Role of IL-17A in Chronic Rhinosinusitis With Nasal Polyp

PURPOSE: Th17-associated inflammation is increased in chronic rhinosinusitis with nasal polyp (CRSwNP), and is associated with disease severity and steroid resistance. Overexpressed interleukin (IL)-17A affects CRSwNP by tissue remodeling, eosinophilic accumulation, and neutrophilic infiltration. We aimed to identify the role of IL-17A in CRSwNP and to evaluate the effects of anti-IL-17A blocking antibody on nasal polyp (NP) formation using a murine NP model. Moreover, we sought to investigate whether the inhibition of mechanistic target of the rapamycin (mTOR) signal pathway could suppress IL-17A expression and NP formation.METHODS: Human sinonasal tissues from control subjects and patients with chronic rhinosinusitis (CRS) were analyzed using immunohistochemistry (IHC) and immunofluorescence staining. The effects of IL-17A neutralizing antibody and rapamycin were evaluated in a murine NP model. Mouse samples were analyzed using IHC, quantitative real-time polymerase chain reaction, and enzyme-linked immunosorbent assay.RESULTS: IL-17A+ inflammatory cells were significantly increased in number in NP from patients with CRSwNP compared to that in uncinate process tissues from control subjects and patients with CRS without NP or CRSwNP. CD68+ M1 macrophages dominantly expressed IL-17A, followed by neutrophils and T helper cells, in NP tissues. Neutralization of IL-17A effectively reduced the number of NPs, inflammatory cytokines, and IL-17A-producing cells, including M1 macrophages. Inhibition of IL-17A via the mTOR pathway using rapamycin also attenuated NP formation and inflammation in the murine NP model.CONCLUSIONS: IL-17A possibly plays a role in the pathogenesis of CRSwNP, the major cellular source being M1 macrophage in NP tissues. Targeting IL-17A directly or indirectly may be an effective therapeutic strategy for CRSwNP.

Strain-Specific Differences in House Dust Mite (Dermatophagoides farinae)-Induced Mouse Models of Allergic Rhinitis

Objectives@#. Limited information is available regarding strain-related differences in mouse models of allergic rhinitis induced by Dermatophagoides farinae (Der f1). In this study, we compared differences between two mouse strains and determined the optimal dose of Der f1 for allergic rhinitis mouse models. @*Methods@#. Forty-eight mice were assigned to the following six groups (n=8 per group): group A (control, BALB/c), group B (Der f1-sensitized BALB/c, 25 µg), group C (Der f1-sensitized BALB/c, 100 µg), group D (control, C57BL/6), group E (Der f1-sensitized C57BL/6, 25 µg), and group F (Der f1-sensitized C57BL/6, 100 µg). Allergic inflammation was induced with Der f1 and alum sensitization, followed by an intranasal challenge with Der f1. Rubbing and sneezing scores, eosinophil and neutrophil infiltration, and immunoglobulin, cytokine, and chemokine levels in the nasal mucosa and from splenocyte cultures were assessed. @*Results@#. Rubbing and sneezing scores were higher in groups B, C, E, and F than in groups A and D, with a similar pattern in both strains (i.e., group B vs. E and group C vs. F). Serum immunoglobulin levels were significantly elevated compared to the control in groups B and C, but not in groups E and F. Eosinophil and neutrophil infiltration increased (all P0.05). BALB/c mice (group B) showed a greater elevation of splenic interleukin (IL)-4 (P<0.01), IL-5 (P<0.01), and IL-6 levels (P<0.05) and nasal IL-4 mRNA levels (P<0.001) than the C57BL/6 mice (group E). Interestingly, mice treated with 100 µg Der f1 showed a weaker allergic response than those treated with 25 µg. @*Conclusion@#. We found 25 µg to be a more appropriate dose for Der f1 sensitization. BALB/c mice are more biased toward a Th2 response and are a more suitable model for allergic rhinitis than C57BL/6 mice. This study provides information on the appropriate choice of a mouse model for allergic rhinitis.

