Maria Antonietta Toscano

Bifidobacterium longum with Fructo-Oligosaccharide (FOS) Treatment in Minimal Hepatic Encephalopathy: A Randomized, Double-Blind, Placebo-Controlled Study

Minimal hepatic encephalopathy (MHE) describes patients with chronic liver disease or cirrhosis who have no clinical symptoms of brain dysfunction but perform worse on psychometric tests compared with healthy subjects. The pathogenesis of hepatic encephalopathy is controversial although ammonia has been found to induce cerebral dysfunction. Increased intestinal ammonia production is due to bacterial urease activity and the production of other toxin methabolities, such as mercaptans, thioles. This study assesses the clinical efficacy of Bifidobacterium longum plus fructo-oligosaccharides (FOS) in the treatment of MHE. A total of 60 cirrhotic patients were randomly and equally divided into two groups receiving Bifidobacterium+FOS (17 males, 13 females; mean age, 46±11 years) or placebo (16 males, 14 females; mean age, 45±12 years), respectively. All patients underwent clinical and laboratory assessment psychometric tests and automated EEG analysis: neurophysiological assessment, liver function assessment, amd neuropsychological assessment. After 90 days of treatment, fasting NH4 serum levels were significantly decreased (P=0.003), performance on Trail Making Test-A was significantly decreased (P=0.000), performance on Trail Making Test-B was significantly decreased (P=0.000), performance on the symbol digit modalities test was significantly improved (P<0.05), performance on block design was significantly improved (P=0.000), and performance on the MMSE test was significantly improved (P=0.000). We conclude that the improvement in biochemical and neuropsychological tests of the group treated with Bifidobacterium longum+FOS are interesting and merit further, close examination.

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.

VEGF receptor-1 involvement in pericyte loss induced by Escherichia coli in an in vitro model of blood brain barrier

The key aspect of neonatal meningitis is related to the ability of pathogens to invade the blood–brain barrier (BBB) and to penetrate the central nervous system. In the present study we show that, in an in vitro model of BBB, on the basis of co‐culturing primary bovine brain endothelial cells (BBEC) and primary bovine retinal pericytes (BRPC), Escherichia coli infection determines changes of transendothelial electrical resistance (TEER) and permeability (Pe) to sodium fluorescein. In the co‐culture model, within BBEC, bacteria are able to stimulate cytosolic and Ca2+‐independent phospholipase A2 (cPLA2 and iPLA2) enzyme activities. In supernatants of E. coli‐stimulated co‐cultures, an increase in prostaglandins (PGE2) and VEGF production in comparison with untreated co‐cultures were found. Incubation with E. coli in presence of AACOCF3 or BEL caused a decrease of PGE2 and VEGF release. SEM and TEM images of BBEC and BRPC showed E. coli adhesion to BBEC and BRPC but only in BBEC the invasion occurs. VEGFR‐1 but not VEGFR‐2 blockade by the specific antibody reduced E. coli invasion in BBEC. In our model of BBB infection, a significant loss of BRPC was observed. Following VEGFR‐1, but not VEGFR‐2 blockade, or in presence of AACOCF3 or BEL, elevated TEER values, reducedpermeability and BRPC loss were found. These data suggest that VEGFR‐1 negatively regulates BRPC survival and its blockade protects the barrier integrity. PGs and VEGF could exert a biological effect on BBB, probably by BRPC coverage ablation, thus increasing BBB permeability. Our results show the role played by the BBEC as well as BRPC during a bacterial attack on BBB. A better understanding of the mechanisms by which E. coli enter the nervous system and how bacteria alter the communication between endothelial cells and pericytes may provide exciting new insight for clinical intervention.

Erythromycin resistance in italian isolates of Streptococcus pyogenes and correlations with pulsed-field gel electrophoresis analysis.

Erythromycin resistance among Streptococcus pyogenes strains has been reported in Italy at high rates during the last few years. A total of 152 erythromycin-resistant isolates of this species from southern Italian regions were characterized for the macrolide-resistance phenotype and screened by PCR for the corresponding genetic determinant. A close correlation was found between these phenotypic/genotypic data concerning macrolide resistance and results of Sma I macrorestriction fragment patterns (PFGE) analysis. In fact, the vast majority of the isolates assigned to individual PFGE classes mostly belonged to a single phenotype of macrolide resistance. All untypeable isolates belonged to the M phenotype. Twenty-two distinct PFGE types were recognized, of which 11 were recorded in only one isolate (one-strain type); about 50% of typeable isolates fell into five type clusters and 70% in seven. The increased erythromycin resistance among Italian isolates of S. pyogenes does not appear to be due to the spread of a single clone, but results indicate that the majority of group A streptococci examined are probably spread from a limited number of clones.

Involvement of PKCα–MAPK/ERK-phospholipase A2 pathway in the Escherichia coli invasion of brain microvascular endothelial cells

Escherichia coli K1 is the most common Gram-negative organism that causes neonatal meningitis following penetration of the blood-brain barrier. In the present study we demonstrated the involvement of cytosolic (cPLA(2)) and calcium-independent phospholipase A(2) (iPLA(2)) and the contribution of cyclooxygenase-2 products in E. coli invasion of microvascular endothelial cells. The traversal of bacteria did not determine trans-endothelial electrical resistance (TEER) and ZO-1 expression changes and was reduced by PLA(2)s siRNA. cPLA(2) and iPLA(2) enzyme activities and cPLA(2) phosphorylation were stimulated after E. coli incubation and were attenuated by PLA(2), PI3-K, ERK 1/2 inhibitors. Our results demonstrate the role of PKCα/ERK/MAPK signaling pathways in governing the E. coli penetration into the brain.

Chlamydia trachomatis Prevalence in Unselected Infertile Couples

Chlamydia (C.) trachomatis, an obligate intracellular bacterium, is responsible for the most common sexual transmitted disease and infertility. The purpose of this study was to evaluate: a) the frequency of chlamydial infection in unselected infertile couples and b) whether chlamydial infection could be identified in the semen sample as effectively as in the urethral swab of infertile patients. To accomplish this, 73 unselected, consecutive infertile couples were enrolled. Both male and female partners underwent a complete work-up to identify the cause of their infertility. A PCR method was used to detect C. trachomatis in urethral swabs and the semen samples of the male partners and in the cervical swabs of the female partners. C. trachomatis infection was found in 6 couples (8.2%). Three couples had both partners infected, 2 couples had only the male partner infected, and 1 only the female partner. C. trachomatis infection was found in the urethral swab of all 5 men infected, whereas the bacterial DNA was found in the semen sample of 2 of them. These findings suggest that C. trachomatis infection is present in about 8% of unselected infertile couples and that the bacterium should be searched in the male partner urethral swab which has a higher sensitivity.

Klebsiella pneumoniae Induces an Inflammatory Response in an In Vitro Model of Blood-Retinal Barrier

ABSTRACT 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 Ca2+-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.

Role of cytosolic and calcium independent phospholipases A2 in insulin secretion impairment of INS-1E cells infected by S. aureus

Cytosolic PLA2 (cPLA2) and Ca2+‐independent PLA2 (iPLA2) play a significant role in insulin β‐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.

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.