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The plaque-forming assay is the standard technique for determining viral titer, and a critical measurement for investigating viral replication. However, this assay is highly dependent on experimental technique and conditions. In the case of human respiratory syncytial virus (RSV) in particular, it can be difficult to objectively confirm the accuracy of plaque-forming assay because the plaques made by RSV are often small and unclear. In recent studies, RT-qPCR methods have emerged as a supportive procedure for assessment of viral titer, yielding highly sensitive and reproducible results. In this report, we compare the viral replication, as determined by plaque-forming assay, and the copy numbers of RSV genes NS1, NS2, N, and F, as determined by RT-qPCR. Two real-time PCR systems, SYBR Green and TaqMan probe, gave highly similar results for measurement of copy numbers of RSV N genes of virus subgroups A. We determined the RSV gene copy numbers in the culture cell supernatant and cell lysate measured at various multiplicities of infection. We found that copy number of the RSV N gene in the culture supernatant and cell lysate was highly correlated with plaque-forming units. In conclusion, RT-qPCR measurement of RSV gene copy number was highly dependent on viral titer, and the detailed comparison between each gene copy number and virus titer should be useful and supportive in confirming RSV plaque-forming assay and virus dynamics. The technique may also be used to estimate the amount of RSV present in clinical specimens.

OBJECTIVES/HYPOTHESIS The aim of this study was to evaluate the utility of 18 F-fluorodeoxyglucose (FDG) positron emission tomography/computed tomography (PET/CT) for accurately diagnosing immunoglobulin G4-related sclerosing sialadenitis (IgG4-SS). STUDY DESIGN Retrospective cohort study. METHODS We reviewed the records of 64 patients with IgG4-SS (35 male and 29 female patients) and 10 patients with clinically suspected IgG4-SS. Pathological diagnoses of patients clinically suspected with IgG4-SS included four cases of malignant lymphoma, one case of multicentric Castleman disease, one case of Sjogren's syndrome, and four cases of sialadenitis. All patients underwent submandibular gland (SMG) biopsies and baseline FDG-PET/CT evaluation. Clinical, serological, pathological, and PET/CT findings were analyzed. We also investigated maximum standardized uptake values (SUVmax) in the salivary glands of 15 patients with malignant disease of the salivary glands during the same period. RESULTS Increased FDG uptake in the SMG and parotid gland was found in 63 (98%) and 23 (35%) patients with IgG4-SS, respectively. FDG uptake of the bilateral SMG and unilateral SMG was recorded in 57 patients (89%) and six patients (9%), respectively. Mean SUVmax in patients with malignant disease of the salivary glands was significantly higher than that in patients with IgG4-SS (P = .035). We defined a positive test for IgG4-SS diagnosis as high SMG FDG uptake and serum IgG4 level ≥135 mg/dL, resulting in a sensitivity, specificity, and accuracy of 96.9%, 90.0%, and 86.4%, respectively. CONCLUSIONS FDG-PET/CT findings in combination with serological and clinical findings may have the capacity to diagnose IgG4-SS and lead to less-invasive biopsy procedures. LEVEL OF EVIDENCE 4. Laryngoscope, 128:1120-1125, 2018.

P63 is a regulator of cell-cell junction complexes in the epidermis. Claudin-4 is regulated via various factors in normal epithelial cells and diseases. We found that claudin-4 was directly regulated via p63 (TAp63 and ΔNp63) in human keratinocytes and nasal epithelial cells. In the epidermis of atopic dermatitis (AD), which contains ΔNp63-deficient keratinocytes, high expression of claudin-4 was observed. In primary keratinocytes, downregulation of ΔNp63 by treatment with short interfering RNA (siRNA)-p63 induced claudin-4 expression. In nasal epithelial cells in the context of rhinitis or nasal polyps, upregulation of TAp63 and downregulation of claudin-4 were observed. In primary nasal epithelial cells transfected with the human telomerase reverse transcriptase gene, knockdown of p63 by siRNAs induced claudin-4 expression. Taken together, these findings indicate that p63 is a negative regulator of claudin-4 expression. Understanding the regulation of claudin-4 via p63 in human epithelial cells may be important for developing therapies for allergies and drug delivery systems.