The Role of IL-17 in a Lipopolysaccharide-Induced Rhinitis Model

PURPOSE: Lipopolysaccharide (LPS) is a cell wall component of Gram-negative bacteria and important for pro-inflammatory mediators. This study aimed to establish a rhinitis model using ovalbumin (OVA) and LPS in order to evaluate the role of interleukin (IL)-17 in the pathogenesis of an LPS-induced non-eosionophilic rhinitis model. METHODS: Mice were divided into 4 groups and each group consisted of 10 mice (negative control group, allergic rhinitis model group, 1-µg LPS treatment group, and 10-µg LPS treatment group). BALB/c mice were sensitized with OVA and 1 or 10 µg of LPS, and challenged intranasally with OVA. Multiple parameters of rhinitis were also evaluated to establish the LPS-induced rhinitis model. IL-17 knockout mice were used to check if the LPS-induced rhinitis model were dependent on IL-17. Eosinophil and neutrophil infiltration, and mRNA and protein expression profiles of cytokine in nasal mucosa or spleen cell culture were evaluated using molecular, biochemical, histopathological, and immunohistological methods. RESULTS: In the LPS-induced rhinitis model, neutrophil infiltration increased in the nasal mucosa, and systemic and nasal IL-17 and interferon-gamma (IFN-γ) levels also increased as compared with the OVA-induced allergic rhinitis model. These findings were LPS-dose-dependent. In IL-17 knockout mice, those phenotypes (neutrophil infiltration, IL-17, and IFN-γ) were reversed, showing IL-17 dependency of LPS-induced rhinitis. The expression of vascular endothelial growth factor (VEGF), an important mediator for inflammation and angiogenesis, decreased in IL-17 knockout mice, showing the relationship between IL-17 and VEGF. CONCLUSIONS: This study established an LPS-induced rhinitis model dependent on IL-17, characterized by neutrophil infiltration and increased expression of IL-17.

Mouse Model of IL-17-Dominant Rhinitis Using Polyinosinic-Polycytidylic Acid

Interleukin (IL)-17 plays an important role in rhinitis and the level thereof correlates with the severity of disease. However, no mouse model for IL-17-dominant rhinitis has yet been developed. Our objective was to establish a mouse model of IL-17-dominant rhinitis via intranasal application of polyinosinic-polycytidylic acid (abbreviated as Poly(I:C)). Mice were divided into 6 groups (n=8 for each group); 1) 1 negative control group, 2) 1 positive control group (OVA/alum model), 3) 2 Poly(I:C) groups (10 or 100 µg), and 4) 2 OVA/Poly(I:C) groups (10 or 100 µg). The positive control group was treated with the conventional OVA/alum protocol. In the Poly(I:C) and OVA/Poly(I:C) groups, phosphate-buffered saline or an OVA solution plus Poly(I:C) were administered. The OVA/Poly(I:C) groups exhibited significantly greater neutrophil infiltration and increased IL-17/interferon γ expression compared with the other groups. However, the levels of total immunoglobulin E (IgE), OVA-specific IgE, eosinophil infiltration, IL-4, IL-5, IL-6, and IL-10 were significantly lower in the OVA/Poly(I:C) groups than in mice subjected to conventional Th2-dominant OVA/alum treatment (the positive control group). IL-17 and neutrophil measurement, chemokine (C-X-C motif) ligand 1 immunohistochemistry, and confocal microscopy revealed increased numbers of IL-17-secreting cells in the nasal mucosa of the OVA/Poly(I:C) groups, which included natural killer cells, CD4 T cells, and neutrophils. In conclusion, we developed a mouse model of IL-17-dominant rhinitis using OVA together with Poly(I:C). This model will be useful in research on neutrophil- or IL-17-dominant rhinitis.

