Discovery of a novel isoxazoline derivative of prednisolone endowed with a robust anti-inflammatory profile and suitable for topical pulmonary administration.

A novel glucocorticoids series of (GCs), 6α,9α-di-Fluoro 3-substituted C-16,17-isoxazolines was designed, synthesised and their structure-activity relationship was evaluated with glucocorticoid receptor (GR) binding studies together with GR nuclear translocation cell-based assays. This strategy, coupled with in silico modelling analysis, allowed for the identification of Cpd #15, an isoxazoline showing a sub-nanomolar inhibitory potency (IC50=0.84 nM) against TNFα-evoked IL-8 release in primary human airways smooth muscle cells. In Raw264.7 mouse macrophages, Cpd #15 inhibited LPS-induced NO release with a potency (IC50=6 nM)>10-fold higher with respect to Dexamethasone. Upon intratracheal (i.t.) administration, Cpd #15, at 0.1 µmol/kg significantly inhibited and at 1 µmol/kg fully counteracted eosinophilic infiltration in a model of allergen-induced pulmonary inflammation in rats. Moreover, Cpd #15 proved to be suitable for pulmonary topical administration given its sustained lung retention (t1/2=6.5h) and high pulmonary levels (>100-fold higher than plasma levels) upon intratracheal administration in rats. In summary, Cpd #15 displays a pharmacokinetic and pharmacodynamic profile suitable for topical treatment of conditions associated with pulmonary inflammation such as asthma and COPD.

A simple circuit for cerebral perfusion during cardiopulmonary bypass surgery of the ascending aorta and the aortic arch.

INTRODUCTION: A circuit was developed to allow for rapid reaction to the needs of perfusion during extracorporeal circulation (ECC) in surgery of the aortic arch and ascending aorta. METHOD: From January 2008 through January 2010, a home-designed circuit was used on 30 patients with aortic dissection who underwent surgery to replace the ascending aorta and the aortic arch and, in some cases, the aortic valve and re-implant of the coronary arteries using Bentall's technique.

Association of increased CCL5 and CXCL7 chemokine expression with neutrophil activation in severe stable COPD.

BACKGROUND: Increased numbers of activated neutrophils have been reported in the bronchial mucosa of patients with stable chronic obstructive pulmonary disease (COPD), particularly in severe disease. OBJECTIVES: To investigate the expression of neutrophilic chemokines and adhesion molecules in bronchial biopsies from patients with stable COPD of different severity (GOLD stages I-IV) compared with age-matched control subjects, smokers with normal lung function and never smokers. METHODS: The expression of CCL5, CXCL1, 5, 6, 7 and 8, CXCR1, CXCR2, CD11b and CD44 was measured in the bronchial mucosa using immunohistochemistry, confocal immunofluorescence, real-time quantitative polymerase chain reaction (RT-QPCR) and Western blotting (WB). RESULTS: The numbers of CCL5+ epithelial cells and CCL5+ and CXCL7+ immunostained cells were increased in the bronchial submucosa of patients with stable severe COPD compared with control never smokers and smokers with normal lung function. This was also confirmed at the level of mRNA expression. The numbers of CCL5+ cells in the submucosa of patients with COPD were 2-15 times higher than any other chemokines. There was no correlation between the number of these cells and the number of neutrophils in the bronchial submucosa. Compared with control smokers, the percentage of neutrophils co-expressing CD11b and CD44 receptors was significantly increased in the submucosa of patients with COPD. CONCLUSION: The increased expression of CCL5 and CXCL7 in the bronchial mucosa of patients with stable COPD, together with an increased expression of extracellular matrix-binding receptors on neutrophils, may be involved in the pathogenesis of COPD.

T helper type 17-related cytokine expression is increased in the bronchial mucosa of stable chronic obstructive pulmonary disease patients.

