Ceftazidime/avibactam-resistant meropenem-susceptible KPC-producing Klebsiella pneumoniae: Analysis of cases and evaluation of in vitro activity of fosfomycin-containing combinations.

OBJECTIVES: Little is known regarding outcomes and optimal therapeutic regimens of infections caused by Klebsiella pneumoniae carbapenemase-producing Klebsiella pneumoniae (KPC-Kp) resistant to ceftazidime/avibactam (CZA) and susceptible to meropenem (MEM). Although susceptible to MEM in vitro, the possibility of developing MEM resistance overtime is a concern. We describe the clinical characteristics of patients with colonization/infection due to KPC variants with a focus on the in vitro activity of fosfomycin (FOS)-containing combinations. METHODS: Patients with colonization/infection due to a KPC variant were included. Fosfomycin susceptibility was performed by agar dilution method. Synergistic activity of FOS-based combinations was evaluated by gradient strip-agar diffusion method. The emergence of in vitro MEM resistance was also tested. RESULTS: Eleven patients were included: eight with infection [four with ventilator-associated pneumonia and four with bloodstream infections] and three with colonization. Previous therapy with CZA was administered to all patients (with a mean cumulative duration of 23 days). All subjects with infection received meropenem, in monotherapy (n = 4) or with amikacin (n = 2) or fosfomycin (n = 2), and achieved clinical cure. A new CZA-susceptible and MEM-resistant KPC-Kp strain was subsequently isolated in three patients (27.3%). Meropenem/vaborbactam (MVB) showed high in vitro activity, while FOS+MEM combination was synergistic in 40% of cases. In vitro resistance to MEM was observed with maintenance of CZA resistance. CONCLUSIONS: Detection of KPC variants may occur within the same patient, especially if CZA has been previously administered. Although clinical success has been obtained with carbapenems, the emergence of MEM resistance is a concern. Fosfomycin plus meropenem is synergistic and may be a valuable combination option for KPC variants, while MVB may be considered in monotherapy. The detection of KPC variants in an endemic setting for KPC-Kp represents a worryingly emerging condition. The optimal therapeutic approach is still unknown and the development of meropenem resistance is of concern, which may lead to therapeutic failure in clinical practice. In these cases, the addition of fosfomycin to meropenem, or a more potent antibiotic, such as meropenem/vaborbactam, may be valuable therapeutic options.

Ethanolic extract of Casearia sylvestris Sw exhibits in vitro antioxidant and antimicrobial activities and in vivo hypolipidemic effect in rats / Extrato etanólico de Casearia sylvestris Sw apresenta atividade antioxidante e antimicrobiana in vitro e efeito hipolipemiante em ratos

ABSTRACT: The Casearia sylvestris Sw (Flacourtiaceae) is a shrub that occurs in forests of Southern Brazil; its leaves are widely used in folk medicine as a depurative, analgesic, anti-inflammatory and antiulcerogenic agent. The objective of this study was to perform the phytochemical description and to evaluate the pharmacological activities (antimicrobial, antifungal, antioxidant and toxicity) of the ethanolic extract (EE) of C. sylvestris Sw. In addition, we also evaluated the effect of the EE of C. sylvestris Sw on the glucose levels and lipid profile in blood serum of rats submitted to a model of streptozotocin-induced diabetes. Material and Methods: In vitro assay: the detection of chemical groups was done through chemical reactions with the development of color or precipitate and by chromatographic profile; the antioxidant activity was measured by the method of reduction of DPPH free radical (2,2-diphenyl-1-picrylhydrazyl); the Minimum Inhibitory Concentration was evaluated by the broth microdilution method, and the Minimum Bactericide Concentration and the Minimum Fungicide Concentration were performed in Petri dishes; the cytotoxic activity was measured by the Artemia salina test. In vivo assay: diabetic and non-diabetic rats were treated with EE of C. sylvestris Sw (300 mg/kg) for 45 days, and the glycaemia and lipid profile were analyzed. Results: The EE showed a Lethal Dose50 of 724.76 μg.mL-1 and important antioxidant, fungicide and fungistatic activities. The EE showed better antimicrobial activity regarding the microorganisms Staphylococcus aureus, Escherichia coli and Salmonella setubal. Conclusion: The EE of C. sylvestris Sw produces a significant decrease in triglycerides, total cholesterol and VLDL levels without any significant alteration in the glycaemia. The EE of C. sylvestris Sw presents antioxidant and antimicrobial activities and it exhibits a potent hypolipidemic effect.

