The endogenous oxytocin after manipulative osteopathic treatment in full-term pregnant women.

OBJECTIVE: The aim of this study is to assess whether the touch of osteopathic manipulative treatment (OMT) can affect the endogenous production of oxytocin in full-term pregnant women and the assessment of well-being following the treatment. PATIENTS AND METHODS: In this study have been enrolled 57 pregnant women at full-term pregnancy (37th-41st week) for evaluation of the concentration of salivary oxytocin 2 minutes before and 2 minutes after a single session of OMT by an osteopath lasting for 30 minutes. Pre-OMT and post-OMT saliva samples were collected with the use of Salivette® salivary swabs. 7 salivary swabs were excluded from the analysis. 50 samples were analyzed with an appropriate ELISA kit. RESULTS: The mean OT salivary concentration pre-OMT was 89.98±16.39, and post-OMT was 100.60±19.13 tends to increase with p=0.0000051. In multivariate analysis, two subgroups show interesting data in the mean difference in OT salivary concentration post-OMT: women with painful contractions (p=0.06) and women under 35 years (p=0.09). CONCLUSIONS: The results of this study demonstrate that the effectiveness of OMT-increasing endogenous oxytocin is statistically significant in full-term pregnant women. The sensation of well-being found in most women indicates that there has been a predominantly central rather than peripheral oxytocin release after OMT.

Molecular characterisation and clonal analysis of group A streptococci causing pharyngitis among paediatric patients in Palermo, Italy.

Group A streptococci (n = 123), isolated consecutively from paediatric patients with pharyngitis from Palermo, Italy, were analysed. The emm and sof genes were sequenced, the presence of the speA and speC genes was investigated, and the macrolide resistance phenotypes and genotypes were determined. A limited number of emm/sof genotypes was found, and the most prevalent types were different from those found in a previous study from Rome. Macrolide resistance was found in the most prevalent clones, suggesting that the spread of mobile antibiotic resistance genes among the fittest clones in the community was the main mechanism influencing macrolide resistance rates in different emm types.

Colorectal carcinomas and PTEN/MMAC1 gene mutations.

PURPOSE: PTEN/MMAC1/TEP1 is a tumor suppressor gene encoding a dual-specificity protein phosphatase with homology to the cytoskeleton proteins, chicken tensin and bovine auxilin. PTEN mutations have been described in several types of human cancer. Recently, mutations at an (A)(6) repeat of PTEN exons 7 and 8 in colorectal cancer (CRC) patients with microsatellite instability have been detected. Moreover, an involvement of the transforming growth factor (TGF)-beta pathway in hereditary colorectal syndromes has been proposed. EXPERIMENTAL DESIGN: In this study, we analyzed the frequency of PTEN gene mutations in 36 CRC patients and 5 colon cancer cell lines. Furthermore, in 16 of 36 patients, microsatellite instability and TGF-beta receptor II analysis was possible. The study was performed by PCR and automated sequencing of the entire coding region of the PTEN gene. RESULTS: About 17% of colon cancer patients and one of five (HSR 320) colon cancer cell lines had mutations. Mutations were detected only among patients with locally advanced or metastatic CRC. PTEN mutations were detected in three of five (60%) patients showing both microsatellite instability and TGF-beta receptor II mutations. These patients presented with advanced or metastatic CRC CONCLUSIONS: Overall, these results show that PTEN alteration together with TGF-beta pathway inactivation could contribute to tumorigenesis and metastatic spread of sporadic and microsatellite unstable CRC.

Group A streptococcal genotypes from pediatric throat isolates in Rome, Italy.

In a study assessing genetic diversity, 114 group A streptococcus (GAS) isolates were recovered from pediatric pharyngitis patients in Rome, Italy. These isolates comprised 22 different M protein gene (emm) sequence types, 14 of which were associated with a distinct serum opacity factor/fibronectin binding protein gene (sof) sequence type. Isolates with the same emm gene sequence type generally shared a highly conserved chromosomal macrorestriction profile. In three instances, isolates with dissimilar macrorestriction profiles had identical emm types; in each of these cases multilocus sequence typing revealed that isolates with the same emm type were clones having the same allelic profiles. Ninety-eight percent of the pharyngeal isolates had emm types previously found to be highly associated with mga locus gene patterns commonly found in pharyngeal GAS isolates.

Stimulation of p38 MAP kinase reduces acidosis and Na(+) overload in preconditioned hepatocytes.

