DNA repair capacity in lymphocytes of nasopharyngeal cancer patients.

Possible hereditary factors in the tumorigenesis of nasopharyngeal cancer (NPC) have not yet been clearly identified. In the present study, the DNA repair capacity of lymphocytes after exposure to the nitrosamine NDEA was quantified in order to elucidate whether this measure may be a factor in susceptibility to NPC. The alkaline single-cell microgel electrophoresis (Comet) assay was used to quantify chemically induced DNA damage and repair capacity in lymphocytes of 30 NPC patients (NPC) and 29 non-tumor donors (NTD). The induction of DNA single strand breaks, alkali labile and incomplete excision repair sites after exposure of lymphocytes to NDEA was assessed as differences between repair intervals of 0 min, 15 min, 30 min and 60 min, respectively. A RC(total) was assessed using the difference between the OTMs of 0 min of repair time and the 60-min repair interval for both groups. Repair capacities (RC) were calculated for the intervals according to the Olive Tail Moment (OTM), a quantitative measure for DNA migration in the Comet assay for the group of NPC patients and the NTD, accordingly. RCs were compared between the two groups using the Mann-Whitney U-Test. RC(15 min), RC(30 min) RC(60 min) and the RC(total) after a 60-min repair interval demonstrated no significant difference between the two groups. Furthermore, when comparing grades of DNA migration (OTM<2, 2-5, 5-10, 10-20, 20-30 and >30), there were no differences evident. In this investigation, rejoining of DNA single strand breaks in lymphocytes of NPC and NTD appeared to be accomplished to an equal degree and in equal time periods. However, the applied method does not give evidence concerning the quality of the single strand break rejoining processes. In this group of patients, tumorigenesis in NPC could not be associated with a decreased DNA repair capacity.

Mono(2-ethylhexyl)phthalate exhibits genotoxic effects in human lymphocytes and mucosal cells of the upper aerodigestive tract in the comet assay.

Phthalic acid esters such as di(2-ethylhexyl)phthalate (DEHP) are widely used as plasticizers in PVC products manufactured for commercial, medical, and consumer purposes. Humans are exposed to phthalates originating, e.g., from blood storage bags, tubing materials, and from food-wrapping. While xenoestrogenic and chronic toxic effects of phthalates have been extensively discussed, there is little data on genotoxic effects in human cells. The alkaline comet assay was used to detect single-strand breaks and alkali labile sites of DNA after incubation of human nasal mucosal cells (n = 11) and peripheral lymphocytes (n = 11) with mono(2-ethylhexyl)phthalate (MEHP), the principal hydrolysis product of DEHP. MEHP showed a dose-dependent enhancement of DNA migration both in human mucosal cells and in lymphocytes. This effect indicates a genotoxic potential of MEHP in human mucosal cells. It confirms previous data obtained on the effect of MEHP on lymphocytes.

Tissue engineering of autologous cartilage grafts in three-dimensional in vitro macroaggregate culture system.

In the field of tissue engineering, techniques have been described to generate cartilage tissue with isolated chondrocytes and bioresorbable or nonbioresorbable biomaterials serving as three-dimensional cell carriers. In spite of successful cartilage engineering, problems of uneven degradation of biomaterial, and unforeseeable cell-biomaterial interactions remain. This study represents a novel technique to engineer cartilage by an in vitro macroaggregate culture system without the use of biomaterials. Human nasoseptal or auricular chondrocytes were enzymatically isolated and amplified in conventional monolayer culture before the cells were seeded into a cell culture insert with a track-etched membrane and cultured in vitro for 3 weeks. The new cartilage formed within the in vitro macroaggregates was analyzed by histology (toluidine blue, von Kossa-safranin O staining), and immunohistochemistry (collagen types I, II, V, VI, and X and elastin). The total glycosaminoglycan (GAG) content of native and engineered auricular as well as nasal cartilage was assayed colorimetrically in a safranin O assay. The biomechanical properties of engineered cartilage were determined by biphasic indentation assay. After 3 weeks of in vitro culture, nasoseptal and auricular chondrocytes synthesized new cartilage with the typical appearance of hyaline nasal cartilage and elastic auricular cartilage. Immunohistochemical staining of cartilage samples showed a characteristic pattern of staining for collagen antibodies that varied in location and intensity. In all samples, intense staining for cartilage-specific collagen types I, II, and X was observed. By the use of von Kossa-safranin O staining a few positive patches-a possible sign of beginning mineralization within the engineered cartilages-were detected. The unique pattern for nasoseptal cartilage is intense staining for type V collagen, whereas auricular cartilage is only weakly positive for collagen types V and VI. Engineered nasal and auricular macroaggregates were negative for anti-elastin antibody (interterritorially). The measurement of total GAG content demonstrated higher GAG content for reformed nasoseptal cartilage compared with elastic auricular cartilage. However, the total GAG content of engineered macroaggregates was lower than that of native cartilage. In spite of the mechanical stability of the auricular macroaggregates, there was no equilibrium of indentation. The histomorphological and immunohistochemical results demonstrate successful cartilage engineering without the use of biomaterials, and identify characteristics unique to hyaline as well as elastic cartilage. The GAG content of engineered cartilage was lower than in native cartilage and the biomechanical properties were not determinable by indentation assay. This study illustrates a novel in vitro macroaggregate culture system as a promising technique for tissue engineering of cartilage grafts. Further long-term in vitro and in vivo studies must be done before this method can be applied to reconstructive surgery of the nose or auricle.

