Klebsiella pneumoniae induces an inflammatory response in an in vitro model of blood-retinal barrier.

Klebsiella pneumoniae has become an important pathogen in recent years. Although most cases of K. pneumoniae endogenous endophthalmitis occur via hematogenous spread, it is not yet clear which microbial and host factors are responsible for the ability of K. pneumoniae to cross the blood-retinal barrier (BRB). In the present study, we show that in an in vitro model of BRB based on coculturing primary bovine retinal endothelial cells (BREC) and primary bovine retinal pericytes (BRPC), K. pneumoniae infection determines changes of transendothelial electrical resistance (TEER) and permeability to sodium fluorescein. In the coculture model, bacteria are able to stimulate the enzyme activities of endothelial cytosolic and Ca(2+)-independent phospholipase A2s (cPLA2 and iPLA2). These results were confirmed by the incremental expression of cPLA2, iPLA2, cyclo-oxygenase-1 (COX1), and COX2 in BREC, as well as by cPLA2 phosphorylation. In supernatants of K. pneumoniae-stimulated cocultures, increases in prostaglandin E2 (PGE2), interleukin-6 (IL-6), IL-8, and vascular endothelial growth factor (VEGF) production were found. Incubation with K. pneumoniae in the presence of arachidonoyl trifluoromethyl ketone (AACOCF3) or bromoenol lactone (BEL) caused decreased PGE2 and VEGF release. Scanning electron microscopy and transmission electron microscopy images of BREC and BRPC showed adhesion of K. pneumoniae to the cells, but no invasion occurred. K. pneumoniae infection also produced reductions in pericyte numbers; transfection of BREC cocultured with BRPC and of human retinal endothelial cells (HREC) cocultured with human retinal pericytes (HRPC) with small interfering RNAs (siRNAs) targeted to cPLA2 and iPLA2 restored the pericyte numbers and the TEER and permeability values. Our results show the proinflammatory effect of K. pneumoniae on BREC, suggest a possible mechanism by which BREC and BRPC react to the K. pneumoniae infection, and may provide physicians and patients with new ways of fighting blinding diseases.

Blood-Brain Barrier in a Haemophilus influenzae Type a In Vitro Infection: Role of Adenosine Receptors A2A and A2B.

The blood-brain barrier (BBB) is mainly made up of tightly connected microvascular endothelial cells (BMECs), surrounded by pericytes (BMPCs) which regulate BBB tightness by providing soluble factors that control endothelial proliferation. Haemophilus influenzae type a (Hia) is able to reach the BBB, crossing it, thus causing meningitis. In this study, by using an in vitro model of BBB, performed with human BMECs and human BMPCs in co-culture, we demonstrated that, after Hia infection, the number of hBMPCs decreased whereas the number of hBMECs increased in comparison with non-infected cells. SEM and TEM images showed that Hia was able to enter hBMECs and reduce TEER and VE-cadherin expression. When the cells were infected in presence of SCH58261 and PSB603 but not DPCPX, an increase in TEER values was observed thus demonstrating that A2A and A2B adenosine receptors play a key role in BBB dysfunction. These results were confirmed by the use of adenosine receptor agonists CGS21680, CCPA, and NECA. In infected co-cultures cAMP and VEGF increased and TEER reduction was counter-balanced by VEGF-R1 or VEGF-R2 antibodies. Moreover, the phosphorylated CREB and Rho-A significantly increased in infected hBMECs and hBMPCs and the presence of SCH58261 and PSB603 significantly abrogated the phosphorylation. In conclusion, this study demonstrated that the infection stimulated A2A and A2B adenosine receptors in hBMECs and hBMPCs thus inducing the pericytes to release large amounts of VEGF. The latter could be responsible for both, pericyte detachment and endothelial cell proliferation, thus provoking BBB impairment.

Towards localization of malignant sites of asymmetry across bilateral mammograms.