RESUMO: Casearia sylvestris Sw (Flacourtiaceae) é uma planta comumente encontrada em florestas do sul do Brasil; suas folhas são amplamente utilizadas na medicina popular como depurativa, analgésica, anti-inflamatória e anti ulcerogênica. O objetivo deste estudo foi apresentar uma descrição fitoquímica e da atividade farmacológica (antimicrobiana, antifúngica, antioxidante e toxicidade) do extrato etanólico (EE) da C. Sylvestris Sw. Adicionalmente, procurou-se avaliar o efeito do EE da C. Sylvestris Sw sobre os níveis séricos de glicose e perfil lipídico de ratos submetidos a um modelo de diabetes induzida por estreptozotocina. A detecção de grupos químicos foi realizada por reações químicas de coloração ou precipitação, e também por cromatografia; a atividade antioxidante foi mensurada pelo método de redução do DPPH (2,2-difenil-1-picril-hidrazil); a concentração mínima inibitória foi realizada pela técnica de micro-diluição, e concentração mínima bactericida e concentração mínima fungicida foram realizadas em placa de Petri; enquanto a atividade citotóxica foi conduzida pelo teste da Artemia salina. Nos ensaios in vivo, ratos diabéticos e não-diabéticos foram tratado com EE da C. Sylvestris Sw (300mg/kg) por 45 dias, e os níveis glicêmico e perfil lipídico foram medidos. A dose Letal50 do EE foi de 724.76 μg.mL-1; mostrando importante atividades antioxidante, fungicida e fungistática e melhor atividade antimicrobiana contra Staphylococcus aureus, Escherichia coli e Salmonella setubal. O EE da C. Sylvestris Sw promoveu diminuição significativa nos níveis de triglicerídeos, colesterol total e VLDL; porém sem efeito significativo nos níveis glicêmicos. O EE da C. Sylvestris Sw, além de apresentar atividade antioxidante e antimicrobiana; possui também potente efeito hipolipidêmico.

Alterations in the cholinesterase and adenosine deaminase activities and inflammation biomarker levels in patients with multiple sclerosis.

Multiple sclerosis (MS) is one of the main chronic inflammatory diseases of the CNS that cause functional disability in young adults. It has unknown etiology characterized by the infiltration of lymphocytes and macrophages into the brain. The aim of this study was to evaluate the acetylcholinesterase (AChE) activity in lymphocytes and whole blood, as well as butyrylcholinesterase (BChE) and adenosine deaminase (ADA) activities in serum. We also checked the levels of nucleotides, nucleosides, biomarkers of inflammation such as cytokines (interleukin (IL)-1, IL-6, interferon (IFN)-γ, tumor necrosis factor-alpha (TNF-α) and IL-10) and C-reactive protein (CRP) in serum from 29 patients with the relapsing-remitting form of MS (RRMS) and 29 healthy subjects as the control group. Results showed that AChE in lymphocytes and whole blood as well as BChE, and ADA activities in serum were significantly increased in RRMS patients when compared to the control group (P<0.05). In addition, we observed a decrease in ATP levels and a significant increase in the levels of ADP, AMP, adenosine and inosine in serum from RRMS patients in relation to the healthy subjects (P<0.05). Results also demonstrated an increase in the IFN-γ, TNF-α, IL-1, IL-6 and CRP (P<0.05) and a significant decrease in the IL-10 (P<0.0001) in RRMS patients when compared to control. Our results suggest that alterations in the biomarkers of inflammation and hydrolysis of nucleotides and nucleosides may contribute to the understanding of the neurological dysfunction of RRMS patients.

Comparative effectiveness of novel oral anticoagulants for atrial fibrillation: evidence from pair-wise and warfarin-controlled network meta-analyses.

