Local production of antigen-specific IgE in different anatomic subsites of allergic fungal rhinosinusitis patients.

OBJECTIVE: Local production of antigen-specific IgE in allergic fungal rhinosinusitis (AFRS) is likely integral to the expression of allergy. This study examines if there are anatomic variations in local IgE expression or if variations among fungal and nonfungal IgE exist. STUDY DESIGN: Cross-sectional study. SETTING: Tertiary medical center. SUBJECTS AND METHODS: Specimens from 11 AFRS, 8 chronic rhinosinusitis without nasal polyps (CRSsNP), and 9 control patients underwent immunohistochemical localization for IgE and evaluation for antigen-specific IgE by ImmunoCAP testing. RESULTS: Inferior turbinate (IT) epithelium had greater IgE staining in AFRS than control (P=0.013) and CRSsNP (P=0.002). A significant difference was also found at the IT subepithelial level for AFRS compared with controls (P=0.001) and CRSsNP (P<0.001). Within AFRS, IgE staining was increased in the subepithelium compared to epithelium (P=0.003). ImmunoCAP analysis on IT tissue from AFRS and controls demonstrated increased antigen-specific IgE for 5 of 14 antigens (P<0.05) and total IgE (P<0.001). There were no significant anatomic differences between IT and sinus IgE staining. CONCLUSION: More fungal and nonfungal IgE is expressed in IT and sinus tissues of AFRS patients, as compared with control and CRSsNP patients.

Chemokine receptor expression in non-melanoma skin cancer.

BACKGROUND: Previous studies suggest that chemokines and chemokine receptors have a role in the metastatic process. A correlation exists between the specific expression of these chemoattractive, pro-inflammatory cytokines and the ability of cancer to disseminate. Prior studies have shown that in metastatic melanoma and squamous cell carcinoma of the head and neck upregulation of CXC (alpha) chemokine receptor (CXCR)4 and CC (beta) chemokine receptor (CCR)7 expression is accompanied by downregulation of the chemokine receptor CCR6. However, the expression patterns of CCR6, CCR7 and CXCR4 in non-melanoma skin cancer have yet to be elucidated. METHODS: The expression patterns of CCR6, CCR7 and CXCR4 were determined using an immunohistochemical approach on formalin-fixed, paraffin-embedded normal, pre-cancerous actinic (solar) keratosis, squamous cell carcinoma and basal cell carcinoma tissues. RESULTS: Analysis of chemokine receptor expression showed downregulation of CCR6 and upregulation of CCR7 and CXCR4 in potentially metastatic non-melanoma skin cancer, invasive squamous cell carcinoma, but this pattern did not exist in non-melanoma skin cancer with no metastatic potential, basal cell carcinoma; or actinic keratosis, when compared with normal skin. CONCLUSIONS: Chemokine receptor expression may influence the biological behavior of non-melanoma skin cancer. The exact mechanism by which this occurs requires further study.

Autocrine motility-stimulatory pathways of oral premalignant lesion cells.

Patients with premalignant oral lesions have varying levels of risk of developing oral squamous cell carcinoma (OSCC), whose aggressiveness requires increased motility. Not known is if and how premalignant oral lesion cells acquire the increased motility characteristic of OSCC. This was addressed by immunohistochemical analysis of banked premalignant lesion tissues and by functional analyses using cultures established from premalignant oral lesions and OSCC. These studies showed premalignant oral lesion cells and OSCC to be more motile than normal keratinocytes. Concomitantly, levels of ceramide were reduced. The activity of the protein phosphatase PP-2A, which restricts motility and which can be activated by ceramide, was also diminished. This was due to IL-10 released from premalignant lesion cells. Treatment with a membrane-permeable ceramide restored PP-2A activity and blocked migration. These studies show an autocrine motility-stimulatory pathway that is mediated in premalignant lesion cells by IL-10 through its reduction of ceramide levels and inhibition of PP-2A activity.

Decreased T-cell proliferation and skewed immune responses in LLC-bearing mice.

