An atlas of cells in the human tonsil.

Palatine tonsils are secondary lymphoid organs (SLOs) representing the first line of immunological defense against inhaled or ingested pathogens. We generated an atlas of the human tonsil composed of >556,000 cells profiled across five different data modalities, including single-cell transcriptome, epigenome, proteome, and immune repertoire sequencing, as well as spatial transcriptomics. This census identified 121 cell types and states, defined developmental trajectories, and enabled an understanding of the functional units of the tonsil. Exemplarily, we stratified myeloid slan-like subtypes, established a BCL6 enhancer as locally active in follicle-associated T and B cells, and identified SIX5 as putative transcriptional regulator of plasma cell maturation. Analyses of a validation cohort confirmed the presence, annotation, and markers of tonsillar cell types and provided evidence of age-related compositional shifts. We demonstrate the value of this resource by annotating cells from B cell-derived mantle cell lymphomas, linking transcriptional heterogeneity to normal B cell differentiation states of the human tonsil.

Mapping Human Immunity and the Education of Waldeyer's Ring.

The development and deployment of single-cell genomic technologies have driven a resolution revolution in our understanding of the immune system, providing unprecedented insight into the diversity of immune cells present throughout the body and their function in health and disease. Waldeyer's ring is the collective name for the lymphoid tissue aggregations of the upper aerodigestive tract, comprising the palatine, pharyngeal (adenoids), lingual, and tubal tonsils. These tonsils are the first immune sentinels encountered by ingested and inhaled antigens and are responsible for mounting the first wave of adaptive immune response. An effective mucosal immune response is critical to neutralizing infection in the upper airway and preventing systemic spread, and dysfunctional immune responses can result in ear, nose, and throat pathologies. This review uses Waldeyer's ring to demonstrate how single-cell technologies are being applied to advance our understanding of the immune system and highlight directions for future research.

A novel cryopreservation and biobanking strategy to study lymphoid tissue stromal cells in human disease.

Nonhematopoietic lymph node stromal cells (LNSCs) regulate lymphocyte trafficking, survival, and function for key roles in host defense, autoimmunity, alloimmunity, and lymphoproliferative disorders. However, the study of LNSCs in human diseases is complicated by a dependence on viable lymphoid tissues, which are most often excised prior to establishment of a specific diagnosis. Here, we demonstrate that cryopreservation can be used to bank lymphoid tissue for the study of LNSCs in human disease. Using human tonsils and lymph nodes (LN), lymphoid tissue fragments were cryopreserved for subsequent enzymatic digestion and recovery of viable nonhematopoietic cells. Flow cytometry and single-cell transcriptomics identified comparable proportions of LN stromal cell types in fresh and cryopreserved tissue. Moreover, cryopreservation had little effect on transcriptional profiles, which showed significant overlap between tonsils and LN. The presence and spatial distribution of transcriptionally defined cell types were confirmed by in situ analyses. Our broadly applicable approach promises to greatly enable research into the roles of LNSCs in human disease.

Podoplanin depletion in tonsil-derived mesenchymal stem cells induces cellular senescence via regulation of the p16Ink4a/Rb pathway.

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 p16Ink4a/Rb pathway activation. CONCLUSION: PDPN may serve as a novel biomarker to mitigate cellular senescence in the clinical application of MSCs.

Multimodal optical mesoscopy reveals the quantity and spatial distribution of Gram-positive biofilms in ex vivo tonsils.

Biofilms are known to be present in tonsils, but little is known about their spatial location and size distribution throughout the tonsil. Studies of the location and distribution of biofilms in tonsil specimens have thus far been limited to either high-magnification methods such as electron microscopy, which enables high-resolution imaging but only from a tiny tissue volume, or lower magnification techniques such as light microscopy, which allow imaging of larger specimens but with poor spatial resolution. To overcome these limitations, we report the use of multimodal optical mesoscopy to visualise and quantify the number and spatial distribution of Gram-positive biofilms in fresh, excised paediatric tonsils. This methodology supports simultaneous imaging of both the tonsil host and biofilms in whole mounts of tissue up to 5 mm × 5 mm × 3 mm with subcellular resolution throughout. A quantitative assessment of 36 tonsil specimens revealed no statistically significant difference between biofilm presence on the tonsil surface and the interior of the tonsil. This new quantitative mesoscale imaging approach may prove useful in understanding the role of biofilms in tonsillar diseases and other infections.

