Early Induction of Cross-Reactive CD8+ T-Cell Responses in Tonsils After Live-Attenuated Influenza Vaccination in Children.

BACKGROUND: Live-attenuated influenza vaccine (LAIV) was licensed for prophylaxis of children 2-17 years old in Europe in 2012 and is administered as a nasal spray. Live-attenuated influenza vaccine induces both mucosal and systemic antibodies and systemic T-cell responses. Tonsils are the lymph nodes serving the upper respiratory tract, acting as both induction and effector site for mucosal immunity. METHODS: Here, we have studied the early tonsillar T-cell responses induced in children after LAIV. Thirty-nine children were immunized with trivalent LAIV (containing A/H1N1, A/H3N2, and B viruses) at days 3, 7, and 14 before tonsillectomy. Nonvaccinated controls were included for comparison. Tonsils and peripheral blood (pre- and postvaccination) were collected to study T-cell responses. RESULTS: Tonsillar and systemic T-cell responses differed between influenza strains, and both were found against H3N2 and B viruses, whereas only systemic responses were observed against A/H1N1. A significant increase in cross-reactive tonsillar CD8+ T cells recognizing conserved epitopes from a broad range of seasonal and pandemic viruses occurred at day 14. Tonsillar T cells showed significant cytokine responses (Th1, Th2, and granulocyte-macrophage colony-stimulating factor). CONCLUSIONS: Our findings support the use of LAIV in children to elicit broadly cross-reactive T cells, which are not induced by traditional inactivated influenza vaccines and may provide protection to novel virus strains.

Correlation between Excessive Daytime Sleepiness (EDS) and self-reported and objective nasal characteristics.

BACKGROUND: The aim of the present study was to explore the relationship between excessive daytime sleepiness (EDS), Peak Nasal Inspiratory Flow (PNIF), and patient reported symptoms from the nose. METHOLOGY: Six hundred and fifty one consecutive patients referred to a hospital on suspicion of sleep related breathing disorder (SRBD) were included in the study. Daytime sleepiness was assessed by Epworth Sleepiness Scale (EpSS). Nasal airflow was measured with Peak Nasal Inspiratory Flow (PNIF). Symptoms of sino-nasal dysfunction and diseases were graded on Visual Analogue Scales (VAS). RESULTS: EpSS score was not correlated with nasal flow as measured by PNIF or nasal obstruction - VAS scores. There were significant associations between daytime sleepiness and patient-reported VAS-scores on nasal discharge, headache, coughing, general health and to some extent sneezing when age, gender, BMI and reported co-morbidity levels were adjusted for. CONCLUSION: A clinical implication of this is that patients with EDS may be evaluated and treated for sino-nasal disease, while medical and surgical measures to open the nose per se may not be effective therapeutic options. A scientific implication is that the relationship between SRBD and sino-nasal disease should be further investigated.

In vitro Monocyte IL-6 Secretion Levels Following Stimulation with Autologous Spheroids Derived from Tumour or Benign Mucosa Predict Long-term Survival in Head and Neck Squamous Cell Carcinoma Patients.

MCP-1/IL-6 in vitro monocyte secretion upon coculture with autologous fragment spheroids was studied in relation to patient 5- and 10-year overall survival rates in head and neck squamous cell carcinoma (HNSCC) patients (n = 65) diagnosed between 1998 and 2005, nine of whom had an human papilloma virus (HPV) tumour infection. The spheroids were harvested from malignant or benign tissue during primary surgery. Two weeks following surgery, freshly isolated autologous monocytes and benign or malignant spheroids were cocultured 24 h in vitro. The IL-6 secretion was expressed as a fraction of the lipopolysaccharide (LPS) response from the same batch of monocytes. HPV status was obtained by employing PCR analyses of primary diagnostic blocks. IL-6/MCP-1 response levels were not found to be dependent on HPV infection status. MCP-1 secretion did not predict prognosis, nor did in vitro IL-6 monocyte background or LPS-stimulated IL-6 secretion. At 5-year observation, dichotomized IL-6 levels following monocyte coculture, with both malignant and benign spheroids, showed a strong trend towards predicting survival, that is a low monocyte malignant coculture response showed a survival of 31 ± 17 versus 58 ± 17% with a high such response (P = 0.057). When studying monocyte IL-6 coculture responses evaluating benign and malignant spheroid results statistically together, a prediction of survival up to 10 years was found (hazard ratio = 0.48; confidence interval = 0.24-0.96; P < 0.05) with double low IL-6 responses. This survival prediction was also present after an adjustment for HPV tumour infection status. In conclusion, monocyte IL-6 in vitro secretion in cocultures with autologous spheroids/serum from HNSCCs predicted 5- and 10-year survivals, both with and without tumour HPV tumour adjustment.

