Resistance to osimertinib in advanced EGFR-mutated NSCLC: a prospective study of molecular genotyping on tissue and liquid biopsies.

BACKGROUND: Resistance to osimertinib in advanced EGFR-mutated non-small cell lung cancer (NSCLC) constitutes a significant challenge for clinicians either in terms of molecular diagnosis and subsequent therapeutic implications. METHODS: This is a prospective single-centre study with the primary objective of characterising resistance mechanisms to osimertinib in advanced EGFR-mutated NSCLC patients treated both in first- and in second-line. Next-Generation Sequencing analysis was conducted on paired tissue biopsies and plasma samples. A concordance analysis between tissue and plasma was performed. RESULTS: Sixty-five advanced EGFR-mutated NSCLC patients treated with osimertinib in first- (n = 56) or in second-line (n = 9) were included. We managed to perform tissue and liquid biopsies in 65.5% and 89.7% of patients who experienced osimertinib progression, respectively. Acquired resistance mechanisms were identified in 80% of 25 patients with post-progression samples, with MET amplification (n = 8), EGFR C797S (n = 3), and SCLC transformation (n = 2) the most frequently identified. The mean concordance rates between tissue and plasma for the EGFR activating mutation and for the molecular resistance mechanisms were 87.5% and 22.7%, respectively. CONCLUSIONS: Resistance to osimertinib demonstrated to be highly heterogeneous, with MET amplification the main mechanism. Plasma genotyping is a relevant complementary tool which might integrate tissue analysis for the study of resistance mechanisms.

Mechanical evaluation of new injection needles for dental anesthesia.

AIM: In this study Authors want to evaluate the static and dynamic flexural behaviour of innovative injection needles for dental anaesthesia comparing to the traditional needles. METHODS: Four kinds of innovative injection needles (Carpule Free Flow-Heraeus Kulzer), different for length and external diameter, have been evaluated in comparison with the traditional ones (Carpule-Heraeus Kulzer), dimensions being equal except for the internal diameter. Static stiffness tests (10 for each kind of needles, resulting in a totality of 80 tests) and fatigue bending test (10 for each kind of needles, each tested at 3 different bending angles, resulting in a totality of 240 tests) have been conducted following the ISO 9626 norms. The final comparison among the obtained data have been conducted using a one way statistical analysis of variance (ANOVA). RESULTS: After stiffness tests the Carpule Free Flow injection needles and the traditional ones showed the same static behaviour, resulting in statistical comparable values of mean deflection at the maximum load allowed by ISO norms tests, whereas the 0.4 mm external diameter showed a statistically significant difference because the Carpule Free Flow needles achieved lower deflection values. After dynamic bending tests at 20 degrees both needle types showed the same behaviour, whereas at 30 degrees bending the Carpule Free Flow injection needles showed a higher number of breaks comparing to normal type, up to a maximum value of breaks (10 out of 10) for the 0.4 mm external diameter needles. After 50 degrees fatigue bending tests the Carpule Free Flow needles came to the break point after lower number of cycles than the Carpule needles. CONCLUSION: This study pointed out that, among anaesthesia injection needles, the Carpule Free Flow needles not only showed better clinical characteristics but also a mechanical behaviour, both static and dynamic, statistically similar to traditional ones, according to international standards. After fatigue bending higher than 20 degrees, exclusively made in this research in order to simulate critical or extreme conditions, all analysed Carpule Free Flow needles have been broken after a lower number of cycles. It is concluded that it is recommended not to repeatedly bend this kind of needles at more than 90 degrees. At the end of this study it may be suggested that this kind of needles cannot be repeatedly bended at angles more than 90 degrees.

An undiagnosed pleural effusion with surprising consequences.

We present the case of a 43-year-old Italian woman with a left undiagnosed pleural effusion, which in subsequent months presented a clinically unexpected evolution with the appearance at first of a right wrist tenosynovitis and subsequently a bilateral lung involvement caused by M. Tuberculosis. With this case report, we would like to underline the importance of making a correct diagnosis of any pleural effusion as soon as possible by at least a thoracocentesis. If untreated, tuberculosis may easily disseminate to other organs. Some considerations and suggestions for antibiotic treatment of pleural effusion will also be given, since many antibiotics have some anti-tuberculosis effect and may delay the diagnosis of this infectious disease.

Effects of Nd:YAG laser on dental enamel.