Characterization of pig tonsils as niches for the generation of Streptococcus suis diversity.

Streptococcus suis is a gram-positive bacterium that causes meningitis, septicemia, endocarditis, and other disorders in pigs and humans. We obtained 42 and 50 S. suis isolates from lesions of porcine endocarditis and palatine tonsils, respectively, of clinically healthy pigs in Japan; we then determined their sequence types (STs) by multilocus sequence typing (MLST), cps genotypes, serotypes, and presence of classical major virulence-associated marker genes (mrp, epf, and sly). The 42 isolates from endocarditis lesions were assigned to a limited number of STs and clonal complexes (CCs). On the other hand, the 50 isolates from tonsils were diverse in these traits and seemingly in the degree of virulence, suggesting that tonsils can accommodate a variety of S. suis isolates. The goeBURST full algorithm using tonsil isolates obtained in this study and those retrieved from the database showed that major CCs as well as many other clusters were composed of isolates originating from different countries, and some of the STs were very similar to each other despite the difference in country of origin. These findings indicate that S. suis with not only different but also similar mutations in the genome have survived in tonsils independently across different geographical locations. Therefore, unlike the lesions of endocarditis, the tonsils of pigs seemingly accommodate various S. suis lineages. The present study suggests that S. suis acquired its diversity by natural mutations during colonization and persistence in the tonsils of pigs.

Expression profile of Toll-like receptors and cytokines in the cecal tonsil of chickens challenged with Eimeria tenella.

Chicken coccidiosis, caused by Eimeria spp., seriously affects the development of the poultry breeding industry. Currently, extensive studies of chicken coccidiosis are mostly focused on acquired immune responses, while information about the innate immune response of chicken coccidiosis is lacking. Toll-like receptor (TLR), the key molecule of the innate immune response, connects innate and adaptive immune responses and induces an immune response against various pathogen infections. Therefore, the quantitative real-time PCR was used to characterize the expression profile of chicken TLRs (chTLRs) and associated cytokines in the cecal tonsil of chickens infected with Eimeria tenella. The results showed that the expression of chTLR1a, chTLR2a, and chTLR5 was significantly upregulated at 3 h post-infection, while chTLR1b, chTLR2b, chTLR3, chTLR7, chTLR15 and chTLR21 was significantly downregulated (p < 0.05). In addition, chTLR1a expression rapidly reached the peaked expression at 3 h post-infection, while chTLR2b and chTLR15 peaked at 168 h post-infection, and chTLR2a expression was highest among chTLRs, peaking at 48 h post-infection (p < 0.05). For cytokines, interleukin (IL)-6 and tumor necrosis factor (TNF)-α peaked at 96 h post-infection, IL-4 and IL-12 peaked at 144 h post-infection, and interferon-γ expression was highest among cytokines at 120 h post-infection. In addition, IL-12 and IL-17 were markedly upregulated at 6 h post-infection (p < 0.05). These results provide insight into innate immune molecules during E. tenella infection in chickens and suggest that innate immune responses may mediate resistance to chicken coccidiosis.

Risk of lymph node metastasis in T1 tonsil squamous cell carcinomas patients according to age stratification at diagnosis.