There are increased numbers of activated T lymphocytes in the bronchial mucosa of stable chronic obstructive pulmonary disease (COPD) patients. T helper type 17 (Th17) cells release interleukin (IL)-17 as their effector cytokine under the control of IL-22 and IL-23. Furthermore, Th17 numbers are increased in some chronic inflammatory conditions. To investigate the expression of interleukin (IL)-17A, IL-17F, IL-21, IL-22 and IL-23 and of retinoic orphan receptor RORC2, a marker of Th17 cells, in bronchial biopsies from patients with stable COPD of different severity compared with age-matched control subjects. The expression of IL-17A, IL-17F, IL-21, IL-22, IL-23 and RORC2 was measured in the bronchial mucosa using immunohistochemistry and/or quantitative polymerase chain reaction. The number of IL-22(+) and IL-23(+) immunoreactive cells is increased in the bronchial epithelium of stable COPD compared with control groups. In addition, the number of IL-17A(+) and IL-22(+) immunoreactive cells is increased in the bronchial submucosa of stable COPD compared with control non-smokers. In all smokers, with and without disease, and in patients with COPD alone, the number of IL-22(+) cells correlated significantly with the number of both CD4(+) and CD8(+) cells in the bronchial mucosa. RORC2 mRNA expression in the bronchial mucosa was not significantly different between smokers with normal lung function and COPD. Further, we report that endothelial cells express high levels of IL-17A and IL-22. Increased expression of the Th17-related cytokines IL-17A, IL-22 and IL-23 in COPD patients may reflect their involvement, and that of specific IL-17-producing cells, in driving the chronic inflammation seen in COPD.

Borderline HER-2 breast cancer cases: histochemical versus real-time PCR analysis and impact of different cut-off values.

Seventy-one cases that had resulted borderline for HER-2 protein expression at conventional immunohistochemical assay (2+) were assessed for HER-2 gene amplification by real-time PCR and by FISH in accordance with the manufacturer's recommendations (gene amplification with ratio >or=2 in both methods). Thirty-three out of 71 cases (47%) resulted amplified at real-time PCR analysis, whereas 15 cases resulted positive at FISH (21%). Apparently, PCR was more sensitive than FISH in HER-2 determination, only 10 cases resulting amplified in both tests. When the mean ratio value obtained in all PCR experiments was adopted as threshold in determining HER-2 gene amplification, the apparent sensitivity of PCR was reduced but correlation between PCR and FISH results was dramatically increased. Furthermore, when the mean PCR ratio value observed in the FISH-positive group was chosen as threshold, the best agreement between PCR and FISH results was achieved. Therefore, we found that the proposed threshold ratio value of >or=2 is not accurate in separating HER-2 amplified and non-amplified cases. We suggest that the threshold ratio value in PCR tests should be determined in each laboratory using FISH controlled cases. Finally, above certain in-lab generated threshold values, PCR might be proposed as a highly predictive positive test in HER-2 assessment.

Nasopharyngeal teratoma and diaphragmatic hernia: a non random association?

We describe a female affected by diaphragmatic hernia and nasopharyngeal teratoma. The case is compared with one already reported and possible diagnoses discussed. These cases appear to represent a new syndrome.

Cellular and molecular mechanisms in chronic obstructive pulmonary disease: an overview.

In the last decade, the analysis of bronchial biopsies and lung parenchyma obtained from chronic obstructive pulmonary disease (COPD) patients compared with those from smokers with normal lung function and non-smokers has provided new insights on the role of the different inflammatory and structural cells, their signalling pathways and mediators, contributing to a better knowledge of the pathogenesis of COPD. This review summarizes and discusses the lung pathology of COPD patients with emphasis on inflammatory cell phenotypes that predominate in different clinical conditions. In bronchial biopsies, a cascade of events takes place during progression from mild-to-severe disease. T lymphocytes, particularly CD8+ cells and macrophages are the prevalent inflammatory cells in the lung of healthy smokers and patients with mild COPD, while total and activated neutrophils predominate in severe COPD. The number of CD4+, CD8+ cells and macrophages expressing nuclear factor-kappa B (NF-kappaB), STAT-4 and IFN-gamma proteins as well as endothelial adhesion molecule-1 in endothelium is increased in mild/moderate disease. In contrast, activated neutrophils (MPO+ cells) and increased nitrotyrosine immunoreactivity develops in severe COPD. In bronchial biopsies obtained during COPD exacerbations, some studies have shown an increased T cell and granulocyte infiltration. Regular treatment with high doses of inhaled glucocorticoids does not significantly change the number of inflammatory cells in bronchial biopsies from patients with moderate COPD. The profile in lung parenchyma is similar to bronchial biopsies. 'Healthy' smokers and mild/moderate diseased patients show increased T lymphocyte infiltration in the peripheral airways. Pulmonary emphysema is associated with a general increase of inflammatory cells in the alveolar septa. The molecular mechanisms driving the lymphocyte and neutrophilic prevalence in mild and severe disease, respectively, needs to be extensively studied. Up-regulation of pro-inflammatory transcription factors NF-kappaB and STAT-4 in mild, activated epithelial and endothelial cells in the more severe disease may contribute to this differential prevalence of infiltrating cells.

