Toll-like receptor 9-positive plasmacytoid dendritic cells promote Th17 immune responses in oral lichen planus stimulated by epithelium-derived cathepsin K.

Oral lichen planus (OLP) is a chronic inflammatory disease associated with T cell infiltration. The crosstalk between oral epithelium and mucosal T cells was considered to be crucial in the pathogenesis of OLP. Here, we selectively extracted the normal epithelium (NE) and lesional epithelium (LE) of buccal mucosa specimens from three patients with OLP by laser capture microdissection due to identify the pathogenic factors. Cathepsin K (CTSK) was identified as one of common upregulated genes in the LE by DNA microarray. Immunohistochemically, CTSK was distinctly detected in and around the LE, while it was rarely seen in the NE. Recent studies showed that CTSK enhanced Toll-like receptor 9 (TLR9) signaling in antigen-presenting cells, leading to Th17 cell differentiation. TLR9 expression mainly co-localized with CD123+ plasmacytoid dendritic cells (pDCs). The number of RORγt-positive cells correlated with that of CTSK-positive cells in OLP tissues. CD123+ pDCs induced the production of Th17-related cytokines (IL-6, IL-23, and TGF-ß) upon stimulation with TLR9 agonist CpG DNA. Moreover, single cell RNA-sequencing analysis revealed that TLR9-positive pDCs enhanced in genes associated with Th17 cell differentiation in comparison with TLR9-negative pDCs. CTSK could induce Th17-related production of CD123+ pDCs via TLR9 signaling to promote the pathogenesis of OLP.

CD163+ M2 Macrophages Promote Fibrosis in IgG4-Related Disease Via Toll-like Receptor 7/Interleukin-1 Receptor-Associated Kinase 4/NF-κB Signaling.

OBJECTIVE: IgG4-related disease (IgG4-RD) is a fibro-inflammatory condition that can affect multiple organs. We previously demonstrated that TLR7-transgenic C57BL/6 mice showed elevated serum IgG1 levels and inflammation with fibrosis in the salivary glands (SGs), lungs, and pancreas. Moreover, we observed extensive Toll-like receptor 7 (TLR-7)-positive CD163+ M2 macrophage infiltration in SGs from IgG4-RD patients. We undertook this study to examine the fibrotic mechanism via the TLR-7 pathway. METHODS: Gene expression in SGs from human TLR7-transgenic mice and IgG4-RD patients was analyzed using DNA microarrays. We extracted the common up-regulated TLR-7-related genes in SGs from TLR7-transgenic mice and IgG4-RD patients. Finally, we investigated the interaction between CD163+ M2 macrophages and fibroblasts before and after stimulation with the TLR-7 agonist loxoribine. RESULTS: In TLR7-transgenic mice and IgG4-RD patients, IRAK3 and IRAK4 were significantly overexpressed. Real-time polymerase chain reaction validated the up-regulation of only IRAK4 in IgG4-RD patients compared with the other groups (P < 0.05). Interleukin-1 receptor-associated kinase 4 (IRAK4) was strongly detected in and around germinal centers in SGs from patients with IgG4-related dacryoadenitis and sialadenitis alone. Double immunofluorescence staining showed that IRAK4-positive cells were mainly colocalized with CD163+ M2 macrophages in SGs (P < 0.05). After stimulation with loxoribine, CD163+ M2 macrophages exhibited significantly enhanced expression of IRAK4 and NF-κB and increased supernatant concentrations of fibrotic cytokines. Finally, we confirmed that the number of fibroblasts was increased by culture with the supernatant of CD163+ M2 macrophages following stimulation with loxoribine (P < 0.05). CONCLUSION: CD163+ M2 macrophages promote fibrosis in IgG4-RD by increasing the production of fibrotic cytokines via TLR-7/IRAK4/NF-κB signaling.

Oral Squamous Cell Carcinoma Contributes to Differentiation of Monocyte-Derived Tumor-Associated Macrophages via PAI-1 and IL-8 Production.