BACKGROUND: The objective of this study is to assess the association between age and lymph nodes metastasis (LNM) in T1 tonsil squamous cell carcinomas (TSCC) patients. METHODS: Patients with T1 TSCC were extracted from the SEER database between 2005 and 2014. Univariate and multivariate logistic regression models were produced to recognize the association between age and risk factors of LNM. RESULTS: A total of 2430 patients were analyzed. Younger patients more frequently presented with LNM compared to their older peers (P < 0.01, respectively.). In multivariate analyses, older age was associated with a significantly lower risk of LNM. Compared to patients aged 29-39-years-old, the hazard ratios for patients aged 40-49, 50-59, 60-69, and 70-88 years old were 0.911 (95 % confidence interval [CI] 0.370-2.245), 0.641 (95 % CI 0.268-1.535), 0.511 (95 % CI 0.212-1.231), and 0.236 (95 % CI 0.095-0.584), respectively. Subgroups analysis shows that the effect of older age was significantly associated with a lower risk of LNM in all groups except for Asian patients (P < 0.05, respectively). CONCLUSION: Our study demonstrates that younger patients with T1 TSCC had a higher risk of LNM than their old peers and the effect of older age was significantly associated with a lower risk of LNM in all groups except for Asian patients. More accurate assessments of LNM and prophylactic neck dissection or prophylactic adjuvant radiation therapy to neck will be imperative for reducing recurrence in younger T1 TSCC.

Predictors of surgical outcomes for limited palatal muscle resection in patients with obstructive sleep apnea.

OBJECTIVE: Limited palatal muscle resection (LPMR) is a modified palatal surgical technique to correct retropalatal obstruction without complications. This study aims to determine the associated factors affecting the success and cure rate of LPMR in patients with obstructive sleep apnea (OSA), thus guiding patient selection and improving surgical outcome. METHODS: Thirty-five OSA patients underwent LPMR were enrolled. All patients received routine physical examination, preoperative drug-induced sleep endoscopy (DISE), and polysomnography (PSG). Clinical, polysomnographic, cephalometric variables, and DISE findings were evaluated. These measurements were compared between the surgical success and failure group based on the results of preoperative and postoperative PSG. Furthermore, we compared the cured and non-cured groups in the surgical success group. RESULTS: Among 35 patients, the overall success rate was 57 % with a cure rate of 31.4 %. Patients with Friedman stage II had a significantly higher success rate (p = 0.032). According to DISE results, tongue base obstruction affected the surgical outcome (p < 0.001). The success rate was 100 % in the no tongue base obstruction during DISE, 72.2 % in the partial obstruction, and 9.1 % in the total obstruction. Tonsil size is also helpful in predicting surgical success rate (p = 0.041). Furthermore, patients with mild AHI were more likely to be surgical cures. when compared with patients with severe AHI (p = 0.044). CONCLUSION: Patients with larger tonsil size and no tongue base obstruction during DISE may have a higher chance of surgical success with LPMR. The lower AHI may be predictors of surgical cure after LPMR.

Post-tonsillectomy bleeding rate decreases with limitation in maximum post-operative ibuprofen dosage: A quality improvement study.

OBJECTIVE: The objective of this study is to investigate the effect of a reduction in the prescribed post-operative ibuprofen dosage on frequency of post-tonsillectomy bleeding. METHODS: A quality improvement study was conducted at a single tertiary care pediatric hospital with patients weighing >40 kg undergoing tonsillectomy. The intervention was limiting the post-operative ibuprofen dosage to a maximum of 400 mg per dose. Data was collected on all patients returning to the hospital with bleeding after tonsillectomy. The primary outcome was the rate of post-tonsillectomy bleeding. Statistical analysis was conducted using nonparametric comparisons and a run chart. RESULTS: A total of 199 tonsillectomy patients >40 kg were included in the study. There were 119 (59.8 %) females and 80 (40.2 %) males total. The pre-intervention group had a total of 56 patients while the post-intervention group had a total of 143 patients. There was no statistical difference in age, weight, or sex between the pre- and post-intervention groups (p > .05). The post-tonsillectomy hemorrhage rate was 11/56 (19.6 %) before the intervention, and 11/143 (7.7 %) after the intervention (p = .016). Children who experienced a bleeding event were significantly older (mean 15.9 years, 95 % CI 14.5-17.3) than those who did not (13.5 years, 95 % CI 12.9-14.1; p = .011). The run chart revealed that the intervention resulted in a nonrandom decrease in rate of post-tonsillectomy bleeding. CONCLUSIONS: Post-tonsillectomy bleeding rate decreased with a ceiling post-operative ibuprofen dose of 400 mg/dose in this quality improvement study. Further research is warranted.

