Socio-economic inequalities in the use of drugs for the treatment of chronic diseases in Italy.

BACKGROUND: Since the use of medicines is strongly correlated to population health needs, higher drug consumption is expected in socio-economical deprived areas. However, no systematic study investigated the relationship between medications use in the treatment of chronic diseases and the socioeconomic position of patients. The purpose of the study is to provide a description, both at national level and with geographical detail, of the use of medicines, in terms of consumption, adherence and persistence, for the treatment of major chronic diseases in groups of population with different level of socioeconomic position.  METHODS: A cross-sectional study design was used to define the "prevalent" users during 2018. A longitudinal cohort study design was performed for each chronic disease in new drug users, in 2018 and the following year. A retrospective population-based study, considering all adult Italian residents (i.e. around 50.7 million people aged ≥ 18 years). Different medications were used as a proxy for underlying chronic diseases: hypertension, dyslipidemia, osteoporosis, diabetes and chronic obstructive pulmonary disease. Only "chronic" patients who had at least 2 prescriptions within the same subgroup of drugs or specific medications during the year were selected for the analysis. A multidimensional measures of socio-economic position, declined in a national deprivation index at the municipality level, was used to identify and estimate the relationship with drug use indicators. The medicine consumption rate for each pharmacological category was estimated for prevalent users while adherence and persistence to pharmacologic therapy at 12 months were evaluated for new users. RESULTS: The results highlighted how the socioeconomic deprivation is strongly correlated with the use of medicines: after adjustment by deprivation index, the drug consumption rates decreased, mainly in the most disadvantaged areas, where consumption levels are on average higher than in other areas. On the other hand, the adherence and persistence indicators did not show the same trend. CONCLUSIONS: This study showed that drug consumption is influenced by the level of deprivation consistently with the distribution of diseases. For this reason, the main levers on which it is necessary to act to reduce disparities in health status are mainly related to prevention. Moreover, it is worth pointing out that the use of a municipal deprivation indicator necessarily generates an ecological bias, however, the experience of the present study, which for the first-time deals with the complex and delicate issue of equity in Italian pharmaceutical assistance, sets the stage for new insights that could overcome the limits.

Extracellular vesicles in blood, milk and body fluids of the female and male urogenital tract and with special regard to reproduction.

Extracellular vesicles (EVs) are released from almost all cells and tissues. They are able to transport substances (e.g. proteins, RNA or DNA) at higher concentrations than in their environment and may adhere in a receptor-controlled manner to specific cells or tissues in order to release their content into the respective target structure. Blood contains high concentrations of EVs mainly derived from platelets, and, at a smaller amount, from erythrocytes. The female and male reproductive tracts produce EVs which may be associated with fertility or infertility and are released into body fluids and mucosas of the urogenital organs. In this review, the currently relevant detection methods are presented and critically compared. During pregnancy, placenta-derived EVs are dynamically detectable in peripheral blood with changing profiles depending upon progress of pregnancy and different pregnancy-associated pathologies, such as preeclampsia. EVs offer novel non-invasive diagnostic tools which may reflect the situation of the placenta and the foetus. EVs in urine have the potential of reflecting urogenital diseases including cancers of the neighbouring organs. Several methods for detection, quantification and phenotyping of EVs have been established, which include electron microscopy, flow cytometry, ELISA-like methods, Western blotting and analyses based on Brownian motion. This review article summarises the current knowledge about EVs in blood and cord blood, in the different compartments of the male and female reproductive tracts, in trophoblast cells from normal and pre-eclamptic pregnancies, in placenta ex vivo perfusate, in the amniotic fluid, and in breast milk, as well as their potential effects on natural killer cells as possible targets.

Cheetah interspecific SCNT followed by embryo aggregation improves in vitro development but not pluripotent gene expression.