T follicular helper (Tfh) cells are involved in specific humoral immunity at initial and recall phases. The fact that the transcription repressors B-cell lymphoma-6 and Blimp-1 determine lineages of Tfh cells and other types of effector CD4(+) T cells, respectively, suggests that there are unique mechanisms to establish Tfh-cell identity. In this study, we found that Tfh cells preferentially express the transcriptional coactivator Bob1. Bob1 of Tfh cells was dispensable for the expression of B-cell lymphoma-6 and the functional property of the cells for B cell help. However, upon initial immunization of foreign antigens, the percentages of Tfh cells in Bob1(-/-) mice were much higher than those in wild-type (WT) mice. In addition, expansion of Tfh cells within Bob1(-/-) CD4(+) T cells transferred into WT mice revealed that the high frequency of Tfh cells was caused by a T-cell-intrinsic mechanism. These findings were further supported by the results of in vitro studies demonstrating that Bob1(-/-) Tfh cells had greater proliferative activity in response to stimuli by CD3/CD28 monoclonal antibody and were also refractory to CD3-induced cell death in comparison to WT Tfh cells. These results suggest that Tfh cells harbor a Bob1-related mechanism to restrict numerical frequency against stimulation of TCRs.

Background Chronic rhinosinusitis (CRS) can be classified into CRS with nasal polyps (CRSwNP) and CRS without nasal polyps (CRSsNP). CRSwNP displays more intense eosinophilic infiltration and the presence of Th2 cytokines. Mucosal eosinophilia is associated with more severe symptoms and often requires multiple surgeries because of recurrence; however, even in eosinophilic CRS (ECRS), clinical course is variable. In this study, we wanted to set objective clinical criteria for the diagnosis of refractory CRS. Methods This was a retrospective study conducted by 15 institutions participating in the Japanese Epidemiological Survey of Refractory Eosinophilic Chronic Rhinosinusitis (JESREC). We evaluated patients with CRS treated with endoscopic sinus surgery (ESS), and risk of recurrence was estimated using Cox proportional hazard models. Multiple logistic regression models and receiver operating characteristics curves were constructed to create the diagnostic criterion for ECRS. Results We analyzed 1716 patients treated with ESS. To diagnose ECRS, the JESREC scoring system assessed unilateral or bilateral disease, the presence of nasal polyps, blood eosinophilia, and dominant shadow of ethmoid sinuses in computed tomography (CT) scans. The cutoff value of the score was 11 points (sensitivity: 83%, specificity: 66%). Blood eosinophilia (>5%), ethmoid sinus disease detected by CT scan, bronchial asthma, aspirin, and nonsteroidal anti-inflammatory drugs intolerance were associated significantly with recurrence. Conclusion We subdivided CRSwNP in non-ECRS, mild, moderate, and severe ECRS according to our algorithm. This classification was significantly correlated with prognosis. It is notable that this algorithm may give useful information to clinicians in the refractoriness of CRS before ESS or biopsy.

Respiratory syncytial virus (RSV) is an important pathogen of bronchiolitis, asthma, and severe lower respiratory tract disease in infants and young children. Matrix metalloproteinases (MMPs) play key roles in viral infection, inflammation and remodeling of the airway. However, the roles and regulation of MMPs in human nasal epithelial cells (HNECs) after RSV infection remain unclear. To investigate the regulation of MMP induced after RSV infection in HNECs, an RSV-infected model of HNECs in vitro was used. It was found that mRNA of MMP-10 was markedly increased in HNECs after RSV infection, together with induction of mRNAs of MMP-1, -7, -9, and -19. The amount of MMP-10 released from HNECs was also increased in a time-dependent manner after RSV infection as was that of chemokine RANTES. The upregulation of MMP-10 in HNECs after RSV infection was prevented by inhibitors of NF-κB and pan-PKC with inhibition of RSV replication, whereas it was prevented by inhibitors of JAK/STAT, MAPK, and EGF receptors without inhibition of RSV replication. In lung tissue of an infant with severe RSV infection in which a few RSV antibody-positive macrophages were observed, MMP-10 was expressed at the apical side of the bronchial epithelial cells and alveolar epithelial cells. In conclusion, MMP-10 induced by RSV infection in HNECs is regulated via distinct signal transduction pathways with or without relation to RSV replication. MMP-10 may play an important role in the pathogenesis of RSV diseases and it has the potential to be a novel marker and therapeutic target for RSV infection.