Unilateral Neck Treatment with either Surgery and/or Radiotherapy for Squamous Cell Carcinoma for the Tonsil.

INTRODUCTION: Strategies for treatment of tonsil carcinoma are under active investigation. Limiting surgical and radiation treatment volumes to the primary tumor and ipsilateral neck in appropriately selected patients are one such approach. Here, we present our institutional experience with treatment through ipsilateral surgical or radiotherapeutic neck management. METHODS: We retrospectively reviewed our institutional database of patients with tonsil carcinoma treated from 2012 to 2020. Patients were included for analysis if they received definitive radiation therapy (RT), definitive surgery (S), or surgery with postoperative radiation therapy (S-PORT) and whose treatment volumes were limited to the primary tumor and involved/elective ipsilateral neck. Patients who received radiation and/or surgery to the contralateral neck (including those with bilateral nodal involvement), as well as patients with metastatic disease, were excluded. Clinical factors including T- and N-stage (AJCC 7th edition), and HPV status (by p16 and/or HPV DNA PCR) were recorded, as were pathologic factors (when applicable) including margin status, extracapsular extension (ECE), lymphovascular invasion (LVSI), and perineural invasion (PNI). Overall survival (OS), progression-free survival (PFS), and locoregional control (LRC) at 2 years were estimated using the Kaplan-Meier method. RESULTS: In total, 71 patients were treated with unilateral neck approaches: S (n = 49), RT (n = 10), and S+PORT (n = 12). Among these patients, 32, 36, and 3 had T1, T2, and T3 disease, respectively. N-stage was N0, N1, N2a, N2b, and N3 in 22, 20, 5, 23, and 1 patient(s), respectively. Concurrent chemotherapy was administered in 12 patients. From those with recorded risk factors, 86% were HPV positive, 20% had LVSI, 7% had PNI, 13% had ECE, and 5% had positive margins. From a median follow-up of 27 months, local, regional, and distant failures occurred in 5, 6, and 5 patients, respectively. No contralateral neck failures were recorded. At 2 years, OS, PFS, and LRC were 92% (95% CI 85-99%), 85% (95% CI 75-95%), and 88% (95% CI 80-98%), respectively. CONCLUSIONS: In patients with early T-stage tonsil carcinoma, treatment of the primary tumor and ipsilateral neck is associated with acceptable OS, PFS, and LRC. In this population, the risk of contralateral neck failure is likely very low regardless of primary treatment modality. Additional prospective studies are needed to determine the impact of limiting treatment extent, either surgical or radiotherapeutic, to the unilateral neck.

Surgical anatomy of the cerebellar tonsils: A cadaveric study.

BACKGROUND: Knowledge of the normal anatomy of the cerebellar tonsils is a prerequisite in various surgeries of the posterior cranial fossa Clinical conditions, as the Chiari I malformations (CIM) alter the normal position of the cerebellar tonsils. OBJECTIVE: Therefore, we aim to better elucidate the surgical anatomy of and around the cerebellar tonsils in regard to the CIM. METHODS: Fifty formalin-fixed adult cadavers injected with colored latex through vertebral arteries underwent craniotomy and durotomy to expose the cerebellar tonsils and related structures. The tonsils and their surrounding anatomy were then studied. RESULTS: Forty cerebellar tonsils were at or above the foramen magnum. Five specimens presented with CIM with the tonsils below (3-5 mm) the FM with a mean tonsillar decent of 7.9 ± 2.3 mm. Of the cadavers without CIM, in forty-two cases, the thickness of the dura mater was within ±3SD ranges. In three cases, the dura mater was thinner at the CVJ and one case; the dura adhered tightly to the inner aspect of the occipital squama. In five CIM cadavers, the dura mater was markedly thicker at the CVJ. The PICA caudal loop was 5.9 ± 1.6 mm long. In CIM cases, the PICA loop was longer, nearer the dura, 1 mm below the superior border of the C1 posterior arch. The distances from the PICA loop were markedly reduced by 3 mm from the spinal accessory nerve and 2 mm from the first spinal nerve. The DN was significantly closer to the tonsillar peduncle in CIM cases. CONCLUSION: These data are important for better understanding the intrinsic and extrinsic anatomy of the cerebellar tonsils in patients with and without CIM. Importantly, tonsillectomy/tonsillar coagulation must consider the close relationship of the dentate nucleus to the base of the cerebellar tonsil to avoid iatrogenic injury.