STAT4 activation in smokers and patients with chronic obstructive pulmonary disease.

Activation of the transcription factor signal transducer and activator of transcription (STAT)-4 is critical for the differentiation of T-helper 1 cells/type-1 cytotoxic T-cells and the production of interferon (IFN)-gamma. Expression of STAT4, phospho-STAT4, IFN-gamma and T-box expressed in T-cells (T-bet) proteins in bronchial biopsies and bronchoalveolar lavage (BAL)-derived lymphocytes, obtained from 12 smokers with mild/moderate chronic obstructive pulmonary disease (COPD) (forced expiratory volume in one second (FEV1) 59 +/- 16% predicted), 14 smokers with normal lung function (FEV1 106 +/- 12% pred) and 12 nonsmoking subjects (FEV1 111 +/- 14% pred), was examined by immunohistochemistry and immunocytochemistry. In bronchial biopsies of COPD patients, the number of submucosal phospho-STAT4+ cells was increased (240 (22-406) versus 125 (0-492) versus 29 (0-511) cells mm(-2)) when compared with both healthy smokers and control nonsmokers, respectively. In smokers, phospho-STAT4+ cells correlated with the degree of airflow obstruction and the number of IFN-gamma+ cells. Similar results were seen in BAL (2.8 (0.2-5.9) versus 1.03 (0.09-1.6) versus 0.69 (0-2.3) lymphocytes x mL(-1) x 10(3)). In all smokers who underwent lavage, phospho-STAT4+ lymphocytes correlated with airflow obstruction and the number of IFNgamma+ lymphocytes. T-bet expression was not altered in bronchial biopsies and BAL-derived lymphocytes between the three groups. In conclusion, this study suggests that stable mild/moderate chronic obstructive pulmonary disease is associated with an active T-helper 1 cell/type-1 cytotoxic T-cell inflammatory process involving activation of signal transducer and activator of transcription 4 and interferon-gamma production.

Isolated PACNS-like presentation of a systemic vasculitis complicating a myelodysplastic syndrome.

Myelodysplastic syndromes (MDS) are a series of haematological malignancies ranging from chronic refractory anaemia to leukaemia. There is increasing recognition of immunological abnormalities in patients with MDS, including few reports of cutaneous vasculitis; in no instance, a cerebral localization has been ascertained. Here, the case of a patient with MDS who presented exclusively with neurological signs that were considered indicative of a primary, isolated central nervous system vasculitis (PACNS) is reported. Although histological findings on brain tissue confirmed a small-vessel vasculitis, this had to be considered in the context of a systemic vasculitis. In fact, at autopsy, an involvement of skin, myocardium, lungs, liver, kidney and bone marrow was also found. An autoimmune vasculitis should be included in the differential diagnosis of acute-onset, isolated, cerebral symptoms complicating the course of MDS.

[Stability of prostaglandin E1 when fractioned into polypropylene syringes]. / Estabilidad de prostaglandina E1 fraccionada en jeringas de polipropileno.

