Measuring and benchmarking the quality of two different organizational ways in delivering infant vaccination.

The aim of this study was the quality of service evaluation of two different organizational ways in delivering infant vaccination according to a Regional Vaccination Plan. Eleven vaccination centres were selected in two Local Health Units (ASLs) belonging to the Regional Health Service of the Lazio Region, Italy. The services offering paediatric vaccinations for children under three years of age, delivered without an appointment (VACP) or with the need for an appointment (VACL), were investigated. The quality aspects under evaluation were communicational efficiency, organisational efficiency and comfort. Subjective data were collected from different stakeholders and involve the elicitation of best and worst feasible performance conditions for the ASLs when delivering VACP/VACL services. Objective data consists in the observation of current performances of the selected vaccination centres. Quality scorecards were obtained from the combination of all data. Benchmarking between VACP and VACL, i.e., two different organisational ways in delivering infant vaccination, can be performed as a result of the probabilistic meaning of the evaluated scores. An expert of vaccination services, i.e., a virtual combination of patients, doctors and nurses, claims the quality of service delivery of the ASLs under investigation with probability 78.03% and 69.67% for VACP and VACL, respectively. In other words, for short, the quality scores of the ASLs were 78.03% for VACP and 69.67% for VACL. Furthermore our results show how to practically improve the current service delivery. The QuaVaTAR approach can result in improvements of the quality of the ASLs for the two different ways of delivering paediatric vaccinations in a simple and intuitive way.

pH-sensitive non-phospholipid vesicle and macrophage-like cells: binding, uptake and endocytotic pathway.

Phospholipid and non-phospholipid vesicles are extensively studied as drug delivery systems to modify pharmacokinetics of drugs and to improve their action in target cells. It is believed that the major barrier to efficient drug delivery is entrapment of drugs in the endosomal compartment, since this eventually leads to its degradation in lysosomes. For these reasons, the knowledge of internalization pathway plays a fundamental role in optimizing drug targeting. The aim of this work is to characterize pH-sensitive Tween 20 vesicles, their interaction with macrophage-like cells and their comparison with pH-sensitive liposomes. The effect of different amounts of cholesteryl hemissucinate on surfactant vesicle formation and pH-sensitivity was studied. To evaluate the initial mode of internalization in Raw 264.7 and the intracellular fate of neutral and pH-sensitive formulations, flow cytometry in presence and in absence of selected inhibitors and fluorescence microscopy in absence and presence of specific fluorescent endocytotic markers were used. The obtained results showed that the surfactant vesicle pH-sensitivity was about two or three fold higher than that obtained with pH-sensitive liposomes in the presence of serum in vitro. The uptake mechanism of surfactant vesicles, after incubation with macrophage-like cells, is comparable to that of liposomes (clathrin-mediated endocytosis).

Glycosyl and polyalcoholic prodrugs of lonidamine.

Polyhydric alcohol derivatives of the anticancer agent lonidamine (LND) have been synthesized. The increased water solubility showed by prodrugs 4, 7, and 25 together with their logP values (2.19, 2.55, and 2.54, respectively) and chemical stability might be beneficial for prodrugs absorption after oral administration. Moreover, the new prodrugs undergo enzymatic hydrolysis in plasma and release LND demonstrating that they are promising candidates for in vivo investigations.

Increase of skin-ceramide levels in aged subjects following a short-term topical application of bacterial sphingomyelinase from Streptococcus thermophilus.

Several studies have demonstrated that ceramides play an essential role in both the barrier and water-holding functions of healthy stratum corneum, suggesting that the dysfunction of the stratum corneum associated with ageing as well that observed in patients with several skin diseases could result from a ceramide deficiency. In a previous study our group reported a significant increase in skin ceramide levels in healthy subjects after treatment in vivo with a cream containing a preparation of Streptococcus thermophilus. The presence of high levels of neutral sphingomyelinase activity in this organism was responsible for the observed increase of stratum corneum ceramide levels, thus leading to an improvement in barrier function and maintenance of stratum corneum flexibility. The aim of the present work is to investigate the effects of the topical treatment of a Streptococcus thermophilus-containing cream on ceramide levels of stratum corneum of healthy elderly women. The ceramide levels, transepidermal water loss and capacitance were evaluated on stratum corneum sheets from the forearms of 20 healthy female subjects treated with a base cream or the same cream containing a sonicated preparation of the lactic acid bacterium Streptococcus thermophilus. A 2-week topical application of a sonicated Streptococcus thermophilus preparation led to significant and relevant increase of stratum corneum ceramide levels. Moreover, the hydration values of the treated forearm of each subject was significantly higher than control sites. These results suggest that the experimental cream was able to improve the lipid barrier and to increase a resistance against ageing-associated xerosis.

