Effectiveness of a new method of disinfecting the root canal, using Er, Cr:YSGG laser to kill Enterococcus faecalis in an infected tooth model.

Some lasers have demonstrated to provide effective disinfection when used as adjunctive device to the conventional treatment. The aim of this in vitro study was to determine the effectiveness of the erbium, chromium:yttrium scandium gallium garnet (Er, Cr:YSGG) laser by measuring its bactericidal effect inside the root canal experimentally colonized with Enterococcus faecalis. The laser was tested at different irradiation times (30 and 60 s) and energy of impulses (75 and 25 mJ). A total of 52 single-rooted extracted human teeth were endodontically prepared with rotary instrumentation. All were sterilized and inoculated with a suspension of E. faecalis (105 bacteria/ml). The teeth were randomized into three treatment (group 1, group 2, and group 3) and one control groups. In all groups, teeth were chemically irrigated with 5.25% sodium hypochlorite and 17% ethylenediaminetetraacetic acid. Groups 1 and 2 were also irradiated at 30 and 60 s, respectively, with an Er, Cr:YSGG laser at 75 mJ. Teeth of group 3 were treated with laser for 60 s at 25 mJ. Samples were processed to detect the presence of E. faecalis. For all groups, a bactericidal effect was observed. The use of laser at 75 mJ with an irradiation time of 30 and 60 s eliminated a percentage of 92.3 and 100% of E. faecalis, respectively. In the control group, a reduction of 92.3% was observed. Lower percentage of reduction (46.1%) was obtained in teeth treated with laser at 25 mJ for 60 s. No statistical differences were observed between the groups (P = 0.543, Fisher's exact test). The results indicated a bactericidal effect of Er, Cr:YSGG laser irradiation at the settings used in this study. The highest bactericidal effect of this laser was observed at 60 s of irradiation time, using an energy pulse of 75 mJ.

tst1-positive ST22-MRSA-IVa in healthy Italian preschool children.

A survey was performed in May 2013 to assess methicillin-resistant Staphylococcus aureus (MRSA) nasal colonization in healthy children attending 26 municipal daycare centres in Palermo, Italy. Of the 500 children, ten (2 %) tested positive. Eight MRSA isolates were tst1-positive ST22-MRSA-IVa, spa t223; the other two isolates were identified as ST1-IVa and ST398-V, respectively. tst1-positive ST22-MRSA, spa t223 has been previously identified only in the Middle Eastern area.

Isolation, expression and immunological characterization of a calcium-binding protein from Parietaria pollen.

The diagnosis and therapy of allergic disorders are usually performed with crude extracts which are a heterogeneous mixture of proteins with different allergenic potency. The knowledge of the allergenic composition is a key step for diagnostic and therapeutic options. Parietaria judaica pollen represents one of the main sources of allergens in the Mediterranean area and its major allergens have already been identified (Par j 1 and Par j 2). In addition, inhibition studies performed using a calcium-binding protein (CBP) from grass pollen (Phl p 7) showed the presence of a homologue of this cross-reactive allergen in the Parietaria extract. Screening of a cDNA library allowed us to isolate a 480bp cDNA containing the information for an 87 AA long protein with high level of homology to calcium-binding proteins from other allergenic sources. It was expressed as a recombinant allergen in Escherichia coli and purified by affinity chromatography. Its expression allowed us to study the prevalence of this allergen in a population of allergic patients in southern Europe. Immunoblotting and inhibition studies showed that this allergen shares a pattern of IgE epitopes in common with other 2-EF-hand calcium-binding proteins from botanically non-related species. The immunological properties of the Pj CBP were investigated by CD63 activation assay and CFDA-SE staining. In conclusion, DNA recombinant technology allowed the isolation, expression and immunological characterization of a cross-reactive calcium-binding protein allergen from Parietaria judaica pollen.

Allergens of Parietaria judaica pollen--I. Purification and characterization of a hapten and a low molecular weight allergenic peptide.