UNLABELLED: Prostaglandin E1 is indicated for the temporary maintenance of patent ductus arteriosus in newborns with ductus-dependent congenital heart defects. Since the standard daily dose is smaller (0.2-0.4 ml) than one ampoule (1 ml), we performed a chemical stability study of prostaglandin E1 when it is fractioned into polypropylene syringes. Three concentrations were studied: 500 mg/ml (original), and 1:2 and 1:4 dilutions in sodium chloride 0.9%. Syringes were kept at 4 degrees C for 30 days. Prostaglandin E1 levels were determined by using high-pressure chromatography. In all three formulations, the concentrations underwent no statistically significant changes over time (p>0.05). CONCLUSION: PGE1 is chemically stable at 4 degrees C for 30 days when fractioned into polypropylene syringes, both in the original and the 1:2 and 1:4 diluted formulations.

Increased expression of nuclear factor-kappaB in bronchial biopsies from smokers and patients with COPD.

The expression of nuclear factor (NF)-kappaB is an indicator of cellular activation and of inflammatory mediator production. The aim of the present study was to characterise the expression and localisation of p65, the major subunit of NF-kappaB, in the bronchial mucosa of patients with chronic obstructive pulmonary disease (COPD), and to examine the relationship between p65 expression and disease status. Bronchial biopsies were obtained from 14 smokers with COPD, 17 smokers with normal lung function and 12 nonsmokers with normal lung function. The number of p65 positive (+) cells was quantified by immunohistochemistry and the expression of p65 in bronchial biopsies from the three groups was examined by Western blotting (WB). Smokers with normal lung function and patients with COPD had increased numbers of p65+ cells in the epithelium and increased p65 nuclear expression. In COPD patients the number of epithelial p65+ cells correlated with the degree of airflow limitation. WB analysis showed an increase in p65 in smokers with normal lung function and COPD patients (p<0.05). Bronchial biopsies in smokers with normal lung function and chronic obstructive pulmonary disease patients show increased expression of p65 protein, predominantly in the bronchial epithelium. Disease severity is associated with an increased epithelial expression of nuclear factor-kappaB.

Increased cyclooxygenase-2 expression is associated with chemotherapy resistance and poor survival in cervical cancer patients.

PURPOSE: To investigate the expression of cyclooxygenase (COX-2) and its association with clinicopathologic parameters and clinical outcome in patients with cervical cancer. PATIENTS AND METHODS: The study included 84 patients with stage IB to IVA cervical cancer. Patients with early-stage cases (n = 21) underwent radical surgery, whereas patients with locally advanced cervical cancer (LACC) (n = 63) were first administered neoadjuvant cisplatin-based treatment and subjected to surgery in case of response. Immunohistochemical analysis was performed on paraffin-embedded sections with rabbit antiserum against COX-2. RESULTS: COX-2--integrated density values in the overall population ranged from 1.2 to 82.3, with mean plus minus SE values of 27.4 plus minus 2.4. According to the chosen cutoff value, 36 (42.9%) of 84 patients were scored as COX-2 positive. COX-2 levels were shown to be highly associated with tumor susceptibility to neoadjuvant treatment. COX-2 showed a progressive increase from mean plus minus SE values of 19.9 plus minus 8.0 in complete responders through 31.5 plus minus 3.5 in partial responses to 44.8 plus minus 3.9 in patients who were not responsive (P =.0054). When logistic regression was applied, only advanced stage and COX-2 positivity retained independent roles in predicting a poor chance of response to treatment. COX-2--positive patients had a shorter overall survival (OS) rate than COX-2--negative patients. In patients with LACC, the 2-year OS rate was 38% in COX-2--positive versus 85% in COX-2--negative patients (P =.0001). In the multivariate analysis, only advanced stage and COX-2 positivity retained independent negative prognostic roles for OS. CONCLUSION: The assessment of COX-2 status could provide additional information to identify patients with cervical cancer with a poor chance of response to neoadjuvant treatment and unfavorable prognosis.

Decreased T lymphocyte infiltration in bronchial biopsies of subjects with severe chronic obstructive pulmonary disease.