Prognostic implications of hemoglobin levels before and after surgery as well as before and after radiochemotherapy for head and neck tumors.

Several studies have shown that tumor cells may develop resistance to radiotherapy, proliferating under hypoxic conditions. Following surgery, patients may develop low hemoglobin levels, which may cause low oxygen conditions. This retrospective analysis was undertaken to determine the impact of low hemoglobin levels in patients with head and neck tumors treated with combined-modality therapy (surgery and postoperative radiochemotherapy). We studied 120 patients with mostly advanced head and neck tumors (88% stage III/IV) who had undergone macroscopically complete resections of their primary tumors and lymph node metastases. At 20-277 days after surgery (median: 51.3 days), these patients received postoperative chemoradiotherapy (56.7 Gy of radiation over 28-49 days and cisplatin 6 mg/m(2) body surface area on radiation treatment days with a cumulative dose of 96 mg/m(2)). Normal hemoglobin levels were considered to be 12 g/dl for females and 13 g/dl for males. Decreased hemoglobin levels before or after surgery and before or after chemoradiotherapy were correlated with the prognosis. Preoperatively, 99 of 114 patients (87%) had normal levels of hemoglobin compared with only 20 of 107 patients (19%) postoperatively. At the onset of radiochemotherapy, the hemoglobin levels of 82 of 116 patients (71%) were within the normal range. After radiochemotherapy, however, 62 of 114 patients (54%) had normal hemoglobin levels. Univariate analysis (Kaplan-Meier method and log-rank test) showed that patients with decreased pre- or postoperative hemoglobin levels had significantly worse locoregional control ( P=0.032 and P=0.0001, respectively) and lower overall survival ( P=0.0013 and P=0.0002, respectively) than patients with normal hemoglobin levels. The 3-year locoregional control rates in patients with preoperative hemoglobin levels that were normal, were reduced by 1-2 g/dl or were reduced by more than 3 g/dl, respectively, were 78%, 55% and 50%. Correlated with normal and diminished postoperative hemoglobin levels, the 3-year locoregional control rates were 90%, 84% and 50%, respectively. There was no correlation between prognosis and hemoglobin level at the onset or after radiochemotherapy. On multivariate analysis, only the postoperative hemoglobin level remained a prognostic factor for locoregional control ( P=0.0241) and overall survival ( P=0.0080). We conclude that low postoperative hemoglobin levels resulting from blood loss may influence the efficacy of postoperative radiochemotherapy in patients with head and neck cancer. Early intervention to raise the postoperative hemoglobin level may result in better tumor control and overall survival after combined-modality therapy.

Health-Related Quality of Life in rhino surgery.

BACKGROUND: Health-Related Quality of Life (HRQL) measures the impact of a pathologic condition on patient's daily life. Besides from disease-related symptoms, it includes a wide spectrum of daily life activities such as physical and social activities, emotional problems, general feeling, and so on. HRQL of patients with nasal diseases is known to correlate only moderately to conventional clinical markers. HRQL data about patients undergoing rhino surgery are not available to date. OBJECTIVE: The purpose of this study was to study HRQL in patients undergoing nasal surgery and to measure therapeutic effects 3 months postoperatively. METHODS: We used a specific health profile HRQL questionnaire with 25 items summarized in 6 symptom groups: sleep; nonnasal, nasal, and emotional symptoms; headache; and practical problems (symptom score 1 to 4). A visual analog scale (0 to 10) was given to measure the patient's general feeling related to their nasal disease. One hundred eighty-one patients undergoing nasal surgery for various reasons were included preoperatively. One hundred seven of them could be interviewed 3 months postoperatively to study therapeutic effects of our surgical intervention. RESULTS: Patient's pre- and postoperative HRQL status could be determined, and differences in disease-related subgroups could be identified. Comparing pre- and postoperatively gained HRQL data revealed a significant improvement in symptom score of 23 of 25 items and according to the following in all symptom groups: sleep preoperatively 2.46 versus postoperatively 1.98 (P <.01), nonnasal symptoms preoperatively 2.13 versus postoperatively 1.91 (P <.01), headache preoperatively 2.17 versus postoperatively 1.72 (P <.01), practical problems preoperatively 2.47 versus postoperatively 2.06 (P <.01), nasal symptoms preoperatively 2.39 versus postoperatively 1.90 (P <.01), and emotional problems preoperatively 2.01 versus postoperatively 1.81 (P <.01). The general feeling score improved from preoperatively 6.47 to postoperatively 3.95 (P <.01) as well. CONCLUSIONS: We could measure patient's HRQL status pre- and postoperatively, could work out peculiarities of the studied subgroups, and showed therapeutic benefits of our surgical intervention.