The aim of this study was to evaluate the capacity of domestic cat (Dc, Felis silvestris) oocytes to reprogram the nucleus of cheetah (Ch, Acinonyx jubatus) cells by interspecies SCNT (iSCNT), by using embryo aggregation. Dc oocytes were in vitro matured and subjected to zona pellucida free (ZP-free) SCNT or iSCNT, depending on whether the nucleus donor cell was of Dc or Ch respectively. ZP-free reconstructed embryos were then cultured in microwells individually (Dc1X and Ch1X groups) or in couples (Dc2X and Ch2X groups). Embryo aggregation improved in vitro development obtaining 27.4, 47.7, 16.7 and 28.3% of blastocyst rates in the Dc1X, Dc2X, Ch1X and Ch2X groups, respectively (P<0.05). Moreover, aggregation improved the morphological quality of blastocysts from the Dc2X over the Dc1X group. Gene expression analysis revealed that Ch1X and Ch2X blastocysts had significantly lower relative expression of OCT4, CDX2 and NANOG than the Dc1X, Dc2X and IVF control groups. The OCT4, NANOG, SOX2 and CDX2 genes were overexpressed in Dc1X blastocysts, but the relative expression of these four genes decreased in the Dc2X, reaching similar relative levels to those of Dc IVF blastocysts. In conclusion, Ch blastocysts were produced using Dc oocytes, but with lower relative expression of pluripotent and trophoblastic genes, indicating that nuclear reprogramming could be still incomplete. Despite this, embryo aggregation improved the development of Ch and Dc embryos, and normalized Dc gene expression, which suggests that this strategy could improve full-term developmental efficiency of cat and feline iSCNT embryos.

Tiger, Bengal and Domestic Cat Embryos Produced by Homospecific and Interspecific Zona-Free Nuclear Transfer.

The aim of this study was to evaluate three different cloning strategies in the domestic cat (Felis silvestris) and to use the most efficient to generate wild felid embryos by interspecific cloning (iSCNT) using Bengal (a hybrid formed by the cross of Felis silvestris and Prionailurus bengalensis) and tiger (Panthera tigris) donor cells. In experiment 1, zona-free (ZP-free) cloning resulted in higher fusion and expanded blastocyst rates with respect to zona included cloning techniques that involved fusion or injection of the donor cell. In experiment 2, ZP-free iSCNT and embryo aggregation (2X) were assessed. Division velocity and blastocyst rates were increased by embryo aggregation in the three species. Despite fewer tiger embryos than Bengal and cat embryos reached the blastocyst stage, Tiger 2X group increased the percentage of blastocysts with respect to Tiger 1X group (3.2% vs 12.1%, respectively). Moreover, blastocyst cell number was almost duplicated in aggregated embryos with respect to non-aggregated ones within Bengal and tiger groups (278.3 ± 61.9 vs 516.8 ± 103.6 for Bengal 1X and Bengal 2X groups, respectively; 41 vs 220 ± 60 for Tiger 1X and Tiger 2X groups, respectively). OCT4 analysis also revealed that tiger blastocysts had higher proportion of OCT4-positive cells with respect to Bengal blastocysts and cat intracytoplasmic sperm injection blastocysts. In conclusion, ZP-free cloning has improved the quality of cat embryos with respect to the other cloning techniques evaluated and was successfully applied in iSCNT complemented with embryo aggregation.

DNA fragmentation, transgene expression and embryo development after intracytoplasmic injection of DNA-liposome complexes in IVF bovine zygotes.