Involvement of cPLA2 inhibition in dexamethasone-induced thymocyte apoptosis.

Various molecular mechanisms have been suggested to be involved in dexamethasone induced thymocyte apoptosis. In this study we show that pharmacological inhibition of cytoplasmic PLA2 in mouse thymocytes for 18 h with arachidonyl trifluoromethyl ketone (AACOCF3) (10 microM) and palmitoyl trifluoromethyl ketone (PACOCF3) (10 microM) induced a drastic increase of thymocyte apoptosis comparable to that observed following Dex (10(-7) M) treatment, while inhibition of secretory PLA2 with p-bromophenacyl bromide (pBPB) (20 microM) did not. AACOCF3-induced thymocyte apoptosis, similarly to Dex-induced thymocyte apoptosis, was eliminated by cell pre-treatment with the PI-PLCbeta inhibitor, U73122, but not by the PC-PLC inhibitor D609. These observations were corroborated by the ability of AACOCF3, like Dex, to induce a rapid and transient increase in DAG generation. In addition, AACOCF3-induced apoptosis involved the activation of the acidic sphingomyelinase (aSMase) but not of the neutral sphingomyelinase (nSMase), as evaluated by measurements of enzyme activity in cell extracts following thymocyte exposure to AACOCF3 and by the ability of monensin to inhibit AACOCF3-induced thymocyte apoptosis. In addition, the AACOCF3 apoptotic effect resulted in an early increase of ceramide levels. AACOCF3-induced thymocyte apoptosis involved the activation of caspase 3, and cell pre-treatment with a caspase 3 inhibitor prevented AACOCF3-induced apoptosis. These observations suggest that cPLA2 inhibition may have a role in Dex-induced thymocyte apoptosis and highlight the importance of cPLA2 activity in thymocyte survival.

VSL#3 probiotic upregulates intestinal mucosal alkaline sphingomyelinase and reduces inflammation.

BACKGROUND: Alkaline sphingomyelinase, an enzyme found exclusively in bile and the intestinal brush border, hydrolyzes sphingomyelin into ceramide, sphingosine and sphingosine-1-phosphate, thereby inducing epithelial apoptosis. Reduced levels of alkaline sphingomyelinase have been found in premalignant and malignant intestinal epithelia and in ulcerative colitis tissue. Probiotic bacteria can be a source of sphingomyelinase. OBJECTIVE: To determine the effect of VSL#3 probiotic therapy on mucosal levels of alkaline sphingomyelinase, both in a mouse model of colitis and in patients with ulcerative colitis. METHODS: Interleukin-10 gene-deficient (IL10KO) and wild type control mice were treated with VSL#3 (10(9) colony-forming units per day) for three weeks, after which alkaline sphingomyelinase activity was measured in ileal and colonic tissue. As well, 15 patients with ulcerative colitis were treated with VSL#3 (900 billion bacteria two times per day for five weeks). Alkaline sphingomyelinase activity was measured through biopsies and comparison of ulcerative colitis disease activity index scores obtained before and after treatment. RESULTS: Lowered alkaline sphingomyelinase levels were seen in the colon (P=0.02) and ileum (P=0.04) of IL10KO mice, as compared with controls. Treatment of these mice with VSL#3 resulted in upregulation of mucosal alkaline sphingomyelinase activity in both the colon (P=0.04) and the ileum (P=0.01). VSL#3 treatment of human patients who had ulcerative colitis decreased mean (+/- SEM) ulcerative colitis disease activity index scores from 5.3+/-1.8946 to 0.70+/-0.34 (P=0.02) and increased mucosal alkaline sphingomyelinase activity. CONCLUSION: Mucosal alkaline sphingomyelinase activity is reduced in the intestine of IL10KO mice with colitis and in humans with ulcerative colitis. VSL#3 probiotic therapy upregulates mucosal alkaline sphingomyelinase activity.

Simultaneous quantification of Gemcitabine and Irinotecan hydrochloride in rat plasma by using high performance liquid chromatography-diode array detector.