Tumor-associated macrophages (TAMs) promote cancer cell proliferation and metastasis, as well as anti-tumor immune suppression. Recent studies have shown that tumors enhance the recruitment and differentiation of TAMs, but the detailed mechanisms have not been clarified. We thus examined the influence of cancer cells on the differentiation of monocytes to TAM subsets, including CD163+, CD204+, and CD206+ cells, in oral squamous cell carcinoma (OSCC) using immunohistochemistry, flow cytometry, and a cytokine array. Furthermore, we investigated the effect of OSCC cells (HSC-2, SQUU-A, and SQUU-B cells) on the differentiation of purified CD14+ cells to TAM subsets. The localization patterns of CD163+, CD204+, and CD206+ in OSCC sections were quite different. The expression of CD206 on CD14+ cells was significantly increased after the co-culture with OSCC cell lines, while the expressions of CD163 and CD204 on CD14+ cells showed no change. High concentrations of plasminogen activator inhibitor-1 (PAI-1) and interleukin-8 (IL-8) were detected in the conditioned medium of OSCC cell lines. PAI-1 and IL-8 stimulated CD14+ cells to express CD206. Moreover, there were positive correlations among the numbers of CD206+, PAI-1+, and IL-8+ cells in OSCC sections. These results suggest that PAI-1 and IL-8 produced by OSCC contribute to the differentiation of monocytes to CD206+ TAMs.

Activated M2 Macrophages Contribute to the Pathogenesis of IgG4-Related Disease via Toll-like Receptor 7/Interleukin-33 Signaling.

OBJECTIVE: IgG4-related disease (IgG4-RD) is a unique inflammatory disorder in which Th2 cytokines promote IgG4 production. In addition, recent studies have implicated the Toll-like receptor (TLR) pathway. This study was undertaken to examine the expression of TLRs in salivary glands (SGs) from patients with IgG4-RD. METHODS: SGs from 15 patients with IgG4-RD, 15 patients with Sjögren's syndrome (SS), 10 patients with chronic sialadenitis, and 10 healthy controls were examined histologically. TLR family gene expression (TLR-1 through TLR-10) was analyzed by DNA microarray in the submandibular glands (SMGs). Up-regulation of TLRs was confirmed in SGs from patients with IgG4-RD. Finally, the phenotype of human TLR-7 (huTLR-7)-transgenic C57BL/6 mice was assessed before and after stimulation with TLR agonist. RESULTS: In patients with IgG4-RD, TLR-4, TLR-7, TLR-8, and TLR-9 were overexpressed. Polymerase chain reaction validated the up-regulation of TLR-7 in IgG4-RD compared with the other groups. Immunohistochemical analysis confirmed strong infiltration of TLR-7-positive cells in the SGs of patients with IgG4-RD. Double immunohistochemical staining showed that TLR-7 expression colocalized with CD163+ M2 macrophages. After in vitro stimulation with a TLR-7 agonist, CD163+ M2 macrophages produced higher levels of interleukin-33 (IL-33), which is a Th2-activating cytokine. In huTLR-7-transgenic mice, the focus and fibrosis scores in SMGs, pancreas, and lungs were significantly higher than those in wild-type mice (P < 0.05). Moreover, the concentration of serum IgG, IgG1, and IL-33 in huTLR-7-transgenic mice was distinctly increased upon stimulation with a TLR-7 agonist (P < 0.05). CONCLUSION: TLR-7-expressing M2 macrophages may promote the activation of Th2 immune responses via IL-33 secretion in IgG4-RD.

CD206+ tumor-associated macrophages promote proliferation and invasion in oral squamous cell carcinoma via EGF production.

Tumor-associated macrophages (TAMs) promote tumor progression and inhibit anti-tumor immune response by producing various mediators and preferentially express CD163, CD204, and CD206. However, the role of these TAM subsets in oral squamous cell carcinoma (OSCC) remains unclear. Here we investigated the expression and function of TAM subsets in OSCC, especially in cancer cell proliferation. Biopsy sample from 44 patients with OSCC were examined for the expression of TAM markers and EGF by immunohistochemistry. EGF production of TAM subsets isolated from OSCC patients was assessed by flow cytometry. We also examined the effect of conditioned medium from TAM subsets on the proliferation of OSCC cells. CD163+ cells were detected diffusely all over the tumor and connective tissue area, while CD204+ and CD206+ cells were mainly detected in/around the tumors. Flow cytometric analysis found that CD206+ TAMs strongly produced EGF compared with CD163+ and CD204+ TAMs. Cell proliferation and invasion of OSCC cells cultured with conditioned medium of CD206+ TAMs were strongly enhanced and inhibited by anti-EGFR. The number of CD206+ TAMs positively correlated with worse clinical prognosis. Our results revealed differences in localization and EGF production among these TAM subsets. CD206+ TAMs might play a critical role in the proliferation of OSCC via EGF production.