Age-group-specific associations between adenoid/tonsillar hypertrophy and craniofacial features.

BACKGROUND: Age plays an important role in the association between adenotonsillar hypertrophy and craniofacial morphology. This study aimed to analyse the association of adenoid and tonsillar hypertrophy with craniofacial features in different age groups. METHODS: Lateral cephalograms were obtained from 942 patients aged 6-15 years (433 boys, 509 girls). They were divided into three age groups: 6-9 years (n = 189), 9-12 years (n = 383), and 12-15 years (n = 370). According to the different sites of pharyngeal obstruction, they were classified as control group (CG), adenoid hypertrophy group (AG), tonsillar hypertrophy group (TG) and adenotonsillar hypertrophy group (ATG). Cephalometric measurements were performed on each enrolled participant. Comparisons between groups and correlations between these cephalometric variables and obstruction sites were evaluated. RESULTS: At 6-9 years of age, ATG and TG correlated with increased mandibular height (B = 2.2, p = 0.029; B = 2.6, p = 0.042, respectively). At the age of 9-12 years, AG showed a steep growth direction (B = 1.5, p = 0.002), TG showed a higher probability of Class III skeletal pattern (smaller SNB, ANB and SGn/FH, larger Go-Me) and ATG manifested a higher proportion of Class III skeletal pattern. At 12-15 years of age, there was no significant association between cephalometric measurements and pharyngeal lymphoid tissue enlargement. CONCLUSIONS: Children with isolated adenoid hypertrophy have a vertical growth direction at 9-12 years of age. Isolated adenoid hypertrophy correlated with longer mandibular body, more anterior mandible and horizontal skeletal Class III pattern at 6-12 years. Combination of obstructive adenoids and tonsils manifested similarly to children with isolated tonsil hypertrophy.

Patient with actinomycosis of the cervicofacial and abdominal area, case report. / Wielopostaciowosc promienicy ­ jednoczesna aktynomikoza obszaru szyjno-twarzowego i brzusznego, opis przypadku.

Actinomycosis is a very rare, infectious disease, which is especially difficult to diagnose due to non-specific symptoms and the ability to emulate neoplasms or inflammatory changes. Due to those facts, it is often misdiagnosed or diagnosed too late to be successfully treated. This article presents the case of 31-year-old Caucasian female with recurrent upper respiratory tract infections and tonsillitis as the potential risk factors of actinomycosis. Upon examination of material collected through the course of tonsillectomy, the patient was diagnosed with actinomycosis of the left palatine tonsil. Despite the introduction of antibiotic therapy, initial progression was noted with the appearance of numerous, hypodense changes in the liver and the spleen, which regressed during further antibiotic treatment. According to our team's knowledge, this is the first described case of a patient with actinomycosis occurring simultaneously in the cervico-facial and abdominal area. The unusual localization and potential dissemination of actinomycosis should be considered in clinical practice.

Stress Triggers Expression of Bovine Herpesvirus 1 Infected Cell Protein 4 (bICP4) RNA during Early Stages of Reactivation from Latency in Pharyngeal Tonsil.