BACKGROUND AND OBJECTIVES: The analysis of patterns of asymmetry between the left and right mammograms of a patient can provide meaningful insights into the presence of an underlying tumor in its early stage. However, the identification of breast cancer by investigating bilateral asymmetry is difficult to perform due to the indistinct and borderline nature of the asymmetric signs as they appear on mammograms. METHODS: In this study, to increase the positive-predictive value of asymmetry in mammographic screening, a novel computerized approach for the automatic localization of malignant sites of asymmetry in mammograms is proposed. The sites of anatomical correspondence between the right and left regions of each radiographic projection were extracted by means of two bilateral masking procedures, inspired by radiologists' criteria in interpreting mammograms and based on the use of detected landmarking structures. Relative variations of spatial patterns of intensity values and of orientations of directional components within each site were quantified by combining multidirectional Gabor filters and indices of structural similarity. The localization of the sites of malignant asymmetry was performed by coupling two quadratic discriminant analysis classifiers, one for each masking procedure, that assigned the likelihood of malignancy to each site of correspondence. RESULTS: The performance of the proposed method was assessed on 94 mammographic images from two publicly available databases and containing at least one asymmetric site. Sensitivity, specificity and balanced accuracy levels of 0.83 (0.09), 0.75 (0.06), and 0.79 (0.04), respectively were obtained in the classification of malignant asymmetric sites vs benign/normal sites using cross-validation. In addition, a further blind test on a dataset of Full Field Digital Mammograms achieved levels of sensitivity, specificity, and balanced accuracy of 0.86, 0.65, and 0.75, respectively. CONCLUSIONS: The achieved performance indicates that the proposed system is effective in localizing sites of malignant asymmetry and it is expected to improve computer-aided diagnosis of breast cancer.

Chronic peripheral arteriopathy is associated with seropositivity to Chlamydia pneumoniae.

The study was carried out to clarify the correlation between Chlamydia pneumoniae infection and peripheral arterial disease (PAD). The level of specific antibodies of the 133 consecutive patients suffering from PAD at 2nd stage of Leriche's classification were compared with 60 healthy controls by using a commercial Micro-IF Test. A higher incidence of serological evidence of C. pneumoniae infection was found in the patients (106/133) than in controls (6/60). These results are in agreement with other findings that measured the infection in atheromasic plaques. A strong cause-effect relationship between bacterial infection and peripheral arterial disease was not found, but the raised seropositivity level could be considered as a target for medical therapy of PAD.

Role of cytosolic and calcium independent phospholipases A(2) in insulin secretion impairment of INS-1E cells infected by S. aureus.

Cytosolic PLA2 (cPLA2) and Ca(2+)-independent PLA2 (iPLA2) play a significant role in insulin ß-cells secretion. Bacterial infections may be responsible of the onset of diabetes. The mechanism by which Staphylococcus aureus infection of INS-1 cells alters glucose-induced insulin secretion has been examined. After acute infection, insulin secretion and PLA2 activities significantly increased. Moreover, increased expressions of phospho-cPLA2, phospho-PKCα and phospho-ERK 1/2 were observed. Chronic infection causes a decrease in insulin release and a significant increase of iPLA2 and COX-2 protein expression. Moreover, insulin secretion in infected cells could be restored using specific siRNAs against iPLA2 isoform and specific COX-2 inhibitor.

Asthenozoospermia and membrane remodeling enzymes: a new role for phospholipase A2.

Phosholipase A2 (PLA2 ) activity in the seminal plasma and in sperm heads is closely related to sperm motility and male fertility. Therefore, the purpose of this study was to investigate the possible involvement of different isoforms of phospholipase in asthenozoospermia. To accomplish this, cPLA2 , phospho-cPLA2 , iPLA2 , and sPLA2 were evaluated by immunofluorescence and immunoblot analyses in spermatozoa obtained from 22 normozoospermic men and 28 asthenozoospermic patients. We found significant differences in cPLA2 and its phosphorylated/activated form, iPLA2 , and sPLA2 content and distribution in normal and asthenozoospermic patients. cPLA2 was localized in heads, midpieces, and tails of all spermatozoa as constitutive enzyme, less expressed in the tail of spermatozoa with low progressive motility. While active phospho-cPLA2 distribution was homogeneous throughout the cell body of control-donor spermatozoa, lower levels were detected in the tails of asthenozoospermic patients, as opposed to its strong presence in heads. Low immunofluorescence signal for iPLA2 was found in astenozoospermic patients, whereas sPLA2 was significantly lower in the heads of asthenozoospermic patients. Spermatozoa with low progressive motility showed differences both in terms of total specific activity and of intracellular distribution. cPLA2 , iPLA2 , and sPLA2 specific activities correlated positively and in a significantly manner with sperm progressive motility both in normozoospermic men and asthenozoospermic patients. In conclusion, PLA2 s are expressed in different areas of human spermatozoa. Spermatozoa with low motility showed differences in total specific activity and enzyme distributions. We speculated that PLA2 expression and/or different distribution could be potential biomarkers of asthenozoospermia, one of the major causes of male factor infertility.