Deficits in immune cell responses have been reported in cancer patients. We used the murine Lewis lung carcinoma (LLC) model to better understand these deficits. The goal of this study was to determine if the immune responses of LLC tumor-bearing (TB) mice differ from control mice and whether the difference could be attributed to either antigen-presenting cells (FPC) or to T cells. Tumors were first allowed to grow in vivo for approximately 2 weeks. Splenocytes were then isolated for in vitro proliferation and cytokine release studies. The results showed a decrease in mitogen-stimulated proliferation by unfractionated splenocyte cultures from TB mice when compared to control mice in response to concanavalin A (Con A), a T-cell mitogen. Decreased responses were also observed when the APC spleen cell fraction from TB mice was cultured with normal T cells, although proliferation was more prominently reduced in cultures of TB T cells plus normal APC. Also, splenocytes from TB mice secreted significantly increased levels of IFN-gamma, IL-4, and IL-10. Admixing APC from control mice with TB T cells significantly decreased levels of IL-4 and IL-10 secretion as compared to the levels secreted by cocultures of TB T cells and TB APC. The decreased cytokine profile in the presence of normal APC despite the presence of TB T cells suggests that APC contributes to the immune dysfunction, including Th skewing of tumor bearers, possibly through their influence on T-cell expansion and cytokine production. Finally, our assessment of the APC population contributing to the observed immune dysfunction--i.e., dendritic cells or macrophages--showed that the proliferation of TB T cells was decreased regardless of the APC population with which they were cocultured. However, normal T-cell proliferation was only reduced by the addition of TB macrophages and not by the addition of TB dendritic cells. In conclusion, our results demonstrate that LLC TB mice have a skewed immune response characterized by a decreased proliferative response with both T cells and APC affected by the presence of tumor.

Combination docetaxel plus vitamin D(3) as an immune therapy in animals bearing squamous cell carcinomas.

OBJECTIVE: Background tumor growth results in the mobilization of immune inhibitory CD34(+) progenitor cells. However, vitamin D(3) can differentiate the CD34(+) cells into immune stimulatory dendritic cells. This study determined if docetaxel treatment could increase the impact of the vitamin D(3) to generate dendritic cells. METHODS: The murine squamous cell carcinoma model, SCC VII/SF, which is often used as a head and neck cancer model, was used to determine the immunological effects of two cycles of docetaxel plus vitamin D(3). RESULTS: Vitamin D(3) with or without docetaxel was similarly effective in reducing CD34(+) cell levels within the spleen, lymph nodes, and tumor. Dendritic cell levels were similarly enhanced in the lymph nodes by vitamin D(3) alone or combined with docetaxel. However, the combination treatment caused a prominent increase in intratumoral levels of active T cells, which was not observed by the individual treatments. CONCLUSION: Incorporating docetaxel treatment with vitamin D(3) differentiation-inducing treatment enhances intratumoral immune responsiveness.

Incomplete Th2 skewing of cytokines in plasma of patients with squamous cell carcinoma of the head and neck.

Levels of cytokines, and in particular those that reflect Th1 or Th2 bias, were measured in the plasma of patients with head and neck squamous cell carcinomas (HNSCC). Compared with plasma cytokine levels of age-matched controls, cytokine levels in HNSCC patients suggested a shift to a Th2 bias as levels of the Th2 cytokines interleukin-4 (IL-4), IL-6, and IL-10 were increased, and levels of the Th1 cytokine interferon-gamma (IFN-gamma) were decreased. However, levels of the Th1 cytokines IL-2 and granulocyte macrophage-colony-stimulating factor (GM-CSF) were increased, which is not consistent with full Th2 skewing. Assessment of cytokine levels in patients with malignancies other than HNSCC demonstrated many similarities to HNSCC patients, but HNSCC patients exhibited a more pronounced increase in GM-CSF levels and a decline in IFN-gamma levels. For most cytokines there was no association between the shifts in cytokine levels in HNSCC patients and either the extent of tumor burden or extent of metastasis. However, patients with large HNSCC tended to be the population that demonstrated increased levels of IL-4 and IL-6. These results suggest skewing toward a Th2 bias in HNSCC patients, with the Th2 shift being incomplete and indicative of the presence, rather than the extent, of malignant disease.

Modulation of Th1 and Th2 cytokine profiles and their association with advanced head and neck squamous cell carcinoma.