Bovine herpesvirus 1 (BoHV-1), an important pathogen of cattle, establishes lifelong latency in sensory neurons within trigeminal ganglia (TG) after acute infection. The BoHV-1 latency-reactivation cycle, like other alphaherpesvirinae subfamily members, is essential for viral persistence and transmission. Notably, cells within pharyngeal tonsil (PT) also support a quiescent or latent BoHV-1 infection. The synthetic corticosteroid dexamethasone, which mimics the effects of stress, consistently induces BoHV-1 reactivation from latency allowing early stages of viral reactivation to be examined in the natural host. Based on previous studies, we hypothesized that stress-induced cellular factors trigger expression of key viral transcriptional regulatory genes. To explore this hypothesis, RNA-sequencing studies compared viral gene expression in PT during early stages of dexamethasone-induced reactivation from latency. Strikingly, RNA encoding infected cell protein 4 (bICP4), which is translated into an essential viral transcriptional regulatory protein, was detected 30 min after dexamethasone treatment. Ninety minutes after dexamethasone treatment bICP4 and, to a lesser extent, bICP0 RNA were detected in PT. All lytic cycle viral transcripts were detected within 3 h after dexamethasone treatment. Surprisingly, the latency related (LR) gene, the only viral gene abundantly expressed in latently infected TG neurons, was not detected in PT during latency. In TG neurons, bICP0 and the viral tegument protein VP16 are expressed before bICP4 during reactivation, suggesting distinct viral regulatory genes mediate reactivation from latency in PT versus TG neurons. Finally, these studies confirm PT is a biologically relevant site for BoHV-1 latency, reactivation from latency, and virus transmission. IMPORTANCE BoHV-1, a neurotropic herpesvirus, establishes, maintains, and reactivates from latency in neurons. BoHV-1 DNA is also detected in pharyngeal tonsil (PT) from latently infected calves. RNA-sequencing studies revealed the viral infected cell protein 4 (bICP4) RNA was expressed in PT of latently infected calves within 30 min after dexamethasone was used to initiate reactivation. As expected, bICP4 RNA was not detected during latency. All lytic cycle viral genes were expressed within 3 h after dexamethasone treatment. Conversely, bICP0 and the viral tegument protein VP16 are expressed prior to bICP4 in trigeminal ganglionic neurons during reactivation. The viral latency related gene, which is abundantly expressed in latently infected neurons, was not abundantly expressed in PT during latency. These studies provide new evidence PT is a biologically relevant site for BoHV-1 latency and reactivation. Finally, we predict other alphaherpesvirinae subfamily members utilize PT as a site for latency and reactivation.

Removal of the Erns RNase Activity and of the 3' Untranslated Region Polyuridine Insertion in a Low-Virulence Classical Swine Fever Virus Triggers a Cytokine Storm and Lethal Disease.

In this study, we assessed the potential synergistic effect of the Erns RNase activity and the poly-U insertion in the 3' untranslated region (UTR) of the low-virulence classical swine fever virus (CSFV) isolate Pinar de Rio (PdR) in innate and adaptive immunity regulation and its relationship with classical swine fever (CSF) pathogenesis in pigs. We knocked out the Erns RNase activity of PdR and replaced the long polyuridine sequence of the 3' UTR with 5 uridines found typically at this position, resulting in a double mutant, vPdR-H30K-5U. This mutant induced severe CSF in 5-day-old piglets and 3-week-old pigs, with higher lethality in the newborn (89.5%) than in the older (33.3%) pigs. However, the viremia and viral excretion were surprisingly low, while the virus load was high in the tonsils. Only alpha interferon (IFN-α) and interleukin 12 (IL-12) were highly and consistently elevated in the two groups. Additionally, high IL-8 levels were found in the newborn but not in the older pigs. This points toward a role of these cytokines in the CSF outcome, with age-related differences. The disproportional activation of innate immunity might limit systemic viral spread from the tonsils and increase virus clearance, inducing strong cytokine-mediated symptoms. Infection with vPdR-H30K-5U resulted in poor neutralizing antibody responses compared with results obtained previously with the parent and RNase knockout PdR. This study shows for the first time the synergistic effect of the 3' UTR and the Erns RNase function in regulating innate immunity against CSFV, favoring virus replication in target tissue and thus contributing to disease severity. IMPORTANCE CSF is one of the most relevant viral epizootic diseases of swine, with high economic and sanitary impact. Systematic stamping out of infected herds with and without vaccination has permitted regional virus eradication. However, the causative agent, CSFV, persists in certain areas of the world, leading to disease reemergence. Nowadays, low- and moderate-virulence strains that could induce unapparent CSF forms are prevalent, posing a challenge for disease eradication. Here, we show for the first time the synergistic role of lacking the Erns RNase activity and the 3' UTR polyuridine insertion from a low-virulence CSFV isolate in innate immunity disproportional activation. This might limit systemic viral spread to the tonsils and increase virus clearance, inducing strong cytokine-mediated symptoms, thus contributing to disease severity. These results highlight the role played by the Erns RNase activity and the 3' UTR in CSFV pathogenesis, providing new perspectives for novel diagnostic tools and vaccine strategies.

