Search Results (15)

Symptoms of respiratory tract infections (RTI) caused by viral or bacterial triggers include sore throat and cough. Plantago lanceolata (ribwort plantain) is used in herbal medicine to treat symptoms of RTI. This study evaluated the pharmacological actions of P. lanceolata extract (PL) and StrepCough Plantain-Dry cough syrup (PLS) in in vitro models of sore throat and cough. Human tonsil epithelial cells (HTEpiC) cells were stimulated with a combination of lipoteichoic acid and peptidoglycan in the presence or absence of PL or PLS. Levels of TNF-α, IL-6, PGE₂, and COX-2 were measured using ELISA, while phospho-p65 protein levels were measured using Lumit^® immunoassay. The effects of PL and PLS on bradykinin-induced inflammation and increased intracellular Ca²⁺ were investigated in bronchial epithelial cells (BEAS-2B), cells over-expressing TRPV1. Results showed that the pre-treatment of HTEpiC cells with PL and PLS resulted in a significant (p < 0.05) reduction in the LTA + PGN-induced increased production of TNFα, IL-6, PGE₂, as well as COX-2 and phospho-p65 protein levels. Bradykinin-induced increased levels of TNFα, IL-6, and PGE₂ in BEAS-2B cells over-expressing TRPV1 were also reduced by PL and PLS, while reducing levels of intracellular Ca²⁺. This study has demonstrated that extract of P. lanceolata and StrepCough Plantain-Dry cough syrup reduce inflammation in human tonsil epithelial cells. The extract and syrup also reduced TRPV1-mediated inflammation and increased intracellular Ca²⁺ in BEAS-2B bronchial epithelial cells. This study has provided pharmacological evidence for the use of P. lanceolata in healthcare interventions to treat symptoms of respiratory tract infection. Full article

Background/Objective: Human papillomavirus (HPV) represents the most widespread sexually transmitted infection globally, with high-risk strains such as HPV16 driving a rising incidence of oropharyngeal squamous cell carcinoma (OPSCC), particularly in developed countries like the United States and United Kingdom. In the U.S., HPV16-associated OPSCC has surpassed cervical cancer as the most common HPV-related malignancy. Despite the availability of preventive vaccines, uptake remains suboptimal among adolescents and shifting sexual behaviors have contributed to increased disease burden. Early detection remains a major clinical challenge due to the absence of defined precursor lesions and the extended latency between viral exposure and disease onset. Most patients present with advanced-stage disease and no prior clinical history of pre-malignancy, limiting access to early-stage samples and hindering biomarker discovery. Methods: To address these limitations, we developed an in vitro HPV16 oral cancer model, using the three-dimensional organotypic raft culture system that simulates the progression of HPV16-transfected oral epithelium from precancerous states to malignant phenotypes. Results: Using HPV16-transfected human tonsil keratinocytes, we generated stratified and differentiated epithelia that mimic the biochemical and structural changes observed in vivo. This system enables detailed monitoring of epithelial differentiation, biochemical shifts, viral genome status, and key oncogenic and metabolic markers associated with HPV16-driven OPSCC. By aligning expression profiles with clinical datasets, we validated the model through the measurement of virologic markers linked to infection and progression, as well as tissue markers indicative of carcinogenic transformation. Conclusions: This model offers a promising tool for refining early detection strategies and evaluating potential clinical biomarkers, ultimately aiming to improve diagnostic precision and therapeutic outcomes in HPV-associated OPSCC. Full article

Porcine hemagglutinating encephalomyelitis virus (PHEV) replicates in the upper respiratory tract and tonsils of pigs. Using an air–liquid interface porcine respiratory epithelial cells (ALI-PRECs) culture system, we demonstrated that PHEV disrupts respiratory epithelia homeostasis by impairing ciliary function and inducing antiviral, pro-inflammatory cytokine, and chemokine responses. This study explores the mechanisms driving early innate immune responses during PHEV infection through host transcriptome analysis. Total RNA was collected from ALI-PRECs at 24, 36, and 48 h post inoculation (hpi). RNA-seq analysis was performed using an Illumina Hiseq 600 to generate 100 bp paired-end reads. Differential gene expression was analyzed using DeSeq2. PHEV replicated actively in ALI-PRECs, causing cytopathic changes and progressive mucociliary disruption. Transcriptome analysis revealed downregulation of cilia-associated genes such as CILK1, DNAH11, LRRC-23, -49, and -51, and acidic sialomucin CD164L2. PHEV also activated antiviral signaling pathways, significantly increasing the expression of interferon-stimulated genes (RSAD2, MX1, IFIT, and ISG15) and chemokine genes (CCL5 and CXCL10), highlighting inflammatory regulation. This study contributes to elucidating the molecular mechanisms of the innate immune response to PHEV infection of the airway epithelium, emphasizing the critical roles of the mucociliary, interferon, and chemokine responses. Full article

