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1.
Int J Mol Sci ; 22(16)2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34445351

RESUMO

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.


Assuntos
Diagnóstico por Imagem/métodos , Proteínas/metabolismo , Análise de Célula Única/métodos , Anticorpos/metabolismo , Comunicação Celular , Imunofluorescência/métodos , Corantes Fluorescentes/química , Corantes Fluorescentes/farmacocinética , Formaldeído/química , Peroxidase do Rábano Silvestre/análise , Peroxidase do Rábano Silvestre/metabolismo , Humanos , Técnicas Imunoenzimáticas/métodos , Tonsila Palatina/química , Tonsila Palatina/citologia , Tonsila Palatina/metabolismo , Inclusão em Parafina , Proteínas/análise , Sensibilidade e Especificidade , Coloração e Rotulagem/métodos
2.
Nat Commun ; 12(1): 4372, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34272370

RESUMO

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.


Assuntos
Aloenxertos/imunologia , Linfócitos B/metabolismo , Rejeição de Enxerto/imunologia , Inflamação/metabolismo , Transplante de Rim/efeitos adversos , Animais , Autoanticorpos/imunologia , Linfócitos B/imunologia , Linfócitos B/patologia , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Ontologia Genética , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Imunoglobulina G/imunologia , Rim/imunologia , Rim/metabolismo , Camundongos , Tonsila Palatina/imunologia , Tonsila Palatina/metabolismo , RNA-Seq , Análise de Célula Única , Transplante Homólogo
3.
Int J Mol Sci ; 22(11)2021 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-34071285

RESUMO

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.


Assuntos
Antígenos CD40/metabolismo , Imunomodulação , Células-Tronco Mesenquimais/metabolismo , Tonsila Palatina/metabolismo , Linfócitos T CD4-Positivos , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura , Citocinas/metabolismo , Fator de Transcrição GATA3/metabolismo , Humanos , Imunidade , Indolamina-Pirrol 2,3,-Dioxigenase , Linfócitos T/imunologia
4.
Commun Biol ; 4(1): 563, 2021 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-33980982

RESUMO

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 (ILCFR) that to our knowledge has not been previously identified. ILCFR have a distinct phenotype and transcriptional program when compared to other defined ILCs. Surprisingly, ILCFR inhibit the ability of follicular helper T (Tfh) cells to provide B cell help. The localization of ILCFR 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-ß. 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 ILCFR in regulating GC-Tfh-GC-B cell interactions and propose they expand in chronic inflammatory conditions.


Assuntos
Centro Germinativo/imunologia , Centro Germinativo/fisiologia , Linfócitos/imunologia , Adolescente , Adulto , Linfócitos B/imunologia , Criança , Pré-Escolar , Feminino , Humanos , Imunidade Inata/imunologia , Linfonodos/imunologia , Linfonodos/metabolismo , Ativação Linfocitária/imunologia , Linfócitos/metabolismo , Masculino , Tonsila Palatina/imunologia , Tonsila Palatina/metabolismo , Células T Auxiliares Foliculares/imunologia
5.
Carbohydr Polym ; 264: 117992, 2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-33910730

RESUMO

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.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Quitosana/farmacologia , Células-Tronco Mesenquimais/metabolismo , Tonsila Palatina/citologia , Ciclo Celular/efeitos dos fármacos , Células Cultivadas , Quitosana/química , Ciclina D1/metabolismo , Humanos , Osteogênese/efeitos dos fármacos , Consumo de Oxigênio , Tonsila Palatina/metabolismo , Polímeros/química , Polímeros/farmacologia , Engenharia Tecidual/métodos , Cicatrização/efeitos dos fármacos
6.
Molecules ; 26(8)2021 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-33921211

RESUMO

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.


Assuntos
Amidas/metabolismo , Corantes Fluorescentes/metabolismo , Proteômica , Epitopos/metabolismo , Células HeLa , Peroxidase do Rábano Silvestre , Humanos , Proteínas do Fator Nuclear 90/metabolismo , Tonsila Palatina/metabolismo , Inclusão em Parafina , Análise de Célula Única , Fixação de Tecidos
7.
Life Sci ; 276: 119432, 2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-33794253

RESUMO

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.