Exploring differentially expressed genes of Staphylococcus aureus exposed to human tonsillar cells using RNA sequencing.

BACKGROUND: The nose and the throat are the most predominant colonizing sites of Staphylococcus aureus, and colonization is a risk factor for infection. Nasal colonization is well described; however, we have limited knowledge about S. aureus throat colonization. The main objective of this study was to explore differentially expressed genes (DEGs) in S. aureus throat isolate TR145 exposed to human tonsil epithelial cells (HTEpiC) by using RNA sequencing (RNA-seq) and pathway analysis. DEGs in S. aureus at 1 or 3 hours (h) interaction with its host were explored. RESULTS: S. aureus was co-cultured in absence and presence of tonsillar cells at 1 or 3 h. Over the 3 h time frame, the bacteria multiplied, but still caused only minor cytotoxicity. Upon exposure to tonsillar cell line, S. aureus changed its transcriptomic profile. A total of 508 DEGs were identified including unique (1 h, 160 DEGs and 3 h, 78 DEGs) and commonly shared genes (1 and 3 h, 270 DEGs). Among the DEGs, were genes encoding proteins involved in adhesion and immune evasion, as well as iron acquisition and transport. Reverse transcription qPCR was done on selected genes, and the results correlated with the RNA-seq data. CONCLUSION: We have shown the suitability of using HTEpiC as an in vitro model for investigating key determinants in S. aureus during co-incubation with host cells. Several DEGs were unique after 1 or 3 h exposure to host cells, while others were commonly expressed at both time points. As their expression is induced upon meeting with the host, they might be explored further for future targets for intervention to prevent either colonization or infection in the throat.

Tonsillar transcriptional profiles in atopic and non-atopic subjects.

BACKGROUND: Emerging research suggests that local lymphatic tissue such as tonsils have important role in regulating the immune responses. However, allergen sensitization-induced alterations in transcriptome of tonsils are not known. OBJECTIVES: To examine the key differences in tonsillar gene expression between atopic and non-atopic subjects and further by type of sensitization. METHODS: RNA-sequencing was performed on 52 tonsillar samples from atopic and non-atopic tonsillectomy patients. Sensitization to common food- and aero-allergen was defined by allergen specific IgE. Following groups were studied: (1) aero- and food-allergen sensitized (AS+FS) versus non-sensitized (NS), (2) aeroallergen-sensitized (AS) versus food-allergen sensitized (FS), (3) AS versus NS, (4) FS versus NS. Bioinformatics analysis was done using DESeq2(v3.10.2), WGCNA and GATK pipeline in R software (v3.3.1). Protein-protein interaction network was made from String database. RESULTS: We studied 13 aeroallergen-sensitized, 6 food-allergen sensitized, 4 both food-and aero-allergen-sensitized and 29 non-sensitized tonsillectomy patients. Overall, 697 unique differentially expressed genes (DEGs) were detected in all sensitized subgroups including chemokines (CXCL2, CXCL8, CXCL10, CXCL11), IL-20RA, MUC1 and MUC20. When comparing different groups, the gene expression profiles overlapped except the AS versus FS group comparison, suggesting significantly different gene expression between the two sensitization subgroups. Furthermore, aeroallergen-sensitized subjects had more prominent immune responses compared with non-sensitized and food-allergen sensitized subjects including gene expression for IL-17 pathway and Toll-like receptor signalling pathway. CONCLUSION: Allergic sensitization is associated with extensive tonsillar transcriptomic alterations and changes in immune related genes and pathways. Distinct differences were found between aero-allergen and food-allergen sensitization.