A low mol. wt allergen (Pj-2) and a hapten (Pj-H3) were purified from Parietaria judaica pollen by means of long-term aqueous extraction, dialysis and gel filtrations. The yield of the Pj-2 allergen was 0.94% (w/v) of the total protein present in the aqueous extract of the pollen, while its allergenic activity was about 60% of the total dialyzable activity, as verified by skin prick tests, ELISA- and RAST-inhibition experiments. The homogeneity of this allergen was demonstrated by one single sharp peak on HPLC, one single band on PAGE-SDS and by one single arc on IEF. Its mol. wt, estimated by HPLC and amino acid composition, was 10,400. The amino acid analysis showed 73 amino acid residues, and lysine was predominant, with 20 residues. The hapten Pj-H3 was 0.2% (w/v) of the total protein found in the pollen aqueous extract. It was inactive in skin prick tests even at a protein concn of 2 mg/ml, while it was capable of inhibiting by 60% in ELISA- and RAST-inhibition experiments, suggesting an immunochemical relationship with both IgE and allergens specific to P. judaica. The homogeneity was demonstrated by one single sharp peak on HPLC and one single band on PAGE-SDS. The amino acid analysis showed 10 amino acid residues, with no specific traits, and the mol. wt determined by gel filtration and amino acid composition was 1000. An immunochemical relation between the allergen and the hapten was also suggested by the results of an ELISA-inhibition test, and by the ability of the hapten to partially inhibit the precipitin line between rabbit antibodies to whole P. judaica pollen extract and the Pj-2 allergen. The allergen and the hapten described above, purified at homogeneity and in an antigenically active state, both provide adequate material for further structural and immunological characterizations.

cDNA cloning, sequence analysis and allergological characterization of Par j 2.0101, a new major allergen of the Parietaria judaica pollen.

A clone (P2) coding for an allergen of Parietaria judaica (Pj) pollen has been isolated and sequenced from a cDNA library in lambda ZAP using a pool of 23 sera from Pj-allergic patients. The clone contained an insert of 622 nucleotides with an open reading frame of 133 amino acids (aa) and a putative signal peptide of 31 aa giving a deduced mature processed protein of 102 aa with a molecular mass of 11344 Da. The expressed recombinant protein, named rPar j 2.0101, was a major allergen since it reacted with IgE of 82% (23/28) of the sera of Pj-allergic subjects analyzed. It was shown to be a new allergen since (i) the amino acid sequence homology with the already reported recombinant allergen Par j 1.0101 was 45% and (ii) there was no cross-inhibition between rPar j 2.0101 and rPar j 1.0101. In addition, rPar j 2.0101 inhibited 35% of the specific IgE for 10-14 kDa native allergens and preincubation of sera from Pj-allergic patients with both rPar j 2.0101 and rPar j 1.0101 fully abolished the IgE recognition of the 10-14 kDa native allergen region, suggesting that these two allergens contributed to the region.

cDNA cloning, expression and primary structure of Par jI, a major allergen of Parietaria judaica pollen.

A 659 bp cDNA clone** coding for an allergen of Pj pollen has been isolated from a lambda gt11 library, and its DNA sequence determined. The cDNA insert showed an open reading frame of 429 bp coding for an allergenic protein of 14,866 Da and a deduced amino acid sequence containing 143 residues. The expressed recombinant protein represented the major allergen Par jI since it reacted with 95% of the sera from Pj-allergic patients (n = 22) and with two Par jI-specific monoclonal antibodies. No similarity with other known DNA and protein sequences has been detected.

Inhibitory effect of neuraminidase on SP-induced histamine release and TNF-alpha mRNA in rat mast cells: evidence of a receptor-independent mechanism.

The neuropeptide substance P (SP) is a mediator of neuro-inflammation and can play a role by induction of histamine release (HR) and TNF-alpha. However, its effect on the heterogeneous response of mast cells (MC) has not been completely studied. We have established that the SR can induce 25% of HR in highly purified rat uterine MC at diestrous but not at proestrous phases of the reproductive cycle and 88% of HR in peritoneal mast cells (PMC). We also found 2.2 fold increase in TNF-alpha mRNA at diestrous, in SP stimulated uterine MC versus control and 2.7 fold increase in PMC; RT and competitive PCR were used to amplify the TNF-alpha mRNA. We have thereafter investigated the mechanism whereby the binding of SP to sialic acid on the MC membrane, could trigger secretion of histamine and induction of TNF-alpha mRNA. The neuraminidase pretreatment (0.1 U/ml) inhibited SP-stimulated HR from either uterine MC and PMC (98% and 50%, respectively) and totally inhibited SP-stimulated TNF-alpha mRNA levels. The neuraminidase effect was not toxic, since it was not observed in IgE mediated HR and TNF-alpha mRNA levels. In conclusion, the inhibitory effect of the neuraminidase on the SP-mediated increase of histamine and TNF-alpha mRNA, suggests that the SP-sialic acid interaction could have a role in the MC heterogeneous response.