Assuntos
Citocinas/metabolismo , Células Epiteliais/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Hipóxia/fisiopatologia , Mediadores da Inflamação/metabolismo , Tonsila Palatina/patologia , Proteína SUMO-1/metabolismo , Permeabilidade da Membrana Celular , Células Cultivadas , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/patologia , Tonsila Palatina/imunologia , Tonsila Palatina/metabolismo , Proteína SUMO-1/genética
8.
BMC Vet Res ; 17(1): 88, 2021 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-33618723

RESUMO

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.


Assuntos
Tonsila Palatina/imunologia , Síndrome Respiratória e Reprodutiva Suína/imunologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/classificação , Animais , Genótipo , Imunidade Inata/genética , Tonsila Palatina/citologia , Tonsila Palatina/metabolismo , Tonsila Palatina/virologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/imunologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/isolamento & purificação , Sus scrofa , Suínos , Transcriptoma , Carga Viral/veterinária , Viremia/veterinária , Viremia/virologia
9.
Clin Immunol ; 226: 108697, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33636366

RESUMO

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.


Assuntos
Febre/diagnóstico , Doenças Hereditárias Autoinflamatórias/diagnóstico , Inflamação/diagnóstico , Interleucina-1/metabolismo , Linfadenite/diagnóstico , Faringite/diagnóstico , Estomatite Aftosa/diagnóstico , Complexo CD3/metabolismo , Pré-Escolar , Feminino , Febre/metabolismo , Gastroenteropatias/diagnóstico , Gastroenteropatias/metabolismo , Doenças Hereditárias Autoinflamatórias/metabolismo , Humanos , Inflamação/metabolismo , Linfadenite/metabolismo , Masculino , Tonsila Palatina/metabolismo , Pediatria , Faringite/metabolismo , Estomatite Aftosa/metabolismo , Síndrome , Linfócitos T/metabolismo , Tonsilectomia/métodos
10.
J Clin Pathol ; 74(3): 149-156, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32669366

RESUMO

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.


Assuntos
Fatores de Transcrição Box Pareados/metabolismo , Manejo de Espécimes/métodos , Fixação de Tecidos/métodos , Mama/metabolismo , Dermatologia , Feminino , Fixadores , Formaldeído , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Laboratórios , Fatores de Transcrição Box Pareados/genética , Tonsila Palatina/metabolismo , Inclusão em Parafina , Pele/metabolismo , Coloração e Rotulagem
11.
Appl Immunohistochem Mol Morphol ; 29(1): 76-81, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32134754

RESUMO

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.


Assuntos
Anticorpos Monoclonais Humanizados/química , Antígeno B7-H1/biossíntese , Regulação da Expressão Gênica , Imuno-Histoquímica , Tonsila Palatina/metabolismo , Fixação de Tecidos , Feminino , Humanos , Masculino , Tonsila Palatina/patologia
12.
Sci Rep ; 10(1): 16206, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-33004860

RESUMO

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_g2, and Clostridium_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.


Assuntos
Bactérias/genética , Proteínas de Bactérias/genética , Glomerulonefrite por IGA/microbiologia , Nefropatias/microbiologia , Tonsila Palatina/microbiologia , RNA Ribossômico 16S/análise , Adulto , Bactérias/classificação , Bactérias/isolamento & purificação , Estudos de Casos e Controles , Feminino , Perfilação da Expressão Gênica , Glomerulonefrite por IGA/genética , Humanos , Nefropatias/genética , Masculino , Pessoa de Meia-Idade , Tonsila Palatina/metabolismo , RNA Ribossômico 16S/genética
13.
Sci Rep ; 10(1): 13006, 2020 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-32747802