Health-related quality of life scores in long-term head and neck cancer survivors predict subsequent survival: a prospective cohort study.

OBJECTIVE: To examine the survival prediction of long-term health-related quality of life in patients with head and neck squamous cell carcinoma. DESIGN: Patients with head and neck squamous cell carcinoma diagnosed in the period between July 1992 and October 2001, who had been disease free for a minimum of 1 year following therapy, responded to structured interviews including several validated questionnaires in the period from October 2002 to March 2004. The study ended in June 2009 with a mean observation time of 75 ± 4 months among the survivors. Twenty-four deaths were observed. SETTING: University hospital, referral centre of the Western Norway. PARTICIPANTS: One hundred and thirty-nine cognitive functioning patients. MAIN OUTCOME MEASUREMENTS: Overall survival as of June 2009. This was correlated with various clinical factors and the EORTC QLQ-C30 questionnaire, the Eysenck Personality Inventory and the Coping inventory completed between October 2001 and March 2004. RESULTS: A general symptom sum score was significantly predictive of survival directly and after sequential adjustment for self-reported levels of neuroticism, avoidance focused coping, coping by suppression of competing activity, alcohol consumption, smoking status and heart/lung disease, as well as gender, age, time between diagnosis and inclusion, tumour node metastasis (TNM) stage and tumour site. Similar results were found for the health-related quality of life indices 'fatigue', 'dyspnoea' and 'sleep disturbance'. A dichotomised variable based on the general symptom sum score was calculated, and a high risk group, as to mortality, including less than a quintile of the total patient population was established. A hazard ratio of 5.15 was found for the dichotomised general symptom sum score. CONCLUSION: We have shown a unique and independent survival prediction from long-term EORTC QLQ-C30 scores in successfully treated and cognitive functioning head and neck squamous cell carcinoma patients.

MAPKs ERK and p38, but not JNK phosphorylation, modulate IL-6 and TNF-α secretion following OK-432 in vitro stimulation of purified human monocytes.

Interaction between the immune system and cancer allows for the use of biological response modifiers, e.g. OK-432, in cancer therapy. OK-432, penicillin-killed Streptococcus pyogenes, is used in treating carcinomas, but also lymphangiomas. We have studied the role of monocytes (MOs) in the immune response to OK-432 by examining IL-6 and tumour necrosis factor (TNF)-α secretion after in vitro MO stimulation with OK-432, to some extent in comparison with lipoteichoic acid (LTA) and lipopolysaccharide (LPS). LTA stimulation of whole blood gave IL-6 but not TNF-α secretion, as previously shown with OK-432 stimulation, whereas both cytokines were secreted following LPS stimulation. Addition of the MAPK kinase (MAPKK) MEK inhibitor U0126 inhibited IL-6/TNF-α secretion in a dose-dependent manner. Flow cytometry and to some extent Western blot (Wb) analyses showed that MAPK ERK, located downstream of MEK1/2, is predominantly phosphorylated at isolation from peripheral blood. Addition of the p38 MAP kinase inhibitor SB202190 decreased MO IL-6/TNF-α production upon OK-432 stimulation in a dose-dependent manner. Addition of the MAPK JNK inhibitor SP600125 did not systematically change the MO IL-6/TNF-α OK-432 response. Flow cytometry showed that when stimulating the MOs before isolation from blood, LPS yielded ERK phosphorylation and LPS/LTA p38 phosphorylation, whereas OK-432 had no effects on phosphorylation levels. In conclusion, we have shown that OK-432 resembles TLR2 more than TLR4 stimulation of MOs and depends on MAPKK MEK and MAPK p38, but not on JNK phosphorylation. The MEK and p38 MO OK-432 stimulation dependence is possibly related to the differentiation of cells of the MO lineage.

