory cytokines, and their therapeutic potential is affected. In this study, the antitumor potential of conditioned media (CM), which contains paracrine factors, of mild hyperthermia-stimulated mesenchymal stromal cells derived from lymphoid organ tonsil tissue (T-MSC) was investigated in comparison with CM obtained from T-MSCs grew under normal culture conditions. CM was obtained from T-MSCs that were successfully isolated from palatine tonsil tissue and characterized. The cytotoxic effect of CM on the growth of hematological cancer cell lines at different concentrations (1:1 and 1:2) was demonstrated by methylthiazoldiphenyl-tetrazolium bromide analysis. In addition, the apoptotic effect of T-MSC-CM treatment was evaluated on the cancer cells using Annexin-V/PI detection method by flow cytometry. The pro/anti-apoptotic and cytokine-related gene expressions were also analyzed by real-time polymerase chain reaction post T-MSC-CM treatment. In conclusion, we demonstrated that the factors released from hyperthermia-stimulated T-MSCs induced apoptosis in hematological cancer cell lines in a dose-dependent manner. Importantly, our results at the transcriptional level support that the factors and cytokines released from hyperthermia-stimulated T-MSC may exert antitumoral effects in cancer cells by downregulation of IL-6 that promotes tumorigenesis. These findings reveal that T-MSC-CM can be a powerful cell-free therapeutical strategy for cancer therapy., (© 2022 Wiley Periodicals LLC.)

Human tonsil-derived mesenchymal stem cells (TMSCs) have a superior proliferation rate and differentiation potential compared to adipose-tissue-derived MSCs (AMSCs) or bone-marrow-derived MSCs (BMSCs). TMSCs exhibit a significantly higher expression of the tensin3 gene ( TNS3 ) than AMSCs or BMSCs. TNS is involved in cell adhesion and migration by binding to integrin beta-1 (ITG β1) in focal adhesion. Here, we investigated the roles of four TNS isoforms, including TNS3 and their relationship with integrin in various biological processes of TMSCs. Suppressing TNS1 and TNS3 significantly decreased the cell count. The knockdown of TNS1 and TNS3 increased the gene and protein expression levels of p16, p19, and p21. TNS1 and TNS3 also have a significant effect on cell migration. Transfecting with siRNA TNS3 significantly reduced Oct4, Nanog, and Sox-2 levels. Conversely, when TNS4 was silenced, Oct4 and Sox-2 levels significantly increase. TNS1 and TNS3 promote osteogenic and adipogenic differentiation, whereas TNS4 inhibits adipogenic differentiation of TMSCs. TNS3 is involved in the control of focal adhesions by regulating integrin. Thus, TNS enables TMSCs to possess a higher proliferative capacity and differentiation potential than other MSCs. Notably, TNS3 plays a vital role in TMSC biology by regulating ITGβ1 activity., Competing Interests: The authors indicated no potential conflict of interest.

Amyloid-β (Aβ)-peptide production or deposition in the neuropathology of Alzheimer's disease (AD) was shown to be caused by chronic inflammation that may be induced by infection, but the role of pathogenic-bacteria-related AD-associated Aβ is not yet clearly understood. In this study, we validated the hypothesis that there is a correlation between the Aβ-protein load and bacterial infection and that there are effects of bacteria, Staphylococcus aureus ( S. aureus ), on the Aβ load in the inflammatory environment of human tonsils. Here, we detected Aβ-peptide deposits in human tonsil tissue as well as tissue similar to tonsilloliths found in the olfactory cleft. Interestingly, we demonstrated for the first time the presence of Staphylococcus aureus ( S. aureus ) clustered around or embedded in the Aβ deposits. Notably, we showed that treatment with S. aureus upregulated the Aβ-protein load in cultures of human tonsil organoids and brain organoids, showing the new role of S. aureus in Aβ-protein aggregation. These findings suggest that a reservoir of Aβ and pathogenic bacteria may be a possible therapeutic target in human tonsils, supporting the treatment of antibiotics to prevent the deposition of Aβ peptides via the removal of pathogens in the intervention of AD pathogenesis.

Long non-coding RNAs are emerging as essential regulators of gene expression, but their role in normal and neoplastic B cells is still largely uncharacterized. Here, we report on the expression pattern of the LINC00152 in normal B cells and Chronic Lymphocytic Leukemia B cell clones. Higher LINC00152 levels were consistently observed in memory B cell populations when compared to naïve B cells in the normal tissues analyzed [peripheral blood (PB), tonsils, and spleen]. In addition, independent stimulation via Immunoglobulins (IG), CD40, or Toll-like Receptor 9 (TLR9) upregulated LINC00152 in PB B cells. The expression of LINC00152 in a cohort of 107 early stage Binet A CLL patients was highly variable and did not correlate with known prognostic markers or clinical evolution. TLR9 stimulation, but not CD40 or IG challenge, was able to upregulate LINC00152 expression in CLL cells. In addition, LINC00152 silencing in CLL cell lines expressing LINC00152 failed to induce significant cell survival or apoptosis changes. These data suggest that, in normal B cells, the expression of LINC00152 is regulated by immunomodulatory signals, which are only partially effective in CLL cells. However, LINC00152 does not appear to contribute to CLL cell expansion and/or survival in a cohort of newly diagnosed CLL patients., (© 2021 The Authors. Hematological Oncology published by John Wiley & Sons Ltd.)