BACKGROUND: Studies on the inflammatory process in the large airways of patients with mild/moderate COPD have shown a prevalent T lymphocyte and macrophage infiltration of the bronchial mucosa. However, bronchial inflammation in more severe disease has not been extensively studied. OBJECTIVE: The aim of the present study was to characterize the lymphocyte infiltration in the bronchial mucosa of subjects with severe, compared to mild, COPD, and to examine the relationship between airflow limitation and T lymphocyte numbers in the bronchial mucosa. METHODS: We examined bronchial biopsies obtained from nine smokers with severe airflow limitation, nine smokers with mild/moderate airflow limitation and 14 smokers with normal lung function. Immunohistochemical methods on cryostat sections were used to assess the number of CD3+, CD4+, CD8+ cells and the number of CD3+ cells coexpressing the chemokine receptor CCR5 (CCR5+CD3+) in the subepithelium. RESULTS: Subjects with severe COPD had lower numbers of CD3+, CD8+ and CCR5+CD3+ cells than mild/moderate COPD (P < 0.012, P < 0.02 and P < 0.02, respectively) and control smokers (P < 0.015, P < 0.005 and P < 0.015, respectively). In subjects with airflow limitation the number of CD3+ and CD8+ cells was inversely correlated with the degree of airway obstruction (r = 0.59, P < 0.015 and r = 0.52, P < 0.032, respectively). CONCLUSIONS: Bronchial inflammation in severe COPD is characterized by lower numbers of CD3+ and CD8+ cells and decreased numbers of CD3+ cells coexpressing the chemokine receptor CCR5. T lymphocyte infiltration is inversely correlated with the degree of airflow limitation.

Primary malignant melanoma of the gallbladder in dysplastic naevus syndrome.

A case of gallbladder involvement by malignant melanoma in a 57-year-old woman is reported. The gallbladder, resected for cholelithiasis, harboured a pedunculated polypoid dark mass, which histologically revealed sheets and nests of epithelioid cells with hyperchromatic nuclei in the lamina propria and at the junctional level. These cells were pigmented (with positive reaction with Schmorl's stain and bleaching with peroxide) and showed immunohistochemical positivity for S-100, gp 100 antigen (HMB-45 antibody) and vimentin. The patient, affected by dysplastic naevus syndrome, had a melanoma in situ excised from the scalp 8 years earlier. The features of the investigated lesion address towards a diagnosis of primary gallbladder melanoma. Furthermore, this is the first time that the existence of such a controversial entity is sustained by the ultrastructural investigation of melanosomes, demonstrating the presence of two melanocitary populations, a typical one exclusively junctional and an atypical one both at the junctional level and in the lamina propria.

Bax mutations are an infrequent event in indolent lymphomas and in mantle cell lymphoma.

BACKGROUND AND OBJECTIVES: The Bax gene is one of the most important genes involved in apoptosis regulation. Recently, it has been proposed that inactivating mutations of this death agonist may contribute to the pathogenesis of human tumors. This study was aimed at defining the status of the Bax gene in indolent lymphomas. DESIGN AND METHODS: Fifty paraffin-embedded biopsies from indolent lymphomas (10 small lymphocytic lymphomas, 5 immunocytomas, 20 follicular lymphomas and 15 marginal zone lymphomas) and 10 mantle cell lymphomas ( MCL ) were studied. All six exons of the Bax gene, together with their flanking sequences, underwent mutational analysis by PCR-SSCP followed by direct sequencing of positive cases. Moreover, Bax protein expression was investigated in all samples by immunohistochemical analysis. RESULTS: All analyzed cases showed wild type Bax gene alleles and variable levels of Bax protein expression. INTERPRETATION AND CONCLUSIONS: This study indicates that deregulation of apoptotic control in indolent lymphomas and MCL is not caused by Bax mutations and that other molecular mechanisms must, therefore, be involved.

Early cervical carcinoma: the natural history of lymph node involvement redefined on the basis of thorough parametrectomy and giant section study.