Immunohistochemical Expression and Clinical Significance of Suggested Stem Cell Markers in Hepatocellular Carcinoma

BACKGROUND: Increasing evidence has shown that tumor initiation and growth are nourished by a small subpopulation of cancer stem cells (CSCs) within the tumor mass. CSCs are posited to be responsible for tumor maintenance, growth, distant metastasis, and relapse after curative operation. We examined the expression of CSC markers in paraffin-embedded tissue sections of hepatocellular carcinoma (HCC) and correlated the results with clinicopathologic characteristics. METHODS: Immunohistochemical staining for the markers believed to be expressed in the CSCs, including epithelial cell adhesion molecule (EpCAM), keratin 19 (K19), CD133, and CD56, was performed in 82 HCC specimens. RESULTS: EpCAM expression was observed in 56% of the HCCs (46/82) and K19 in 6% (5/82). EpCAM expression in HCC significantly correlated with elevated alpha-fetoprotein level, microvessel invasion of tumor cells, and high histologic grade. In addition, EpCAM expression significantly correlated with K19 expression. The overall survival and relapsefree survival rates in patients with EpCAM-expressing HCC were relatively lower than those in patients with EpCAM-negative HCC. All but two of the 82 HCCs were negative for CD133 and CD56, respectively. CONCLUSIONS: Our results suggest that HCCs expressing EpCAM are associated with unfavorable prognostic factors and have a more aggressive clinical course than those not expressing EpCAM. Further, the expression of either CD133 or CD56 in paraffin-embedded HCC tissues appears to be rare.

Expression of Cortactin and Focal Adhesion Kinase in Colorectal Adenocarcinoma: Correlation with Clinicopathologic Parameters and Their Prognostic Implication

BACKGROUND: Cortactin and focal adhesion kinase (FAK) are two important components among actin cross-linking proteins that play a central role in cell migration. METHODS: The aims of this study were to evaluate the expression of cortactin and FAK in normal colorectal mucosa and colorectal adenocarcinoma (CRC) using tissue microarray of 2 mm cores to correlate their expression with other clinicopathological factors and, investigate their prognostic significance. RESULTS: Twenty (9%) and 24 cases (11%) of normal colorectal mucosa were immunoreactive for cortactin and FAK. In addition, 184 (84%) and 133 cases (61%) of CRCs were immunoreactive for cortactin and FAK, respectively. Cortactin expression was associated with histologic differentiation and FAK expression. Cortactin, but not FAK expression was also correlated with poor overall and relapse-free survival and served well as an independent prognostic factor for poor survival. CONCLUSIONS: Cortactin expression, in association with FAK expression, may plays an important role in tumor progression. Furthermore, it may also be a satisfactory biomarker to predict tumor progression and survival in CRC patients.

Expression of CHOP in Squamous Tumor of the Uterine Cervix

BACKGROUND: High-risk human papillomavirus (HR-HPV) infection and abnormal p53 expression are closely involved in carcinogenesis of squamous cell carcinoma (SqCC) of uterine cervix. Recent studies have suggested that virus-induced endoplasmic reticulum (ER) stress modulates various cell survival and cell death signaling pathways. The C/EBP homologous protein (CHOP) is associated with ER stress-mediated apoptosis and is also involved in carcinogenesis of several human cancers. We hypothesized that CHOP is involved in the carcinogenesis of uterine cervical cancer in association with HR-HPV and/or p53. METHODS: Immunohistochemistry was used to analyze CHOP and p53 protein expression of tissue sections from 191 patients with invasive cancer or preinvasive lesions of the uterine cervix (61 cases of SqCC, 66 cases of cervical intraepithelial neoplasia [CIN] III, and 64 cases of CIN I). RESULTS: CHOP was expressed in 59.4% of CIN I, 48.5% of CIN III, and 70.5% of SqCC cases. It was also significantly more frequent in invasive SqCC than in preinvasive lesions (p=0.042). Moreover, CHOP expression significantly correlated with HR-HPV infection and p53 expression (p=0.009 and p=0.038, respectively). CONCLUSIONS: Our results suggest that CHOP is involved in the carcinogenesis of the uterine cervix SqCC via association with HR-HPV and p53.