Impaired monocyte function in cancer patients: restoration with a cyclooxygenase-2 inhibitor.

Epidemiological data and animal models have provided evidence that nonsteroidal antiinflammatory drugs (NSAIDs) have an anticancer effect. However, the molecular mechanisms underlying these antineoplastic effects are not well understood. We described previously that expression levels of the chemokine receptor, CCR5, and the beta2-integrin, Mac-1, were down-regulated on primary monocytes after incubation in supernatants from human carcinoma cell lines, and that this down-regulation resulted in impaired monocyte function with respect to migration and adhesion. We now demonstrate that these impairments are also present in vivo. Monocytes from cancer patients displayed significantly reduced CCR5 levels and migration capacities in comparison to cells from healthy donors. Because migration is necessary for the antitumor activity of monocytes/macrophages, these deficits may contribute to the suppressed immune system seen in cancer patients. In a clinical study, we analyzed the effect of a selective COX-2 inhibitor, Rofecoxib, on the migration of monocytes derived from cancer patients. The results revealed significant improvement in migration equal to those levels seen in healthy donors. We conclude that in patients with cancer, the intake of Rofecoxib for 3 wk leads to significant restoration of monocyte function. These data may, at least in part, help explain the anticancer effects of NSAIDs.

Immunochemical and mechanical characterization of cartilage subtypes in rabbit.

Cartilage is categorized into three general subgroups, hyaline, elastic, and fibrocartilage, based primarily on morphologic criteria and secondarily on collagen (Types I and II) and elastin content. To more precisely define the different cartilage subtypes, rabbit cartilage isolated from joint, nose, auricle, epiglottis, and meniscus was characterized by immunohistochemical (IHC) localization of elastin and of collagen Types I, II, V, VI, and X, by biochemical analysis of total glycosaminoglycan (GAG) content, and by biomechanical indentation assay. Toluidine blue staining and safranin-O staining were used for morphological assessment of the cartilage subtypes. IHC staining of the cartilage samples showed a characteristic pattern of staining for the collagen antibodies that varied in both location and intensity. Auricular cartilage is discriminated from other subtypes by interterritorial elastin staining and no staining for Type VI collagen. Epiglottal cartilage is characterized by positive elastin staining and intense staining for Type VI collagen. The unique pattern for nasal cartilage is intense staining for Type V collagen and collagen X, whereas articular cartilage is negative for elastin (interterritorially) and only weakly positive for collagen Types V and VI. Meniscal cartilage shows the greatest intensity of staining for Type I collagen, weak staining for collagens V and VI, and no staining with antibody to collagen Type X. Matching cartilage samples were categorized by total GAG content, which showed increasing total GAG content from elastic cartilage (auricle, epiglottis) to fibrocartilage (meniscus) to hyaline cartilage (nose, knee joint). Analysis of aggregate modulus showed nasal and auricular cartilage to have the greatest stiffness, epiglottal and meniscal tissue the lowest, and articular cartilage intermediate. This study illustrates the differences and identifies unique characteristics of the different cartilage subtypes in rabbits. The results provide a baseline of data for generating and evaluating engineered repair cartilage tissue synthesized in vitro or for post-implantation analysis.

The characterisation of human respiratory epithelial cells cultured on resorbable scaffolds: first steps towards a tissue engineered tracheal replacement.

In this study we have used lectin histochemistry and scanning electron microscopy (SEM) to assess the growth and characterise the differentiation of human respiratory epithelial cells (REC) cultured on two biomaterial scaffolds. The first scaffold, based on a hyaluronic acid derivative, was observed to be non-adhesive for REC. This lack of adhesion was found to be unrelated to the presence of the hyaluronic acid binding domain on the surface of isolated REC. The other scaffold, consisting of equine collagen. was observed to encourage REC spreading and adhesion. Positive Ulex Europaeus agglutinin (UEA) lectin staining of this preparation indicated the presence of ciliated REC on the scaffold surface. However, the marked decrease in peanut agglutinin (PNA) positive staining, relative to that of control cultures and native tissue, indicates a dedifferentiation of the secretory cells of the REC monolayer. SEM analysis of REC cultured on the collagen scaffold confirmed the presence of ciliated cells thereby validating the UEA positive staining. The presence of both established and developing cilia was also verified. This study indicates that collagen biomaterials are appropriate for the tissue engineering of REC. Furthermore, that UEA and PNA staining is a useful tool in the characterisation of cells cultured on biomaterials, therefore helpful in identifying biomaterials that are suitable for specific tissue engineering purposes.