Human rotavirus (HRV) is a leading cause of viral gastroenteritis in children across the globe. The virus has long been established as a pathogen of the gastrointestinal tract, targeting small intestine epithelial cells and leading to diarrhea, nausea, and vomiting. Recently, this classical infection pathway was challenged by the findings that murine strains of rotavirus can infect the salivary glands of pups and dams and transmit via saliva from pups to dams during suckling. Here, we aimed to determine if HRV was also capable of infecting salivary glands and spreading in saliva using a gnotobiotic (Gn) pig model of HRV infection and disease. Gn pigs were orally inoculated with various strains of HRV, and virus shedding was monitored for several days post-inoculation. HRV was shed nasally and in feces in all inoculated pigs. Infectious HRV was detected in the saliva of four piglets. Structural and non-structural HRV proteins, as well as the HRV genome, were detected in the intestinal and facial tissues of inoculated pigs. The pigs developed high IgM antibody responses in serum and small intestinal contents at 10 days post-inoculation. Additionally, inoculated pigs had HRV-specific IgM antibody-secreting cells present in the ileum, tonsils, and facial lymphoid tissues. Taken together, these findings indicate that HRV can replicate in salivary tissues and prime immune responses in both intestinal and facial lymphoid tissues of Gn pigs. Full article

Salmonella Enteritidis is a common foodborne pathogen transmitted through poultry products, which are its main carriers. Poultry are vaccinated against Salmonella Enteritidis in many countries, despite the absence of clinical symptoms, using commercially available live-attenuated vaccines. We previously constructed a highly attenuated temperature-sensitive (ts) Salmonella Enteritidis mutant, 2S-G10. In the present study, we describe the construction and attenuation-associated characteristics of 2S-G10. We infected 1-day-old chicks with 2S-G10 and the parental strains to evaluate the attenuation. One week after infection, 2S-G10 was not detected in the liver, cecum, or cecal tonsil tissues of the orally inoculated chicks, contrary to the parental strain. This indicates that 2S-G10 was highly attenuated when compared to the parental stain. In vitro experiments revealed the inability of 2S-G10 to grow at the normal body temperature of chickens and invade chicken liver epithelial cells. Moreover, single nucleotide polymorphism (SNP) analysis between the complete genome sequence of 2S-G10 and its parental strain revealed SNPs in bcsE, recG, rfaF, and pepD_1 genes, which are involved in epithelial cell invasion and persistence in host systems, growth, lipopolysaccharide core biosynthesis, and cellular survival under heat stress, respectively. These potential characteristics are consistent with the findings of in vitro experiments. Conclusively, chemical treatment–induced random genetic mutations highly attenuated 2S-G10, implying its potential to be developed as a novel live-attenuated vaccine against Salmonella Enteritidis. Full article

Pharyngitis is an inflammation of the pharynx caused by viral, bacterial, or non-infectious factors. In the present study, the anti-inflammatory efficacy of carvacrol was assessed using an in vitro model of streptococcal pharyngitis using human tonsil epithelial cells (HTonEpiCs) induced with Streptococcus pyogenes cell wall antigens. HTonEpiCs were stimulated by a mixture of lipoteichoic acid (LTA) and peptidoglycan (PGN) for 4 h followed by exposure to carvacrol for 20 h. Following exposure, interleukin (IL)-6, IL-8, human beta defensin-2 (HBD-2), epithelial-derived neutrophil-activating protein-78 (ENA-78), granulocyte chemotactic protein-2 (GCP-2), cyclooxygenase-2 (COX-2), tumor necrosis factor-alpha (TNF-α), and prostaglandin (PGE₂) were measured by enzyme-linked immunosorbent assays (ELISA). The levels of pro-inflammatory cytokines, IL-6, IL-8, ENA-78, and GCP-2 were decreased in a carvacrol dose-dependent manner. The production of HBD-2 was significantly suppressed over 24 h carvacrol treatments. PGE₂ and COX-2 levels in the cell suspensions were affected by carvacrol treatment. TNF-α was not detected. The cell viability of all the tested carvacrol concentrations was greater than 80%, with no morphological changes. The results suggest that carvacrol has anti-inflammatory properties, and carvacrol needs to be further assessed for potential clinical or healthcare applications to manage the pain associated with streptococcal pharyngitis. Full article