Caries prevention should be an important goal of the dentistry practitioner. In addition to traditional preventive techniques, laser beam has been a recent suggestion. This study aimed to evaluate Nd:YAG laser treatment effects on dental enamel surfaces, irradiated at different energy levels, by scanning electron microscopy (SEM) observations and surface micro-hardness tests to evaluate if caries prevention can be achieved by laser treatment. Forty human teeth were divided in four groups of 10 specimens each: in three groups the enamel surface was treated with three different laser beam energy levels, 60, 120 and 160 mJ, in one group the enamel was not lased. Five samples from each group were subjected to the Vickers microhardness test and five samples underwent SEM investigation. Results of the microhardness test demonstrated no significant differences between treated and non-treated dental enamel samples. SEM observation demonstrated a rougher enamel surface in all treated groups: the 60 mJ treated group showed tooth surfaces with vertical scratches, the 120 mJ treated group showed the enamel surface covered by craters and cracks, and the 160 mJ treated group showed a completely changed enamel structure with columns separated by voids and with a glass-like surface. These investigations demonstrated that dental enamel laser treatment at low energy levels (not >60 mJ) produces a protective glass-like surface, without the loss of its integrity and could be an advisable technique to prevent caries. Higher energy laser treatment leads to modifications of the enamel morphology such as craters and cracks, even if it does not cause any change in enamel hardness characteristics, which could be more useful in conservative dentistry.

Inhaled corticosteroids in stable COPD patients: do they have effects on cells and molecular mediators of airway inflammation?

STUDY OBJECTIVE: To investigate possible changes in cells and molecular mediators of airway inflammation following inhaled steroid treatment of stable COPD patients. DESIGN: Six-week open preliminary prospective study. SETTING: A university respiratory disease clinic. PATIENTS: : Stable COPD patients with mild disease. INTERVENTION: Six-week treatment with inhaled beclomethasone (1.5 mg die). MEASUREMENTS: The levels of interleukin (IL)-8, myeloperoxidase, eosinophilic cationic protein and tryptase, and cell numbers in bronchial lavage specimens were determined, and the symptom score, the endoscopic bronchitis index, and functional parameters were recorded. RESULTS: After treatment there were significant reductions in the lavage levels of IL-8 ([mean +/- SEM] 1,603.4 +/- 331.2 vs 1,119.2 +/- 265.3 pg/mL, respectively; p = 0. 01) and myeloperoxidase (1,614.5 +/- 682.3 vs 511.2 +/- 144.2 microg/L, respectively; p = 0.05), in cell numbers (250.6 +/- 27.7 vs 186.3 +/- 11.5 cells x 10(3)/mL, respectively; p = 0.04), neutrophil proportion (59.7 +/- 14.3% vs 31.5 +/- 10.1%; p = 0.01), symptom score (4.5 +/- 0.6 vs 1.4 +/- 0.5; p = 0.01), and bronchitis index (8.5 +/- 0.8 vs 5.5 +/- 0.7; p = 0.007). CONCLUSIONS: In stable patients with COPD, inhaled steroid treatment may induce changes on some cellular and molecular parameters of airway inflammation.

Mast cells in bronchiolitis obliterans organizing pneumonia. Mast cell hyperplasia and evidence for extracellular release of tryptase.

Idiopathic bronchiolitis obliterans organizing pneumonia (BOOP) is characterized by air space inflammation and fibrosis of unknown origin. The pathogenesis of the inflammatory reaction and fibrosis in fibrotic lung disorders remains unclear; however, recent attention has focused on the potential role of the mast cell in the genesis of fibrosis. To determine whether mast cells are implicated in the pathogenesis of BOOP, mast cells were identified in BAL fluid and in transbronchial lung biopsy specimens from 11 patients affected by BOOP and 17 control subjects. Mast cells and tryptase were significantly increased in BAL fluid of patients with BOOP (p = 0.001 and p = 0.03, respectively). In lung tissue of patients with BOOP, there was an increased number of mast cells per square millimeter of lung tissue with respect to control group (p = 0.001). Seventy-three percent of mast cells were found in the alveolar septa, 18% within alveoli often plunged in organizing pneumonia, 4% among alveolar lining cells, and 6% along blood vessels. No mast cells were located within alveoli in control subjects. Mast cell degranulation was evident in lung tissue specimens of patients with BOOP but not in those of control subjects (p = 0.01). This study shows the importance of mast cells and mast cell activation in the pathogenesis of BOOP.

Neutrophils infiltrating bronchial epithelium in chronic obstructive pulmonary disease.

