Effect of mast cell chymase on the morphology of thyroid cells in vitro.

We studied the effect of mast cell chymase on the thyroid cells in culture. Rat serosal mast cells, similar functionally to connective tissue mast cells, were obtained after lavage of the peritoneal cavity and lyzed by freezing. The resulting lysate was used as crude enzyme preparation. Mast cell chymase was purified from the crude preparation by anion exchange chromatography. Crude and purified chymase incubated with thyroid cells induced cellular retraction, the appearance of long processes and gradual cell detachment from the substratum. The effect of the enzyme was not cytotoxic. The immunofluorescence studies of thyroid cells showed a decreased amount of polymerized actin and tubulin after incubation with chymase. Neutral protease inhibitor abolished the effect of crude and purified chymase on thyroid cell morphology. The above findings suggest that mast cell chymase may have a function in the control of cell morphology and cell-matrix interaction.     

Lactoperoxidase and human airway host defense

The lactoperoxidase (LPO) antibiotic system is a well-characterized component of mammary and salivary gland secretions. Because LPO has been shown to function in ovine airways, human airway tissue and secretions were examined for the presence of LPO and its substrate, the anion thiocyanate (SCN-). In addition, human airway secretions were tested for LPO-mediated antibacterial activity, and LPO's activity was assessed against some human airway pathogens. The data showed that normal human airway secretions contained LPO enzyme activity (0.65 +/- 0.09 microg/mg secreted protein; n = 17), and Western blots of secretions demonstrated bands of the expected sizes for LPO. LPO mRNA was detected in trachea by sequencing PCR-amplified cDNA. SCN-, LPO's substrate, was present in undiluted airway secretions at concentrations sufficient for LPO catalysis (0.46 +/- 0.19 mM; n = 8), and diluted secretions contained antibacterial activity with LPO-like properties. Immunocytochemistry localized LPO to submucosal glands in human bronchi. Finally, as expected based on the known antibacterial spectrum of the LPO system, airway secretions showed LPO-dependent activity against Pseudomonas aeruginosa. In addition, the airway LPO system was shown to be effective against Burkholderia cepacia and Haemophilus influenzae. Thus, a functional LPO system exists in human airways and may contribute to airway host defense against infection.     

Cyclooxygenase-2 in normal,hyperplastic and neoplastic follicular cells of the human thyroid gland

This study was undertaken to investigate cyclooxygenase-2 (COX-2) expression in follicular cells of the human thyroid. COX-2 expression was studied immunohistochemically in a total of 174 samples. COX-2 immunoreactivity was confined to the cell cytoplasm with the nuclei remaining unlabelled. COX-2 expression was observed in five cases (17.2%) of normal follicular cells and in one case (16.6%) of solid cell nests. Follicular carcinoma expressed COX-2 more frequently than follicular adenoma (93.4% vs 21.1%) (p0.001). A higher percentage of cases of papillary microcarcinomas up-regulated COX-2 in comparison with all papillary carcinomas (p0.05). However, we could not establish any relationships among COX-2, patients ages or lymph node metastases in papillary carcinomas. COX-2 expression was found in 12 (92.3%) poorly differentiated carcinomas and in 13 (92.8%) undifferentiated carcinomas. We found that COX-2 is not always useful as a marker of malignancy. Our results suggest that COX-2 plays a role in progression of all thyroid carcinomas, but in papillary carcinomas, seems more important only in the early stages. COX-2 expression in the undifferentiated carcinoma deserves special consideration due to its prognosis and to the fact that selective COX-2 inhibitors were found to enhance tumour response to radiation in some studies.     

Neural cell adhesion molecule immunoreactivity in Merkel cells and Merkel cell tumours

We have analysed the expression of the neural cell adhesion molecule (NCAM) in normal Merkel cells of pig and human skin, and in nine neuroendocrine carcinomas of the skin (Merkel cell carcinomas). NCAM immunoreactivity was observed in virtually all Merkel cells, both in epidermis and vibrissae of pig snout skin and in human epidermis. Immunostaining surrounded the entire surface of Merkel cells and was not restricted to the contact areas between Merkel cells and nerve terminals. All Merkel cell carcinomas studied were also positive for NCAM. The immunostaining pattern of the tumour cells was similar to that observed in normal Merkel cells; the immunoreactivity was confined to the cell membranes. These results suggest that NCAM may be used as an immunohistochemical marker for both Merkel cells and Merkel cell tumours.     

Looking for ferns: optimization of digestion pretreatment in fluorescence in situ hybridization (FISH) technique on paraffin-embedded tissues.