Choice of psychological coping in laryngectomized, head and neck squamous cell carcinoma patients versus multiple sclerosis patients.

To be treated for cancer must be a frightening experience. Yet quality of life (QoL) of successfully treated cancer patients seems to be relatively similar in comparison with QoL of a general population, with psychological coping partly responsible for this finding. When measuring choice of coping, the nature of coping score levels constituting appropriate scores, and whether score levels rely on the context of the disease has not been settled. We have studied the COPE coping responses as related to disease in successfully treated head and neck squamous cell carcinoma (HNSCC) patient groups (general and laryngectomized), as well as compared to multiple sclerosis (MS) patients. The COPE response patterns have also been compared to the Beck depression inventory (BDI) scores. Age and gender of patients were not directly associated with choice of coping. Within the problem-focused coping indexes, the coping index "active coping" was reported to be most utilized among HNSCC patients, whereas "coping by suppression" and "coping by social support" were most utilized among MS patients. Emotional-focused coping was most prevalent among HNSCC patients and lowest among the MS patients. Level of avoidance coping was similar between the groups. The coping of the general HNSCC patients differed most from the MS patients. An association was shown between increased coping efforts and lowered mood. In particular, avoidance coping was associated with lowered mood. These associations were stronger among the MS patients than HNSCC patients. Drinking to cope was most prevalent among the laryngectomized group, and was correlated with BDI scores in all groups. Furthermore, adequate coping seems to be to limit avoidance coping and promote coping by acceptance. The response pattern of the COPE inventory seems to be valid among HNSCC and MS patients.

Health-related quality of life in long-term survivors after high-dose chemoradiotherapy followed by surgery in esophageal cancer.

Curative treatment of esophageal cancer with definitive or preoperative high-dose chemoradiotherapy inflicts a major strain on the patients with potentially severe physical, emotional, and social consequences. The aim of this study was to assess various aspects of quality of life and fatigue in long-term survivors following such a treatment. Patients undergoing a potentially curative treatment between 1996 and 2007, and still alive (n= 41) completed quality of life questionnaires of the European Organization for Research and Treatment of Cancer core questionnaire (QLQ-C30) and esophageal cancer module (QLQ-OES18). Twenty patients were treated by surgery alone, and 21 patients were scheduled for high-dose chemoradiotherapy followed by surgery. Five of those patients did not undergo planned surgery. Preoperative chemoradiotherapy consisted of three courses of chemotherapy, cisplatin 100 mg/m(2) and 5-fluorouracil 5000 mg/m(2) in each course and concomitant radiotherapy of a median dose 66 Gy. Quality of life in esophageal cancer patients receiving high-dose chemoradiotherapy was compared with that for esophageal cancer patients who received only surgery, head and neck cancer patients, laryngectomized patients, and a random sample of the general Norwegian population. Esophageal cancer patients treated by high-dose chemoradiotherapy had significantly worse global quality of life as reflected by almost all functional scales and higher fatigue compared with esophageal cancer patients who received surgery alone, head and neck cancer patients, and the general Norwegian population. There were no significant differences in quality of life between the esophageal cancer patients receiving high-dose chemoradiotherapy and the laryngectomy patients. Further, the esophageal cancer patients receiving high-dose chemoradiotherapy had higher intensity of other symptoms like general pain, insomnia, nausea/vomiting, diarrhea, and constipation compared with the esophageal cancer patients who received surgery alone, head and neck cancer patients, and the general Norwegian population. High-dose chemoradiotherapy with cisplatin and 5-fluorouracil had a considerable negative long-term effect on global quality of life in patients with resectable esophageal cancer. Fatigue was a prominent long-lasting symptom in these patients.

Co-culture of head and neck squamous cell carcinoma spheroids with autologous monocytes predicts prognosis.