While β-hemolytic streptococcus (β-HS) infections are known to predispose patients to acute poststreptococcal glomerulonephritis, there is evidence that implicates α-hemolytic streptococcus (α-HS) in IgA nephropathy (IgAN). The alternative pathway of the complement system has also been implicated in IgAN. We aimed to explore the association between α-HS and complement activation in human tonsillar mononuclear cells (TMCs) in IgAN. In our study, α-HS induced higher IgA levels than IgG levels, while β-HS increased higher IgG levels than IgA levels with more activation-induced cytidine deaminase, in TMCs in the IgAN group. Aberrant IgA1 O-glycosylation levels were higher in IgAN patients with α-HS. C3 and C3b expression was decreased in IgAN patients, but in chronic tonsillitis control patients, the expression decreased only after stimulation with β-HS. Complement factor B and H (CFH) mRNA increased, but the CFH concentration in culture supernatants decreased with α-HS. The percentage of CD19 + CD35 + cells/complement receptor 1 (CR1) decreased with α-HS more than with β-HS, while CD19 + CD21 + cells/complement receptor 2 (CR2) increased more with β-HS than with α-HS. The component nephritis-associated plasmin receptor (NAPlr) of α-HS was not detected on tonsillar or kidney tissues in IgAN patients and was positive on cultured TMCs and mesangial cells. We concluded that α-HS induced the secretion of aberrantly O-glycosylated IgA while decreasing the levels of the inhibitory factor CFH in culture supernatants and CR1 + B cells. These findings provide testable mechanisms that relate α-HS infection to abnormal mucosal responses involving the alternative complement pathway in IgAN., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Hematuria is an essential symptom of immunoglobulin A nephropathy (IgAN). Although the etiology of hematuria in IgAN has not been fully elucidated, it is thought that the rupture of the glomerular basement membranes caused by intra-capillary leukocyte influx, so-called glomerular vasculitis, is the pathological condition responsible for severe hematuria. Glomerular vasculitis are active lesions that exist in the glomeruli of acute phase IgAN and it is important because it is suspected to make the transition to segmental glomerular sclerosis (SGS) as a repair scar lesion in the chronic phase, and the progression of SGS would eventually lead to glomerular obsolescence. Worsening of hematuria concomitant with acute pharyngitis is common in patients with IgAN; therefore, elucidating the relationship between the immune system of Waldeyer's ring, including the palatine tonsil and epipharyngeal lymphoid tissue, and the glomerular vasculitis may lead to understanding the nature of IgAN. The epipharynx is an immunologically activated site even under normal conditions, and enhanced activation of innate immunity is likely to occur in response to airborne infection. Hyperactivation of innate immunity via upregulation of Toll-like receptors in the interfollicular area of the palatine tonsil and epipharyngeal lymphoid tissue, followed by enhanced fractalkine/CX3CR1 interactions, appears to play an important role in the development of glomerular vasculitis in IgAN. As latent but significant epipharyngitis is present in most patients with IgAN, it is plausible that acute upper respiratory infection may contribute as a trigger for the innate epipharyngeal immune system, which is already upregulated in a chronically inflamed environment. Given that epipharyngitis and its effects on IgAN are not fully understood, we propose that the so-called "epipharynx-kidney axis" may provide an important focus for future research.

Objective: The aim: The aim of the study is to compare the class G antibody content in serum and tissue lysate from tonsils of children with hypertrophy and chronic tonsillitis to: streptolysin-O of Str. haemolyticus, protein-A of S. aureus, proteoglycans of Klebsiela spp., as well as to compare the content of interleukins 1β, 10, TNF-α, γ-IFN and lactoferrin in serum and tissue lysate from tonsils of children with hypertrophy and chronic tonsillitis., Patients and Methods: Materials and methods: We studied tonsils of 33 children aged 4-18 years with hypertrophy of palatine tonsils (HPT) and with chronic tonsillitis (CT). The content of interleukins 1β, 10, TNF-α, γ-IFN and lactoferrin in tonsil lysate and serum was determined by immunofluorescence assay. Antistreptolysin O was studied by neutralization test of micromethod; class G antibodies to protein A of S. aureus and proteoglycans of Klebsiela spp. were studied by treponema pallidum hemagglutination assay. All the results were statistically processed using U-test (Mann-Whitney-Wilcoxon test) and Fisher's z-transformation., Results: Results: The serum and tissue lysate from tonsils of patients with HPT showed significantly high level of antibodies to streptolysin O in comparison with similar studies of substrates from patients with CT. Anti-inflammatory cytokine IL-10 was detected only in the serum of patients with CT. The TNF-α concentration in the lysates of tonsils in the group of patients with HPT was 2 times higher than in the group of patients with CT. The γ-IFN concentration was significantly lower both in the serum and in the lysates of tonsils of patients with CT. The content of lactoferrin in the lysates of patients with CT was 3 times higher (P<0.05) than in the lysates of patients with HPT., Conclusion: Conclusions: The results indicate a significant difference in the state of antibodies to microbial antigens and cytokines production in case of HPT and CT. In tonsils with HPT, there predominate reactions of antibody production to bacterial antigens and antiviral reactions like a high-level cytokines TNF-α and γ-IFN in tissue lysate of palatine tonsils.

Interleukin (IL)-8 has been shown to play an important role in obstructive sleep apnea syndrome (OSAS). However, its role in OSAS development is still controversial. This meta-analysis was to explore the correlation between interleukin (IL)-8 concentration and OSAS. Database (from the inception to July 2021) searches on PubMed, Web of Science, Medline, EMBASE, and Cochrane Library were conducted for studies analyzing the correlation between IL-8 concentration and OSAS, regardless of the language of publication. Standardized mean difference (SMD) and 95% confidence intervals (CI) were used to analyze any prospective association between IL-8 concentration and OSAS. A total of 25 eligible studies, including 2301 participants and 1123 controls, were included in this meta-analysis. The included studies evaluating the association between serum IL-8 concentration and OSAS indicated that adults and children with OSAS had elevated serum concentrations of IL-8 compared with controls (SMD = 0.997, 95% CI = 0.437-1.517, P < 0.001; SMD = 0.431, 95% CI = 0.104-0.759, P = 0.01). Categorization of the study population into subgroups according to body mass index, apnea-hypopnea index (AHI), ethnicity, and sample size also showed that individuals with OSAS had elevated serum concentrations of IL-8 compared with controls. Additionally, the results demonstrated that the higher the AHI, higher was the IL-8 concentration. Similar results were observed in the literature on the association between plasma IL-8 concentration and OSAS. This meta-analysis verified that compared with controls, children and adults with OSAS have significantly elevated IL-8 concentrations.