Expressions of E-cadherin, Cortactin and MMP-9 in Pseudoepitheliomatous Hyperplasia and Squamous Cell Carcinoma of the Head and Neck: Their Relationships with Clinicopathologic Factors and Prognostic Implication

BACKGROUND: E-cadherin, cortactin, and matrix metalloproteinase (MMP)-9 have roles in tumor development or progression, but their expression has not been fully investigated in pseudoepitheliomatous hyperplasia (PEH) and squamous cell carcinoma (SCC) of the head and neck. METHODS: We evaluated the immunohistochemical expression of E-cadherin, cortactin, and MMP-9 in 29 cases of PEH and 97 cases of SCC. Additionally, we evaluated their relationship with clinicopathologic factors and prognostic implications in SCC. RESULTS: Thirty-five cases of SCC showed reduced expression of E-cadherin, whereas none of the PEH did. A total of 20 cases and 11 cases of SCC were immunoreactive for cortactin and MMP-9, respectively, whereas none of the PEH did. In SCC, reduced expression of E-cadherin was correlated with cortactin expression and invasion depth. Cortactin expression was correlated with differentiation, T classification, and recurrence and/or metastasis. MMP-9 expression was correlated with invasion depth. Cortactin expression was correlated with poor overall survival and relapse-free survival and it was an independent prognostic factor. CONCLUSIONS: The reduced expression of E-cadherin and the expression of cortactin may be helpful for the differential diagnosis of PEH and SCC. Furthermore, cortactin expression in association with reduced E-cadherin expression is correlated with poor prognosis in SCC.

Clinicopathologic Study on Combined Hepatocellular Carcinoma and Cholangiocarcinoma: with Emphasis on the Intermediate Cell Morphology

Combined hepatocellular carcinoma and cholangiocarcinoma (combined HCC-CC) is a rare subtype of primary liver cancer. We investigated the histopathologic features of transitional or intermediate areas in 21 combined HCC-CCs and immunophenotypes using different hepatic progenitor cell markers (CK7, CK19, c-kit, NCAM, and EpCAM). Major histologic findings of transitional or intermediate areas of 21 combined HCC-CCs included strands/trabeculae of small, uniform, oval-shaped cells with scant cytoplasm and hyperchromatic nuclei embedded within an abundant stroma, small cells with an antler-like anastomosing pattern, and solid nests of intermediate hepatocyte-like cells surrounded by small cells in periphery, in order of frequency. The intermediate area of one tumor was composed predominantly of spindle cells arranged in short fascicles. Immunophenotype of tumor cells with intermediate morphology suggested a progenitor cell origin for this tumor. Clinical findings of combined HCC-CC showed a closer resemblance with those of HCC than those of CC. In univariate analysis, tumor size, TNM stage, and serum alpha-fetoprotein levels showed a significant association with poor patient survival. Serum alpha-fetoprotein level was an independent prognostic indicator in multivariate analysis. In conclusion, an awareness of the clinicopathologic features, specifically the various morphologic features of intermediate areas in this tumor, is essential for prevention of potential misdiagnosis as another tumor.

The Influence of Alpha-fetoprotein on Natural Suppressor Cell Activity and Ehrlich Carcinoma Growth

The influence of alpha-fetoprotein (AFP) on the bone marrow (BM) natural suppressor (NS) cells of intact Ehrlich carcinoma -bearing CBA mice was studied. Bone marrow NS cells were fractionated into three fractions by isopycnic centrifugation on percoll gradients: NS1 (rho=1.080 g/ml), NS2 (rho=1.090 g/ml) and NS3 (1.100>rho>1.090 g/ml). These fractions were highly different in their sensitivity to known NS cell inductors (interleukin (IL)-2, IL-3 or histamine). None of the NS fractions isolated from the intact mice spontaneously produced antiproliferative activity, however, they showed a high level of NS (antiproliferative and natural killer cell inhibitory) activity under the influence of AFP. A single injection of AFP to intact mice led to an increase of spontaneous NS activity and the inhibition of natural killer cell activity. NS activity, especially NS2, was increased in when tumor cells were subcutaneously inoculated three days after AFP injection. In the AFP-treated mice, the tumor mass at 14 days was 60% larger than that in the untreated mice. Our data confirmed that AFP is a tumor marker that can inhibit cancer immunity and plays a role in cancer pathogenesis.