Co-culture of monocytes with autologous fragment (F) spheroids originating from malignant (M) tumour or benign (B) control mucosa of head and neck squamous cell carcinoma (HNSCC) yields interleukin (IL)-6 and monocyte chemo-attractant protein (MCP)-1 secretion. This study investigates the association between this cytokine co-culture response and prognosis. Analysis of IL-6 and MCP-1 content of supernatants from monocytes in vitro co-culture with autologous MF- or BF-spheroids was investigated in a cohort of HNSCC patients (n = 65) diagnosed between 1998 and 2005, all of whom were treated with curative intent by primary surgery. The IL-6 response was expressed as a fraction of the lipopolysaccharid response of the same batch of monocytes. Recurrence, survival and causes of death were then established following the second part of 2005. MCP-1 levels did not predict prognosis. We found that increased levels of IL-6 from autologous monocytes in co-culture with MF-spheroids predicted recurrence with a hazard ratio (HR) of 1.5 [confidence interval (CI): 1.01-2.60; P = 0.05] and co-culture with BF-spheroids and monocytes predicted recurrence (HR = 4.17; CI: 1.54-11.29; P = 0.005). The same results where obtained in addition with TNM stage of the patients. Simultaneous analysis of BF- and MF-spheroid co-culture IL-6 responses as well as adjustment for age and TNM stage of the patients allowed prediction of total survival (HR = 3.1; CI: 1.11-8.56; P = 0.03) based on BF co-culture levels. IL-6 secreted upon in vitro co-culture with monocytes and BF-spheroids predicts recurrence and prognosis, whereas co-culture with monocytes and MF-spheroids predicts recurrence.

TNF-alpha is secreted by monocytes in transit to become macrophages, but not by peripheral blood monocytes, following OK-432 (lyophilized S. pyogenes) stimulation.

OK-432, penicillin-killed Streptococcus pyogenes, is used in treating lymphangiomas and carcinomas. We have studied proinflammatory interleukin (IL) secretion following OK-432 stimulation of total blood, peripheral blood mononuclear cell (PBMC) and purified monocytes in vitro. OK-432 stimulation of purified monocytes gave IL-1beta, IL-1RA, IL-6, IL-12p40 and tumour necrosis factor (TNF)-alpha response. OK-432 stimulation of cells within blood did, however, not yield TNF-alpha secretion. When PBMC or monocytes were cultured in low-attachment wells a decreased IL secretion was observed compared to adherent cells. Inhibition of Syk kinase with piceatannol, only at high, non-specific doses, but not PI3 kinase inhibition with LY294002 or Wortmannin, decreased monocyte IL response to OK-432. This shows that beta(1-3)-integrin receptor function is not necessary for monocyte OK-432-stimulated TNF-alpha secretion. Direct blockage of the beta(2)-integrin (CD18) receptor by anti-CD18 antibody was also unable to prevent the stimulating effects of OK-432 in human monocytes. On the other hand, Syk phosphorylation is elevated upon adherence of monocytes and this is further increased by OK-432 stimulation, as shown by Western blot. The Fc-receptor was also ruled out as a main receptor of the OK-432 monocyte response. In conclusion, TNF-alpha secretion is only found in monocytes removed from blood. This TNF-alpha secretion is not mediated through the beta(1-3)-integrin receptors. OK-432 may act as a target-seeking substance whereby only monocytes adhered, e.g. to a tumour cell, become cytotoxic in part explaining why OK-432 is well suited as a cancer treatment drug.

The amount of treatment versus quality of life in patients formerly treated for head and neck squamous cell carcinomas.