Mesenchymal stem cells (MSCs) are mesoderm‑originated adult SCs that possess multidirectional differentiation potential. MSCs migrate to injured tissue and secrete a range of paracrine factors that induce regeneration in damaged tissue and exert immune modulation. Because tumor progression is dependent on cross‑talk between the tumor and its microenvironment, MSCs also produce extracellular vesicles (EVs) that mediate information transfer in the tumor microenvironment. However, the effect of MSC‑derived EVs on tumor development and progression is still controversial. To date, tonsil‑derived MSCs (T‑MSCs) have been shown to possess all the defined characteristics of MSCs and show distinctive features of differential potential and immune modulation. To observe the effect of soluble mediators from T‑MSCs on tumor growth, human liver cancer cell line (HepG2) cells were injected into nude mice and HepG2 cell scratch migration assay was performed using conditioned medium (CM) of T‑MSCs. T‑MSC CM inhibited tumor growth and progression and it was hypothesized that EVs from T‑MSCs could inhibit tumor progression. microRNA (miRNA or miR) sequencing using five different origins of T‑MSC‑derived EVs was performed and highly expressed miRNAs, such as miR‑199a‑3p, miR‑214‑3p, miR‑199a‑5p and miR‑199b‑5p, were selected. T‑MSCs inhibited tumor growth and HepG2 cell migration, potentially via miR‑199a‑3p targeting CD151, integrin α3 and 6 in HepG2 cells.

Background: The microRNA (miRNA) expression of the tonsil tissues in patients with immunoglobulin A (IgA) nephropathy (IgAN) has not been reported in the literature., Methods: In this study, the expression of nine miRNAs was measured in the tonsil tissues of patients with IgAN, including miRNA-21-5p, miRNA-29a-3p, miRNA-34a-5p, miRNA-146a-5p, miRNA-146b-5p, miRNA-148b-3p, miRNA-150-5p, miRNA-155-5p, and miRNA-181a-5p. Forty patients with proved primary IgA nephropathy were enrolled in our study, 20 IgAN patients with gross hematuria, which induced by tonsillitis (GH-IgAN group) and 20 IgAN patients without gross hematuria in the history (non-GH-IgAN group). Another 20 patients recruited as the control group (CT group) were chronic tonsillitis without kidney disease., Results: Compared to the CT group, the expression level of miRNA-150-5p in the tonsils was significantly upregulated in the GH-IgAN group, but not in the non-GH-IgAN group (P = 0.031 and P = 0.122, respectively). A correlation analysis was performed between the expression of miRNAs in the tonsils and the clinical data of IgAN patients. The results showed that in the GH-IgAN group, the miRNA-150 expression was positively correlated with systolic blood pressure (β = 2.36, 95% CI 1.11-3.61, P = 0.0016), diastolic blood pressure (β = 1.02, 95% CI 0.22-1.82, P = 0.0224), uric acid (β = 7.43, 95% CI 1.81-13.04, P = 0.0184), leukocyte count (β = 0.22, 95% CI 0.09-0.35, P = 0039), neutrophil count (β = 0.19, 95% CI 0.06-0.32, P = 0.0096), cholesterol (β = 0.09, 95% CI 0.02-0.16, P = 0.0207) and triglyceride level (β = 0.16, 95% CI 0.10-0.22, P < 0.000). Besides, it was negatively correlated with the estimated glomerular filtration rate (eGFR) (β = -2.06, 95% CI: -3.90 - -0.21, P = 0.0421) in the GH-IgAN group; however, no significant correlation was found in the non-GH-IgAN group., Conclusion: The present findings suggest that miRNA-150-5p may be important in the pathogenesis of IgAN, especially in mucosal immunity against the disease., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Modern histopathology is built on the cornerstone principle of tissue fixation, however there are currently no analytical methods of detecting fixation and as a result, in clinical practice fixation is highly variable and a persistent source of error. We have previously shown that immersion in cold formalin followed by heated formalin is beneficial for preservation of histomorphology and have combined two-temperature fixation with ultra-sensitive acoustic monitoring technology that can actively detect formalin diffusing into a tissue. Here we expand on our previous work by developing a predictive statistical model to determine when a tissue is properly diffused based on the real-time acoustic signal. We trained the model based on the morphology and characteristic diffusion curves of 30 tonsil cores. To test our model, a set of 87 different tonsil samples were fixed with four different protocols: dynamic fixation according to our predictive algorithm (C/H:Dynamic, N = 18), gold-standard 24 hour room temperature (RT:24hr, N = 24), 6 hours in cold formalin followed by 1 hour in heated formalin (C/H:6+1, N = 21), and 2 hours in cold formalin followed by 1 hour in heated formalin (C/H:2+1, N = 24). Digital pathology analysis revealed that the C/H:Dynamic samples had FOXP3 staining that was spatially uniform and statistically equivalent to RT:24hr and C/H:6+1 fixation protocols. For comparison, the intentionally underfixed C/H:2+1 samples had significantly suppressed FOXP3 staining (p<0.002). Furthermore, our dynamic fixation protocol produced bcl-2 staining concordant with standard fixation techniques. The dynamically fixed samples were on average only submerged in cold formalin for 4.2 hours, representing a significant workflow improvement. We have successfully demonstrated a first-of-its-kind analytical method to assess the quality of fixation in real-time and have confirmed its performance with quantitative analysis of downstream staining. This innovative technology could be used to ensure high-quality and standardized staining as part of an expedited and fully documented preanalytical workflow., Competing Interests: DB, TL and DC were full-time employees of Roche Tissue Diagnostics during this work and received funding for experiments from Roche Tissue Diagnostics. There are no products in development or marketed products to declare. Roche Tissue Diagnostics has filed patents on the TOF technology and associated algorithms. DB, TL and DC explicitly acknowledge this does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests).