CD163+CD204+ tumor-associated macrophages contribute to T cell regulation via interleukin-10 and PD-L1 production in oral squamous cell carcinoma.

Tumor-associated macrophages (TAMs) promote cancer cell proliferation, invasion, and metastasis by producing various mediators. Although preclinical studies demonstrated that TAMs preferentially express CD163 and CD204, the TAM subsets in oral squamous cell carcinoma (OSCC) remain unknown. In this study, we examined the expression and role of TAM subsets in OSCC. Forty-six patients with OSCC were analyzed for expression of TAMs in biopsy samples by immunohistochemistry. We examined TAM subsets and their production of immune suppressive molecules (IL-10 and PD-L1) in peripheral blood mononuclear cells from three OSCC patients by flow cytometry. CD163 was detected around the tumor or connective tissue, while CD204 was detected in/around the tumors. Flow cytometric analysis revealed that CD163+CD204+ TAMs strongly produced IL-10 and PD-L1 in comparison with CD163+CD204- and CD163-CD204+ TAMs. Furthermore, the number of activated CD3+ T cells after co-culture with CD163+CD204+ TAMs was significantly lower than that after co-culture with other TAM subsets. In clinical findings, the number of CD163+CD204+ TAMs was negatively correlated with that of CD25+ cells and 5-year progression-free survival. These results suggest that CD163+CD204+ TAMs possibly play a key role in the invasion and metastasis of OSCC by T-cell regulation via IL-10 and PD-L1 production.

A pilot study to assess relationship between total IgE and 95% predictive decision points of food specific IgE concentration.

BACKGROUND: False positive test results in children who are tested for food allergies may lead to inappropriate dietary restrictions. OBJECTIVE: The aim of this study was to report our experience with a 3 year-old boy, who presented with "multiple food allergies" and, however, passed the food challenges, and to review our experience regarding management of children with high specific food IgEs with high total IgE. METHODS: Medical records of 16 children with food challenges were reviewed. Median age of subjects was 39 months, with a history of adverse clinical reaction to a food, a specific IgE greater than the decision point, and an elevated total serum IgE level of 500 IU, underwent challenges to the offending food. RESULTS: 13 out of 16 subjects were successfully re-exposed to the suspected foods and continued to tolerate these foods well. CONCLUSION: Our finding suggests a much lower clinical risk with previously defined specific IgE decision points in children with very high levels of total IgE (> 6000 IU/ml).

Reasons for failure to quit: a cross-sectional survey of tobacco use in major cities in Pakistan.

BACKGROUND: Tobacco dependence has been defined as a chronic relapsing disease. Around 5 million annual tobacco-related deaths have been reported worldwide. The majority of smokers want to quit but are not successful. OBJECTIVES: To screen our population for tobacco use, gauge the baseline demographics of tobacco users and assess factors associated with failed attempts to quit. METHODS: Free health camps supervised by a physician were held across two major cities of Pakistan. All consenting participants were administered a questionnaire and had their exhaled carbon monoxide (CO) levels measured. RESULTS: Of 12 969 participants successfully enrolled, the mean age was 31.4 ± 10.0 years. More than three quarters were aged 20-40 years (n = 10 168, 78.4%). The overall average CO level was 12.0 ± 8.0 ppm. The majority of the participants wanted to quit, and nearly everyone had received advice about quitting. The majority had tried smoking cessation pharmacotherapy. Friends/peer pressure (n = 1554, 12%), anxiety (n = 681, 5.3%), tobacco dependence (n = 1965, 15.2%) and stress/mood changes (n = 390, 3.0%) were the most widely observed reasons for failure to quit in study participants. CONCLUSION: The information provided by this study can guide the development of more targeted intervention programmes for smokers who wish to quit.

Particle formation and characterization of mackerel reaction oil by gas saturated solution process.