The aim of the present study was to investigate the association between the self-reported quality of life (QoL) versus the initial TNM stage and amount of primary and recurrent tumor therapy given in a population of formerly treated head and neck squamous cell carcinoma (HNSCC) patients. We determined QoL by the European Organization for Research and Treatment of Cancer-Quality of Life Questionnaire (EORTC-QLQ) C30/H&N35 by structured interview. One hundred and twenty-two patients less than 80 years old, who had been diagnosed with HNSCC in western Norway in the period from 1992 to1997, and who had survived until 2000, were identified. Of these patients, 106 were eligible to be included. Ninety-six of these patients agreed to be interviewed. For TNM stage as well as the type of therapy given (local surgery, neck dissection or radiation therapy), T stage predicted the general QoL scores. Both increased TNM stage and all given tumor therapy seemingly caused lower H&N symptom QoL scores. Of the various tumor treatments employed, neck radiation therapy and neck dissection were indicated to be the most closely associated with the H&N QoL scores. Having neck dissection performed seemingly caused impairment beyond what was explained by the initial TNM stage. In conclusion, tumor therapy to HNSCC should not be restricted due to general QoL considerations. Further study of how and when to perform neck treatment is suggested in order to avoid unnecessary reduced H&N QoL.

Psychological coping style versus disease extent, tumour treatment and quality of life in successfully treated head and neck squamous cell carcinoma patients.

OBJECTIVES: To study self-reported coping style by the COPE questionnaire and the association to tumour node distant metastasis (TNM) stage, received treatment and health-related quality of life (HRQoL) in a population of successfully treated head and neck squamous cell carcinoma (HNSCC) patients. DESIGN: Disease free HNSCC patients were interviewed during a regular outpatient visit to the Department. SETTING: All patients <80 years who had been diagnosed with HNSCC, in Western Norway, in the period from 1992 to 1997 and who were disease free in the year 2000 were included. MAIN OUTCOME MEASURES: The HNSCC patients were studied by the European organization for research and treatment of cancer quality of life (QoL) questionnaire and by the COPE questionnaire from which coping styles profiles were extracted. Clinical and socio-demographic variables were also gathered. RESULTS: Level of problem focused coping style was associated with the T stage and whether or not the patient was given neck radiotherapy. An avoidance-focused coping style was generally associated with lowered HRQoL. Employment of emotional focused coping was associated with low HRQoL among the patients treated with surgery only, and associated with increased HRQoL among patients treated with radiation therapy only. The associations between HRQoL and coping style levels were stronger for HRQoL indexes reflecting cognitive/emotional function than for HRQoL indexes reflecting physical function. CONCLUSIONS: In former HNSCC patients, level of problem focused coping style was associated with the T stage and if given neck radiotherapy. Level of avoidance coping was inversely associated with the HRQoL, and level of emotional coping was associated with HRQoL in a complex manner.

The personality and quality of life in HNSCC patients following treatment.

The aim of the present study was to investigate the association between self-reported quality of life (QoL) and personality in successfully treated primary head and neck squamous cell carcinoma (HNSCC) patients. We determined QoL using the European Organisation for Research and Treatment of Cancer Quality of life Questionnaire (EORTC-QLQ) C30/H and N35, and personality by the Eysenck Personality Inventory (EPI). All patients younger than 80 years who had been diagnosed with HNSCC in Western Norway in the period from 1992 to 1997, and who had survived until 1999, were sampled. 96 patients (a 90% response rate) were included. Questionnaires were also mailed to all Norwegian laryngectomised patients; 104 patients returned the questionnaires (a 50% response rate). The neuroticism scores were test re-test reliable as determined by the neuroticism scores measured at the primary HNSCC diagnosis for a sub-sample (N=22) of the included patients. High neuroticism was associated with a low QoL in both patient samples. The neuroticism score was associated with the QLQ-C30 scales (common variance: 17-25%) and all QoL scores in the laryngectomised group (common variance: 11-25%), and the H and N35 symptom scores in the laryngectomised sample. The associations could still be shown when adjustments were made for gender, age, marital status, educational level, number of children and level of treatment. Extraversion was associated with general QoL, physical and emotional scores in the HNSCC patient sample. Radiation therapy in the HNSCC sample was associated with the H and N35 symptom scores, but different ones to those associated with neuroticism. In conclusion, high neuroticism, but not extraversion, is associated with a lowered QoL.

Mechanisms for monocyte activation in co-culture with autologous tumor spheroids.