Mother-to-child transmission (MTCT) of human immunodeficiency virus type 1 (HIV-1) and human cytomegalovirus (HCMV) may occur during pregnancy, labor, or breastfeeding. These viruses from amniotic fluid, cervicovaginal secretions, and breast milk may simultaneously interact with oropharyngeal and tonsil epithelia; however, the molecular mechanism of HIV-1 and HCMV cotransmission through the oral mucosa and its role in MTCT are poorly understood. To study the molecular mechanism of HIV-1 and HCMV MTCT via oral epithelium, we established polarized infant tonsil epithelial cells and polarized-oriented ex vivo tonsil tissue explants. Using these models, we showed that cell-free HIV-1 and its proteins gp120 and tat induce the disruption of tonsil epithelial tight junctions and increase paracellular permeability, which facilitates HCMV spread within the tonsil mucosa. Inhibition of HIV-1 gp120-induced upregulation of mitogen-activated protein kinase (MAPK) and NF-κB signaling in tonsil epithelial cells, reduces HCMV infection, indicating that HIV-1-activated MAPK and NF-κB signaling may play a critical role in HCMV infection of tonsil epithelium. HCMV infection of tonsil epithelial cells also leads to the disruption of tight junctions and increases paracellular permeability, facilitating HIV-1 paracellular spread into tonsil mucosa. HCMV-promoted paracellular spread of HIV-1 increases its accessibility to tonsil CD4 T lymphocytes, macrophages, and dendritic cells. HIV-1-enhanced HCMV paracellular spread and infection of epithelial cells subsequently leads to the spread of HCMV to tonsil macrophages and dendritic cells. Our findings revealed that HIV-1- and HCMV-induced disruption of infant tonsil epithelial tight junctions promotes MTCT of these viruses through tonsil mucosal epithelium, and therapeutic intervention for both HIV-1 and HCMV infection may substantially reduce their MTCT. IMPORTANCE Most HIV-1 and HCMV MTCT occurs in infancy, and the cotransmission of these viruses may occur via infant oropharyngeal and tonsil epithelia, which are the first biological barriers for viral pathogens. We have shown that HIV-1 and HCMV disrupt epithelial junctions, reducing the barrier functions of epithelia and thus allowing paracellular penetration of both viruses via mucosal epithelia. Subsequently, HCMV infects epithelial cells, macrophages, and dendritic cells, and HIV-1 infects CD4 + lymphocytes, macrophages, and dendritic cells. Infection of these cells in HCMV- and HIV-1-coinfected tonsil tissues is much higher than that by HCMV or HIV-1 infection alone, promoting their MTCT at its initial stages via infant oropharyngeal and tonsil epithelia.

In mucosa such as tonsil, antibody-producing plasmocytes (PCs) lie in sub-epithelium space, which is thought to provide a suitable environment for their survival. A proliferation inducing ligand (APRIL) is one key survival factor for PCs present in this area. According to in situ staining, apical epithelial cells produced APRIL, and the secreted product had to migrate all through the stratified surface epithelium to reach basal cells. A similar process also occurred in the less-organized crypt epithelium. Tonsil epithelial cells captured secreted APRIL, thanks to their surface expression of the APRIL coreceptor, either syndecan-1 or -4 depending on their differentiation stage. In the most basal epithelial cells, secreted APRIL accumulated inside secretory lamp-1 + vesicles in a polarized manner, facing the sub-epithelium. The tonsil epithelium upregulated APRIL production by apical cells and secretion by basal cells upon Toll-like receptor stimulation. Furthermore, LPS-stimulated epithelial cells sustained in vitro PC survival in a secreted APRIL-dependent manner. Taken together, our study shows that the tonsil epithelium responds to pathogen sensing by a polarized secretion of APRIL in the sub-epithelial space, wherein PCs reside., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Sturm, Quinterot, Guyot, Righini, Daamen, van Kuppevelt and Huard.)

Background: Neurological disorders are considered one of the greatest burdens to global public health and a leading cause of death. Stem cell therapies hold great promise for the cure of neurological disorders, as stem cells can serve as cell replacement, while also secreting factors to enhance endogenous tissue regeneration. Adult human multipotent stem cells (MSCs) reside on blood vessels, and therefore can be found in many tissues throughout the body, including palatine tonsils. Several studies have reported the capacity of MSCs to differentiate into, among other cell types, the neuronal lineage. However, unlike the case with embryonic stem cells, it is unclear whether MSCs can develop into mature neurons., Methods: Human tonsillar MSCs (T-MSCs) were isolated from a small, 0.6-g sample, of tonsillar biopsies with high viability and yield as we recently reported. Then, these cells were differentiated by a rapid, multi-stage procedure, into committed, post-mitotic, neuron-like cells using defined conditions., Results: Here we describe for the first time the derivation and differentiation of tonsillar biopsy-derived MSCs (T-MSCs), by a rapid, multi-step protocol, into post-mitotic, neuron-like cells using defined conditions without genetic manipulation. We characterized our T-MSC-derived neuronal cells and demonstrate their robust differentiation in vitro., Conclusions: Our procedure leads to a rapid neuronal lineage commitment and loss of stemness markers, as early as three days following neurogenic differentiation. Our studies identify biopsy-derived T-MSCs as a potential source for generating neuron-like cells which may have potential use for in vitro modeling of neurodegenerative diseases or cell replacement therapies., (© 2021. The Author(s).)