In order to characterize neutrophil and eosinophil presence in the airways of patients with chronic obstructive pulmonary disease (COPD), bronchoscopy with bronchial washings and bronchial biopsies was performed in 12 smoking stable COPD subjects and 18 normal non-smoking control subjects. Bronchial biopsies were examined by light microscopy using plastic embedding and histochemical techniques to identify different cell types. Bronchial washing fluid of COPD patients was characterized by a predominance of neutrophils (P = 0.001), and a slight, but significant (P = 0.03), increase of eosinophil fraction. Subjects with COPD had higher number of neutrophils in the epithelium (P = 0.01), and eosinophils in the lamina propria (P = 0.01) than did control subjects. The thickness of reticular basement membrane was increased for COPD patients in comparison to control subjects (P = 0.01). The present study provides evidence of neutrophil infiltration both in bronchial washing and bronchial epithelium of patients with COPD, suggesting that the source of neutrophils in airway lumen may be the bronchial mucosa. Although less common than in asthma, airways of COPD subjects reveal eosinophil presence and airway remodelling.

Bronchoalveolar lavage in intensive care units.

Pneumonia is common in those patients placed in intensive care units, especially in mechanically ventilated patients. The high mortality rate of ventilator-associated pneumonia requires a rapid initiation of the appropriate antibiotic treatment. Patients who do not respond to initial antibiotic regimens could have the additional benefit of the use of invasive techniques such as bronchoalveolar lavage. Moreover, BAL is of clinical use to identify several non-infectious pulmonary conditions that may mimic pneumonia in these patients. Such conditions include pulmonary haemorrhages, acute eosinophilic pneumonia, malignancy, drug-induced toxicity, adult respiratory distress syndrome and cardiogenic pulmonary oedema. It is important to distinguish these conditions from pneumonia because the management and prognosis of these entities is quite different.

Mechanical and ultrastructural evaluation of quartz post-endodontic reconstructions.

Aesthetics is a very important element in dentistry, but requires the support of good mechanical performance. Quartz fiber used in post-endodontic reconstruction is an aesthetic material, although there is little research concerning its mechanical properties. This study evaluated the retentive property of post-endodontic reconstruction, composed of a quartz fiber post. Different thermal stresses were applied in vitro to post-endodontic reconstructions, in order to simulate oral thermal action on post-system dental structure linkage. We chose 30 human extracted teeth, endodontically treated and restored, and then divided them into three groups of 10 teeth. A different treatment was applied to each group before mechanical testing: in the 1st group no treatment was done (controls); in the 2nd group teeth were subjected, in a climatic chamber, to 10 thermo-cycles between 4 degrees C and 58 degrees C; in the 3rd group teeth were stored in a saline solution at 37 degrees C for 48 hr. The teeth then underwent tensile shear stress tests at break point using a computerized electronic dynamometer. After mechanical testing, two teeth from each group were longitudinally half-sectioned, sputter-coated in gold and observed by scanning electron microscopy (SEM). The extracted quartz fiber post of each tooth also underwent SEM observation. Mechanical test results demonstrated that thermal cyclic variations could affect bond stability between dental structures and posts in quartz fiber reconstructions, whereas their bond strength seemed unaffected by humidity increases. Quartz fiber post SEM observation demonstrated a homogeneous structure and a regular fiber disposition. Dental root canal morphology SEM images always showed a different thickness in the cement layer. (Journal of Applied Biomaterials & Biomechanics 2004; 2: 156-61).

Lymphocyte activation markers in peripheral blood before and after natural exposure to allergen in asthmatic patients.

Activated T cells and their cytokine products are involved in the pathogenesis of asthma. However, little is known about changes in circulating T cell subsets in allergic asthma during natural exposure to allergens. We examined whether natural allergen exposure of patients with atopic asthma is associated, in vivo, with changes of lymphocyte subtypes and activation markers in peripheral blood. Ten patients with atopic mild asthma sensitized only to grass pollen had peripheral venous blood lymphocyte analyses before and during the pollen season. No significant changes were observed. There was an inversion in the CD4/CD8 ratio in the peripheral blood both before (p < 0.05) and during (p < 0.01) seasonal exposure when compared to a group of healthy, age-matched control subjects. Evaluation of T cells expressing CD25 activation marker also demonstrated a significant reduction of CD4+25+ cells and a significant increase of CD+25+ cells compared to the controls. CD23+ cells (B lymphocytes with low affinity Fc IgE receptor) in the asthmatic group out of the pollen season correlated negatively with hyperreactivity to methacholine (p < 0.05). We conclude that in mild allergic asthmatic patients sensitized to grass pollen, blood lymphocyte subsets and their activation markers do not reflect seasonal exposure. Moreover, our findings show that these patients have higher proportions of CD8+ cells expressing higher levels of CD25 in their blood compared to normal subjects both before and during the pollen season.