Summary This study was designed to evaluate the quality and viability of bovine embryos produced by in vitro fertilization (IVF), after intracytoplasmic injection of pCX-EGFP-liposome complexes or pBCKIP2.8-liposome complexes (plasmids that codify the human insulin gene). Cleavage, blastocysts and expanded blastocysts rates of these both groups were not different from that of controls (IVF or IVF embryos injected with liposomes alone; IVF-L). The percentage of EGFP-positive (EGFP+) blastocysts was 41.8%. In Experiment 2, the blastocysts obtained after injection of pCX-EGFP-liposome complexes that did or did not express the transgene, were analyzed by TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labelling) assay at days 6, 7 and 8 of culture in vitro(Bd6, Bd7 and Bd8), in order to evaluate DNA fragmentation. The EGFP+ blastocysts showed different proportions of TUNEL-positive cells (T+) at Bd6, Bd7 and Bd8 (91, 73.7 and 99.5%, respectively) while blastocysts without EGFP expression (EGFP-) showed statistically lower numbers of fragmented nuclei (0, 44.6 and 85%, respectively; P < 0.05). There was no evidence of DNA fragmentation in either Bd6 or Bd7 IVF and IVF-L control blastocysts, but T+ nuclei were detected at Bd8 in both groups (66.4 and 85.8% respectively). Finally, IVF blastocysts (n = 21) injected with insulin-liposome complexes, cultured for 6, 7 and 8 days, were transferred to recipient cows. Pregnancy rates of 18.2% (2/11) and 40% (2/5) resulted from the transfer of Bd6 and Bd7 cells, respectively. Two pregnancies developed to term but they were not transgenic for the insulin gene. In conclusion, EGFP expression affects DNA integrity but not embryo development. Moreover, additional transfers are required in order to overcome the drawbacks generated by in vitro culture length and transgene expression.

Evaluation of cheetah and leopard spermatozoa developmental capability after interspecific ICSI with domestic cat oocytes.

The ICSI procedure is potentially of great value for felids, and it has not been extensively studied in these species. The objectives of this work were to determine the best conditions for ICSI in the domestic cat (DC) to generate interspecific embryos by injecting cheetah (Ch) and leopard (Leo) spermatozoa. Firstly, DC oocytes were matured with insulin-transferrin-selenium (ITS) or without it (MM) and cultured using atmospheric (21%) or low (5%) oxygen tension after ICSI. The group ITS-5%O2 showed the highest blastocyst rate (p < 0.05), 20.9% vs 8.7%, 7% and 6.5%, for MM-21%O2 , MM-5%O2 and ITS-21%O2 , respectively. The best conditions were used to generate the interspecific embryos, together with ionomycin activation (Io) after ICSI. Interspecific embryos resulted in high rates of blastocysts that were not positively affected by Io activation: 32.6% vs 21% for Ch and Ch-Io, 9.8% vs 21% for Leo and Leo-Io, and 20% vs 17.4% for DC and DC-Io. We also evaluated DNA-fragmented nuclei of experiment 1 and 2 blastocysts, using TUNEL assay. The fragmented nucleus proportion was higher in the ITS-5%O2 group, 67.6%. Surprisingly, interspecific blastocysts showed the lowest fragmented nucleus proportion: 27% and 29.9% for Ch and Leo, respectively. We concluded that ITS and 5%O2 improve blastocyst formation in DC, although with a concomitant increase in DNA fragmentation. Most importantly, cheetah and leopard spermatozoa were able to generate blastocysts without artificial activation, which suggests that developmental capacity of wild felid spermatozoa can be evaluated by interspecific ICSI. This technique should be used to assist wild felid reproduction.

Systemic absorption of rifamycin SV MMX administered as modified-release tablets in healthy volunteers.

The new oral 200-mg rifamycin SV MMX modified-release tablets, designed to deliver rifamycin SV directly into the colonic lumen, offer considerable advantages over the existing immediate-release antidiarrheic formulations. In two pharmacokinetics studies of healthy volunteers, the absorption, urinary excretion, and fecal elimination of rifamycin SV after single- and multiple-dose regimens of the new formulation were investigated. Concentrations in plasma of >2 ng/ml were infrequently and randomly quantifiable after single and multiple oral doses. The systemic exposure to rifamycin SV after single and multiple oral doses of MMX tablets under fasting and fed conditions or following a four-times-a-day (q.i.d.) or a twice-a-day (b.i.d.) regimen could be considered negligible. With both oral regimens, the drug was confirmed to be very poorly absorbable systemically. The amount of systemically absorbed antibiotic excreted by the renal route is far lower than 0.01% of the administered dose after both the single- and multiple-dose regimens. The absolute bioavailability, calculated as the mean percent ratio between total urinary excretion amounts (ΣXu) after a single intravenous injection and after a single oral dose under fasting conditions, was 0.0410±0.0617. The total elimination of the unchanged rifamycin SV with feces was 87% of the administered oral dose. No significant effect of rifamycin SV on vital signs, electrocardiograms, or laboratory parameters was observed.