Multiplexed single-cell analysis of proteins in their native cellular contexts holds great promise to reveal the composition, interaction and function of the distinct cell types in complex biological systems. However, the existing multiplexed protein imaging technologies are limited by their detection sensitivity or technical demands. To address these issues, here, we develop an ultrasensitive and multiplexed in situ protein profiling approach by reiterative staining with off-the-shelf antibodies and cleavable fluorescent tyramide (CFT). In each cycle of this approach, the protein targets are recognized by antibodies labeled with horseradish peroxidase, which catalyze the covalent deposition of CFT on or close to the protein targets. After imaging, the fluorophores are chemically cleaved, and the antibodies are stripped. Through continuous cycles of staining, imaging, fluorophore cleavage and antibody stripping, a large number of proteins can be quantified in individual cells in situ. Applying this method, we analyzed 20 different proteins in each of ~67,000 cells in a human formalin-fixed paraffin-embedded (FFPE) tonsil tissue. Based on their unique protein expression profiles and microenvironment, these individual cells are partitioned into different cell clusters. We also explored the cell-cell interactions in the tissue by examining which specific cell clusters are selectively associating or avoiding each other.

Intrarenal B cells in human renal allografts indicate transplant recipients with a poor prognosis, but how these cells contribute to rejection is unclear. Here we show using single-cell RNA sequencing that intrarenal class-switched B cells have an innate cell transcriptional state resembling mouse peritoneal B1 or B-innate (Bin) cells. Antibodies generated by Bin cells do not bind donor-specific antigens nor are they enriched for reactivity to ubiquitously expressed self-antigens. Rather, Bin cells frequently express antibodies reactive with either renal-specific or inflammation-associated antigens. Furthermore, local antigens can drive Bin cell proliferation and differentiation into plasma cells expressing self-reactive antibodies. These data show a mechanism of human inflammation in which a breach in organ-restricted tolerance by infiltrating innate-like B cells drives local tissue destruction., (© 2021. The Author(s).)

Biofunctional polymers have been widely used to enhance the proliferation and functionality of stem cells. Here, we report the development of a new biofunctional polymer, octanoyl glycol chitosan (OGC), and demonstrate its effects on the cell cycle and stem cell function using tonsil-derived mesenchymal stem cells (TMSCs). OGC treatment (100 μg/mL) significantly increased the proliferation of TMSCs, which could be attributed to cyclin D1 up-regulation in the G1 phase of the cell cycle. Additionally, OGC enhanced the ability of TMSCs to differentiate into adipocytes, chondrocytes, and osteoblasts. Taken together, this new biofunctional polymer, OGC, can promote stemness and osteogenesis, as well as induce stem cell proliferation by enhancing the intracellular metabolic rate and regulating the cell cycle. Thus, in the future, OGC could be a potential therapeutic additive for improving stem cell function., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Introduction: SOCS1, a negative regulator of JAK/STAT signaling, is among the most frequently mutated genes in DLBCL and classical Hodgkin lymphoma. The C-terminal SOCS box domain, mediating the degradation of phospho-JAK2, is often affected or even lacking. The analysis of such variants is hampered by the lack of a SOCS1-specific monoclonal antibody recognizing the C-terminus of SOCS1. As this C-terminus is often lost or mutated in B-cell lymphomas, staining with amino-terminal targeting antibodies in a lymphoma setting might be misleading., Methods: BALB/c mice were immunized with a truncated SOCS1 C-terminal protein. The supernatant of generated hybridoma cells was screened by ELISA and, immunohistochemically, on formalin-fixed and paraffin-embedded tonsil. After antibody purification by affinity chromatography, epitope mapping and cross-reactivity check followed via substitution scans. SOCS1 protein expression was investigated on cell cultures and cytoblocks of SOCS1 WT stably transfected HEK293T cells, lymphoma cell lines and lymphoid tissues., Results: Procedures resulted in one monoclonal IgG1 anti-SOCS1 antibody, 424C, that recognizes and strongly binds to the C-terminal region of SOCS1 in immunoblot and immunohistochemistry analyses., Conclusion: This new anti-SOCS1 monoclonal antibody is a valuable tool to detect SOCS1 expression dependent on an existing SOCS1 box and, therefore, indicating a full-length SOCS1 protein., (© 2021 The Authors. European Journal of Haematology published by John Wiley & Sons Ltd.)

Background: Adenoid hypertrophy (AH) can cause harmful effects on untreated children, which include mouth breathing, chronic intermittent hypoxia, sleep disordered breathing (SDB), and even some behavioral problems. However, the molecular mechanisms underlying this pathophysiological process have remained poorly understood., Methods: In this study, SUMO was induced silencing and overexpression using RNAi and lentiviral-mediated vector. FITC-Dextran and TEER were performed to examine the role of SUMO in cell permeability. Co-immunoprecipitation (Co-IP) assay was performed to examine the interaction between SUMO1 and HIF-1α. Immunohistochemistry staining was used to examine the expressions of ZO-1, Claudin-1 and occluding respectively., Results: We found that a hypoxic condition caused a dramatic upregulation of SUMO-1 expression in a time-dependent manner, a member of the ubiquitin-like protein family. Knockdown of SUMO-1 deeply suppressed the secretions of pro-inflammation cytokines including IL-6, IL-8, and TNF-α, and decreased the permeability of HTECs. Moreover, the HIF-1α inhibitor 2-MeOE2 abolished the function of SUMO-1 in HTECs. Furthermore, results obtained from CO-IP had suggested that SUMO-1 interacted with HIF-1α, and prevented its ubiquitination and degradation in HTECs by sumoylating. Importantly, our data showed that hypoxia-induced inflammation was markedly inhibited by M2 macrophages that possess potent anti-inflammatory function., Conclusion: Our results suggest that selectively inhibiting the SUMO-1-HIF-1α signaling pathway leads to anti-inflammatory responses in human tonsil epithelial cells, which might be a novel therapeutic approach for managing hypoxia-induced SDB resulting from AH., (Copyright © 2021. Published by Elsevier Inc.)