BACKGROUND: Although parametrectomy is the most difficult step in the surgical treatment of cervical carcinoma and is the main cause of postoperative complications, little attention has been given to the patterns of parametrial spread. METHODS: Sixty-nine patients with previously untreated cervical carcinoma (Fédération Internationale de Gynécologie et d'Obstétrique [FIGO] Stage IB1, 49 patients [71%]; Stage IB2, 8 patients [12%]; and Stage IIA, 12 patients [17%]; squamous, 59 patients [86%]; and adenocarcinoma, 10 patients [14%]) underwent radical hysterectomy and pelvic +/- aortic lymphadenectomy. Hysterectomy specimens were processed with the giant section technique. To obtain a thorough three-dimensional assessment of the paracervical tissue, both the superficial and deep layers of the cervicovesical ligament (anterior parametrium) and the uterosacral ligament (posterior parametrium) were separated from the uterus and submitted for pathologic evaluation. After resection of the lateral parametrium with hemoclips, the lympho-fatty tissue remaining around the pudendal vessels was removed carefully and referred to as "the distal part of the lateral parametrium." RESULTS: When analyzing all the parametria, lymph nodes were present in 64 patients (93%). Clinically undetected parametrial involvement was found by pathologic examination in 15 Stage IB1 patients (31%), 5 Stage IB2 patients (63%), and 7 Stage IIA patients (58%). Metastases were found in the cardinal, cervicovesical, and sacrouterine ligaments and principally were comprised of lymph node and vascular space invasion. Twenty-five patients (36%) had pelvic lymph node metastases whereas concomitant parametrial involvement was observed in all patients. The overall 5-year survival was 91%, being higher for parametria and lymph node negative patients (100%) than for those with lymph node and/or parametrial metastases (78%). CONCLUSIONS: A three-dimensional pathologic assessment showed that subclinical parametrial spreading of the so-called "early" tumors (Stage IB-IIA) occurred in approximately 30-60% of these patients, and metastasis to the pelvic lymph nodes always was associated with parametrial disease. A better understanding of the patterns of parametrial diffusion will improve knowledge of the natural history of cervical carcinoma and in the future may influence the treatment of these patients. Furthermore, pathologic assessment of cervical carcinoma should be modified to evaluate correctly the parametrial status of each patient. The current routine pathologic evaluation of the parametria makes it very difficult to detect lymph node metastases and tumor emboli.

Quercetin inhibits p21-RAS expression in human colon cancer cell lines and in primary colorectal tumors.

Immunocytochemical studies have revealed that 10 microM quercetin reduced the steady state levels of p21-ras proteins in both colon cancer cell lines and primary colorectal tumors. These findings were confirmed by Western blot and flow cytometric analysis showing that the inhibition of p21-ras expression by quercetin was time- and concentration-dependent. Twenty-four-hour treatment with 10 microM quercetin reduced p21-ras levels to about 50% of control values. Quercetin was similarly effective in inhibiting the expression of K-, H-, and N-ras proteins. Moreover, the effect of quercetin on ras oncogene expression was not dependent on the cell cycle position of colon cancer cells and appeared to be specific and not merely a consequence of overall inhibition of protein synthesis. Northern blot analysis revealed that quercetin produced in colon cancer cells an early (30 min) reduction of the steady state levels of K-, H-, and N-ras mRNAs. This reduction was also present after 6 hr of flavonoid treatment. These effects of quercetin suggest a possible chemopreventive role for this compound in colorectal carcinogenesis.

Morphometric prognostic index in breast cancer.

OBJECTIVE: To assess the ability of the morphometric prognostic index (MPI) in predicting clinical outcome in a group of breast cancer patients with short-term follow-up and to assess the relationship between MPI and other prognosticators. STUDY DESIGN: The study group consisted of 63 cases of breast cancer. Follow-up data were available for 48 patients. MPI values were calculated, and degree of nuclear and tubular differentiation was investigated in each tumor. S-phase fraction (SPF), estrogen and progesterone receptors were also studied. RESULTS: The group of patients with MPI values < 0.60 had percent values of disease-free survival significantly higher than did those with MPI values > or = 0.60. Furthermore, significant direct correlations were found between MPI and degree of nuclear atypia and between MPI and SPF. Significant inverse relationships were found between MPI and tumor progesterone receptor levels and between MPI and degree of histologic tubular differentiation. CONCLUSION: The validity of MPI as a prognosticator in breast cancer was confirmed, even in a limited number of patients observed in short-term follow-up. MPI seems to be a reliable and economical prognosticator in selecting breast cancer patients for adjuvant chemotherapy.