Cortexolone 17α-propionate 1% cream, a new potent antiandrogen for topical treatment of acne vulgaris. A pilot randomized, double-blind comparative study vs. placebo and tretinoin 0·05% cream.

BACKGROUND: Acne vulgaris is a disorder of the pilosebaceous unit in which the androgens contribute to its onset and persistence. The use of antiandrogens is therefore potentially effective; however, antiandrogens for topical use are not available on the market. Cortexolone 17α-propionate (CB-03-01; Cosmo S.p.A, Lainate, Italy) is a new potent topical antiandrogen potentially useful in acne vulgaris. OBJECTIVES: To evaluate the safety and the topical efficacy of CB-03-01 1% cream in acne vulgaris as compared with placebo and with tretinoin 0·05% cream (Retin-A® ; Janssen-Cilag). METHODS: Seventy-seven men with facial acne scored 2-3 according to Investigator's Global Assessment (IGA) were randomized to receive placebo cream (n = 15), or CB-03-01 1% cream (n = 30), or tretinoin 0·05% cream (n = 32) once a day at bedtime for 8 weeks. Clinical efficacy was evaluated every 2 weeks including total lesion count (TLC), inflammatory lesion count (ILC), acne severity index (ASI) and IGA. Safety assessment included local irritancy score, laboratory tests, physical examination, vital signs and recording of adverse events. RESULTS: CB-03-01 1% cream was very well tolerated, and was significantly better than placebo regarding TLC (P = 0·0017), ILC (P = 0·0134) and ASI (P = 0·0090), and also clinically more effective than comparator. The product also induced a faster attainment of 50% improvement in all the above parameters. CONCLUSIONS: This pilot study supports the rationale for the use of topical antiandrogens in the treatment of acne vulgaris. CB-03-01 1% cream seems to fit with the profile of an ideal antiandrogen for topical use.

Short communication: reference values for blood parameters in Holstein dairy cows: effects of parity, stage of lactation, and season of production.

Confidence intervals for blood parameters used for nutritional and metabolic profile testing in cattle were calculated for clinically normal lactating Holstein cows, taking into account the effects of parity, stage of lactation, and season. Blood samples were collected from 740 cows in 33 Italian dairy herds according to a predefined protocol. Herds were visited during summer and the following winter, sampling 12 lactating cows at each visit (4 primiparous and 8 multiparous). Six cows were selected from the early-lactation group (days in milk: 10 to 89) and the other 6 were selected from the mid-lactation group (days in milk: 90 to 215). Cow selection criteria excluded animals clinically exposed to periparturient diseases as well as animals not considered in good health by a veterinary clinical examination. For each blood variable, outliers were identified and discarded. Data were then analyzed for their Gaussian distribution and variables with not normal distribution were log-transformed to adjust for lack of normality. Herd mean values were calculated for each blood parameter according to 3 main classification factors: parity (primiparous vs. multiparous), stage of lactation (early vs. mid) and season of production (summer vs. winter). The resulting data set was statistically analyzed using a mixed model with the fixed effects of these factors, their interactions, and the random effect of herd. General 95% confidence intervals were calculated for blood variables that showed a relevant herd variance component such as albumin, triglycerides, aspartate, urea, glucose, alanine aminotransferase, lactate dehydrogenase, direct and total bilirubin, calcium, magnesium, and potassium. For the remaining parameters, specific confidence intervals were calculated for each level of the significant main factors. Parity affected blood concentration of total protein, globulin, creatinine, alkaline phosphatase, gamma glutamyl transferase, creatinine kinase, and phosphorus. Blood nonesterified fatty acids, aspartate aminotransferase, gamma glutamyl transferase, creatinine kinase and cholesterol were influenced by stage of lactation. The season of production had a significant effect on total protein, globulin, creatinine, alkaline phosphatase, phosphorus, sodium, and chlorine. The outcomes of this work will improve the accuracy of the biochemical profile as a tool for dairy practitioners to assess the metabolic status of lactating Holstein cows.