In this manuscript we aimed at the simultaneous separation and quantification of Gemcitabine and Irinotecan hydrochloride (injected both as single components and in combination) from Sprague Dawley rat plasma by using a validated method obtained through the use of a High Performance Liquid Chromatography (HPLC)-diode array detector (DAD). Gemcitabine and Irinotecan hydrochloride were detected and quantified using a Zorbax Extend C-18 column (250 mm × 4.6 mm; 5 µm particle size) in gradient elution mode. The chromatographic analyses were carried out in 15 min. The analytical mode was calibrated and validated in the concentration range from 0.1 to 18 µg/mL both for Gemcitabine and Irinotecan hydrochloride. Sprague Dawley rat plasma was used to perform the analysis. 3-methylxanthine was the internal standard. The weighted-matrix matched standard curves of Gemcitabine and Irinotecan hydrochloride showed a good linearity up to 18 µg/mL. Parallelism tests were also performed to evaluate whether the over-range samples could be analyzed after dilution without affecting the analytical performance. The intra- and inter-day precision (RSD%) values of Gemcitabine and Irinotecan hydrochloride were ≤7.14% and ≤11.5%, respectively. The intra- and inter-day trueness (Bias%) values were in the range from -11.5% to 1.70% for both drugs. The analytical mode performance was further tested after collecting Sprague Dawley rat plasma following a single-dose administration of chemotherapeutics or their association. The validated HPLC-DAD method allowed the simultaneous quantification of Gemcitabine and Irinotecan hydrochloride in the rat plasma, besides the evaluation of the pharmacokinetic parameters and drug delivery.

Effect of Bifidobacterium infantis on Interferon- gamma- induced keratinocyte apoptosis: a potential therapeutic approach to skin immune abnormalities.

Current management of atopic dermatitis is mainly directed to the reduction of cutaneous inflammation. Since patients with atopic dermatitis show abnormalities in immunoregulation, a therapy aimed to adjust their immune function could represent an alternative approach, particularly in the severe form of the disease. Indeed, T-lymphocytes constitute a large population of cellular infiltrate in atopic/allergic inflammation and a dysregulated T-cell induced keratinocyte apoptosis appears to be an important pathogenetic factor of the eczematous disease. In recent years, attention has been focused on the interaction between host and probiotics which may have anti-inflammatory properties and immunomodulatory activities. The aim of the present work is to investigate the effect of a selected probiotic extract, the Bifidobacterium infantis extract, on a human keratinocyte cell line (HaCaT) abnormal apoptosis induced by activated-T-lymphocyte. An in vitro model of atopic dermatitis was used to assess the ability of the probiotic extract to protect HaCaT from apoptosis induced by soluble factors (IFN-gamma and CD95 ligand) released by human T-lymphocytes in vitro activated with anti-CD3/CD28 mAbs or Phytohemoagglutinin. Evidence is given that the bacterial extract treatment was able to totally prevent T lymphocyte-induced HaCaT cell apoptosis in vitro. The mechanism underlying this inhibitory effect has been suggested to depend on the ability of the bacterial extract to significantly reduce anti-CD3/CD28 mAbs and mitogen-induced T-cell proliferation, IFN-gamma generation and CD95 ligand release. These preliminary results may represent an experimental basis for a potential therapeutic approach mainly targeting the skin disorders-associated immune abnormalities.

Designing novel pH-sensitive non-phospholipid vesicle: characterization and cell interaction.

In this work, we report the preparation, the characterization and interaction with cells of novel pH-sensitive non-phospholipid vesicle formulations, from a non-ionic surfactant mixed with cholesterol (CHOL) and his derivative cholesteryl hemisuccinate (CHEMS), as pH-sensitive molecule. This molecule, can destabilize the vesicle lipid bilayer when exposed to an acidic environment, with a subsequent release of vesicular content, enhancing the cytoplasmatic delivery of drugs to target cells. Vesicles were characterized by static and dynamic light scattering, in order to evaluate their dimensions, bilayer thickness and vesicle stability. Membrane permeability changes were determined by the release of entrapped hydroxypyrene-1,3,6-trisulfonic acid (HPTS). Also diphenylhesatriene (DPH) fluorescence anisotropy and zeta potential measurements were used to evidence the pH sensitivity. Furthermore vesicles were characterized by means of electronic microscopy after freeze-fracture. The interaction of non-lipid vesicles containing different fluorescent dyes with Raw 264.7, mouse monocite macrophage, were analyzed by flow cytometric analysis. The obtained results indicate that the pH-sensitive vesicular structures show good plasma stability and relevant pH-sensitivity. Moreover this formulation was able to interact with target membranes (i.e. plasma or endosomal membrane) and to release the encapsulated material into the cytoplasm.

Autoamplification of apoptosis following ligation of CD95-L, TRAIL and TNF-alpha.