Effect of substance P on uterine mast cell cytokine release during the reproductive cycle.

There is increasing evidence that neuropeptides, steroid hormones and inflammatory cytokines influence the immune response during the reproductive cycle. In the present study, we focus on the effects of neuropeptide Substance P (SP) during the pre-implantation stage of embryo development (day 4 of pregnancy), at pro-estrus and di-estrus (two phases with different hormonal states). We found heterogeneous responses to SP and anti-IgE by the rat uterine mast cells (MCs), as detected by ELISA. In fact, MCs purified from uteri on day 4 of pregnancy released histamine, granulocyte macrophage-colony stimulating factor (GM-CSF) and tumor necrosis factor-alpha (TNF-alpha) in response to anti-IgE, but not to SP. When pre-incubated with SP, the release to anti-IgE was significantly enhanced compared to anti-IgE alone. Exposure of SP to antibodies to SP, prior to pre-incubation with MCs, negated the SP effect on IgE-mediated release. At the pro-estrus phase SP showed similar behavior as on day 4 of pregnancy, whereas at the di-estrus phase SP alone was capable of inducing release of histamine and cytokines from purified uterine MCs. Moreover, non-quantitative RT-PCR analysis of the TNF-alpha mRNA level suggested an SP stimulation at the di-estrus phase, but neither on day 4 of pregnancy nor at the pro-estrus phase. Taken together, these data strongly suggest that SP can modulate IgE-mediated uterine MC release of histamine and inflammatory cytokines in different ways, depending on the phase of the reproductive cycle.

Mast cell production of TNF-alpha induced by substance P evidence for a modulatory role of substance P-antagonists.

Unregulated increasing of Tumor necrosis factor-alpha (TNF-alpha) could be pathogenic in inflammatory diseases. The aim of this study was to investigate the anti-inflammatory role of the Substance P-antagonists (SPAs) through the inhibition of histamine release (HR) and TNF-alpha production from mast cell. Rat peritoneal mast cells (PMC) stimulated with Substance P (SP), in the presence of SPAs or not, were analyzed for HR and TNF-alpha protein production. Competitive Polymerase Chain Reaction, with an internal standard competing with target cDNA for the same primers, was used to determine the TNF-alpha mRNA expression. We show that the increase of either HR and TNF-alpha levels in peritoneal (PMC) after induction with SP was inhibited by pre-incubation with SPA or with the Peptide 101 (P101), while the [D-Pro2, D-Phe7, D-Trp9]-SP (dSP) had no effect. Neuraminidase treatment suggests that dSP, as well as SP, interacts with sialic acid residues on the cell surface. Moreover, SPA and P101 also inhibit the release of histamine and TNF-alpha induced by dSP suggesting that a receptor-independent mechanism is involved. These data could be useful to better understand the mechanisms involved in the mast cell activation and TNF-alpha production in the inflammatory diseases where SP is involved.

Immunosuppressive effect of early pregnancy factor on early expression of cell surface membrane IgG.

We have adopted a new assay to investigate the influence of early pregnancy factor (EPF) on the modulation of lymphocyte activity. Lymphocytes were attached to the plastic surfaces of microplates in serum-free medium in the presence of Sepharose-Con A. After 2-3 days incubation with EPF, and ELISA assay was used to detect the expression of surface membrane IgG (smIgG); this was done in the same microplates used for the culture, thus avoiding cell manipulation. Using only a few picograms of EPF a significant inhibition (in the range 26-40%) was obtained. The variation in the inhibition observed was mainly due to the different sources of lymphocytes used. Unrelated proteins and hormones, tested at the same concentration as EPF, did not show any inhibitory activity. Using the F(ab)2 fragment of anti-human IgG instead of the whole molecule the same levels of inhibition were obtained, suggesting that the observed inhibition by EPF was not due to a non-specific interaction between the anti-human IgG and the Fc receptors on the cell. Such inhibitory activity detected in vitro by this method provides additional support for a suppressive role for EPF during pregnancy.