Efficient transgene expression in IVF and parthenogenetic bovine embryos by intracytoplasmic injection of DNA-liposome complexes.

Transgenic animals constitute an important tool with many biotechnological applications. Although there have been advances in this field, we propose a novel method that may greatly increase the efficiency of transgenic animal production and thereby its application. This new technique consists of intracytoplasmic injection of liposomes, in bovine oocytes and zygotes, to introduce exogenous DNA. In the first experiment, we evaluated embryo development and EGFP expression in In Vitro Fertilization (IVF) embryos injected with different concentrations of exogenous DNA-liposome complexes (0.5, 5, 50, 500 ng pCX-EGFP/µl). The highest EGFP-embryos rates were obtained using 500 ng pCX-EGFP/µl. In the second experiment, we evaluated embryo development and EGFP expression following the injection of DNA-liposome complexes into pre-fertilized oocytes and presumptive zygotes, 16 and 24 h post-fertilization. Approximately 70% of the cleaved embryos and 50% of the blastocysts expressed EGFP, when egfp-liposome was injected 16 h post-fertilization. The percentages of positive embryos for the 24-h post-fertilization and pre-fertilization groups were 30.1 and 6.3, respectively. Blastocysts that developed from injected zygotes were analysed by PCR, confirming the presence of transgene in all embryos. Finally, we examined the embryo development and EGFP expression of parthenogenetic embryos that resulted from the injection of egfp-liposome complexes into pre-activated oocytes, and 3 and 11 h post-activated oocytes. The group with the highest expression rate (48.4%) was the one injected 3 h post-activation. In summary, this study reports the efficient, reproducible and fast production of IVF and parthenogenetic embryos expressing EGFP, by the intracytoplasmic injection of liposomes to introduce the foreign DNA.

Downregulation of E-cadherin in pluripotent stem cells triggers partial EMT.

Epithelial to mesenchymal transition (EMT) is a critical cellular process that has been well characterized during embryonic development and cancer metastasis and it also is implicated in several physiological and pathological events including embryonic stem cell differentiation. During early stages of differentiation, human embryonic stem cells pass through EMT where deeper morphological, molecular and biochemical changes occur. Though initially considered as a decision between two states, EMT process is now regarded as a fluid transition where cells exist on a spectrum of intermediate states. In this work, using a CRISPR interference system in human embryonic stem cells, we describe a molecular characterization of the effects of downregulation of E-cadherin, one of the main initiation events of EMT, as a unique start signal. Our results suggest that the decrease and delocalization of E-cadherin causes an incomplete EMT where cells retain their undifferentiated state while expressing several characteristics of a mesenchymal-like phenotype. Namely, we found that E-cadherin downregulation induces SNAI1 and SNAI2 upregulation, promotes MALAT1 and LINC-ROR downregulation, modulates the expression of tight junction occludin 1 and gap junction connexin 43, increases human embryonic stem cells migratory capacity and delocalize ß-catenin. Altogether, we believe our results provide a useful tool to model the molecular events of an unstable intermediate state and further identify multiple layers of molecular changes that occur during partial EMT.

Ultrasound imaging of the sural nerve: ultrasound anatomy and rationale for investigation.