RESUMO

The aim of this study was to examine T cell function in tonsils of patients with recurrent acute tonsillitis (RAT) or peritonsillar abscess (PTA) by analyzing the cytokine production following T cell receptor (TCR) and co-receptor stimulation with a combination of anti-CD3 and anti-CD28 antibodies. The release of IFN-γ, TNF-α, IL-2, IL-4, IL-6, IL-10 and IL-17A from isolated, stimulated T cells of 27 palatine tonsils (10 RAT, 7 PTA, 10 tonsils without inflammation) was measured via a bead-based flow cytometric analysis. The results were compared with the cytokine release of isolated peripheral T cells in a subset of the same patients (6 PTA, 4 patients without signs of inflammation in the blood). TCR stimulation increased the concentration of released cytokines in tonsil and blood as well as in different forms of inflammation and tissue with no inflammation. Stimulation increased the pro-inflammatory cytokines TNF-α, IFN-γ, and IL-2 more than the anti-inflammatory cytokines IL-4 and IL-10 in tonsil and blood samples in RAT, PTA, and samples without inflammation. Blood of patients with PTA showed a higher pro-inflammatory cytokine level compared to the samples of patients without inflammation. T cells in tonsils are fully responsive and competent for antigen-induced cytokine production in RAT and PTA. One should be aware that tonsillectomy, if indicated, might remove a functioning immune organ. Tonsillotomy might be an alternative even in adults to maintain immunological function.


Assuntos
Citocinas/biossíntese , Tonsilite/metabolismo , Doença Aguda , Adulto , Feminino , Humanos , Ativação Linfocitária , Masculino , Tonsila Palatina/metabolismo , Recidiva , Linfócitos T/imunologia , Tonsilite/sangue , Tonsilite/imunologia
14.
Int J Mol Med ; 46(3): 1166-1174, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32582998

RESUMO

Bone marrow (BM) transplantation (BMT) represents a curative treatment for various hematological disorders. Prior to BMT, a large amount of the relevant anticancer drug needed to be administered to eliminate cancer cells. However, during this pre­BMT cytotoxic conditioning regimen, hematopoietic stem cells in the BM and thymic epithelial cells were also destroyed. The T cell receptor (TCR) recognizes diverse pathogen, tumor and environmental antigens, and confers immunological memory and self­tolerance. Delayed thymus reconstitution following pre­BMT cytotoxic conditioning impedes de novo thymopoiesis and limits T cell­mediated immunity. Several cytokines, such as RANK ligand, interleukin (IL)­7, IL­22 and stem cell factor, were recently reported to improve thymopoiesis and immune function following BMT. In the present study, it was found that the co­transplantation of tonsil­derived mesenchymal stromal cells (T­MSCs) with BM­derived cells (BMCs) accelerated the recovery of involuted thymuses in mice following partial pre­BMT conditioning with busulfan­cyclophosphamide treatment, possibly by inducing FMS­like tyrosine kinase 3 ligand (FLT3L) and fibroblast growth factor 7 (FGF7) production in T­MSCs. The co­transplantation of T­MSCs with BMCs also replenished the CD3+ cell population by inhibiting thymocyte apoptosis following pre­BMT cytotoxic conditioning. Furthermore, T­MSC co­transplantation improved the recovery of the TCR repertoire and led to increased thymus­generated T cell diversity.


Assuntos
Transplante de Medula Óssea/métodos , Células-Tronco Mesenquimais/citologia , Tonsila Palatina/citologia , Linfócitos T/citologia , Timo/citologia , Animais , Complexo CD3 , Feminino , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Masculino , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Tonsila Palatina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/metabolismo
15.
FASEB J ; 34(7): 9269-9284, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32413173