Ischemic preconditioning has been shown to improve liver resistance to hypoxia/reperfusion damage. A signal pathway involving A(2A)-adenosine receptor, G(i)-proteins, protein kinase C and p38 MAP kinase is responsible for the development of hypoxic preconditioning in hepatocytes. However, the coupling of this signal pathway with the mechanisms responsible for cytoprotection is still unknown. We have observed that stimulation of A(2A)-adenosine receptors or of p38 MAPK by CGS21680 or anisomycin, respectively, appreciably reduced intracellular acidosis and Na(+) accumulation developing during hypoxia. These effects were reverted by p38 MAPK inhibitor SB203580 as well as by blocking vacuolar proton ATPase with bafilomycin A(1). SB203580 and bafilomycin A(1) also abolished the cytoprotective action exerted by both CGS21680 and anisomycin. We propose that the stimulation of p38 MAPK by preconditioning might increase hepatocyte resistance to hypoxia by activating proton extrusion through vacuolar proton ATPase, thus limiting Na(+) overload promoted by Na(+)-dependent acid buffering systems.

Signal pathway involved in the development of hypoxic preconditioning in rat hepatocytes.

Ischemic preconditioning improves liver resistance to hypoxia and reduces reperfusion injury following transplantation. However, the intracellular signals that mediate the development of liver hypoxic preconditioning are largely unknown. We have investigated the signal pathway leading to preconditioning in freshly isolated rat hepatocytes. Hepatocytes were preconditioned by 10-minute incubation under hypoxic conditions followed by 10 minutes of reoxygenation and subsequently exposed to 90 minutes of hypoxia. Preconditioning reduced hepatocyte killing by hypoxia by about 35%. A similar protection was also obtained by preincubation with chloro-adenosine or with A(2A)-adenosine receptor agonist CGS21680, whereas A(1)-adenosine receptor agonist N-phenyl-isopropyladenosine (R-PIA) was inactive. Conversely, the development of preconditioning was blocked by A(2)-receptor antagonist 3,7-dimethyl-1-propargylxanthine (DMPX), but not by A(1)-receptor antagonist 8-cyclopenthyl-1, 3-dipropylxanthine (DPCPX). In either preconditioned or CGS21680-treated hepatocytes a selective activation of delta and epsilon protein kinase C (PKC) isoforms was also evident. Inhibition of heterotrimeric G(i) protein or of phospholypase C by, respectively, pertussis toxin or U73122, prevented PKC activation as well as the development of preconditioning. MEK inhibitor PD98509 did not interfere with preconditioning that was instead blocked by p38 MAP kinase inhibitor SB203580. The direct activation of p38 MAPK by anisomycin A mimicked the protection against hypoxic injury given by preconditioning. Consistently, an increased phosphorylation of p38 MAPK was observed in preconditioned or CGS21680-treated hepatocytes, and this effect was abolished by PKC-blocker, chelerythrine. We propose that a signal pathway involving A(2A)-adenosine receptors, G(i)-proteins, phospholypase C, delta- and epsilon-PKCs, and p38 MAPK, is responsible for the development of liver ischemic preconditioning.

Ethanol potentiates hypoxic liver injury: role of hepatocyte Na(+) overload.

Centrilobular hypoxia has been suggested to contribute to hepatic damage caused by alcohol intoxication. However, the mechanisms involved are still poorly understood. We have investigated whether alterations of Na(+) homeostasis might account for ethanol-mediated increase in hepatocyte sensitivity to hypoxia. Addition of ethanol (100 mmol/l) to isolated rat hepatocytes incubated under nitrogen atmosphere greatly stimulated cell death. An increase in intracellular Na(+) levels preceded cell killing and Na(+) levels in hepatocytes exposed to the combination of ethanol and hypoxia were almost twice those in hypoxic cells without ethanol. Na(+) increase was also observed in hepatocytes incubated with ethanol in oxygenated buffer. Ethanol addition significantly lowered hepatocyte pH. Inhibiting ethanol and acetaldehyde oxidation with, respectively, 4-methylpyrazole and cyanamide prevented this effect. 4-methylpyrazole, cyanamide as well as hepatocyte incubation in a HCO(3)(-)-free buffer or in the presence of Na(+)/H(+) exchanger blocker 5-(N,N-dimethyl)-amiloride also reduced Na(+) influx in ethanol-treated hepatocytes. 4-methylpyrazole and cyanamide similarly prevented ethanol-stimulated Na(+) accumulation and hepatocyte killing during hypoxia. Moreover, ethanol-induced Na(+) influx caused cytotoxicity in hepatocytes pre-treated with Na(+), K(+)-ATPase inhibitor ouabain. Also in this condition 4-methylpyrazole and 5-(N,N-dimethyl)-amiloride decreased cell killing. These results indicate that ethanol can promotes cytotoxicity in hypoxic hepatocytes by enhancing Na(+) accumulation.