BACKGROUND: Damage to the sural nerve (SuN) may arise from surgical stripping or thermal ablation of the small saphenous vein (SSV). OBJECTIVE: This study aims to demonstrate that visualisation of the SuN and its point of contact with the SSV ('risk point') using ultrasound imaging can be achieved in routine clinical practice. TYPE OF STUDY: This is a cohort study. PATIENTS: Fifteen normal subjects and five patients with chronic venous insufficiency (CVI) (two with a dilated, incompetent SSV). METHOD: The SuN was identified using high-resolution ultrasound imaging using 14- and 18-MHz probes. Two manoeuvres were found to improve visualisation: (1) the contrast of the nerve was increased compared with the other tissues by varying the angle of insonation; and (2) the transducer was moved up and down the limb for a short distance during transverse imaging of the calf. The muscles and other soft tissues appeared 'out of focus', whereas the SuN retained both shape and echogenicity. Once the nerve has been identified, proceeding proximally, the point of separation of the two components is often detectable. It is then possible to follow the two different nerves observing the medial sural cutaneous nerve (MSCN) inside the 'triangle' of connective tissue below the SSV joining the tibial nerve and the lateral sural cutaneous nerve (LCSN) joining the common peroneal nerve, which runs inside a tiny fascial duplication. The extent of nerves, which were identified, was recorded in each limb as well as their anatomical distribution. RESULTS: The SuN and the point at which it might be at risk were identified on ultrasound images in 39 of 40 limbs (97%) studied. In transverse section, it was readily identified within the saphenous compartment. It lies in close proximity to the SSV only in the distal third of the limb, where the two components of the nerve: MSCN, a branch of the tibial nerve; and LSCN, a branch of the common peroneal nerve join together. The relationship between the SuN and the SSV is very variable, with the nerve running separately or in close contact with the vein for variable distances, in many different combinations. CONCLUSIONS: The SuN and 'risk point' can be identified by ultrasonography (US). We propose that this technique could be used to prevent damage to the SuN during surgical or thermal ablation of the SSV and during Achilles tendon surgery.

Caspase-3/-8/-9, Bax and Bcl-2 expression in the cerebellum, lymph nodes and leukocytes of dogs naturally infected with canine distemper virus.

Canine distemper is an immunosuppressive disease caused by the canine distemper virus (CDV). Pathogenesis mainly involves the central nervous system and immunosuppression. Dogs naturally infected with CDV develop apoptotic cells in lymphoid tissues and the cerebellum, but this apoptotic mechanism is not well characterized. To better understand this process, we evaluated the expression of Bax, Bcl-2, and caspase-3, -8 and -9, by evaluating mRNA levels in the peripheral blood, lymph nodes and cerebellum of CDV-infected (CDV+) and uninfected (CDV-) dogs by real-time polymerase chain reaction (PCR). Blood samples from 12 CDV+ and 8 CDV- dogs, diagnosed by reverse transcription-PCR, were subjected to hematological analysis and apoptotic gene expression was evaluated using real-time-PCR. Tissues from the cerebellum and lymph nodes of four CDV+ and three CDV-dogs were also subjected to real time-PCR. No significant differences were found between CDV+ and CDV- dogs in the hemotological results or in the expression of caspase-3, -8, -9, Bax, and Bcl-2 in the peripheral blood. However, expression of Bax, caspase-3, -8 and -9 was significantly higher in the cerebellum of CDV+ compared to CDV- dogs. Expression of caspase-3 and -8 was significantly higher in the lymph nodes of CDV+ compared to CDV- dogs. We concluded that infection with CDV induces apoptosis in the cerebellum and lymph nodes in different ways. Lymph node apoptosis apparently occurs via caspase-3 activation, through the caspase-8 pathway, and cerebellum apoptosis apparently occurs via caspase-3 activation, through the caspase-8 and mitochondrial pathways.

Biochemical and immunohistochemical evidence that in cartilage an alkaline phosphatase is a Ca2+-binding glycoprotein.