RESUMO

Monocytic cells perform crucial homeostatic and defensive functions. However, their fate and characterization at the transcriptomic level in human tissues are partially understood, often as a consequence of the lack of specific markers allowing their unequivocal identification. The 6-sulfo LacNAc (slan) antigen identifies a subset of non-classical (NC) monocytes in the bloodstream, namely the slan+ -monocytes. In recent studies, we and other groups have reported that, in tonsils, slan marks dendritic cell (DC)-like cells, as defined by morphological, phenotypical, and functional criteria. However, subsequent investigations in lymphomas have uncovered a significant heterogeneity of tumor-infiltrating slan+ -cells, including a macrophage-like state. Based on their emerging role in tissue inflammation and cancer, herein we investigated slan+ -cell fate in tonsils by using a molecular-based approach. Hence, RNA from tonsil slan+ -cells, conventional CD1c+ DCs (cDC2) and CD11b+ CD14+ -macrophages was subjected to gene expression analysis. For comparison, transcriptomes were also obtained from blood cDC2, classical (CL), intermediate (INT), NC, and slan+ -monocytes. Data demonstrate that the main trajectory of human slan+ -monocytes infiltrating the tonsil tissue is toward a macrophage-like population, displaying molecular features distinct from those of tonsil CD11b+ CD14+ -macrophages and cDC2. These findings provide a novel view on the terminal differentiation path of slan+ -monocytes, which is relevant for inflammatory diseases and lymphomas.


Assuntos
Amino Açúcares/metabolismo , Células Dendríticas/metabolismo , Macrófagos/metabolismo , Monócitos/metabolismo , Tonsila Palatina/metabolismo , Tonsilite/genética , Estudos de Casos e Controles , Células Cultivadas , Células Dendríticas/citologia , Perfilação da Expressão Gênica , Humanos , Macrófagos/citologia , Monócitos/citologia , Tonsila Palatina/citologia , Tonsilite/metabolismo , Tonsilite/patologia
16.
Cells ; 9(3)2020 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-32155780

RESUMO

Mesenchymal stromal cells (MSCs) from various sources exhibit different potential for stemness and therapeutic abilities. Recently, we reported a unique MSCs from human palatine tonsil (TMSCs) and their superior proliferation capacity compared to MSCs from other sources. However, unique characteristics of each MSC are not yet precisely elucidated. We investigated the role of stanniocalcin-1 (STC1), an anti-oxidative hormone, in the functions of TMSCs. We found that STC1 was highly expressed in TMSC compared with MSCs from bone marrow or adipose tissue. The proliferation, senescence and differentiation of TMSCs were assessed after the inhibition of STC1 expression. STC1 inhibition resulted in a significant decrease in the proliferation of TMSCs and did not affect the differentiation potential. To reveal the anti-oxidative ability of STC1 in TMSCs themselves or against other cell types, the generation of mitochondrial reactive oxygen species (ROS) in TMSC or ROS-mediated production of interleukin (IL)-1ß from macrophage-like cells were detected. Interestingly, the basal level of ROS generation in TMSCs was significantly elevated after STC1 inhibition. Moreover, down-regulation of STC1 impaired the inhibitory effect of TMSCs on IL-1ß production in macrophages. Taken together, these findings indicate that STC1 is highly expressed in TMSCs and plays a critical role in proliferating and ROS-regulatory abilities.


Assuntos
Glicoproteínas/metabolismo , Células-Tronco Mesenquimais/metabolismo , Tonsila Palatina/metabolismo , Proliferação de Células , Humanos , Tonsila Palatina/citologia , Espécies Reativas de Oxigênio , Transfecção
17.
Tissue Eng Regen Med ; 17(1): 105-119, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32002842