Background: Enterovirus (EV) infections are associated with a broad range of diseases. Since the first experimental infection of primates with poliovirus (PV), tonsils and the Peyer’s patches (PPs) have been believed to be the primary replication sites of EVs. Our aim was to localize different viral markers in the small intestines (SI) of coxsackievirus B (CVB) orally and intraperitoneally (i.p.) infected mice. Methods: Transverse sections of SIs of both infected and control male outbred mice were collected at different intervals post-infection (p.i) and analyzed for presence of interferon-alpha (IFN-α) and viral protein VP1 by immunohistochemistry and in situ hybridization (ISH). Fluorescent marker, eGFP, was identified in cryosections of mice infected with eGFP-CVB3. Results: In the infected SIs, we observed enlarged germinating centers (GCs) in the PPs; IFN-α was detected in the PPs and mucosal layer of the SIs. However, VP1, viral RNA and the eGFP were absent in the GCs of PPs at all stages of infection irrespective of the virus strains used. Conclusions: Virus was present in the epithelial cells but not in GCs of the PPs of the murine SIs. Our results do not support the hypothesis of EV replication in the PP especially in the GCs. Full article

Human papillomavirus (HPV) is a virus with the potential to infect human epithelial cells and an etiological agent of many types of cancer, including head and neck cancer. The aim of the study was to determine the prevalence of HPV infection in patients with head and neck squamous cell carcinoma (HNSCC), patients with chronic tonsillitis, and healthy individuals, and to establish high- and low-risk HPV genotypes in these groups. The objectives also comprised the delineation of the relationship between the infection with high- or low-risk HPV subtypes and clinicopathological and demographic characteristics of the study groups. This study was composed of 76 patients diagnosed with HNSCC, 71 patients with chronic tonsillitis, and 168 cases without either of these conditions (the control group). HPV detection and identification of subtypes were performed on isolated DNA using a test which allowed detection of 33 common high-risk and low-risk HPV subtypes. The prevalence of HPV infection was 42.1%, 25.4%, and 37.5% in HNSCC, chronic tonsillitis, and control groups, respectively. HPV 16 was the most prevalent genotype in all groups and the non-oncogenic HPV 43/44 was frequent in HNSCC patients. This analysis provides insight into the prevalence of oral oncogenic and non-oncogenic HPVs in patients with head and neck cancer, patients with chronic tonsillitis and healthy individuals, and leads to the conclusion that further investigations are warranted to examine a larger cohort of patients focusing on high- and low-risk HPV genotypes. Efforts should be focused on screening and prevention strategies, and therefore, it is important to introduce tools for effective detection of HPV genotypes. Furthermore, given the role of vaccines against oral HPV infection, our observations lead to the suggestion that HPV vaccination should be of considerable importance in public health strategies. Full article

Mother-to-child transmission (MTCT) of HIV-1 may occur during pregnancy, labor, and breastfeeding; however, the molecular mechanism of MTCT of virus remains poorly understood. Infant tonsil mucosal epithelium may sequester HIV-1, serving as a transient reservoir, and may play a critical role in MTCT. Innate immune proteins human beta-defensins 2 (hBD-2) and -3 may inactivate intravesicular virions. To establish delivery of hBD-2 and -3 into vesicles containing HIV-1, we tagged hBDs with the protein transduction domain (PTD) of HIV-1 Tat, which facilitates an efficient translocation of proteins across cell membranes. Our new findings showed that hBD-2 and -3 proteins tagged with PTD efficiently penetrated polarized tonsil epithelial cells by endocytosis and direct penetration. PTD-initiated internalization of hBD-2 and -3 proteins into epithelial cells led to their subsequent penetration of multivesicular bodies (MVB) and vacuoles containing HIV-1. Furthermore, PTD played a role in the fusion of vesicles containing HIV-1 with lysosomes, where virus was inactivated. PTD-initiated internalization of hBD-2 and -3 proteins into ex vivo tonsil tissue explants reduced the spread of virus from epithelial cells to CD4+ T lymphocytes, CD68+ macrophages, and CD1c+ dendritic cells, suggesting that this approach may serve as an antiviral strategy for inactivating intraepithelial HIV-1 and reducing viral MTCT. Full article