Palmoplantar pustulosis (PPP) is an autoimmune disease characterized by psoriasis-like erythematous lesions on palms and/or soles due to an abnormal humoral immune response. Tonsillectomy is effectively employed for the treatment of PPP; however, how tonsils are involved in the aetiology of PPP remains unclear. Here we analysed surgically resected palatine tonsils from 36 cases of PPP as well as usual recurrent tonsillitis (RT) as a control. Histological examination revealed that a unique lesion, with lymphoid follicles surrounded by reticular crypt epithelial cells, was more frequently observed in tonsils of patients with PPP than in those with RT (p < 0.0001; PPP vs RT). Interestingly, crypt epithelial cells in primary cultures derived from PPP tonsils showed marked production of interleukin-6 (IL-6). Moreover, these epithelial cells from PPP tonsils expressed p53-related transcription factors in their nuclei that were found to contribute to the up-regulation of IL-6 gene expression. These findings suggest that, at least in part, the specialized lymphoepithelial symbiosis of PPP tonsils, under the control of p53-related factors, may be relevant to the generation of the impaired micro-environment underlying the aberrant production of autoantibodies.

BACKGROUND/AIMS: Transforming growth factor-beta (TGF-beta) initiates and maintains epithelial-mesenchymal transition (EMT), which causes disassembly of tight junctions and loss of epithelial cell polarity. In mature hepatocytes during EMT induced by TGF-beta, changes in the expression of tight junction proteins and the fence function indicated that epithelial cell polarity remains unclear. METHODS: In the present study, using primary cultures of adult rat hepatocytes at day 10 after plating, in which epithelial cell polarity is well maintained by tight junctions, we examined the effects of 0.01-20 ng/ml TGF-beta on the expression of the integral tight junction proteins, claudin-1, -2 and occludin, as well as the fence function. RESULTS: In adult rat hepatocytes, TGF-beta induced EMT, which was indicated as upregulation of Smad-interacting protein-1 (SIP1) and Snail and down-regulation of E-cadherin. Down-regulation of claudin-1 and upregulation of occludin were observed beginning from a low dose of TGF-beta, whereas upregulation of claudin-2 was observed at a high dose of TGF-beta. Furthermore, treatment with TGF-beta caused disruption of the fence function, which was closely associated with the expression of claudin-1 via p38 mitogen-activated protein kinase (MAPK), phosphoinositide-3 kinase and protein kinase C but not MAPK signalling pathways. CONCLUSION: These results suggest that in mature hepatocytes in vitro, TGF-beta induces EMT by down-regulation of claudin-1 and the fence function via distinct signalling pathways.

Recurrent otitis media are frequently intractable during childhood. It is unclear whether recurrent otitis media is caused by etiological bacteria colonization or by new infections. Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis were isolated from the nasopharynx of 7 otitis-prone and 2 non-prone children with recurrent otitis media. Plural bacterial species and strains were found in all children while affected by otitis media. The same strain was repeatedly isolated from all otitis-prone children even after administration of antibiotics but was not from the non-prone children. Antibiotic susceptibility did not differ significantly among the same repeatedly isolated strains. This pilot study suggests that the etiological bacteria tend to colonize and is hard to eliminate in otitis-prone children.

Alloiococcus otitidis has been found to be associated with otitis media with effusion. In this study we investigated whether TLR2 and collectins, surfactant protein A (SP-A) and mannose-binding lectin (MBL), interacted with A. otitidis. Both SP-A and MBL bound to A. otitidis in a Ca(2+)-dependent manner. A. otitidis induced IL-8 secretion from U937 cells and NF-kappaB activation in TLR2-transfected HEK293 cells. However, the cells transfected with the mutant TLR2(P681H) did not respond to A. otitidis. In addition, A. otitidis co-sedimented a recombinant soluble form of the extracellular TLR2 domain, indicating direct binding of the bacterium to TLR2. SP-A and MBL augmented the phagocytosis of A. otitidis by J774A.1 cells. The collectin-stimulated phagocytosis of A. otitidis was significantly attenuated when fucoidan and polyinosinic acid were co-incubated. Immunoblotting analysis revealed that MBL was present in the middle ear effusion from patients with otitis media. These results demonstrate that A. otitidis is a ligand for the collectins and TLR2, and that the collectins enhance the phagocytosis of A. otitidis by macrophages, suggesting important roles of the collectins and TLR2 in the innate immunity of the middle ear against A. otitidis infection.