Biopsies from carcinoma tissue and benign control mucosa from head and neck squamous cell carcinoma patients were used to establish fragment (F)-spheroids in vitro. We have previously shown that autologous monocytes co-cultured with F-spheroids in vitro secrete interleukin (IL)-6 upon 24h in co-culture. Presently, the aim was to study the mechanisms of this monocyte secretion. Paraformaldehyde (0.1% for 2min) or actinomycin-D (1 microg/ml for 24h) pre-treatment of the F-spheroids abolished the monocyte IL-6 co-culture response. Addition of glucose (100mM) or mannose (100mM), and to some extent galactose (100mM), but not fructose (100mM) to the co-cultures, partly inhibited the monocyte IL-6 co-culture response, but such addition did not inhibit the in vitro monocyte lipopolysaccharide (LPS)-generated IL-6 secretion. When mannose was added to the co-cultures, monocyte IL-6 mRNA expression was eradicated in malignant co-cultures and reduced to a low level in benign co-cultures. Addition of mouse anti-human beta(1)-integrin (anti-CD29) antibody (2 microg/ml) diminished the IL-6 co-culture response but not the monocyte LPS-generated IL-6 response. In conclusion, the monocyte IL-6 co-culture response is dependent on live spheroids and to some extent on direct contact with the F-spheroids, possibly via lectin-like receptor(s), the mannose receptor and beta(1)-integrin.

Relation between mood, social support and the quality of life in patients with laryngectomies.

Two hundred laryngectomized members of the Norwegian Society of Laryngectomies (NSL), a subsidiary of the Norwegian Cancer Society, were invited to answer the EORTC QLQ-C30 (version 3.0) and QLQ-H&N35 QOL questionnaires to assess their quality of life (QOL). The Beck Depression Inventory (BDI) scores, their levels of social support and their marital and educational statuses were also determined. In addition, the activity levels of each patient within the NSL were assessed. The questionnaires were returned anonymously by 104 patients. The results of this sample were compared with the responses to the EORTC QLQ C30/H&N35 by all of the survivors of treatment for head and neck squamous cell carcinoma (HNSCC) in western Norway between 1992 and 1997. This sample included 96 of 106 eligible patients. The QLQ-C30 symptom scores include, e.g., dyspnea, smell and taste. However, neither the QLQ-C30 functional scores nor the disease-specific scores of the people with laryngectomies differed from the general HNSCC-treated population. The level of social support by family, friends and neighbors was not associated with the QOL, whereas high BDI scores were associated with reduced QOL by most measured indexes. Furthermore, a positive association was determined between the level of activity within the NSL and QOL. This relation was to some extent secondary to differential BDI scores. In conclusion, the QOL of people with laryngectomies is relatively similar to a general population of patients treated because of HNSCC, is related to the activity level within a patient interest organization and is associated with a lower mood level.

Monocyte and monocyte-derived macrophage secretion of MCP-1 in co-culture with autologous malignant and benign control fragment spheroids.

This study was performed in order to determine how monocytes and macrophages in co-culture with autologous head and neck squamous cell carcinoma (HNSCC) tumor tissue regulate the secretion of monocyte chemotactic protein-1 (MCP-1). The levels of MCP-1 were measured when autologous monocytes or monocyte-derived macrophages (MDMs) were co-cultured in vitro with autologous fragment (F)-spheroids established from HNSCC tumors or benign mucosa serving as control. MCP-1 secretion from co-culture stimulated monocytes and MDMs was increased compared to spontaneous MCP-1 secretion. With prolonged co-culture, MDMs showed a steady-state MCP-1 secretion above background levels for up to 96 h, even with change of co-culture media every 24 h. Addition of an anti-MCP-1 antibody to the medium decreased co-culture-induced monocyte IL-6 secretion. Addition of lipopolysaccharide (LPS) (1 [microg/ml) reduced MCP-1 secretion compared to spontaneous secretion in monocyte cultures. F-spheroids also secrete MCP-1, but at insignificant levels compared to the MCP-1 secretion from monocytes and MDMs. MCP-1 secretion from monocytes/MDMs is regulated differently when co-culture stimulation is compared to LPS-stimulation. Monocytes and MDMs expressed MCP-1 mRNA at a high level in all tested conditions: stimulated in co-culture, not stimulated or stimulated with LPS, indicating post-transcriptional regulation of MCP-1 secretion. The secretion of MCP-1 from tumor-derived F-spheroids, and the maintenance of co-culture MCP-1 secretion from MDMs in vitro, suggests that tumor-associated macrophages are a source of MCP-1 in HNSCC tumors.