Specific immunotherapy downregulates peripheral blood CD4 and CD8 T-lymphocyte activation in grass pollen-sensitive asthma.

Several lines of evidence indicate that specific immunotherapy may act by modifying the immune responses of T-lymphocytes to the antigen. To evaluate the effect of specific immunotherapy on the activation of T-lymphocytes by cluster of differentiation cells (CD4+ and CD8+) in peripheral blood, the expression of two surface activation markers, the p55 interleukin-2 receptor (CD25) and human leucocyte antigen (HLA)-DR, was studied prospectively on circulating CD4+ and CD8+ T-cell subsets in subjects with grass-pollen sensitive asthma before and after 1 yr of treatment with specific immunotherapy. Twenty five asthmatic patients with pollen sensitivity other than grass, studied out of their pollen season, served as the control group. Specific immunotherapy improved clinical indices of disease activity including symptom scores and medication use during the pollen season of the treatment year. It had a marked effect in reducing the expression of the two activation markers, CD25 and HLA-DR, in both CD4+ (p=0.002 and p=0.005, respectively) and CD8+ (p=0.01 and p=0.01, respectively) T-cell subsets, in parallel with a significant decrease in CD23 expression on B-cells (p=0.008) and in grass-specific immunoglobulin E levels (p=0.01) in the peripheral blood of subjects with grass pollen-sensitive asthma. The decreased T-lymphocyte activation observed in immunotherapy-treated subjects after the treatment year was significant (p=0.05) in comparison with the control group. These data add to the view that the efficacy of specific immunotherapy may be attributed to the downregulation of T-cell responses.

Superoxide anion production by monocytes of corticosteroid-treated asthmatic patients.

Reactive oxygen species play an important role in promoting inflammation. Blood monocytes have been described to release higher amounts of superoxide anion in uncontrolled and untreated asthmatics. Corticosteroids are widely used in asthma, but little is known about their molecular mechanism of action. The aim of our study was to analyse the ex vivo effect of corticosteroid treatments on superoxide anion release by blood monocytes. Superoxide anion release was measured by a spectrophotometric method based on the superoxide dismutase (SOD) -inhibitable reduction of ferricytochrome C by blood monocytes from untreated patients and asthmatics treated with i.v., inhaled and oral corticosteroids. Monocytes from uncontrolled and untreated asthmatics, released high amounts of superoxide anions. After short-term treatment with i.v., corticosteroids, this release was found to decrease significantly (1.410 versus 0.340 nM, p<0.05). Cells from asthmatics who had undergone long-term treatment with inhaled or oral steroids presented low amounts of superoxide anion production, with a significant difference as compared to untreated asthmatics (0.375 nM p<0.01 and 0.620 nM p<0.02 respectively). In general, patients with controlled asthma (treated with short-term oral steroids, or with long-term inhaled steroids) released lesser amounts of superoxide anion than uncontrolled and untreated asthmatics. In the case of steroid-dependent asthmatics there was no difference between patients with a controlled or uncontrolled disease.

Influence of atmospheric nitric oxide concentration on the measurement of nitric oxide in exhaled air.

BACKGROUND: Measurement of nitric oxide (NO) in exhaled air shows promise as a non-invasive method of detecting lung inflammation. However, variable concentrations of NO are measured in environmental air. The aim of this study was to verify a possible relationship between exhaled NO and atmospheric NO values during high atmospheric NO days. METHOD: Exhaled air from 78 healthy non-smokers of mean age 35.3 years was examined for the presence of NO using a chemiluminescence NO analyser and NO levels were expressed as part per billion (ppb). The exhaled air from all the subjects was collected into a single bag and into two sequential bags. Before each test atmospheric NO was measured. RESULTS: The mean (SE) concentration of exhaled NO collected into the single bag was 17.1 (0.6) ppb while the mean values of exhaled NO in bags 1 and 2 were 16.7 (1.3) ppb and 13.8 (1.2) ppb, respectively. The atmospheric NO concentrations registered before each test varied from 0.4 to 71 ppb. There was a significant correlation between exhaled NO in the single bag and atmospheric NO (r = 0.38, p = 0.001). The atmospheric NO concentration also correlated with exhaled NO both in bag 1 (r = 0.44, p = 0.0001) and in bag 2 (r = 0.42, p = 0.0001). These correlations disappeared with atmospheric NO concentrations lower than 35 ppb. CONCLUSIONS: These results indicate a relationship between atmospheric NO and NO levels measured in exhaled air, therefore exhaled NO should not be measured on very high atmospheric NO days.