BACKGROUND Non-Hodgkin lymphoma (NHL) represents a heterogeneous group of tumors that vary with regard to their biologic aggressiveness and clinical course. In in vitro studies, matrix metalloproteinase 9 (MMP9) was reportedly expressed by human NHL cells and elevated levels of MMP9 have been observed in a subset of patients with high-grade NHL. METHODS The expression of MMP2 and MMP9 was evaluated in 158 patients with NHL and the relation between the expression of these proteins and clinicopathologic factors was analyzed. All but 1 patient had received radiation therapy and 92 patients also were treated with intensive combination chemotherapy. RESULTS Nearly all the patients with extranodal natural killer NK/T-cell lymphoma nasal type and anaplastic large cell lymphoma, T-cell/null cell type expressed MMP9. In contrast, only a small fraction of the patients with mucosa-associated lymphoid tissue (MALT) lymphomas and follicular lymphomas expressed MMP9. Approximately 50% of the diffuse large B-cell lymphoma (DLBCL) cases expressed MMP9. The expression of MMP2 was noted in some of the patients with DLBCL and nasal NK/T-cell lymphoma. The overall survival rates of patients who expressed MMP9 were significantly lower than that of those who did not. Such a correlation was not demonstrated in MMP2 expression. When MMP9 expression was analyzed in DLBLC patients, the overall survival rates of patients who expressed MMP9 were significantly lower than those who did not express MMP9. Chemotherapy was associated with better overall survival in DLBCL patients who expressed MMP9. Overall survival rates of T-cell/NK-cell lymphoma patients who expressed MMP9 appeared to be lower than that in those who did not express MMP9. However, chemotherapy was not found to improve overall survival in patients who expressed MMP9. CONCLUSIONS MMP9 expression was observed in patients with aggressive NHL and was characterized by poor overall survival. Cancer 2004;100:356–65.© 2003 American Cancer Society.

DO11.10 transgenic mice, expressing an ovalbumin (OVA)-specific alphabeta T-cell receptor (TCR), have been used as a model of various immune diseases associated with T lymphocytes. Some studies of immunoresponse in lung have involved adoptive transfer of DO11.10 mice. As of yet, however, there have been no studies of the adoptive transfer model in the upper airway. The purpose of this study was to establish an animal model to clarify the recruitment mechanism and the roles of Th2 cells in allergic rhinitis. In accordance with the adoptive transfer system, we generated Th0, Th1 and Th2 cells from DO11.10 mice and transferred them into wild type BALB/c mice. Following nasal OVA challenge to DO11.10 mice or to the BALB/c mice into which antigen-specific Th2 cells had been transferred, the number of local antigen-specific TCR-positive cells accompanying the local eosinophilia had significantly increased. However, nasal OVA challenge to BALB/c mice into which antigen-specific Th0 or Th1 cells were transferred failed to increase the number of local OVA-specific TCR positive cells. These observations suggest that an antigen-specific homing mechanism of Th2 cells may exist in nasal mucosa. Analysis of this model will assist in the development of new therapeutic strategy, which targets Th2 cells in allergic rhinitis.

Summary Background The cysteinyl leukotrienes (CysLT) are lipid mediators that have been implicated in the pathogenesis of allergic diseases. Pharmacological studies using CysLTs indicate that two classes of receptors, named CysLT1 and CysLT2 receptor, exist. The former is sensitive to the CysLT1 antagonist currently used to treat asthma and allergic rhinitis. Recently, the cDNA for human CysLT1 and CysLT2 receptor have been cloned, making it now possible to study the gene expression of CysLTs receptors. Objective We have used reverse transcription and polymerase chain reaction (RT-PCR) to study the gene expression of CysLT1 and CysLT2 receptor and in situ hybridization to determine the distribution of CysLT1 receptor mRNA in human nasal mucosa. In addition, the distribution of the CysLT1 receptor protein was studied by immunohistochemistry. Methods Human turbinates were obtained after turbinectomy from six patients with nasal obstruction refractory to medical therapy. Total RNA was isolated from human nasal mucosa and both CysLT1 and CysLT2 receptor mRNA was detected in these tissues by using RT-PCR. For in situ hybridization study of human nasal mucosa, we used biotin-labelled oligonucleotides probes encoding human CysLT1 receptor cDNA. To identify the cells expressing the CysLT1 receptor protein, double immunostaining was performed by using anti-CysLT1 receptor antibody and monoclonal antileucocyte antibodies. Results RT-PCR analysis of total nasal RNA demonstrated the expression of both CysLT1 receptor and CysLT2 receptor mRNA. In situ hybridization indicated high levels of CysLT1 receptor hybridization in blood vessels and the interstitial cells, but a sparse signal in airway epithelium and submucosal glands. The immunohistochemical studies revealed that anti-CysLT1 receptor antibody labelled eosinophils, mast cells, macrophages, neutrophils and vascular endothelial cells in the nasal mucosa. Conclusion The results may have an important clinical implication and also promote further investigation of the regulation of CysLT1 receptor in health and disease.