Most of the health benefits of fish oil can be attributed to the presence of omega-3 fatty acids like Docosahexenoic acid (DHA) and Eicosapentaenoic acid (EPA). There are few dietary sources of EPA and DHA other than oily fish. EPA and DHA have great potential effect on human health. In this research, Supercritical carbon dioxide (scCO2) extracted mackerel oil was reacted by enzyme at different systems to improve the EPA and DHA. Different types of immobilize enzyme TL-IM, RM-IM, Novozyme 435 were assessed for improving PUFAs. Best result was found at non-pressurized system using TL-IM. Reacted oil particle were obtained with polyethylene glycol by gas saturated solution process (PGSS). Different parameters like temperature, pressure, agitation speed and nozzle size effect on particle formulation were observed. SEM and PSA analysis showed, small size non spherical particles were obtained. It was found that after particle formation poly unsaturated fatty acids (PUFAs) were present in particle as same in oil. PUFAs release from particle was almost linear against constant time duration. Oil quality in particle not change significantly, in this contrast this study will be helpful for food and pharmaceutical industry to provide high EPA and DHA containing powder.

Composition of Asarum heterotropoides var. mandshuricum radix oil from different extraction methods and activities against human body odor-producing bacteria.

In this study, oils from Asarum heterotropoides were extracted by traditional solvent extraction and supercritical CO2 (SC-CO2) extraction methods and their antioxidant activities along with antimicrobial and inhibitory activities against five human body odor-producing bacteria (Staphylococcus epidermidis, Propionibacterium freudenreichii, Micrococcus luteus, Corynebacterium jeikeium, and Corynebacterium xerosis) were evaluated. The oil was found to contain 15 components, among which the most abundant component was methyl eugenol (37.6%), which was identified at every condition studied in different extraction methods. The oil extracted with n-hexane and ethanol mixture exhibited a strong antioxidant activity (92% ± 2%) and the highest ABTS and 2,2-diphenyl-1-picrylhydrazyl scavenging activities (89% ± 0.2%). The highest amounts of total phenolic content and total flavonoid content were 23.1±0.4 mg/g and 4.9±0.1 mg/g, respectively, in the traditional method. In the SC-CO2 method performed at 200 bar/50°C using ethanol as an entrainer, the highest inhibition zone was recorded against all the aforementioned bacteria. In particular, strong antibacterial activity (38±2 mm) was found against M. luteus. The minimum inhibitory concentration (MIC) for the oil against bacteria ranged from 10.1±0.1 µg/mL to 46±2 µg/mL. The lowest MIC was found against M. luteus. Methyl eugenol was found to be one of the major compounds working against human body odor-producing bacteria.

Cigarette smoking among adolescent females in Pakistan.

SETTING: Tobacco use among adolescent females is gaining importance as they take up smoking in increasing numbers. Recent studies show that sex differences in tobacco use are disappearing and that tobacco companies are agressively targeting females in developing countries. OBJECTIVE: To determine the factors influencing the use of cigarettes among female high-school students in Karachi, Pakistan, and hence to identify areas for future intervention. METHOD: A two-stage cluster sample of 644 females was drawn from government and private schools in a locality in Karachi. A self-administered questionnaire was used for data collection. RESULTS: The mean age was 15.29 years. The prevalence of ever-smokers was found to be 16.3%. Univariate analysis indicated that private school attendance, underage sale of tobacco at shops, tobacco use by family members and exposure to cigarette advertisements predicted smoking among girls. Knowledge assessment of the subjects showed that most girls had good knowledge about the hazards of smoking. CONCLUSION: Our study has identified several risk factors for smoking among young girls. Appropriate interventions are needed in the form of public education programmes, effective anti-tobacco laws and stricter enforcement of existing laws to restrict the number of young female smokers.

Asthma knowledge and approach among Pakistani family physicians and the impact of an educational programme.