Long-Term Tropical Residency Diminishes Central Sudomotor Sensitivities in Male Subjects

Tropical natives (TROP) are capable of tolerating tropical heat because of their long-term adaptation to tropical environments. When exposed to heat stress, these natives tend to respond with lower sweat output, which is generally thought to be the result of heat acclimatization. The main objective of this study was to clarify central mechanisms inherent to suppressed thermal sweating in tropical natives (Malaysians) by comparing their sweating responses to those of temperate native (TEMP) (Koreans). This experiment was conducted in a thermoneutral climatic chamber (24+/-0.5 degrees C, 40+/-3% relative humidity). Heat loads were applied to each subject by the immersion of their lower legs in a hot water bath (43 degrees C for 30 min). Sweat onset-time and sweat volume were compared between TROP and TEMP. The sweat onset-times on four selected points on the body ranged from 10.25 to 13.47 min in TEMP subjects, and from 16.24 to 17.83 min in TROP subjects (p<0.001). The local sweat volumes at the same sites ranged from 4.30 to 9.74 mg/cm2 in TEMP subjects, and from between 1.80 to 4.40 mg/cm2 in TROP subjects (p<0.001). These results demonstrated a significant difference between TROP and TEMP subjects with regard to the manner in which they regulate their body temperatures when exposed to heat loads, and verified that long-term thermal adaptation blunts sweating sensitivities.

Comparison of Interleukin-8 Levels in Long-Distance Runners and Healthy Sedentary Non-Athletic Control Subjects

We have previously demonstrated that the level of leukocytes and neutrophils significantly increased immediately and 30 min after exercise. Interleukin-8 (IL-8) is an inflammatory cytokine that acts as a chemokine on neutrophils. In the present study, we evaluated the correlation between the number of neutrophils and leukocytes, and between the number of neutrophils and plasma IL-8 level. Long-distance trained runners (TRs, n = 10) and untrained sedentary control subjects (SEDs, n = 10) ran for one hour at 70% of heart rate reserve. In the TR, the number of neutrophils correlated significantly with the number of leukocytes in the blood. However, there was no correlation between the number of neutrophils and the plasma IL-8 concentration in both groups. Expressions of IL-8 protein and mRNA were markedly higher in the TRs as compared to the SEDs at three time intervals (pre-exercise, immediately after exercise, and post exercise). In conclusion, our results show that 1) the neutrophil level was dependent on the level of leukocytes 2) there was no correlation between the neutrophils count and plasma IL-8 concentration and 3) a higher plasma IL-8 level in athletes may be a unique characteristic of intensive training.

Sweating by Exercise Controls Body Temperature through Increase of Interleukin-1beta

This study was designed to investigate the expression and production of interleukin-1beta (IL-1beta) in human peripheral blood of trained runners and untrained controls after temporary moderate intensity exercise. Male long-distance trained runners (TR) and untrained sedentary control subjects (SED) ran for 1 h at 70% of heart rate reserve (HRR). IL-1beta gene and protein expressions were significantly higher in TR than those with SED at all 3 intervals examined independently. Significant increases in total sweat volume and oral temperature were observed after exercise in both groups, however, there were some differences between the groups. We conclude, therefore, that sweating due to exercise is associated with increase of IL-1beta and it is correlated with decrease of oral temperature.

Differential protein expression of etoposide-treated CaSki cervical carcinoma cells / 부인종양