Identification of a histamine-releasing factor secreted by human pre-implantation embryos grown in vitro.

Human pre-implantation stage embryos cultured in vitro spontaneously secreted a factor capable of inducing histamine-release from human blood basophils. The embryo-derived histamine-releasing factor (EHRF) has been isolated from the culture medium by means of heparin-Sepharose affinity chromatography. The factor bound to the column and was then eluted by increasing the buffer molarity to 1.5 M NaCl. EHRF was detected using an enzymatic-isotopic microassay and sensitized basophils known to undergo release with anti-IgE. The EHRF-induced histamine-release was calcium and temperature dependent and the relatively slow kinetics (10 min) were similar to those obtained with anti-IgE. EHRF caused the release of a substantial amount of histamine (48%, n = 18) in a dose-dependent manner. The equivalent fraction isolated from medium containing unfertilized oocytes gave less than 10% of histamine-release using the same source of basophils, suggesting that EHRF was secreted after fertilization. EHRF was very stable since it was resistant to boiling, lyophilization, and to several freeze and thaw treatments. The histamine-releasing activity induced by EHRF was measured in vitro also by means of purified leukocytes containing sensitized basophils. EHRF could represent a message sent by the embryo to the mother to induce histamine release at the time of implantation.

Dispersal of rat uterine mast cells and their functional response to an embryo-derived histamine releasing factor: a possible model for embryo implantation.

Rat uterine tissue was dissociated by enzymatic digestion with collagenase and viable mast cells were obtained. Their viability was assessed by the ability to exclude trypan blue dye and to respond functionally to different stimuli. Challenge with anti-IgE gave a calcium-dependent histamine release of 49%, whilst the undigested uterine fragments gave 23%. Moreover, they were capable of releasing histamine on challenge with the compound 48/80, suggesting a similarity with connective tissue mast cells. This similarity was further supported by their insensitivity to aldehyde blocking of dye binding. The final dispersed cell preparation contained 3 X 10(5) mast cells/g of uterine tissue, representing about 2% of total nucleated cells. The total histamine content of the undigested uterus was 2.5 micrograms/g of tissue, whilst after digestion the histamine determined was 1.2 pg per mast cell with a yield of 14%. The total histamine content of the uterus changed throughout the reproductive cycle, increasing before ovulation, reaching a maximum during ovulation and then decreasing after embryo implantation. This suggests that the implanting embryo, interacting with the uterus, may be capable of inducing the release of histamine. The embryo-derived histamine releasing factor (EHRF) that we have described previously is capable of inducing 22% histamine-release on uterine mast cells, thus supporting this hypothesis.

Substance P selectively activates TNF-alpha mRNA in rat uterine immune cells: a neuroimmune link.

Substance P (SP) is a neuropeptide which influences the interaction between the nervous and immune systems. It is an important modulator of cytokines, including tumour necrosis factor-alpha (TNF-alpha) whose role during the reproductive processes has been established. We have investigated the effects of SP on TNF-alpha mRNA expression in macrophages and mast cells (MC) isolated from rat peritoneum and uterus. Cell supernatants were analysed for their histamine content as a measure of stimulation. SP alone increased TNF-alpha expression in peritoneal MC but not in peritoneal macrophages. The addition of SP resulted in a six-fold enhancement of TNF-alpha expression in uterine MC whereas no stimulation was observed in macrophages as determined by competitive polymerase chain reaction (PCR).

Evidence that brain mast cells can modulate neuroinflammatory responses by tumour necrosis factor-alpha production.

Tumour necrosis factor-alpha (TNF-alpha) levels in mammalian brain increase during neuroinflammatory diseases. We used the competitive polymerase chain reaction (PCR) to quantify the amount of TNF-alpha in stimulated and unstimulated brain mast cells (BMC). A cDNA fragment shortened by a deletion of 56 bp was used as an internal TNF-alpha-specific standard. The immunological stimulus resulted in enhanced TNF-alpha mRNA expression and increased release of histamine and TNF-alpha. This is the first time that BMC showing functional FCepsilonRI-bound IgE receptors have been purified. Our results support the hypothesis that BMC mediators might induce an initial response in neuroinflammatory diseases.

Histamine and tumor necrosis factor-alpha production from purified rat brain mast cells mediated by substance P.