BACKGROUND A prospective, randomized study was performed to determine the efficacy of neoadjuvant chemotherapy over radiotherapy alone in patients with locally advanced nasopharyngeal carcinoma. METHODS From January 1991 to December 1998, 80 patients were enrolled in this study. Patients with locoregional carcinoma of the nasopharynx were randomized to receive two courses of chemotherapy, consisting of cisplatin and 5-fluorouracil (CDDP-5FU), that were administered before radiation therapy (CT arm) or radiotherapy alone. The patients who received neoadjuvant chemotherapy were treated with radiation therapy, which was scheduled to commence 2 weeks after the second course chemotherapy. RESULTS With a median follow-up of 49 months, a trend toward improved overall survival or disease free survival favoring the CT arm was observed (5-year overall survival rate, 60% vs. 48%; 5-year disease free survival rate, 55% vs. 43%), although this difference was not significant. There were no differences in locoregional failure free survival between the two arms. However, metastasis free survival favored the CT arm, although this difference was not significant. The results also demonstrated that most patients in the CT arm who experienced recurrent disease developed locoregional recurrences before distant metastases, suggesting that improvements in locoregional control may lead to improved disease free survival. CONCLUSIONS The use of CDDP-5FU chemotherapy prior to radiotherapy in patients with nasopharyngeal carcinoma did not result in a significant improvement in disease free survival or overall survival. However, there was a positive tendency in favor of the CT arm for distant metastasis free survival, although there was no improvement in the locoregional recurrence free survival rate. Cancer 2002;94:2217–23. © 2002 American Cancer Society. DOI 10.1002/cncr.10473

It is well known that the frequency of upper respiratory infection is clinically increased after radiotherapy of the head and neck region. This study found higher antibacterial secretory immunoglobulin A (S-IgA) activity against three indigenous streptococci (Streptococcus mitis, S. salivarius, and S. sanguis I) and S. pneumoniae in patients who had undergone radiation therapy of the head and neck region than in control subjects. This showed no relation to the extent of the radiation field. Compared with before radiotherapy, the S-IgA titer against S. pneumoniae and its ratio to the activities against the indigenous streptococci were significantly higher in patients with fully irradiated major salivary glands. These results indicated that the radiotherapy promoted the antigen-specific S-IgA production of virulent streptococci in most patients with head and neck cancer, even more than 6 months after radiotherapy. The resulting altered balance in the S-IgA system of normal indigenous streptococci may also impair the ability to maintain the stable bacterial interference between normal indigenous and virulent streptococci in the oropharyngeal cavity.

A pyriform sinus fistula can cause acute thyroiditis or recurrent infection in the neck. This fistula is believed to be a remnant of the branchial apparatus, although its origin has yet to be pinpointed. The spatial distribution of C cells in the thyroid gland was mapped by immunohistologic method in four patients with a pyriform sinus fistula. The C cells were identified immunohistologically with anticalcitonin antibody. The stained calcitonin-positive cells also crossreacted with the antibodies to carcinoembryonic antigen, chromogranin A, and neuron-specific enolase. The C cells were mainly distributed near the end of the fistula, and in three patients their concentration per unit volume of thyroid tissue was found to be inversely proportional to the distance from the end of the fistulas. Comparison of distant locations of the left-sided thyroid lobe in patients and the same region in control subjects showed a similar number of C cells. Thus this limited distribution of C cells suggested that the pyriform sinus fistula was either a remnant of the ultimobranchial body, the result of disturbed migration of the C cell in the fetus, or both. (OTOLARYNGOL HEAD NECK SURG 1995;112:268-73.)

was published in 1953, Miku-licz’s disease (MD) has been considered a part of pri-mary Sjogren’s syndrome (SS). However, certain clinicaldifferences exist between MD and SS. The characteristics ofMD include persistent swelling of the lacrimal and salivaryglands, undetectable or slight secretory dysfunction, andresponsiveness to steroid treatment. With regard to serolog-ical findings, MD patients exhibit a lack of anti-SS-A andanti-SS-B antibodies.

Sera from 85 patients with head and neck cancer including laryngeal cancer, hypopharyngeal cancer, nasopharyngeal cancer, and maxillary cancer were assayed for immune complexes (IC) by solid-phase anti-C3 enzyme immunoassay and for squamous cell carcinoma-related (SCC) antigen by radioimmunoassay. The positive rates of IC and SCC antigen in head and neck cancer patients were 29.4% and 34.1%, respectively. Their serum levels and positive rates were found to be elevated according to the degree of advancement of the disease stage, showing their good clinical correlations. With individual patients there was no significant relationship between IC and SCC antigen.