A glycoprotein that exhibits alkaline phosphatase activity and binds Ca2+ with high affinity has been extracted and purified from cartilage matrix vesicles by fast protein liquid chromatography. Antibodies against this glycoprotein were used to analyze its distribution in chondrocytes and in the matrix of calcifying cartilage. Under the light microscope, using immunoperoxidase or immunofluorescence techniques, the glycoprotein is localized in chondrocytes of the resting zone. At this level, the extracellular matrix does not show any reaction. In the cartilage plate, between the proliferating and the hypertrophic region, a weak immune reactivity is seen in the cytoplasm, whereas in the intercolumnar matrix the collagen fibers appear clearly stained. Stained granular structures, distributed with a pattern similar to that of matrix vesicles, are also visible. Calcified matrix is the most stained area. These results were confirmed under the electron microscope using both immunoperoxidase and protein A-gold techniques. In parallel studies, enzyme activity was also analyzed by histochemical methods. Whereas resting cartilage, the intercellular matrix of the resting zone, and calcified matrix do not exhibit any enzyme activity, the zones of maturing and hypertrophic chondrocytes are highly reactive. Some weak reactivity is also shown by chondrocytes of the resting zone. The observation that this glycoprotein (which binds Ca2+ and has alkaline phosphatase activity) is synthesized in chondrocytes and is exported to the extracellular matrix at the time when calcification begins, suggests that it plays a specific role in the process of calcification.

Purification, biochemical and functional characterization of miliin, a new thiol-dependent serine protease isolated from the latex of Euphorbia milii.

Miliin, a new thiol-dependent serine protease purified from the latex of Euphorbia milii possesses a molecular weight of 79 kDa, an isoelectric point of 4.3 and is optimally active at 60 degrees C in the pH range of and 7.5-11.0. Activity tests indicate that milliin is a thiol-dependent serine protease.

Bone marrow mesenchymal stem cells as nuclear donors improve viability and health of cloned horses.

INTRODUCTION: Cell plasticity is crucial in cloning to allow an efficient nuclear reprogramming and healthy offspring. Hence, cells with high plasticity, such as multipotent mesenchymal stem cells (MSCs), may be a promising alternative for horse cloning. In this study, we evaluated the use of bone marrow-MSCs (BM-MSCs) as nuclear donors in horse cloning, and we compared the in vitro and in vivo embryo development with respect to fibroblasts. MATERIALS AND METHODS: Zona-free nuclear transfer was performed using BM-MSCs (MSC group, n=3432) or adult fibroblasts (AF group, n=4527). Embryos produced by artificial insemination (AI) recovered by uterine flushing and transferred to recipient mares were used as controls (AI group). RESULTS: Blastocyst development was higher in the MSC group than in the AF group (18.1% vs 10.9%, respectively; p<0.05). However, pregnancy rates and delivery rates were similar in both cloning groups, although they were lower than in the AI group (pregnancy rates: 17.7% [41/232] for MSC, 12.5% [37/297] for AF and 80.7% [71/88] for AI; delivery rates: 56.8% [21/37], 41.5% [17/41] and 90.1% [64/71], respectively). Remarkably, the gestation length of the AF group was significantly longer than the control (361.7±10.9 vs 333.9±8.7 days), in contrast to the MSC group (340.6±8.89 days). Of the total deliveries, 95.2% (20/21) of the MSC-foals were viable, compared to 52.9% (9/17) of the AF-foals (p<0.05). In addition, the AF-foals had more physiological abnormalities at birth than the MSC-foals; 90.5% (19/21) of the MSC-delivered foals were completely normal and healthy, compared to 35.3% (6/17) in the AF group. The abnormalities included flexural or angular limb deformities, umbilical cord enlargement, placental alterations and signs of syndrome of neonatal maladjustment, which were treated in most cases. CONCLUSION: In summary, we obtained 29 viable cloned foals and found that MSCs are suitable donor cells in horse cloning. Even more, these cells could be more efficiently reprogrammed compared to fibroblasts.

Methylene blue MMX® tablets for chromoendoscopy. Bioavailability, colon staining and safety in healthy volunteers undergoing a full colonoscopy.