The effect of cytokines and neuropeptides on neuroimmune functions has not been completely elucidated and recent evidence suggests an important role for these molecules linking the neuroimmune system and inflammatory events. The aim of this study was to analyse the effect of substance P (SP) on a pure population of hypothalamic brain mast cell (BMC). A pure population of BMC challenged with 10(-8) M SP gave 78% histamine release (HR) and secreted 600 pg/ml of tumor necrosis factor-alpha (TNF-alpha) as determined by ELISA. The production of TNF-alpha mRNA, measured by a competitive RT-PCR, was 14 times higher than that in unstimulated cells. The secretion of histamine and TNF-alpha from BMC after stimulation with SP supports the hypothesis that these mediators could induce an initial response in neuroinflammatory diseases.

Biochemical studies of a natural antioxidant isolated from rosemary and its application in cosmetic dermatology.

Oxidative damage to DNA, RNA, proteins and cell membranes occurs when the cellular concentration of reactive oxygen species exceeds the capacity of the cell to eliminate them. Aerobic prokaryotic and eukaryotic organisms have developed a set of cell defense systems to mitigate the damaging effects of reactive oxygen species. Epithelial surfaces contain antioxidants that could be expected to provide a defence against environmental stress caused by reactive oxygen and nitrogen species. Skin, which has a highly differentiated and complex structure, is particularly vulnerable to free radical damage because of its contact with oxygen and with other environmental stimuli. Fruit and vegetables contain several classes of compounds that when ingested can potentially contribute to endogenous modulation of antioxidant defences. The purpose of this study was to investigate the effectiveness of a natural extract derived from rosemary to protect free radical-induced skin damage. We provide evidence that an alcoholic extract of rosemary leaves, Rosm1, is endowed with strong antioxidant activity and, as evaluated by both in vitro and in vivo systems, is capable of inhibiting oxidative alterations to skin surface lipids. The present study provides a preclinical perspective on the interface between the biochemical properties of a natural extract isolated from rosemary leaves, a better understanding of the endogenous antioxidant potential of skin and the real validity of natural antioxidant biotechnology in antiaging skin management.

Regulation of heat shock protein synthesis in human skin fibroblasts in response to oxidative stress: role of vitamin E.

Skin plays an important role in protection against oxidative stressors such as ultraviolet radiation, ozone and chemicals. Chronic sun exposure causes degenerative changes in the skin that are recognized as photoaging. Oxidative stress has been shown to alter the expression of mammalian antioxidant enzymes as well as to enhance numerous transcription factors, including nuclear factor kappaB, stress-activated protein kinase and heat shock factor This latter is the transcription factor for the synthesis of heat shock proteins, which have been known to protect against a wide variety of toxic conditions, including extreme temperatures, oxidative stress and cytotoxic drugs. In this study we investigated the role of oxidative stress in the induction of heat shock protein (HSP) 70 in human skin fibroblasts and the effect of vitamin E. We found that significant HSP70 induction occurred after exposure to HOOH and that this was associated with a significant perturbation in protein and nonprotein sulfhydryl groups, and with a significant increase in protein carbonyl levels. Treatment with vitamin E conferred significant protection against stress-induced modifications of cellular sulfhydryl and carbonyl content, while maintaining functional levels of cytoprotective HSP70. Our results point to the possible involvement of redox mechanisms in the heat shock signal transduction pathway, which may play an important regulatory role in the genetic mechanisms of tolerance to oxidative stress. Exogenous antioxidant supplementation with vitamin E could have cosmetic benefits and may be an efficient tool to mitigate the consequences of free radical-induced skin damage.

Successful control of an outbreak of colonization by Klebsiella pneumoniae carbapenemase-producing K. pneumoniae sequence type 258 in a neonatal intensive care unit, Italy.

This article reports an outbreak of colonization by Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-Kp) sequence type (ST) 258 in a neonatal intensive care unit (NICU) in Palermo, Italy. KPC-Kp ST258 was detected by an active surveillance culture programme. Between 18th September and 14th November 2012, KPC-Kp was isolated from 10 out of 54 neonates admitted in the outbreak period. No cases of infection were recorded. Male sex was associated with colonization, whereas administration of ampicillin- sulbactam plus gentamicin was protective. Infection control interventions interrupted the spread of KPC-Kp without the need to close the NICU to new admissions.