OBJECTIVES: To assess asthma knowledge and approach among Pakistani family physicians and the impact of an educational programme. DESIGN: A total of 89 family physicians attending a continuing medical education programme were evaluated using a 17-item questionnaire before and after a lecture and seminar-based educational programme. Three scores -- total score (TSc), knowledge subscore (KSc) and approach subscore (ASc) -- were calculated before and after the course. RESULTS: A total of 82 (92%) family physicians completed the questionnaire. A minimum TSc of > or =9 was achieved by 57% physicians before the course; this increased to 78% after the course. Physicians with < or =10 years since graduation achieved a higher baseline TSc than those who had graduated > or =20 years previously. The latter group showed a larger post-course improvement. Analysis of individual questions disclosed that 79% of family physicians were unable to assess the empirical risk of asthma in an unborn child, 61% were unable to correctly classify/treat Stage 2 asthma and a similar number peak expiratory flow rate (PEFR) (l/min) based acute asthma exacerbation. Reassuringly, 97% correctly identified clinical signs and 87% life-threatening features of asthma. CONCLUSION: Of all family physicians who responded to the questionnaire, 57% achieved acceptable baseline standards. Deficiencies regarding areas such as genetic counselling and disease severity assessment were observed. Strengths in identifying clinical signs and life-threatening features were evident. Continuing medical education resulted in a significant improvement in the post-course assessment, regardless of the years since graduation.

Foreign body aspiration in adults.

Foreign body aspiration is not an uncommon problem, particularly in children, the elderly, and those with predisposing factors like neurological impairment. In adults, regional differences exist in the type of foreign body aspirated. We report four cases of foreign body aspiration in South Asian adults, three of which were related to the widespread habit of chewing stimulant seeds such as betel nuts (two) and tamarind seed (one). It is important for clinicians to maintain a high index of suspicion for the diagnosis of foreign body inhalation in the appropriate clinical setting. Bronchoscopy is necessary not only for the diagnosis but also for the removal of foreign body from the bronchial tree. Once the foreign body is removed, the improvement in symptoms is usually dramatic, making it rewarding for any physician to manage.

Increasing social and psychological information conveyed to General Practitioners after bad news consultations by use of separate letters from the Lung Cancer Nurse Specialist.

Multidisciplinary teams (MDT) now review all cases of lung cancer. These teams include a Lung Cancer Nurse Specialist (LCNS). These Nurses help support the patient and should facilitate communication and liaise with other services. The LCNS is present when the diagnosis is given to the patient but also usually spends time afterwards with the patient and their family. We postulated that a separate letter from the LCNS to the General Practitioner (GP) after the consultations would convey extra information to the GP. In 58 new lung cancer patients reviewed in the clinic, the LCNS and Physician independently wrote separate letters after the consultation in which the diagnosis of lung cancer was given. The GPs were asked by questionnaire about the usefulness of the letter from the LCNS. This letter was considered by the GP to provide extra information in: (i) 69% concerning the patients reaction to the diagnosis; (ii) 85% concerning who attended the clinic with the patient; (iii) 85% about what referrals were made to community services; (iv) 86% about who the patient was living with; (v) 81% about who the patients carers were; (vi) 81% information on the patients condition; (vii) 70% concerning the information given to patients about benefits. Ninety-seven percent of the GPs found the LCNS letter useful or very useful and 92% of the GPs thought that the information in the letter would be useful or very useful when they next saw the patient. Separate and independent letters from the LCNS after "bad news" consultation in lung cancer provides added useful information for GPs. Ninety-one percent of the GPs wanted the letters from the LCNS to continue.

Telemedicine for multidisciplinary lung cancer meetings.

According to recent UK guidelines on the management of lung cancer, all cases should be reviewed prospectively by a lung cancer multidisciplinary team (MDT) and a thoracic surgeon should be readily available to liaise with the MDT. However, there is a shortage of thoracic surgeons in the UK. Over a one-year period, 28 MDT meetings were held at a district general hospital in Southend, at which 62 patients were presented to a tertiary cardiothoracic centre in London, 80 km away, via ISDN videoconferencing at 384 kbit/s. The annual resection rate increased by 30% following the introduction of the telemedicine MDTmeetings, and the mean time from first being seen in the clinic to surgery was reduced from 69 to 54 days.We estimate that the telemedicine meetings saved over three working weeks of thoracic surgical time during the year.

Documenting clinicopathologic consultations.

The role of the clinical pathologist as a provider of services reimbursable under Medicare Part B is increasingly being challenged. This article reports how a group of pathologists is successfully providing consultations that meet the guidelines of Medicare and the Joint Commission on Accreditation of Hospitals.