RESUMO

BACKGROUND: We first determined the efficacy of lesional injection of tonsil-derived MSCs (mesenchymal stem cells) for the treatment of 5-fluorouracil induced oral mucositis. METHODS: Oral mucositis was induced in hamsters by administration of 5-fluorouracil (day 0, 2, 4) followed by mechanical trauma (day 1, 2, 4). The experimental groups included MT (mechanical trauma only), 5-FU + MT (mechanical trauma with 5-fluorouracil administration), TMSC (mechanical trauma with 5-fluorouracil administration, tonsil-derived mesenchymal stem cells injection), DEXA (mechanical trauma with 5-fluorouracil administration, dexamethasone injection), and saline (mechanical trauma with 5-fluorouracil administration, saline injection). RESULTS: On day 10, gross and histologic analyses showed that nearly complete healing and epithelialization of the cheek mucosa of the TMSC group, whereas the other groups showed definite ulcerative lesions. Compared with the MT and DEXA groups, CD31 expression was greater in the TMSC group on days 10 and 14. Tendency towards a decrease in MMP2 expression with the time in the TMSC group was observed. In addition, the TMSC group showed higher expression of TGF-ß, and NOX4 on day 10 compared with the other groups. Scratch assay demonstrated that the conditioned media harvested from tonsil-derived MSCs significantly increased migratory efficacy of NIH3T3 cells. Transwell assay showed that the preferential migration of tonsil-derived MSCs to the wound area. CONCLUSION: Intralesional administration of tonsil-derived MSCs may accelerate wound healing of 5-fluorouracil induced oral mucositis by upregulating neovascularization and effective wound contraction. In addition, tonsil-derived MSCs might contribute to oral ulcer regeneration via the stimulation of fibroblast proliferation and migration.


Assuntos
Fluoruracila/efeitos adversos , Tonsila Palatina/metabolismo , Estomatite/induzido quimicamente , Cicatrização/efeitos dos fármacos , Cicatrização/fisiologia , Animais , Criança , Cricetinae , Meios de Cultivo Condicionados , Modelos Animais de Doenças , Feminino , Humanos , Imuno-Histoquímica , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Células-Tronco Mesenquimais/metabolismo , Camundongos , Mucosa Bucal/metabolismo , Células NIH 3T3 , Úlceras Orais , Tonsila Palatina/patologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Células-Tronco/efeitos dos fármacos , Estomatite/patologia , Fator de Crescimento Transformador beta
18.
PLoS One ; 15(2): e0228327, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32059005

RESUMO

Chronic wasting disease (CWD) continues to spread or be recognized in the United States, Canada, and Europe. CWD is diagnosed by demonstration of the causative misfolded prion protein (PrPCWD) in either brain or lymphoid tissue using immunodetection methods, with immunohistochemistry (IHC) recognized as the gold standard. In recent years, in vitro amplification assays have been developed that can detect CWD prion seeding activity in tissues, excreta, and body fluids of affected cervids. These methods potentially offer earlier and more facile detection of CWD, both pre- and post-mortem. Here we provide a longitudinal profile of CWD infection progression, as assessed by both real-time quaking-induced conversion (RT-QuIC) and IHC on serial biopsies of mucosal lymphoid tissues of white-tailed deer orally exposed to low doses of CWD prions. We report that detection of CWD infection by RT-QuIC preceded that by IHC in both tonsil and recto-anal lymphoid tissue (RAMALT) in 14 of 19 deer (74%). Of the 322 biopsy samples collected in post-exposure longitudinal monitoring, positive RT-QuIC results were obtained for 146 samples, 91 of which (62%) were concurrently also IHC-positive. The lower frequency of IHC positivity was manifest most in the earlier post-exposure periods and in biopsies in which lymphoid follicles were not detected. For all deer in which RT-QuIC seeding activity was detected in a tonsil or RAMALT biopsy, PrPCWD was subsequently or concurrently detected by IHC. Overall, this study (a) provides a longitudinal profile of CWD infection in deer after low yet infectious oral prion exposure; (b) illustrates the value of RT-QuIC for sensitive detection of CWD; and (c) demonstrates an ultimate high degree of correlation between RT-QuIC and IHC positivity as CWD infection progresses.


Assuntos
Imuno-Histoquímica , Técnicas de Amplificação de Ácido Nucleico/métodos , Doença de Emaciação Crônica/patologia , Administração Oral , Animais , Cervos , Progressão da Doença , Estudos Longitudinais , Tecido Linfoide/metabolismo , Tecido Linfoide/patologia , Tonsila Palatina/metabolismo , Tonsila Palatina/patologia , Proteínas Priônicas/genética , Proteínas Priônicas/metabolismo , Príons/administração & dosagem , Doença de Emaciação Crônica/metabolismo
19.
Mucosal Immunol ; 13(3): 460-470, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31896761