CD95-L, TNF-alpha and TRAIL are death-inducing ligands (DILs) which may signal apoptosis via crosslinking of their cognate receptors. The present study shows that treatment of cells with agonistic mAB alpha APO-1 (CD95), recombinant TRAIL or TNF-alpha leads to enhanced mRNA and protein expression of each DIL with concomitant death in target cells. Immunoprecipitation of CD95-L protein from supernatant as well as neutralizing antibodies suggest DIL proteins to be cooperatively acting mediators of these cytotoxic activity. Autoamplification of the death signal was blocked in cells with a defect in apoptosis signaling either due to a dysfunctional FADD molecule or to the failure to activate JNK/SAPKs. Phosphorylation and enhanced binding of cJun and ATF-2 to DIL promoters suggest JNK/SAPKs as activators of these transcription factors following death receptor triggering. In consequence, autocrine production of DILs allows the spread of death signals to sensitive target cells. Oncogene (2000) 19, 4255 - 4262

Gel-embedded niosomes: preparation, characterization and release studies of a new system for topical drug delivery.

In the present paper physical gels, prepared with two polysaccharides, Xanthan and Locust Bean Gum, and loaded with non-ionic surfactant vesicles, are described. The vesicles, composed by Tween20 and cholesterol or by Tween85 and Span20, were loaded with Monoammonium glycyrrhizinate for release experiments. Size and zeta (ζ)-potential of the vesicles were evaluated and the new systems were characterized by rheological and dynamo-mechanical measurements. For an appropriate comparison, a Carbopol gel and a commercial gel for topical applications were also tested. The new formulations showed mechanical properties comparable with those of the commercial product indicating their suitability for topical applications. In vitro release experiments showed that the polysaccharide network protects the integrity of the vesicles and leads to their slow release without disruption of the aggregated structures. Furthermore, being the vesicles composed of molecules possessing enhancing properties, the permeation of the loaded drugs topically delivered can be improved. Thus, the new systems combine the advantages of matrices for a modified release (polymeric component) and those of an easier permeability across the skin (vesicle components). Finally, shelf live experiments indicated that the tested gel/vesicle formulations were stable over 1 year with no need of preservatives.

Effect of the lactic acid bacterium Streptococcus thermophilus on ceramide levels in human keratinocytes in vitro and stratum corneum in vivo.

The effects of Streptococcus thermophilus on ceramide levels either in vitro on cultured human keratinocytes or in vivo on stratum corneum, have been investigated. In vitro, Streptococcus thermophilus enhanced the levels of ceramides in keratinocytes in a time-dependent way. The presence of high levels of neutral, glutathione-sensitive, sphingomyelinase in Streptococcus thermophilus could be responsible for the observed ceramide increase. The application of a base cream containing sonicated Streptococcus thermophilus in the forearm skin of 17 healthy volunteers for 7 d also led to a significant and relevant increase of skin ceramide amounts, which could be due to the sphingomyelin hydrolysis through bacterial neutral sphingomyelinase. Indeed, similar results were obtained with a base cream containing purified bacterial neutral sphingomyelinase. In addition, the inhibition of bacterial neutral sphingomyelinase activity through glutathione blocked the skin ceramide increase observed after the treatment. The topical application of a sonicated Streptococcus thermophilus preparation, leading to increased stratum corneum ceramide levels, could thus result in the improvement of lipid barrier and a more effective resistance against xerosis.

Do inert beta-lactamase inhibitors act as synergizers of beta-lactam antibiotics? Utility of boric and boronic acids.

Boric and boronic acids were used as inhibitors of beta-lactamases produced by two Citrobacter diversus strains and by one strain of Pseudomonas aeruginosa; all strains were clinical isolates. The beta-lactamases produced by the two Citrobacter diversus strains were inhibited by both borates and boronates, using cephazolin as substrate. The enzyme from Pseudomonas aeruginosa was inhibited only by boronates, using benzylpenicillin as substrate. These inhibitors were also used in combination with selected beta-lactams so as to determine if a synergism of antimicrobial activity occurred. All data reported in the present paper indicate that the minimum inhibitory concentration (MIC) values were lowered in the presence of these inhibitors for the two Citrobacter diversus strains. In the Pseudomonas aeruginosa strains the MIC values were not significantly altered, thus indicating the presence of a permeability barrier for 3-aminophenylboronic acid.

Inhibitors and inactivators of beta-lactamase from Mycobacterium fortuitum.