Diagnostic value of cytokine assays in cerebrospinal fluid in culture-negative, polymerase chain reaction-positive bacterial meningitis.

Analysis of bacterial DNA using a polymerase chain reaction performed with broad-range eubacterial 16S rDNA primers may yield a diagnosis of bacterial meningitis in cases where Gram staining of cerebrospinal fluid (CFS), antigen detection techniques or culture fail. Since this PCR technique occasionally gives false-positive results due to contamination of samples or laboratory reagents, a study was performed to establish the diagnostic value of assaying concentrations of tumour necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) in 90 CSF samples. A high correlation was found between a positive PCR result and the concentrations of TNF-alpha and IL-10, indicating that cytokine assays may be used as a confirmatory test. The findings suggested that a combination of the PCR technique, amplicon sequencing and assay of TNF-alpha and IL-10 concentrations in CSF is a reliable and cost-effective procedure for diagnosis of culture-negative bacterial meningitis.

Alterations of Na(+) homeostasis in hepatocyte reoxygenation injury.

Reperfusion injury represents an important cause of primary graft non-function during liver transplantation. However, the mechanism responsible for cellular damage during reoxygenation has not yet been completely understood. We have investigated whether changes in intracellular Na(+) distribution might contribute to cause hepatocyte damage during reoxygenation buffer after 24 h of cold storage. Hepatocyte reoxygenation resulted in a rapid increase in cellular Na(+) content that was associated with cytotoxicity. Na(+) accumulation and hepatocyte death were prevented by the omission of Na(+) from the incubation medium, but not by the addition of antioxidants. Blocking Na(+)/H(+) exchanger and Na(+)/HCO(3)(-) co-transporter by, respectively, 5-(N,N-dimethyl)-amiloride or omitting HCO(3)(-) from the reoxygenation medium significantly decreased Na(+) overload and cytotoxicity. Stimulation of ATP re-synthesis by the addition of fructose also lowered Na(+) accumulation and cell death during reoxygenation. A significant protection against Na(+)-mediated reoxygenation injury was evident in hepatocytes maintained in an acidic buffer (pH 6.5) or in the presence of glycine. The cytoprotective action of glycine or of the acidic buffer was reverted by promoting Na(+) influx with the Na(+)/H(+) ionophore monensin. Altogether, these results suggest that Na(+) accumulation during the early phases of reoxygenation might contribute to liver graft reperfusion injury.

Ischemic preconditioning reduces Na(+) accumulation and cell killing in isolated rat hepatocytes exposed to hypoxia.

Short periods of ischemia followed up by reperfusion are known to protect the heart against injury caused by a subsequent sustained ischemia. This phenomenon, known as ischemic preconditioning, has also been recently shown to reduce ischemic liver damage, but the mechanisms involved are still unknown. By using isolated hepatocytes as an in vitro model of liver preconditioning, we have investigated the possible effect of preconditioning on intracellular pH and Na(+) homeostasis. Freshly isolated rat hepatocytes were preconditioned by 10 minutes of incubation under hypoxic conditions followed up by 10 minutes of reoxygenation and subsequently exposed to 90 minutes of hypoxia. Although preconditioning did not ameliorate adenosine triphosphate (ATP) depletion, preconditioned hepatocytes exhibited an increased resistance to cell killing during hypoxic incubation. Intracellular acidosis and Na(+) accumulation developing during hypoxia were appreciably reduced in preconditioned cells. The effects of preconditioning on intracellular pH, Na(+) homeostasis, and cytotoxicity were mimicked by stimulating protein kinase C (PKC) with 4beta-phorbol-12-myristate-13-acetate (PMA) or 1,2 dioctanoyl-glycerol (1,2 DOG). Conversely, inhibiting PKC with chelerythrine or blocking vacuolar proton ATPase (V-ATPase) with bafilomycin A(1) abolished the protection given by preconditioning or by PMA treatment on hypoxic acidosis, Na(+) overload, and hepatocyte killing. Similarly, the addition of Na(+) ionophore monensin also reverted the cytoprotection exerted by preconditioning. This indicated that ischemic preconditioning of isolated hepatocytes decreased cell killing during hypoxia by preventing intracellular Na(+) accumulation. We propose that, after preconditioning, the stimulation of PKC might activate proton extrusion through V-ATPase, thus, limiting intracellular acidosis and Na(+) overload promoted by Na(+)-dependent acid buffering systems.