Human autologous monocytes and monocyte-derived macrophages in co-culture with carcinoma F-spheroids secrete IL-6 by a non-CD14-dependent pathway.

The secretion of interleukin (IL)-1 beta, IL-6 and tumour necrosis factor (TNF)-alpha were compared when freshly isolated autologous monocytes or monocytederived macrophages (MDMs) were co-cultured in vitro with autologous fragment (F)-spheroids established from a series of head and neck squamous cell carcinoma (HNSCC) patients. F-spheroids were generated from the malignant tumour (M-spheroids) or from benign mucosa (B-spheroids) from which the tumour originated control. If monocytes maturated towards MDMs before co-culture, the IL-6 secretion declined dependent on the extent of the MDM maturation by both M- and B-spheroid stimulation. When MDMs maturated in continuous co-culture, a steady-state secretion of IL-6 continued for several days but diminished when the culture medium was changed every 24 h. No co-culture-induced IL-1 beta or TNF-alpha was determined. Both the cytokine secretion and the mRNA gene expression revealed a different monocyte/MDM activation when co-culture and lipopolysaccharide (LPS)-stimulation were compared. Addition of anti-CD14 (10 microg/ml) decreased monocyte LPS-stimulated, but increased monocyte co-culture stimulated IL-6 secretion. In conclusion, M- and B-spheroids similarly stimulated monocytes and to a lesser extent MDMs. MDMs that maturated with F-spheroids present, retained responsiveness at the monocyte level. Co-culture-induced monocyte stimulation, as measured by IL-6 secretion, was not dependent on activation via the CD14 molecule.

Monocytes secrete interleukin-6 when co-cultured in vitro with benign or malignant autologous fragment spheroids from squamous cell carcinoma patients.

Biopsies from tumour and benign mucosa were removed from patients with head and neck squamous-cell carcinoma (HNSCC), chopped into cubes and transferred to a nonadhesive culture system where in vitro fragment (F)-spheroids were established. The F-spheroids stabilized within 14 days of culture in vitro with epithelial cells and fibroblasts on the surface. F-spheroids were co-cultured with freshly isolated autologous monocytes. The monocytes of 10 of 11 patients secreted interleukin (IL)-6 at a level similar to that of the average monocyte endotoxin-stimulated response. Secreted IL-1beta or tumour necrosis factor-alpha (TNF-alpha) levels greater than 0.1 times the endotoxin-stimulated secretion were determined in one and two of the 11 co-culture experiments, respectively. This different monocyte response to F-spheroids compared with endotoxin stimulation was also present at the mRNA expression level. HNSCC monocytes secreted no IL-6 after co-culture with autologous fibroblasts. When monocytes and F-spheroids were cultured separated by a semipermeable membrane, the IL-6 supernatant level was only approximately 25% of that observed during co-culture with direct contact. F-spheroids secreted only trace amounts of IL-6. In conclusion, monocytes of HNSCC patients generally secrete IL-6, but not IL-1beta or TNF-alpha, after stimulation with epithelial-associated components of F-spheroids upon direct contact and in part by a soluble substance.

Peripheral blood T-lymphocyte and monocyte function and survival in patients with head and neck carcinoma.