Objectives: Tonsil tissue is a very important component of the human immunity system, contributing to the functioning of the cellular and humoral defence system, especially in childhood. The endoplasmic reticulum (ER) is an organelle that has a very important function in the balanced functioning of cells, in which the accumulation of a cellular protein called ER stress occurs in case of dysfunction. ER stress influences the pathogenesis of many diseases and immune system functions. We aimed to investigate the relation between the diseases of tonsil tissue and ER stress response to elucidate the mechanisms of diseases related with the immune system., Methods: A prospective study was conducted in 46 children aged between 2 and 16 years who underwent tonsillectomy for chronic tonsillitis or tonsillar hypertrophy. Tonsil tissue was separated into two groups according to their size and evaluated in terms of ER stress markers and apoptosis markers by Real-time PCR and Western blot analysis., Results: The ΔCT levels of ER stress markers (ATF4, ATF6, CHOP, GRP78, EIF2AK3, ERN1, GRP94) were greater in children with chronic tonsillitis (p < 0.005). In contrast, the tonsillar hypertrophy group had greater ΔCT levels of apoptosis markers (BAX, BCL-2) according to the Real-time PCR method (p < 0.005). According to the Western blot analysis, the normalized levels of ATF4, ATF6, CHOP, GRP78, and ERN1 genes were found greater in the chronic tonsillitis group than the tonsillar hypertrophy group. There was no difference between the two groups in terms of normalized BCL-2 and BAX levels by Western blot analysis., Conclusion: This is the first study in the literature investigating the effect of the ER stress pathway on the etiopathogenesis of tonsil diseases. It was concluded that the ER stress pathway plays a role in the etiopathogenesis of chronic tonsillitis. Investigating the relationship between ER stress and structures such as the tonsil tissue that make up the immune system can help create new treatment strategies., Clinical Trial Registration: Trial Registration ClinicalTrials.gov Identifier: NCT04653376., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Background: Tonsil-derived mesenchymal stem cells (T-MSCs) were reported to have suppressive effect on T cells, yet much remains unknown about the underlying mechanisms supporting this effect. We investigated the underlying mechanism of the immunomodulatory effect of T-MSCs on immune cell proliferation and cytokine production. Methods: We isolated T-MSCs from human palatine tonsil and evaluated the immunomodulatory capacity using RT-PCR, ELISA, and flow cytometry. Additionally, we assessed the expression of various soluble factors and several costimulatory molecules to detect the priming effect on T-MSCs. Results: T-MSCs significantly inhibited the immune cell proliferation and cytokine expression (TNF-α and IFN-γ) in the direct co-culture, but there was no suppressive effect in indirect co-culture. Additionally, we detected a remarkably higher expression of indoleamine 2,3-dioxygenase (IDO) in the primed T-MSCs having co-expression CD40. Moreover, immune cells or CD4 + T cells showed lower TNF-α, IFN-γ, and IL-4 expression when the primed T-MSC were added; whereas those findings were reversed when the inhibitor for IDO (not IL-4) or CD40 were added. Furthermore, T-bet and GATA3 levels were significantly decreased in the co-cultures of the primed T-MSCs and CD4 + T cells; whereas those findings were reversed when we added the neutralizing anti-CD40 antibody. Conclusions: Primed T-MSCs expressing IDO and CD40 may have immunomodulatory capacity via Th1-mediated and Th2-mediated immune response.

Background: The application of extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) requires customized materials to target disease or cell damage. We hypothesized that EVs exert different inflammatory effects on one recipient cell, although stem cells of different origins in humans have similar payloads., Results: Here, the payload of EVs released by crosstalk between MSCs and human middle ear epithelial cells (HMEECs) extracted from adipose tissue, bone marrow and tonsils significantly increased the level of anti-inflammatory factors. EVs derived from the co-culture medium decreased TNF-α, COX-2, IL-1β, and IL-6 levels to approximately zero within 3 h in HMEECs. Expression of miR-638 and amyloid-β A4 precursor protein-binding family A member 2 was analyzed using microarrays and gene ontology analysis, respectively., Conclusions: In conclusion, stem cells of different origins have different payloads through crosstalk with recipient-specific cells. Inducing specific factors in EVs by co-culture with MSCs could be valuable in regenerative medicine.

Innate Lymphoid Cells (ILCs) are immune cells typically found on mucosal surfaces and in secondary lymphoid organs where they regulate the immune response to pathogens. Despite their key role in the immune response, there are still fundamental gaps in our understanding of ILCs. Here we report a human ILC population present in the follicles of tonsils and lymph nodes termed follicular regulatory ILCs (ILC FR ) that to our knowledge has not been previously identified. ILC FR have a distinct phenotype and transcriptional program when compared to other defined ILCs. Surprisingly, ILC FR inhibit the ability of follicular helper T (Tfh) cells to provide B cell help. The localization of ILC FR to the germinal centers suggests these cells may interfere with germinal center B cell (GC-B) and germinal center Tfh cell (GC-Tfh) interactions through the production of transforming growth factor beta (TGF-β. Intriguingly, under conditions of impaired GC-Tfh-GC-B cell interactions, such as human immunodeficiency virus (HIV) infection, the frequency of these cells is increased. Overall, we predict a role for ILC FR in regulating GC-Tfh-GC-B cell interactions and propose they expand in chronic inflammatory conditions.