Fluorescence in situ hybridization (FISH) is a useful cytogenetic technique for the detection of chromosome aberrations. However, applying this technique routinely on paraffin-embedded tissue is hampered by technical problems. The efficiency of hybridization is influenced by formalin fixation time, and this may vary considerably between specimens. We present a simple method for improving hybridization by microscopically monitoring the time of enzymatic digestion. To establish optimal digestion time, enzymatic digestion was stopped at 3-minute intervals for biopsies and 10-minute intervals for autopsies in 24 paraffin-embedded samples. At every stop, tissue morphology was examined under light microscopy to determine if observed changes could be correlated with subsequent FISH results. The appearance of fernlike formations was found to mark the optimal digestion time that produced the strongest hybridization signals. Using this method of digestion time control, an additional 41 cases were evaluated for FISH with various types of probe. Monitoring under the microscope could be more spaced if the morphology did not change after the first visual control and could be adapted to the type of sample (in general, endoscopic samples, total digestion time of about 10 min; routine biopsies, 15 to 30 min; autopsy samples, 20 to 40 min). In every case, the appearance of the fernlike pattern correlated with proper hybridization signal. Monitoring digestion time for the appearance of fernlike structures is a useful method for improving reproducibility of FISH technique on paraffin-embedded samples. It is particularly useful when dealing with samples under heterogeneous fixation conditions (consultations, autopsies, etc.), because it eliminates the need for repetition.     

Elevated Tissue Kallikrein Activity in Airway Secretions from Patients with Tracheobronchitis Associated with Prolonged Mechanical Ventilation

The clinical course of patients undergoing prolonged mechanical ventilation is often complicated by the development of purulent tracheobronchitis. The purpose of this study was to assess whether ventilator-associated hypersecretion is associated with elevated levels of tissue kallikrein (TK) activity. TK can induce marked bronchial inflammation in animal models and TK activity is increased in the airway secretions of symptomatic asthmatics. It has not been studied in conditions with predominantly neutrophilic bronchial secretions, although animal data indicate that neutrophil elastase may stimulate TK activity. We measured TK activity in airway secretions of patients undergoing mechanical ventilation for more than 4 weeks (PMV group) and in two comparator groups: patients with cystic fibrosis, who were colonized with Pseudomonas aeruginosa (CF group) and patients undergoing mechanical ventilation for less than one week who did not have clinical evidence of purulent airway secretions (acute mechanical ventilation, AMV group). We also compared the level of neutrophil elastase (NE) activity, an index of neutrophil activation, in the three patient groups. TK and NE activity in the sol phase were measured by the degradation of chromogenic substrates (DL Val-Leu-Arg pNA and N-Methoxy Succinyl Ala-Ala-Pro-Val pNA, respectively). Intergroup differences in cell counts were not significant. However, TK activity was significantly less in the AMV group than in the PMV and cystic fibrosis patients (Kruskal-Wallis ANOVA, p < 0.05). Elastase activity was significantly greater in the CF group (p < 0.05) than in the other two groups. Compared to patients undergoing short-term mechanical ventilation (AMV group), TK activity was elevated in patients with purulent tracheobronchitis associated with prolonged mechanical ventilation (PMV group). The elevation in TK activity in these patients is comparable to levels in sputum from patients with cystic fibrosis (CF group), although the latter had a significantly higher level of NE activity. The observation of increased TK activity in patients with neutrophilic airway inflammation suggests that TK may play a role in modulating inflammation in ventilator-associated tracheobronchitis and may be worthy of further study to determine its source and significance.     

Left atrial strain in the assessment of diastolic function: providing new insights into primary myocardial dysfunction in Marfan syndrome

The International Journal of Cardiovascular Imaging - Previous studies using conventional echocardiographic measurements have reported subclinical left ventricular (LV) diastolic abnormalities in...     

Combined cytoplasmic and membranous EGFR and p53 overexpression is a poor prognostic marker in early stage oral squamous cell carcinoma

J Oral Pathol Med (2012) 41 : 559–567 Objective: Our aim was to evaluate the expression of several molecules that regulate growth, the cell cycle and signalling pathways including EGFR, p53, p16 and p27 in oral squamous cell carcinomas (OSCC). We examined their utility as prognostic markers by relating to clinicopathological characteristics and the clinical outcome. Patients and methods: Using tissue microarray technology, we analysed 67 primary OSCC and examined immunohistochemical expression of EGFR, p53, p16 and p27. Multivariate analysis was conducted to examine their role in survival. Results: Many of the markers were highly expressed in these cancers. Membranous EGFR expression in 95.2%, both membrane and cytoplasm expression in 35%, p53 expression in 61.6%, p27 expression in 89.5% and p16 expression in 27.9% of cases. In the multivariate analysis, independent prognostic influence of a lower overall survival was determined only for advanced tumour stage (P < 0.001), p53 overexpression (P = 0.004), EGFR cytoplasm and membrane co‐expression location (P = 0.002) and p16 reduced expression (P = 0.002). When considering a subgroup of early stage tumours, p53 overexpression (P = 0.028) and combined membranous and cytoplasm EGFR co‐expression (P = 0.039) were indicators of a lower overall survival. For disease‐free survival, in addition to these three factors, the histological grade (P = 0.011) showed independent prognostic values. Conclusion: The independent value of EGFR subcellular location (cytoplasm and membrane) and p53 overexpression in overall survival even in early stages of OSCC suggests that these markers may serve as reliable biological markers to identify high‐risk subgroups and to guide therapy.