Intracellular Na+ accumulation and hepatocyte injury during cold storage.

BACKGROUND: The mechanisms responsible for liver damage during cold storage are still not completely understood. We have investigated the role played by alterations of Na+ homeostasis in cell injury during cold hypoxia. METHODS: The changes in Na+ distribution were investigated in isolated rat hepatocytes stored at 4 degrees C under hypoxic conditions. RESULTS: Hepatocyte cold stored up to 72 hr in Krebs-Henseleit-Hepes buffer showed a progressive increase in intracellular Na+ content that preceded the loss of cell viability. Na+ accumulation and cell death were prevented using Na+-free, acidic (pH 6.5) or glycine-supplemented storage media. The Na+ ionophore monensin reverted the cytoprotection exerted by glycine and by the acidic medium, but not that given by Na+-free Krebs-Henseleit-Hepes. A low Na+ content was also important for the cytoprotection observed using University of Wisconsin solution. CONCLUSIONS: Na+ overload might contribute to liver graft injury occurring during cold storage.

Glycine protects against hepatocyte killing by KCN or hypoxia by preventing intracellular Na+ overload in the rat.

Glycine has been shown to prevent hepatocyte death induced by anoxia and by several toxic agents. However, the mechanisms responsible for such a cytoprotective effect have not yet been entirely clarified. We have previously shown that an uncontrolled increase in intracellular Na+ is critical for hepatocyte killing induced by adenosine triphosphate (ATP) depletion. We herein report that protection by glycine (2 mmol/L) against cytotoxicity induced in isolated rat hepatocyte by potassium cyanide (KCN) or hypoxia was associated with the prevention of cytosolic Na+ accumulation. The addition of the Na+ ionophore, monensin, abolished the effects of glycine on both Na+ increase and cytotoxicity. Pretreating hepatocytes with the glycine-receptor antagonist, strychnine (1 mmol/L), similarly prevented Na+ overload and cell killing. Glycine at high concentrations and strychnine are known to block Cl- channels in many cell types. Consistently, we have observed that glycine and strychnine prevented the increase of intracellular Cl- levels caused by hypoxia or KCN. Incubation of hepatocytes in a Cl(-)-free medium, obtained by substituting chloride with membrane-impermeable gluconate, significantly reduced Na+ accumulation and cell killing triggered by hypoxia or KCN. Both these effects were abolished by the addition of monensin. The cytoprotective action exerted by hepatocyte incubation in the Cl(-)-free medium was, however, lost when membrane-permeable nitrate, which allowed Na+ accumulation, was used instead to replace chloride. Altogether, these results indicate that glycine inhibition of Cl- conductance protects against hepatocyte killing induced by KCN and hypoxia by interfering with intracellular Na+ accumulation triggered by ATP depletion.

Role of Na+/Ca2+ exchanger in preventing Na+ overload and hepatocyte injury: opposite effects of extracellular and intracellular Ca2+ chelation.

We have previously shown that an increase of intracellular Na+ occurs in isolated rat hepatocytes undergoing ATP depletion and that Na+ accumulation is associated with an uncontrolled influx of Ca2+ through the activation in reverse mode of the Na+/Ca2+ exchanger. In the present study we have investigated the relationship between alterations of Na+ and Ca2+ homeostasis and hepatocyte killing using treatments which differentially chelate extracellular or intracellular Ca2+. Chelation of extracellular Ca2+ by ethylene glycol bis-(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) potentiated Na+ overload and cell killing induced in isolated rat hepatocytes by hypoxia or menadione. Similar effects were also observed when Na+ accumulation was induced by the combined addition of Na+ ionophore monensin and the inhibition of plasma membrane Na+/K+ ATPase by ouabain. Conversely, the use of the intracellular Ca2+ chelator EGTA acetoxymethyl ester (EGTA/AM) reduced Na+ overload and hepatocyte death induced by hypoxia or cell treatment with menadione or monensin plus ouabain. The effects of EGTA/AM were reverted in the presence of bepridil, an inhibitor of Na+/Ca2+ exchanger. Altogether these results indicated that differential chelation of intracellular or extracellular Ca2+ influences in opposite ways hepatocyte killing due to ATP depletion by modulating intracellular Na+ levels through the reversed activity of the Na+/Ca2+ exchanger.