Autoinflammatory disorders of the innate immune system present with recurrent episodes of inflammation often beginning in early childhood. While there are now more than 30 genetically-defined hereditary fever disorders, many patients lack a clear diagnosis. Many pediatric patients are often grouped with patients with periodic fever, aphthous stomatitis, pharyngitis, and adenitis (PFAPA) syndrome despite failing to meet diagnostic criteria. Here, we categorize these patients as syndrome of undifferentiated recurrent fever (SURF), and identify the unique features which distinguish them from the PFAPA syndrome. SURF patients were more likely to report gastrointestinal symptoms of nausea, vomiting and abdominal pain, and experienced inconsistent responses to on-demand steroid therapy compared to PFAPA patients. For this previously undefined cohort, an optimal course of therapy remains uncertain, with medical and surgical therapies largely driven by parental preference. A subset of patients with SURF underwent tonsillectomy with complete resolution. Flow cytometric evaluation demonstrates leukocytic populations distinct from PFAPA patients, with reduced CD3+ T cell numbers. SURF patient tonsils were predominantly characterized by an IL-1 signature compared to PFAPA, even during the afebrile period. Peripheral blood signatures were similar between groups suggesting that PFAPA and SURF patient tonsils have localized, persistent inflammation, without clinical symptoms. These data suggest that SURF is a heterogenous syndrome on the autoinflammatory disease spectrum., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Understanding the composition, function and regulation of complex cellular systems requires tools that quantify the expression of multiple proteins at their native cellular context. Here, we report a highly sensitive and accurate protein in situ profiling approach using off-the-shelf antibodies and cleavable fluorescent tyramide (CFT). In each cycle of this method, protein targets are stained with horseradish peroxidase (HRP) conjugated antibodies and CFT. Subsequently, the fluorophores are efficiently cleaved by mild chemical reagents, which simultaneously deactivate HRP. Through reiterative cycles of protein staining, fluorescence imaging, fluorophore cleavage, and HRP deactivation, multiplexed protein quantification in single cells in situ can be achieved. We designed and synthesized the high-performance CFT, and demonstrated that over 95% of the staining signals can be erased by mild chemical reagents while preserving the integrity of the epitopes on protein targets. Applying this method, we explored the protein expression heterogeneity and correlation in a group of genetically identical cells. With the high signal removal efficiency, this approach also enables us to accurately profile proteins in formalin-fixed paraffin-embedded (FFPE) tissues in the order of low to high and also high to low expression levels.

Mesenchymal stromal cells (MSCs) are considered as a promising therapeutic tool for liver fibrosis, a main feature of chronic liver disease. Because small extracellular vesicles (sEVs) harboring a variety of proteins and RNAs are known to have similar functions with their derived cells, MSC-derived sEVs carry out the regenerative capacities of MSCs. Human tonsil-derived MSCs (T-MSCs) are reported as a novel source of MSCs, but their effects on liver fibrosis remain unclear. In the present study, we investigated the effects of T-MSC-derived sEVs on liver fibrosis. The expression of profibrotic genes decreased in human primary hepatic stellate cells (pHSCs) co-cultured with T-MSCs. Treatment of T-MSC-sEVs inactivated human and mouse pHSCs. Administration of T-MSC-sEVs ameliorated hepatic injuries and fibrosis in chronically damaged liver induced by carbon tetrachloride (CCl 4 ). miR-486-5p highly enriched in T-MSC-sEVs targeting the hedgehog receptor, smoothened (Smo), was upregulated, whereas Smo and Gli2, the hedgehog target gene, were downregulated in pHSCs and liver tissues treated with T-MSC-sEVs or miR-486-5p mimic, indicating that sEV-miR-486 inactivates HSCs by suppressing hedgehog signaling. Our results showed that T-MSCs attenuate HSC activation and liver fibrosis by delivering sEVs, and miR-486 in the sEVs inactivates hedgehog signaling, suggesting that T-MSCs and their sEVs are novel anti-fibrotic therapeutics for treating chronic liver disease., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Aims: Though formalin remains to be the gold standard fixative in pathology departments, analytical challenges persist for nucleic acid evaluations. In our laboratory, formalin fixation of skin samples in particular impairs diagnostic accuracy and demands repetition of biopsies and analytical procedures. PAXgene Tissue Systems may be an alternative; however, according to manufacturer specifications it only allows fixation for 48 hours before having to add a stabiliser. This may be a challenge in laboratories, which are closed in weekends and bank holidays. Our aim was to validate this alternative fixative for dermatological samples with prolonged fixation times using standard laboratory protocols developed for formalin-fixed specimens. We compared the results with gold standard formalin fixation., Methods: Skin specimens were formalin or PAXgene fixed for either 2 hours, 24 hours, 3 days or 7 days, paraffin-embedded, analysed and scored by observers., Results: Generally, formalin outperformed PAXgene fixation in H&E stains and fluorescence in situ hybridisation (FISH), but both seem usable for diagnostics. Time of PAXgene fixation did not have an impact on alcian blue-Van Gieson (ABVG), H&E (p=0.48), nor immunohistochemistry (p=0.74). There was a tendency towards best PAXgene performance at 24 hours of fixation for FISH, and for DNA integrity analysis 24 hours or 3 days., Conclusions: Prolonging PAXgene fixation time to 3 days before adding stabiliser does not seem to have major impact on performance of general diagnostic analysis, but our preliminary results show optimisation of internal protocols are needed. PAXgene is an expensive alternative and may be confined to some dermatological samples., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Porcine reproductive and respiratory syndrome (PRRS) is a threat to pig production worldwide. Our objective was to understand mechanisms of persistence of PRRS virus (PRRSV) in tonsil. Transcriptome data from tonsil samples collected at 42 days post infection (dpi) were generated by RNA-seq and NanoString on 51 pigs that were selected to contrast the two PRRSV isolates used, NVSL and KS06, high and low tonsil viral level at 42 dpi, and the favorable and unfavorable genotypes at a genetic marker (WUR) for the putative PRRSV resistance gene GBP5., Results: The number of differentially expressed genes (DEGs) differed markedly between models with and without accounting for cell-type enrichments (CE) in the samples that were predicted from the RNA-seq data. This indicates that differences in cell composition in tissues that consist of multiple cell types, such as tonsil, can have a large impact on observed differences in gene expression. Based on both the NanoString and the RNA-seq data, KS06-infected pigs showed greater activation, or less inhibition, of immune response in tonsils at 42 dpi than NVSL-infected pigs, with and without accounting for CE. This suggests that the NVSL virus may be better than the KS06 virus at evading host immune response and persists in tonsils by weakening, or preventing, host immune responses. Pigs with high viral levels showed larger CE of immune cells than low viral level pigs, potentially to trigger stronger immune responses. Presence of high tonsil virus was associated with a stronger immune response, especially innate immune response through interferon signaling, but these differences were not significant when accounting for CE. Genotype at WUR was associated with different effects on immune response in tonsils of pigs during the persistence stage, depending on viral isolate and tonsil viral level., Conclusions: Results of this study provide insights into the effects of PRRSV isolate, tonsil viral level, and WUR genotype on host immune response and into potential mechanisms of PRRSV persistence in tonsils that could be targeted to improve strategies to reduce viral rebreaks. Finally, to understand transcriptome responses in tissues that consist of multiple cell types, it is important to consider differences in cell composition.