Biochemical and functional characterization of skeletal muscle cells differentiated from tonsil-derived mesenchymal stem cells.

INTRODUCTION/AIMS: Human tonsils are a readily accessible source of stem cells for the potential treatment of skeletal muscle disorders. We reported previously that tonsil-derived mesenchymal stem cells (TMSCs) can differentiate into skeletal muscle cells (SKMCs), which renders TMSCs promising candidates for cell therapy for skeletal muscle disorders. However, the functional properties of the myocytes differentiated from mesenchymal stem cells have not been clearly evaluated. In this study we investigated whether myocytes differentiated from TMSCs (skeletal muscle cells derived from tonsil mesenchymal stem cells [TMSC-SKMCs]) exhibit the functional characteristics of SKMCs. METHODS: To test the insulin reactivity of TMSC-SKMCs, the expression of glucose transporter 4 (GLUT4) and phosphatidylinositol 3-kinase/Akt was analyzed after the cells were treated for 30 minutes with 100 nmol/L insulin in normal or high-glucose medium. We also examined whether these cells formed a neuromuscular junction (NMJ) when cocultured with motor neurons, and whether they were stimulated by electrical signals using whole-cell patch clamping. RESULTS: Skeletal muscle cells derived from tonsil mesenchymal stem cells expressed SKMC markers, such as MYOD, MYH3, MYH8, TNNI1, and TTN, at high levels, and exhibited a multinucleated cell morphology and a myotube-like shape. The expression of the acetylcholine receptor and GLUT4 was confirmed in TMSC-SKMCs. In addition, these cells exhibited insulin-mediated glucose uptake, NMJ formation, and transient changes in cell membrane action potential, all of which are representative functions of human SKMCs. DISCUSSION: Tonsil-derived mesenchymal stem cells can be functionally differentiated into SKMCs and may have potential for clinical application for the treatment of skeletal muscle disorders.

Age-related hypertrophy of adenoid and tonsil with its relationship with craniofacial morphology.

BACKGROUND: When analyzing the relationship between adenotonsillar hypertrophy and craniofacial morphology, researchers generally regarded hypertrophied adenoids and tonsils as a whole. It remains unclear whether different enlarged sites of pharyngeal lymphoid tissue would correlate with multiple craniofacial subtypes. We hypothesized there would be craniofacial subtypes correlated with different locations of hypertrophied adenoid and tonsil. METHODS: Lateral cephalometric radiographs were obtained from 466 children (171 boys and 295 girls, aged 12.27 ± 2.69 years). They were divided into four groups according to different sites of enlarged pharyngeal lymphoid tissue: adenoid hypertrophy group (AG, n = 126), tonsillar hypertrophy group (TG, n = 59), adenotonsillar hypertrophy group (ATG, n = 69) and control group (CG, n = 212). Five commonly used angles for cephalometric measurements were investigated: SNA (Sella-Nasion-Point A), SNB (Sella-Nasion-Point B), ANB (Point A-Nasion-Point B), mandibular plane angle (MP/SN) and Y-axis angle (SGn/FH). RESULTS: Children with isolated tonsillar hypertrophy correlated with increased SNA (unstandardized regression coefficient B = 1.38, p = 0.009) and SNB (B = 1.99, p = 0.001) compared with controls. However, children with isolated adenoid hypertrophy correlated with decreased SNB (B=-0.94, p = 0.036), increased ANB (B = 0.74, p = 0.014) and increased MP/SN (B = 2.22, p < 0.001). Similarly, children with adenotonsillar hypertrophy correlated with decreased SNB (B=-1.36, p = 0.015), increased ANB (B = 1.35, p < 0.001) and increased MP/SN (B = 2.64, p = 0.001). CONCLUSIONS: Isolated adenoid hypertrophy correlated with a retrognathic mandible, an increased maxillo-mandibular sagittal discrepancy, and an increased mandibular plane angle. Isolated tonsillar hypertrophy correlated with maxillary and mandibular protrusion. Adenotonsillar hypertrophy did not show a superimposed craniofacial pattern of the above two but showed the same craniofacial pattern as isolated adenoid hypertrophy.