OBJECTIVE: Plasma cytokine concentrations from patients with head and neck squamous cell carcinoma (HNSCC) were measured to determine whether the potential modulation of host Th1 vs Th2 immune responses are associated with advanced clinical disease. STUDY DESIGN AND SETTING: The concentrations of IL-4, IL-6, IL-10, and IL-12 were measured in the plasma of 58 patients with histologically proven HNSCC. These data were examined with respect to the histologic size (T-stage) of the primary tumor, and presence of nodal metastasis. RESULTS: The concentrations of IL-12 were greater from patients without nodal metastasis, and with T(1)/T(2)-stage tumors. IL-10 levels were greater from patients with nodal metastasis, and with T(3)/T(4)-stage tumors. The concentrations of IL-6 were greater from patients with T(3)/T(4)-stage tumors. CONCLUSIONS: Using parameters of primary tumor size and presence of nodal metastasis, patients with advanced HNSCC have significantly less plasma IL-12 levels, and greater plasma IL-10 and IL-6 levels. SIGNIFICANCE: Patients with advanced HNSCC have a potentially diminished Th1 immune response, and a stronger potential Th2 immune response when compared to that of patients with less advanced disease. EBM RATING: D-5.

Local IgE production in nonatopic nasal polyposis.

INTRODUCTION: Chronic rhinosinusitis with nasal polyposis (CRSwNP) represents an eosinophilic T-helper 2 inflammatory response. Local production of IgE within nasal polyps (NPs) has been demonstrated, suggesting a role for local IgE in the pathogenesis of NP in atopic CRS patients. We hypothesized that local IgE specific to inhalant allergens may also play a role in the genesis of NP in nonatopic CRS patients. METHODS: Sinus and inferior turbinate tissue was obtained from nonatopic CRSwNP patients (n = 7), chronic rhinosinusitis without nasal polyps (CRSsNP) patients (n = 15), and healthy controls (n = 9) at the time of surgery. ImmunoCAP analysis (Phadia AB, Portage, MI) for 14 common inhalant antigens was performed on tissue homogenates to determine the antigen-specific response. RESULTS: Total IgE levels did not differ in sinus or turbinate tissue between CRSwNP, CRSsNP, or control patients. CRSwNP sinus tissue had higher levels of specific IgE for cockroach and plantain (p = .03) than other groups and elevated Alternaria IgE levels when compared with CRSsNP sinus tissue (p < .05). No significant differences were found for any of the other antigen-specific IgE levels. Fifty-seven percent of CRSwNP polyps demonstrated a polyclonal IgE response, whereas the other 43% had no demonstrable antigen-specific IgE. In contrast, only 17% of CRSsNP patients demonstrated a polyclonal response within sinus tissue, whereas 67% had no detectable antigen-specific IgE. There was no significant difference in levels of IgE in inferior turbinate tissue between the groups (p > .05). CONCLUSIONS: Localized mucosal IgE specific to common inhalant allergens appears to play a role in a subset of CRSwNP patients without evidence of systemic atopy.

Tumors skew endothelial cells to disrupt NK cell, T-cell and macrophage functions.

INTRODUCTION: Patients and mice with solid tumors, such as Lewis lung carcinoma (LLC), have defects in functions of immune effector cells. Endothelial cells, a component of the tumor vasculature, are potential regulators of immune cell functions. Therefore, these studies examined the impact of exposure to LLC tumor on the ability of endothelial cells to modulate immune cell functions. MATERIALS AND METHODS: Endothelial cells were pre-treated with LLC tumor-conditioned medium (Endo(T-sup)) for 24 h. Control endothelial cells that were exposed to medium (Endo(Media)) epithelial cell-conditioned medium or (Endo(Epi-sup)). After the initial 24 h incubation, endothelial cells were washed and fresh media was added. Cells were allowed to incubate for an additional 24 h. Supernatants from Endo(Media), Endo(Epi-sup) or Endo(T-sup) were collected and assayed for immune modulatory products and for immune modulatory activity. RESULTS: Supernatant from Endo(T-sup) contained increased levels of PGE2, IL-6 and VEGF as compared to Endo(Media) and Endo(Epi-sup) controls. NK cell activity, as measured by TNF-alpha and IFN-gamma secretion, was increased following exposure to media conditioned by Endo(Media) and Endo(Epi-sup) Exposure of NK cells to supernatants of Endo(T-sup), also increases TNF-alpha and IFN-gamma secretion, but to a lesser extent than by Endo(Media) and Endo(Epi-sup). Examination of macrophage functions demonstrated that supernatant from Endo(T-sup) decreased microbead phagocytosis and increased production of the immune suppressive mediators, IL-10 and PGE2. Lastly, T-cell responses to stimulation with anti-CD3 in the presence of supernatants from Endo(T-sup) were examined. IFN-gamma production by CD8+ T-cells was reduced after exposure to Endo(T-sup)-conditioned medium, as compared to cells treatments with medium or control conditioned medium. Production of IFN-gamma by CD4+ T-cells exposed to Endo(T-sup) was not altered. CONCLUSIONS: Taken together, these studies demonstrate that tumors skew endothelial cells to disrupt NK cell, T-cell and macrophages functions, and represents a novel mechanism of tumor-induced immune suppression.