Inhaled beclomethasone dipropionate downregulates CD4 and CD8 T-lymphocyte activation in peripheral blood of patients with asthma.

BACKGROUND: Previous studies demonstrated a downregulation of T-lymphocyte (CD3+ cells) activation in peripheral blood after treatment with inhaled corticosteroids in patients with asthma. OBJECTIVE: This study was carried out to evaluate the effect of inhaled corticosteroids on CD4 and CD8 T-lymphocyte activation, respectively. METHODS: We examined the expression of three surface activation markers (CD25, HLA-DR, and very late activation antigen 1) on circulating CD4+ and CD8+ T-cell subsets in subjects with asthma (n = 23) before and 8 weeks after treatment with inhaled beclomethasone dipropionate dry powder (daily dose, 800 microg). RESULTS: Beclomethasone dipropionate treatment had a marked effect in reducing the expression of the activation marker CD25 (p < 0.01) in both CD4+ and CD8+ T-cell subsets in peripheral blood of patients with asthma. However, no correlation was found between the downregulation of CD4 and CD8 T-lymphocyte activation and the improvement in physiologic indices of disease activity. CONCLUSIONS: These data add to the view that CD4+ and CD8+ T lymphocytes in peripheral blood of patients with asthma are in an activated state that is downregulated by inhaled corticosteroids.

Seasonal variations of T-cell cytokine pattern in peripheral blood from atopic subjects.

Previous studies on the effect of seasonal exposure to the sensitizing antigen on T-cell cytokine pattern from atopic subjects evaluated T-cell cytokine production by titration in the serum or culture supernatants. The purpose of this study was to determine the seasonal variations of T-cell cytokine pattern from atopic subjects at the single-cell level. We examined the interleukin-4 (IL-4) and interferon-gamma expression in peripheral blood CD4 + and CD8 + T cells from 11 subjects with grass-pollen-sensitive allergy before and during the 1999 grass pollen season using a flow cytometric method of intracellular cytokine detection. Eight healthy volunteers served as the control group. Flow cytometric analysis of peripheral blood lymphocytes showed no seasonal variations of IL-4- and interferon-gamma-producing T cells in atopic subjects. However, there was a decreased percentage of IL-4-producing cells among peripheral blood CD4+ and CD8+ T cells from the atopic subjects both during and outside the pollen season in comparison to the controLs. We did not find seasonal variations of T-cell cytokine pattern in peripheral blood from atopic subjects. However, we observed a decreased percentage of IL-4-producing T cells in peripheral blood from these subjects in comparison to healthy controls. These data add to the view of a continuous migration of T helper 2 (TH2) cells from the blood to the tissues of primary allergen exposure.

Effects of glucocorticoids on endogenous and transcellular metabolism of eicosanoids in asthma.

BACKGROUND: Human blood polymorphonuclear cells, which biosynthesize eicosanoids from the 5-lipoxygenase (5-LO) pathway, are likely to be involved in asthma, in which glucocorticoids represent the first line of therapy. Their effects on leukotriene release after a short course of treatment, which have been reported in several studies, are controversial. OBJECTIVE: We sought to investigate whether long-term oral glucocorticoids inhibit lipid mediators from the 5-LO pathway. METHODS: Twelve normal control subjects, 29 asthmatic subjects, and 50 glucocorticoid-dependent asthmatic subjects were included in the study. Polymorphonuclear cells were studied for endogenous and transcellular metabolism of eicosanoids. RESULTS: Total leukotriene B(4) production was significantly lower in cells from glucocorticoid-dependent asthmatic subjects (mean +/- SD, 177 +/- 26 ng/10(7) cells) than in control subjects (406 +/- 27), untreated asthmatic subjects (421 +/- 34), and asthmatic subjects treated with inhaled glucocorticoids (290 +/- 56). When incubated with arachidonic acid, these polymorphonuclear cells released very low amounts of 5(S)- and 12(S)-hydroxy-eicosatetraenoic acid (HETE), whereas endogenous 15(S)-HETE was found in substantial amounts. The transformation of exogenous 15(S)-HETE into 5(S),15(S)-diHETE and lipoxins was significantly more important in untreated asthmatic subjects than in control subjects and glucocorticoid-dependent asthmatic subjects. CONCLUSION: This study showed that long-term oral corticotherapy affects the 5-LO activity and leads to a decrease production of all metabolites in contrast to short-term or inhaled glucocorticoids. This study also questions the site of action of glucocorticoids in regulating the availability of arachidonic acid and potential eicosanoid regulation, as previously held in phospholipase A2 studies.