New epidemiological data on resistance to netilmicin and other aminoglycosides.

In previous investigations we considered the increasing resistance of Gram-negative organisms to aminoglycosides over time. We therefore performed further studies to evaluate eventual variations in the incidence of bacterial resistance to aminoglycosides over the last four years. We tested Gram-negative microorganisms recently isolated from pathological materials of various clinical origins with gentamicin, amikacin, tobramycin and netilmicin. Our data show evidence that there was no significant increasing resistance to aminoglycosides, and that netilmicin continues to be active as an anti-Gram-negative antibiotic.

Estimation of the breast skin-line in mammograms using multidirectional Gabor filters.

Segmentation of the breast region is a fundamental step in any system for computerized analysis of mammograms. In this work, we propose a novel procedure for the estimation of the breast skin-line based upon multidirectional Gabor filtering. The method includes an adaptive values-of-interest (VOI) transformation, extraction of the skin-air ribbon by Otsu's thresholding method and the Euclidean distance transform, Gabor filtering with 18 real kernels, and a step for suppression of false edge points using the magnitude and phase responses of the filters. On a test set of 361 images from different acquisition modalities (screen-film and full-field digital mammograms), the average Hausdorff and polyline distances obtained were 2.85 mm and 0.84 mm, respectively, with reference to the ground-truth boundaries provided by an expert radiologist. When compared with the results obtained by other state-of-the-art methods on the same set of images and with respect to the same ground-truth boundaries, our method mostly outperformed the other approaches. The results demonstrate the effectiveness and robustness of the proposed algorithm.

Probiotics in the gastrointestinal diseases of the elderly.

Changes of the gut microflora in elderly appear to involve a reduction in numbers of healthy bacteria (lactobacilli and bifidobacteria) and an increase in numbers of potentially pathogenic species. These changes are generally described as gastrointestinal disorders and infections. This review analyses benefits of probiotics in old people, with particular interesting for the latest researches relevant to elderly people, e.g. trials examining enteric infections, antibiotic-associated diarrhea and Clostridium difficile associated diarrhea, functional bowel problems (constipation and irritable bowel syndrome), inflammatory bowel diseases, stimulation of the immune system and prevention of cancer. A growing number of researches indicates that some probiotic strains may help to maintain the health in old people, suggesting both health and cost-saving benefits in offering fermented dairy products. These benefits include: establishment of balanced intestinal microflora; improving colonization resistance and or prevention of diarrhea; reduction of fecal enzymes; reduction of serum cholesterol; reduction of potential mutagenes; reduction of lactose intolerance; synthesis of vitamins; predigestion of proteins.

[Efficacy and tolerance of azithromycin in respiratory tract infections in children]. / Efficacia e tollerabilità dell'azitromicina nelle infezioni respiratorie del bambino.

This study was carried out to determine the clinical efficacy and the clinical safety of azithromycin in a group of children with acute respiratory tract infections. The study involved 82 children treated with a single daily 10 mg/kg dose of azithromycin for three consecutive days. 7 days later, the overall clinical response was 100% (cure and improvement): bacteriological cure was achieved in 97.5% of the patients treated. Recurrences were never observed. Side effects not requiring interruption of therapy were observed in 3 patients (3.6%). The side effects were gastrointestinal disturbances. In conclusion azithromycin showed a remarkably clinical efficacy for treatment of acute respiratory infections in children. Tolerability and therapeutic compliance were excellent.