Effects of housing density on weight gain, immune function, behavior, and plasma corticosterone concentrations in BALB/c and C57BL/6 mice.

The Guide for the Care and Use of Laboratory Animals contains recommended housing densities for rodent species that are commonly used by the scientific community. However, at the time of the Guide's publication, housing density recommendations were based heavily on the professional judgment of qualified scientists. Some scientists therefore question whether rodents can be housed at greater densities, whereas others wonder whether the space currently provided for rodents is sufficient. The present study was designed to determine the effect of housing adult female BALB/c- and C57BL/6-mice in standard 75-in(2) (484-cm(2)) ventilated cages at various housing densities (n = 2, 5, and 10 mice/cage). Measures of weight gain, plasma corticosterone, behavior, and immune parameters were evaluated at 7, 28, and 70 d after housing allocation. Housing BALB/c mice at 10/cage had negative effects on weight gain, corticosterone, behavior, and immune parameters. Housing C57BL/6 mice at 10/cage did not affect immune function or weight gain, although behavior and corticosterone showed statistical trends implying a negative effect Differences associated with housing densities of 2 and 5 mice/cage were less robust for all variables measured. We conclude that housing female BALB/c mice at 10 mice/cage (that is, at twice the Guide-recommended density) affects their physiology. We also conclude that mice vary in their responses in the parameters measured. These observations support the conclusion that it will be extremely challenging to scientifically determine an optimal cage density standard that can be uniformly applied across all mouse strains.

Antigen-specific IgE in sinus mucosa of allergic fungal rhinosinusitis patients.

BACKGROUND: Local tissue production of antigen-specific immunoglobulin E (IgE) has been shown in patients with allergic rhinitis and in patients with chronic rhinosinusitis (CRS) with nasal polyps. In allergic fungal rhinosinusitis (AFRS), specific IgE has been established in nasal lavage fluid and eosinophilic mucin. In this study, local production of antigen-specific IgE within sinus mucosa of AFRS patients was evaluated. METHODS: Sinus mucosa homogenates from 11 AFRS patients, 8 patients with CRS without nasal polyps (CRSsNP), and 9 nonrhinosinusitis control patients were assessed for IgE localization by immunohistochemistry. AFRS and control tissue homogenates were also evaluated for antigen-specific IgE to 14 common antigens by ImmunoCAP testing (Phadia AB, Portage, MI). RESULTS: There was a significant increase in IgE staining in AFRS sinus epithelium and subepithelium compared with controls and with patients with CRSsNP (p

Immunolocalization of dendritic cells and pattern recognition receptors in chronic rhinosinusitis.

BACKGROUND: Dendritic cell (DC) activation and antigen presentation to T cells are critical to innate and adaptive immunity. Toll-like receptors (TLRs) are known to bind pathogen-associated molecular patterns in addition to sinonasally secreted surfactant proteins (SP) such as SP-A and SP-D. TLR binding is known to activate DCs. Based on these observations, we sought to establish the presence, in sinonasal mucosa, of DC and the pattern recognition receptors (PRRs), CD14, TLR2, and TLR4. METHODS: Sinonasal biopsy specimens were taken from patients with eosinophilic nonatopic nasal polyposis (n = 4), allergic fungal sinusitis (n = 1), and nondiseased patients undergoing cerebrospinal fluid leak repair or pituitary tumor resection (n = 2). Tissue samples were stained immunohistochemically for PRR (CD14, TLR2, and TLR4), mature DC marker (CD208), iDC marker (CD209), or isotype controls. RESULTS: Immature and mature DC were immunolocalized to the subepithelial stroma and ciliated epithelial surface, respectively. Diffuse staining of CD14 was observed throughout the stroma with additional staining in the ciliated epithelium. The TLR markers showed no staining in the ciliated epithelium. TLR2 primarily localized in stroma immediately deep to the ciliated epithelial surface. TLR4 immunolocalized to submucosal seromucinous gland ductal epithelium. Data from nondiseased patients were mixed, with one patient showing minimal staining of any of the tested cellular markers. CONCLUSION: This study indicates progressive DC activation and emigration of mature antigen-presenting cells from the epithelial surfaces of sinonasal mucosa. The presence of TLR known to bind SP-A and SP-D suggests a link between SP expression and immune response in sinonasal mucosa.