Objective: Oropharyngeal/laryngeal carcinoma are common cancers of the upper aerodigestive system. Human papillomavirus (HPV) is described as the most frequent in the cancer of unknown primary. The presence of Helicobacter pylori (HP) in the oral cavity is discussed in some papers. The aim of study: To analyze the incidence of HPV and HP in oropharyngeal/laryngeal cancer persons versus persons with chronic tonsillar inflammation and healthy persons. Methods: The samples were taken in three groups: (1) tissue of oropharynx/larynx cancer (103 specimens); (2) tissue of palatine tonsils (85 specimens); and (3) healthy control group (50 specimens). We analyzed the presence of HP (PCR) and HPV genomic DNA (Sacace HPV High-Risk Screen Real-TM Quant) in the samples. Results: HP was detected in 86 samples (83.5%) and high-risk HPV in 62 samples (60.2%). We found a very high incidence of HP. In the cancer group, HP was detected in 82.5% cases and HPV positivity in 57.8%. In total, 7.2% of the cancer patients were negative for HP and HPV together. In turn, 53.6% of the cancer patients were positive for HP and HPV together. Four cases (4.2%) were positive for HPV only. VacA positivity was detected in 82 (79.6%) of the cancer cases and VacA negativity in 21 (20.4%) if the cancer cases. The incidence of HP in chronic inflammation (n = 85) was 65 cases (76.5%) and the incidence of HPV was 38 cases (44.7%). VacA positivity was detected in 59 (69.4%) of the chronic inflammation cases and VacA negativity was found in 26 (30.6%) of the chronic inflammation cases. Regarding the control group, we found HP positivity in 5 cases (11.1%) and HPV positivity in 19 cases (42.2%). There was VacA positivity in 6 cases (50.0%) of the control group. Statistically significantly lower prevalence of HP (p < 0.001) and HPV (p = 0.006) was found in the control group. Conclusions: We suggest that the palatine tonsils are colonized by HP. In our study, HP was present in oropharyngeal cancer in more cases in comparison with HPV infection. The presence of VacA from HP can have an influence on the human epithelial and immune cells’ regulation ways. Our results do not support idea that the CagA-positive HP is a primary carcinogen in oropharyngeal area. Full article

Multiorgan tropism of SARS-CoV-2 has previously been shown for several major organs. We have comprehensively analyzed 25 different formalin-fixed paraffin-embedded (FFPE) tissues/organs from autopsies of fatal COVID-19 cases (n = 8), using histopathological assessment, detection of SARS-CoV-2 RNA using polymerase chain reaction and RNA in situ hybridization, viral protein using immunohistochemistry, and virus particles using transmission electron microscopy. SARS-CoV-2 RNA was mainly localized in epithelial cells across all organs. Next to lung, trachea, kidney, heart, or liver, viral RNA was also found in tonsils, salivary glands, oropharynx, thyroid, adrenal gland, testicles, prostate, ovaries, small bowel, lymph nodes, skin and skeletal muscle. Viral RNA was predominantly found in cells expressing ACE2, TMPRSS2, or both. The SARS-CoV-2 replicating RNA was also detected in these organs. Immunohistochemistry and electron microscopy were not suitable for reliable and specific SARS-CoV-2 detection in autopsies. These findings were validated using in situ hybridization on external COVID-19 autopsy samples (n = 9). Apart from the lung, correlation of viral detection and histopathological assessment did not reveal any specific alterations that could be attributed to SARS-CoV-2. In summary, SARS-CoV-2 and its replication could be observed across all organ systems, which co-localizes with ACE2 and TMPRSS2 mainly in epithelial but also in mesenchymal and endothelial cells. Apart from the respiratory tract, no specific (histo-)morphologic alterations could be assigned to the SARS-CoV-2 infection. Full article

CTLA-4 (cytotoxic T-lymphocyte-associated protein 4) was originally defined as a T-lymphocyte antigen and was used as a target in cancer immunotherapy. Unfortunately, the existence of CTLA-4 in cells other than T-lymphocytes is often overlooked. The goal of the present study was to analyze the distribution pattern of CTLA-4 in the human tonsils using a panel of anti–CTLA-4 antibodies of different clones. We found that CTLA-4 was expressed in T-lymphocyte cells of various geneses, including hematopoietic cells and their derivatives (monocytes, macrophages, dendritic, plasma cells, mast cells, and neutrophils), as well as stromal cells of mesodermal (mesenchymal) origin and reticular epithelial cells of ectodermal origin. The expression of CTLA-4 in cells of different origins supports the proposition that CTLA-4 is not restricted to the lymphoid cell lineage and can provide broader effects of CTLA-4 on immune regulation. Full article