OBJECTIVE: This study was designed to examine the pharmaco-dynamic pattern of proteomic expression in cervical carcinoma cells (CaSki cell line; HPV-16 positive) after in vitro treatment by the etoposide. METHODS: We analyzed proteomic profiling in cervical carcinoma cells after etoposide treatment using two-dimensional gel electrophoresis (2-DE) with MALDI-TOF-MS used for protein identification. Then, we tested the several experimental methods for verification and functional identification, including MTT assay, PI staining, DNA fragmentation assay, FDA, FACS and Western blot analysis. RESULTS: Etoposide inhibited the CaSki cervical cancer cell growth in a dose-dependent manner and the optimal concentration of etoposide is 2micrometer(IC50) in the CaSki cervical cancer cells. The etoposide induced apoptosis, as determined by DNA fragmentation assay, FACS, and Western blot. The etoposide increased the protein expression of Fas (Apo-1/CD95), p53, pRb and caspase-3, but decreased the level of Bcl-2 and caspase-3 precursor and subsequently triggered the mitochondrial apoptotic pathway (release of cytochrome c and activation of caspase-9). To this end, we analyzed CaSki cancer cells using 2-DE. Eight proteins (XAP-5, HXC-36, serine/threonine protein phosphatase 2B catalytic subunit, G2/mitotic-specific cyclin B1, T-box transcription factor TBX20, diacylglycerol kinase, amiloride-sensitive amine oxidase, HEF-like protein, ras-related protein Rab-20) were down-regulated and nine proteins (RNA 3'-terminal phosphate cyclase-like protein, late endosomal/lysosomal Mp1 interacting protein, glia maturation factor, replication protein A 14 kDa subunit, mago sashi protein homolog, 14 kDa phosphohistidine phosphatase, protein C14 or f48, cyclin-dependent kinase 4 inhibitior A, retinoic acid-binding protein II) were up-regulated in etoposide-treated CaSki cells when compared with non-treated cells. CONCLUSION: Our results clearly indicate that etoposide induced cell death by apoptosis. These findings may provide insights into the mechanisms underlying the apparent anti-tumoral effects of etoposide.

Proteome Analysis of Differential Protein Expression in Cervical Cancer Cells after Paclitaxel Treatment / Journal of the Korean Cancer Association, 대한암학회지

@#PURPOSE: It is well known that infection with HPV (human papillomavirus) is the main cause of cervical cancer and certain types of HPV are recognized as carcinogens. At present, there is little information regarding the antineoplastic mechanism of paclitaxel against cervical carcinoma cells. We thus tried to analyze differential protein expression and antineoplastic mechanism-related proteins after paclitaxel treatment on cervical cancer cells by using a proteomic analysis and to investigate the mechanism of action. MATERIALS AND METHODS: Using proteomics analysis including 2-DE and MALDI-TOF-MS, we detected the antineoplastic mechanism-related proteins. Then, we performed western blot analysis for apoptosis- and transformation- related proteins to confirm expression patterns derived from proteome analysis after paclitaxel treatment. RESULTS: We identified several cellular proteins that are responsive to paclitaxel treatment in HeLa cells using proteomics methods. Paclitaxel treatment elevated main-ly apoptosis, immune response and cell cycle check point- related proteins. On the other hand, paclitaxel treatment diminished growth factor/oncogene-related proteins and transcription regulation-related proteins. Also, in the HPV-associated cervical carcinoma cells, paclitaxel demonstrated anti-proliferative activity through the membrane death receptor-mediated apoptotic pathway and the mitochondrial-mediated pathway. CONCLUSION: Identification and characterization of functionally modulated proteins involved in anti-cancer regulatory events should lead to a better nderstanding of the long-term actions of paclitaxel at the molecular level and will contribute to the future development of novel therapeutic drug treatments based upon current therapies.

Economical Sweating Function in Africans: Quantitative Sudomotor Axon Reflex Test

People in tropics have the ability to tolerate heat by residential permanence in the tropics. Previously, we have shown that African and Thai subjects who lived for whole their lives in only their respective countries sweat less under hot conditions than South Koreans who also lived whole their lives in Korea. The difference in sweating responses was attributed to the dissimilar central and peripheral sweating mechanisms operating in people from both groups. In the present study, acetylcholine (ACh), the primary transmitter for the sudomotor functions, was iontophoretically administered to South Koreans and Africans to determine the characteristic sudorific responses of their acclimatized biologic make-up to their respective environments. Using quantitative sudomotor axon reflex test (QSART), direct (DIR) and axon reflex (AXR) responses were evaluated. The findings revealed that the sweat onset-time among South Koreans was 0.91 min earlier than among Africans (P< 0.01). The axon reflex sweat volume of nicotine receptor activity AXR (1) and sweat volume of muscarinic receptor activity DIR (2) among South Koreans were 79% and 53% greater (P< 0.01), respectively. These results indicate that the reduced thermal sweating among Africans is at least in part attributed to the diminished sensitivity of sweat glands to ACh.