Mechanisms of tumor growth and metastasis in head and neck squamous cell carcinoma.

The formation and progression of head and neck squamous cell carcinoma (HNSCC) is multisystemic and involves the immune system, vascularization, and dissemination. Immune involvement includes the subversion of anti-tumor defenses. Vascularization involves both angiogenesis and vasculogenesis. Dissemination involves local tumor invasion as well as distant metastasis through processes including angiogenesis and lymphangiogenesis. Current studies in the dysregulation of various processes, including genetic stability, angiogenesis, lymphangiogenesis, immune regulation, and immune function, are opening opportunities for the development of targeted tumor therapies. The interrelationship of these processes in HNSCC development will be explored in this review.

Increased aberrance of cytokine expression in plasma of patients with more advanced squamous cell carcinoma of the head and neck.

Patients with head and neck squamous cell carcinoma (HNSCC) are biased toward the Th2 phenotype as they have increased levels of the Th2 cytokines IL-4, IL-6 and IL-10 and diminished levels of the Th1 cytokine IFN-gamma. However, this bias is incomplete since levels of the Th1 cytokines IL-2 and GM-CSF are increased. This study examined the interrelationship among the plasma levels of Th1 and Th2 cytokines in 101 HNSCC patients and 40 age-matched controls without a known malignancy. Control subjects showed extensive interrelationships among levels of IL-1, IL-2, IL-4, and IFN-gamma, as well as between GM-CSF and TGF-beta. HNSCC patients showed an interrelationship in levels of IL-2, IL-4, IFN-gamma and GM-CSF, but to a less significant degree. What was prominent was a decline in correlation among cytokine levels with increased disease burden, such that there were no relationships among any of the cytokines in stage T4 patients. Nodal involvement also was associated with cytokine levels being more independent of each other, although the impact of nodal disease was less prominent than tumor burden. These results show a partial Th2 cytokine bias in HNSCC patients and a progressively more aberrant expression of cytokines with more advanced disease.

Phase 1B study to improve immune responses in head and neck cancer patients using escalating doses of 25-hydroxyvitamin D3.

Patients with head and neck squamous cell carcinoma (HNSCC) have profound immune defects. These defects are associated with a poor prognosis and are mediated, in part, by immune inhibitory CD34(+) progenitor cells, whose numbers are increased in the peripheral blood of HNSCC patients. Immune inhibitory CD34(+) cells are also present within HNSCC tumors. A phase IB clinical trial was conducted with HNSCC patients to determine if treatment with the differentiation-inducer 25-hydroxyvitamin D(3) could diminish CD34(+) cell levels and improve a panel of immune parameters. Here we present the results of treatment with orally administered escalating doses (20, 40, 60 microg) of 25-hydroxyvitamin D(3), with an emphasis on the six patients who received the maximum dosage of 60 microg per day. Peripheral blood was collected at 0, 1, 2, 4, and 6 weeks, and assessed for markers of immune activity. Although no clinical responses were observed, results of this pilot study demonstrated that treatment of HNSCC patients with 25-hydroxyvitamin D(3 )reduces the number of immune suppressive CD34(+) cells, increases HLA-DR expression, increases plasma IL-12 and IFN-gamma levels, and improves T-cell blastogenesis. In contrast, 25-hydroxyvitamin D(3) treatment did not modulate plasma IL-1beta, IL-2, IL-4, IL-6, IL-10, GM-CSF, or TGF-beta levels.