Protective humoral memory forms in secondary lymphoid organs where B cells undergo affinity maturation and differentiation into memory or plasma cells. Here, we provide a comprehensive roadmap of human B cell maturation with single-cell transcriptomics matched with bulk and single-cell antibody repertoires to define gene expression, antibody repertoires, and clonal sharing of B cell states at single-cell resolution, including memory B cell heterogeneity that reflects diverse functional and signaling states. We reconstruct gene expression dynamics during B cell activation to reveal a pre-germinal center state primed to undergo class switch recombination and dissect how antibody class-dependent gene expression in germinal center and memory B cells is linked with a distinct transcriptional wiring with potential to influence their fate and function. Our analyses reveal the dynamic cellular states that shape human B cell-mediated immunity and highlight how antibody isotype may play a role during their antibody-based selection., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Follicular helper CD4 T (Tfh) cells play an essential role in the formation of germinal centers (GCs), where mature B cells proliferate, differentiate, and provide long-term protective humoral responses. Despite the extensive phenotypic characterization and identification of human Tfh cell subsets, their spatial positioning at tissue level is not well understood. Here, we describe a quantitative multiplexed immunofluorescence approach allowing for the comprehensive in situ characterization of Tfh cells in human tonsils and lymph nodes (LNs) from individuals with angioimmunoblastic T-cell lymphoma (AITL). We have developed eight multiplexed panels comprising a spectrum of Tfh cell markers, like PD-1, CXCR5, and ICOS, along with transcription factors (Bcl6, Tbet, GATA3), to assess their expression, frequencies, spatial distribution and co-localization in a quantitative manner. Combined analysis of relevant markers revealed the presence of several Tfh cell subsets at tissue level based on the differential expression of surface receptors, nuclear factors as well as their distinct localization within the follicular areas. Interestingly, we found a considerable amount of tonsillar Tfh cells expressing high levels of the Th2 regulator GATA3. The co-expression of GATA3, CXCR5, and BCL6, points to an important role of GATA3 for the generation of effector human Tfh cells. Furthermore, our data revealed significantly different Tfh cell profile signatures between health and disease. Therefore, our imaging platform generates meaningful information for the in situ characterization of human Tfh cells and could provide the base for future studies aiming to a comprehensive understanding of Tfh cell tissue heterogeneity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ioannidou, Ndiaye, Noto, Fenwick, Fortis, Pantaleo, Petrovas and de Leval.)

Humanized antibodies targeting programmed death receptor 1 (PD-1) or its ligand (PD-L1) have been approved for the treatment of different cancers. Some of these antibodies show a correlation between the tissue expression of PD-L1 and response. Evaluation of PD-L1 expression presents multiple challenges, but some preanalytical issues such as tissue fixation have been scarcely evaluated. With the hypothesis that immunohistochemical staining of PD-L1 may be impacted by the time of specimen fixation, we evaluated differences in its expression in tonsil samples exposed to predefined fixation times. Random nontumoral tonsillectomy specimens were blindly evaluated in tissue microarray slides after staining with SP142 and SP263 antibodies. With fixation times ranging from 12 to 72 hours, between 2.8% and 6.1% of the samples were considered to be suboptimally stained, with no differences between the 2 antibodies within these fixation times. A significantly higher proportion of samples exposed to a fixation time of 96 hours presented suboptimal immunostaining (15.6%, P<0.0001). In addition, suboptimally stained spots were 20.8% using SP142 and 10.4% using SP263 after 96 hours of fixation (P=0.046). In conclusion, the quality of staining for PD-L1 in tonsil samples decreased with overfixation of the specimen at times >72 hours. Samples exposed to formaldehyde for longer periods presented suboptimal results for both clones, but the SP142 antibody presented a significantly lower tolerance to formalin overexposure than SP263. These results indicate the relevance of a controlled preanalytical processing of samples and particularly the length of fixation of tumor specimens.

Immunoglobulin A nephropathy (IgAN) involves repeated events of gross haematuria with concurrent upper airway infections. The mucosal immune system, especially the tonsil, is considered the initial site of inflammation, although the role of the tonsillar microbiota has not been established in IgAN. In this study, we compared the tonsillar microbiota of patients with IgAN (n = 21) and other glomerular diseases (n = 36) as well as, healthy controls (n = 23) from three medical centres in Korea. The microbiota was analysed from tonsil swabs using the Illumina MiSeq system based on 16S rRNA gene. Tonsillar bacterial diversity was higher in IgAN than in other glomerular diseases, although it did not differ from that of healthy controls. Principal coordinates analysis revealed differences between the tonsillar microbiota of IgAN and both healthy and disease controls. The proportions of Rahnella, Ruminococcus&#95;g2, and Clostridium&#95;g21 were significantly higher in patients with IgAN than in healthy controls (corrected p < 0.05). The relative abundances of several taxa were correlated with the estimated glomerular filtration rate, blood urea nitrogen, haemoglobin, and serum albumin levels. Based on our findings, tonsillar microbiota may be associated with clinical features and possible immunologic pathogenesis of IgAN.