Chicken Salmonella enterica serovars are enteric bacteria associated with massive public health risks and economic losses. There is a widespread antimicrobial resistance among S. enterica serotypes, and innovative solutions to antibiotic resistance are needed. We aimed to use probiotics to reduce antibiotic resistance and identify the major probiotic players that modify the early interactions between S. enterica and host cells. One-day-old cobb broiler chicks were challenged with S. typhimurium after oral inoculation with different probiotic strains for 3 days. The adherence of different probiotic strains to Caco-2 intestinal epithelial cells was studied in vitro. Lactobacillus (Lacticaseibacillus) casei ATTC334 and Bifidobacterium breve JCM1192 strains attached to Caco-2 cells stronger than B. infantis BL2416. L. casei ATTC334 and B. breve JCM1192 reduced S. typhimurium recovery from the cecal tonsils by competitive exclusion mechanism. Although B. infantis BL2416 bound poorly to Caco-2 epithelial cells, it reduced S. typhimurium recovery and increased IFN-γ and TNF-α production. L. casei ATTC334, B. breve JCM1192 and B. infantis BL2416 improved body weight gain and the food conversion rate in S. typhimurium-infected broilers. B. longum Ncc2785 neither attached to epithelial cells nor induced IFN-γ and TNF-α release and consequently did not prevent S. typhimurium colonization in broiler chickens. In conclusion, probiotics prevented the intestinal colonization of S. typhimurium in infected chickens by competitive exclusion or cytokine production mechanisms. Full article

Streptococcus pyogenes is well documented as a multi-virulent and exclusively human pathogen. The LuxS-based signaling in these bacteria has a crucial role in causing several infections through pathways that are pathogenic. This study evaluated the individual and synergistic effects of citral and phloretin against S. pyogenes in relation to major virulence traits. The in vitro synergy of citral and phloretin was evaluated by the checkerboard method. The fractional inhibitory concentration (FIC) values were calculated to determine the interactions between the inhibitors. The bacteria’s virulence properties were tested in the presence of the molecules, individually as well as in combination. Molecules’ cytotoxicity was tested using human tonsil epithelial cells. The synergistic effects of the molecules on the expression of biofilm and quorum sensing genes were tested using quantitative real-time polymerase chain reaction (qRT-PCR). The molecules were also tested for their impact on LuxS protein by molecular docking, modeling, and free-energy calculations. When the two molecules were assessed in combination (synergistic effect, FIC Index of 0.5), a stronger growth inhibitory activity was exhibited than the individual molecules. The cell surface hydrophobicity, as well as genes involved in quorum sensing and biofilm formation, showed greater suppression when the molecules were tested in combination. The in silico findings also suggest the inhibitory potential of the two molecules against LuxS protein. The binding orientation and the binding affinity of citral and phloretin well support the notion that there is a synergistic effect of citral and phloretin. The data reveal the combination of citral and phloretin as a potent antibacterial agent to combat the virulence of S. pyogenes. Full article

Tumor cell metabolism differs from that of normal cells, conferring tumors with metabolic advantages but affording opportunities for therapeutic intervention. Accordingly, metabolism-targeting therapies have shown promise. However, drugs targeting singular metabolic pathways display limited efficacy, in part due to the tumor’s ability to compensate by using other metabolic pathways to meet energy and growth demands. Thus, it is critical to identify novel combinations of metabolism-targeting drugs to improve therapeutic efficacy in the face of compensatory cellular response mechanisms. Our lab has previously identified that the anti-cancer activity of propranolol, a non-selective beta-blocker, is associated with inhibition of mitochondrial metabolism in head and neck squamous cell carcinoma (HNSCC). In response to propranolol, however, HNSCC exhibits heightened glycolytic activity, which may limit the effectiveness of propranolol as a single agent. Thus, we hypothesized that propranolol’s metabolic effects promote a state of enhanced glucose dependence, and that propranolol together with glycolytic inhibition would provide a highly effective therapeutic combination in HNSCC. Here, we show that glucose deprivation synergizes with propranolol for anti-cancer activity, and that the rational combination of propranolol and dichloroacetate (DCA), a clinically available glycolytic inhibitor, dramatically attenuates tumor cell metabolism and mTOR signaling, inhibits proliferation and colony formation, and induces apoptosis. This therapeutic combination displays efficacy in both human papillomavirus-positive (HPV(+)) and HPV(−) HNSCC cell lines, as well as a recurrent/metastatic model, while leaving normal tonsil epithelial cells relatively unaffected. Importantly, the combination significantly delays tumor growth in vivo with no evidence of toxicity. Additionally, the combination of propranolol and DCA enhances the effects of chemoradiation and sensitizes resistant cells to cisplatin and radiation. This novel therapeutic combination represents a promising treatment strategy which may overcome some of the limitations of targeting individual metabolic pathways in cancer. Full article