The inhibiting or inactivating effects of some beta-lactam antibiotics on beta-lactamase from Mycobacterium fortuitum were studied. Among all substrates tested, clavulanic acid and sulbactam were the strongest competitive inhibitors of the enzyme although the latter was slightly hydrolyzed. Imipenem and cefoxitin scarcely inhibited the beta-lactamase yet expressed good activity against the microorganism in vitro, suggesting that the effectiveness of these drugs on M. fortuitum might be due to high permeation through the cell wall. All the isoxazolylpenicillins tested and methicillin inactivated the enzyme of M. fortuitum by a first rapid phase of acylation followed by a steady-state process of enzyme reactivation (deacylation). Clavulanic acid and sulbactam showed Ki values for the enzyme inactivation closely corresponding to hematic concentrations achievable in vivo during antibiotic treatment.

Alcohol consumption and blood pressure. An Italian study.

We studied the relationship between alcohol consumption and arterial pressure in 1,190 subjects of both sexes aged between 18 and 63 years who were examined during the course of a program of preventive medicine organized by Centro Diagnostico Italiano. In 711 subjects who were not requested to alter their usual alcohol consumption we found a significant relationship between alcohol consumption and systolic arterial pressure, b + SE(b), 4.6 +/- 2.1 mmHg/100 g ethanol/day. In particular, males who were heavy drinkers (greater than or equal to 50 g ethanol/day) presented significantly higher systolic pressure levels than the other men, d +/- SE(d), 3.7 +/- 1.6 mmHg, whereas no significant differences were observed among the various classes of women subdivided according to alcohol intake (only 4.6% of the women consumed greater than 50 g ethanol/day). On the other hand, in 479 subjects who were requested to abstain from alcohol consumption during the three days preceding the examination, no significant relation was found between alcohol intake and arterial pressure. The difference between the systolic pressure levels of the male heavy drinkers and those of the male moderate and non-drinkers was only 0.1 mmHg. Excessive alcohol consumption, in this case, mainly in the form of wine, was therefore associated with higher systolic pressure levels. However, it seems that abstaining from alcohol for even a brief period may modify this relation considerably.

Coffee consumption and blood pressure: an Italian study.

The relation between habitual coffee consumption and blood pressure was studied in 500 Italian subjects, males and females, aged 18-62 years. After allowing for sex, age and weight, the pressure levels showed a significant decrease with increasing coffee consumption. Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were respectively 130.4 +/- 1.8 (SE) mmHg and 81.5 +/- 1.1 mmHg for non-coffee drinkers, 129.4 +/- 1.4 and 82.2 +/- 0.9 mmHg for 1 cup per day, 128.4 +/- 0.8 and 81.4 +/- 0.5 mmHg for 2-3 cups per day, 124.9 +/- 1.1 and 78.8 +/- 0.7 mmHg for 4-6 cups per day, and 124.1 +/- 2.5 and 78.7 +/- 1.6 mmHg for more than 6 cups of coffee daily (analysis of covariance: SBP F = 3.46, 4 df, P less than 0.01; DBP F = 3.46, 4 df, P less than 0.01). Even after correcting pressure levels for habitual alcohol intake and cigarette smoking, we observed a mean reduction in SBP and DBP of 0.80 mmHg and 0.48 mmHg respectively per cup per day.

Effect of cigarette smoking and coffee consumption on apolipoprotein B levels.

Apolipoprotein B levels were studied in relation to cigarette smoking, coffee and alcohol consumption, physical activity, age and body mass index in 253 men aged 21-61 years. The mean apolipoprotein B level was 7.3 +/- 3.2 mg/dl and was higher for smokers compared with non-smokers. Considering the smokers of over 20 cigarettes/day and the non-smokers, this difference reached 12.6 +/- 4.3 mg/dl. A significant increase of 7.2 +/- 3.5 mg/dl in apolipoprotein B levels was observed in the subjects who drank over 3 cups of coffee/day compared with the remaining subjects, but the increase was only 4.3 +/- 3.7 mg/dl when we made a correction for cigarette consumption. Furthermore, for cigarette smoking and coffee consumption, there is apparently an interactive effect with BMI and/or age (vs apolipoprotein B levels). However, with a stepwise selection among explicative variables [age, BMI, smoking (yes/no) and coffee consumption (less than or equal to 3, greater than 3 cups/day)] and all their interactions of first order, only the interaction between BMI and smoking (BMI*smoking: b +/- ES (b) = 0.3029 +/- 0.0303), and age and BMI (age*BMI), are significantly and positively related to serum levels of apolipoprotein B. Thus cigarette smoking, interacting with high BMI, appear related to higher apolipoprotein B levels.