OBJECTIVES/HYPOTHESIS: To determine if the T-lymphocyte and monocyte functions of peripheral blood mononuclear cells (PBMCs) from patients with head and neck squamous cell carcinomas (HNSCC) are predictive factors for outcome. STUDY DESIGN: A prospective study describing the outcome, as to total survival and death by disease after at least 40 months observation, of 81 previously untreated male HNSCC patients in relation to PBMC T-lymphocyte and monocyte function. METHODS: T-lymphocyte mitogenesis and the cytokine level in culture supernatants of PBMC as well as monocytes were analyzed. These parameters were related to survival by Cox regression and Kaplan-Meier survival analysis. RESULTS: When patients with high versus low T-lymphocyte mitogen-stimulated proliferations were compared, a decreased proliferation was seen to be related to worse outcome. The predictive value of T-lymphocyte proliferation was shown to be an independent prognostic factor when adjusted for stage and stratified for anatomic location in survival analysis. The predictive value was also retained when the serum values of the major serum proteins and hormones and scores based on the smoking and alcohol history were added to the survival analysis with lymphocyte proliferation. Supernatant levels of gamma-interferon, interleukin (IL)-2, or IL-4 in PBMC cultures were not related to outcome. Monocyte function measured by endotoxin-stimulated IL-1beta, IL-6, IL-12, and tumor necrosis factor-alpha secretion did not relate to outcome of the patients. CONCLUSION: The PBMC T-lymphocyte-stimulated proliferation is an independent prognostic factor for male HNSCC patients.

Ex vivo interleukin (IL)-1 beta, IL-6, IL-12 and tumor necrosis factor-alpha responsiveness with monocytes from patients with head and neck carcinoma.

Seventy newly diagnosed Caucasian male patients with head and neck squamous cell carcinomas (HNSCC) were included in the study. All patients were less than 80 years of age, with no cachexia or auto-immune disease, and they were not taking immuno-active medications. Monocytes from these patients were cultured in vitro and supplemented with autologous serum under ex vivo conditions or cultured with serum-free medium. Comparison was made to monocytes from 59 patients with benign HN diseases. Similar physical activity levels prior to testing as well as a minimum stress load were ensured in both groups. Increased monocyte supernatant levels were determined under ex vivo conditions of interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6) and tumor necrosis factor (TNF)-alpha, but not of interleukin-12 (IL-12) with endotoxin stimulated monocytes of HNSCC patients compared to control conditions. Increased monokine levels were not present with mononuclear cell cultures stimulated with a T-cell general stimulatory agent or with purified monocytes not specifically stimulated. With endotoxin-stimulated monocytes under in vitro conditions, an increased supernatant was shown for TNF-alpha, but not IL-6. With serum from the different patients cultured with monocytes employed from a healthy control, no difference between the groups was shown in the IL-6 and TNF-alpha response to endotoxin stimulation. The differences in IL-1 beta and TNF-alpha, but not IL-6 levels were differentiated statistically from the smoking and alcohol histories of the patients. These findings suggest that the function of monocytes in general, and thus possibly all mononuclear phagocyte system cells in HNSCC patients, are altered.

Peripheral blood mononuclear cell (PBMC) responsiveness in patients with head and neck cancer in relation to tumour stage and prognosis.

We have previously shown an increased T lymphocyte and monocyte responsiveness in peripheral blood mononuclear cells (PBMC) from patients with head and neck squamous cell carcinoma (HNSCC) compared with PBMC from control patients. This study reports T lymphocyte function of PBMC of 81 patients with HNSCC dependent on disease stage and prognosis. Males with HNSCC under 80 years of age without cachexia, with no auto-immune disease or previous cancer and on no immuno-active medication were included at the time of diagnosis of disease. The follow-up was for at least 18 months. When cells from patients with early vs late stage disease according to the T, N or T + N stage of HNSCC were compared, decreased in vitro mitogen-stimulated and spontaneous T cell proliferation was seen with increasing tumour stage. When patients were studied according to disease-specific survival, a decreased T lymphocyte mitogen-stimulated proliferation was observed to be associated with a poorer prognosis. No changes in prognosis were noticed related to decreased gamma-IFN, IL-2 or IL-4 level of the supernatants of the T lymphocyte-stimulated PBMC in vitro cultures. With stratification for disease stage, we determined that PBMC in vitro T lymphocyte-stimulated proliferation predicted outcome for the HNSCC patients. The results were similar for both laryngeal and oral cavity/pharyngeal cancers. The present investigation provides evidence to support the idea that the relationship between HNSCC and the immune system of the host may provide clinically useful information about prognosis.