Methylene blue-MMX® tablets are proposed as an aid for detection and visualisation of adenomas and carcinomas in patients undergoing colonoscopy, by improving their detection rate and highlighting the presence of the intestinal dysplastic lesions. Single total doses of 100 and 200 mg were administered to healthy volunteers undergoing a bowel cleansing preparation and a full colonoscopy to investigate the colonic staining. The pharmacokinetics of methylene blue and the safety after exposure to the tablets were also investigated. With 200 mg, the best staining, assessed as the sum of acceptable and good staining, was achieved in the ascending colon and rectosigmoid (75% subjects each), the transverse and the descending colon (approximately 63% each). Absence of staining or overstaining were reported for no colonic region of interest in any subject. Similar results were observed in the 100 mg dose group. Methylene blue blood concentrations reached a peak (Cmax) in a median time (Tmax) of 12 h with 100 mg and 16 h with 200 mg. AUC0-t was 10.7 ±â€¯6.7 µg/mLxh after 100 mg and 25.2 ±â€¯7.4 µg/mLxh after 200 mg. Half-life ranged between 9 and 22 h after the lower dose and between 6 and 26 h after the higher dose. The cumulative urinary excretion was about 28% after 100 mg and about 39% after 200 mg up to 60 h post-dose. The overall frequency of adverse events after single dose of the test product administered along with a bowel cleansing preparation was 39%, but only one was related to the test product: abnormal transaminases. The most frequent adverse event was a transient polyuria (17%). One serious adverse event (gastrointestinal haemorrhage) led the subject to study discontinuation and hospitalisation and another subject withdrew the study due to one adverse event (haematemesis). Either event was not related to methylene blue.

Role of biochemical markers of bone remodeling in clinical practice.

Bone tissue is subject to remodeling throughout the lifetime of an individual. Through a continuous remodeling cycle, actuated via the so-called 'bone remodeling units', old bone is resorbed by osteoclasts with the formation of cavities that are subsequently filled by osteoblasts. Bone loss observed in old age and in women after menopause is due to an imbalance between bone resorption and formation. Biochemical markers provide a dynamic view of the remodeling process, which covers rate of turnover and pathogenesis, and should improve fracture risk prediction. Furthermore, they can be used to monitor the short-term effects of therapy, and indicate if an excessive slowing of the remodeling process is occurring. When searching for markers of bone remodeling, biochemists have focused mainly on skeletal molecules that can be dosed in plasma and/or urine, as indicators of osteoblast function (i.e. bone alkaline phosphatase, osteocalcin, procollagene I C- and N-terminal propeptides) or osteoclast function (i.e. pyridinium crosslinks, collagen I C- and N-terminal telopeptides). The clinical significance of any marker for bone remodeling depends on two fundamental characteristics: specificity and variability. If the objective is to monitor therapeutic efficacy, it seems most rational to use a resorption marker for drugs that act principally on osteoclast, such as estrogens or bisphosphonates, while for drugs that act principally on osteoblast, such as PTH-peptides a marker for bone formation would be more appropriate.

An antimicrobial stewardship program reduces antimicrobial therapy duration and hospital stay in surgical wards.

We report a quasi-experimental study of the implementation of an antimicrobial stewardship program in two surgical wards, with a pre-intervention period with just assessment of prescription and an intervention period with a prospective audit on antibiotic prescription model. There was a significant reduction of length of stay and the total days of antimicrobial administration. There were no differences in mortality between groups. The antimicrobial stewardship program led to the early detection of inappropriate empirical antibiotic treatment and was associated with a significant reduction in length of stay and the total duration of antimicrobial therapy.

The glycosides of hydroxylysine are final products of collagen degradation in humans.

Glucosyl-galactosyl-hydroxylysine (GGHYL) and galactosyl-hydroxylysine (GHYL) are metabolites derived from collagen degradation. They are useful biochemical markers provided they are not further processed. Experiments were run to test the activity of alpha- and beta-glycosidases in human kidney cortex preparations. Results allow to exclude the presence of the specific enzymes, in contrast with what is reported for the rat kidney tissue.