RESUMO

The human nasopharynx is frequently exposed to microbial pathogens, including superantigen-producing Staphylococcus aureus (SAg-Sau), which activates potent pro-inflammatory T cell responses. However, cellular mechanisms that control SAg-Sau-driven T cell activation are poorly understood. Using human nasopharynx-associated lymphoid tissue (NALT), we show that SAg-Sau drove a strong Th17 activation, which was associated with an impaired CD4+ T cell-mediated immune regulation. This impairment of immune control correlated with a significant downregulation of interleukin-35 (IL-35) expression in tonsillar CD4+ T cells by SAg-Sau. Supplementing recombinant IL-35 suppressed SAg-Sau-activated Th17 responses, and this IL-35-mediated suppression positively correlated with the level of Th17 activation. Interestingly, SAg-Sau stimulation induced Foxp3+ Treg expansion and interleukin-10 (IL-10) production, which effectively suppressed the Th1 response, but failed to control the activation of Th17 cells. Overall, our results reveal an aberrant T cell regulation on SAg-Sau-driven Th17 activation and identify IL-35 as a critical cytokine to control superantigenic S.aureus-activated Th17 responses.


Assuntos
Interleucinas/metabolismo , Tecido Linfoide/imunologia , Nasofaringe/imunologia , Staphylococcus aureus/imunologia , Superantígenos/imunologia , Células Th17/imunologia , Células Th17/metabolismo , Antígenos de Bactérias/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Humanos , Interleucina-10/metabolismo , Linfonodos/imunologia , Linfonodos/metabolismo , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Tonsila Palatina/imunologia , Tonsila Palatina/metabolismo , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo
20.
Q J Nucl Med Mol Imaging ; 64(3): 299-306, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30221906

RESUMO

BACKGROUND: The aim of this article was to evaluate the usefulness of sequential dual-time-point 2-deoxy-2-[18F]fluoro-D-glucose positron emission tomography/computed tomography (DTP [18F]FDG PET/CT) in distinguishing physiologic, inflammatory and malignant palatine tonsils as difficult to differentiate in the oncological practice. METHODS: A total of 90 patients before the treatment underwent sequential DTP [18F]FDG PET/CT examinations. We analyzed 104 structures in 90 patients: 31 physiologic tonsils, 28 histopathologically confirmed inflammatory tonsils of non-specified origin, 31 histopathologically confirmed palatine tonsils cancer and 14 non-malignant contralateral tonsils in patients with histopathologically confirmed unilateral palatine tonsil malignancy. Patients underwent sequential [18F]FDG PET/CT examinations at 60 and 90 minutes post-injection of the [18F]FDG. We analyzed the SUVmax and SUVmean values at 60 and 90 minutes post-injection changes over time and the Retention Index (RI-SUVmax). To find the predictive SUV value and the RI cut-off between physiology, inflammatory and malignancy, we used the ROC analysis. RESULTS: The average SUVmax values at 60 and 90minutes post-injection within physiologic palatine tonsils were 1.36±0.26 and 1.31±0.26, respectively, P>0.05. The average SUVmax values at 60 and 90 minutes post-injection within inflammatory and malignant tonsils were 3.74±1.45, 3.80±1.47 (P>0.05) and 5.19±2.19, 5.81±2.50 (P<0.05), respectively. The RI-SUVmax fluctuation over time were 5±28% within physiologic, -4±11% within contralateral non-malignant tonsils in patients with one tonsil involved, 2±11% within inflammatory and 13±13% within malignant tonsils. CONCLUSIONS: The sequential dual-time-point [18F]FDG PET/CT examinations may increase the sensitivity and the specificity of the PET/CT method in differential palatine tonsils diagnosis.


Assuntos
Fluordesoxiglucose F18 , Ácido Glucárico/metabolismo , Tonsila Palatina/diagnóstico por imagem , Tonsila Palatina/metabolismo , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Adulto , Idoso , Idoso de 80 Anos ou mais , Transporte Biológico , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Masculino , Pessoa de Meia-Idade , Curva ROC , Adulto Jovem
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