INTRODUCTION: Novel oral anticoagulants have been tested against warfarin for atrial fibrillation, yet no direct comparison is available. We thus aimed to perform pair-wise (direct) and warfarin-adjusted network (i.e. indirect) meta-analyses of novel oral anticoagulants for atrial fibrillation. METHODS: Databases were searched for randomized warfarin-controlled trials of novel anticoagulants for non-valvular atrial fibrillation. The primary end-point was long-term stroke/systemic embolism. Odds ratios (95% intervals) were computed with RevMan and WinBUGS. RESULTS: Seven trials (52701 patients) were included, focusing on apixaban, dabigatran, edoxaban and rivaroxaban. Pair-wise meta-analysis showed that after a weighted average of 23 months these novel anticoagulants lead to significant reductions in the risk of stroke/systemic embolism (odds ratio=0.81 [0.71-0.92], I2=23%) and all cause death (odds ratio=0.88 [0.82-0.95], I2=0%) in comparison to warfarin. Network meta-analysis showed that apixaban and dabigatran proved similarly superior to warfarin in preventing stroke/systemic embolism (odds ratio=0.78 [0.62-0.96] for apixaban vs warfarin; odds ratio=0.66 [0.52-0.84] for high-dose dabigatran vs warfarin; odds ratio for apixaban vs high-dose dabigatran=1.17 [0.85-1.63]), but apixaban was associated with fewer major bleedings (odds ratio=0.73 [0.57-0.93]) and drug discontinuations (odds ratio=0.64 [0.52-0.78]) than dabigatran. Rivaroxaban did not reduce stroke/systemic embolism (odds ratio=0.87 [0.71-1.07]) or major bleedings in comparison to warfarin (odds ratio=0.87 [0.71-1.07]) and was associated with more major bleedings in comparison to apixaban (odds ratio=1.52 [1.19-1.92]). Data for edoxaban were inconclusive. CONCLUSIONS: Novel oral anticoagulants appear as a very promising treatment option for atrial fibrillation.

Mild hyperhomocysteinemia alters extracellular adenine metabolism in rat brain.

Since homocysteine (Hcy) is considered a risk factor to cerebral diseases and adenine nucleotides are important molecules to brain normal function, in the present study we investigated the effect of chronic mild hyperhomocysteinemia on ectonucleotidase activities and expression in rat cerebral cortex. The levels of ATP, ADP, AMP and adenosine (Ado) in cerebrospinal fluid (CSF) of adult rats also were evaluated by high-performance liquid chromatography. For the chronic chemically induced mild hyperhomocysteinemia, Hcy (0.03 µmol/g of body weight) was administered subcutaneously from the 30th to the 60th day of life. Control rats received saline solution in the same volumes. Results showed that Hcy significantly decreased nucleotide hydrolysis in the synaptosomal fraction and increased E-NTPDase1 and ecto-5'-nucleotidase transcripts in rat cerebral cortex. ATP levels were significantly increased, while Ado decreased in CSF of Hcy-treated rats. These findings suggest that the unbalance in ATP and Ado levels may be, at last in part, involved in the cerebral toxicity of mild hyperhomocysteinemia.

Gadolinium increases the vascular reactivity of rat aortic rings

Gadolinium (Gd) blocks intra- and extracellular ATP hydrolysis. We determined whether Gd affects vascular reactivity to contractile responses to phenylephrine (PHE) by blocking aortic ectonucleoside triphosphate diphosphohydrolase (E-NTPDase). Wistar rats of both sexes (260-300 g, 23 females, 7 males) were used. Experiments were performed before and after incubation of aortic rings with 3 µM Gd. Concentration-response curves to PHE (0.1 nM to 0.1 mM) were obtained in the presence and absence of endothelium, after incubation with 100 µM L-NAME, 10 µM losartan, or 10 µM enalaprilat. Gd significantly increased the maximum response (control: 72.3 ± 3.5; Gd: 101.3 ± 6.4 percent) and sensitivity (control: 6.6 ± 0.1; Gd: 10.5 ± 2.8 percent) to PHE. To investigate the blockade of E-NTDase activity by Gd, we added 1 mM ATP to the bath. ATP reduced smooth muscle tension and Gd increased its relaxing effect (control: -33.5 ± 4.1; Gd: -47.4 ± 4.1 percent). Endothelial damage abolished the effect of Gd on the contractile responses to PHE (control: 132.6 ± 8.6; Gd: 122.4 ± 7.1 percent). L-NAME + Gd in the presence of endothelium reduced PHE contractile responses (control/L-NAME: 151.1 ± 28.8; L-NAME + Gd: 67.9 ± 19 percent AUC). ATP hydrolysis was reduced after Gd administration, which led to ATP accumulation in the nutrient solution and reduced ADP concentration, while adenosine levels remained the same. Incubation with Gd plus losartan and enalaprilat eliminated the pressor effects of Gd. Gd increased vascular reactivity to PHE regardless of the reduction of E-NTPDase activity and adenosine production. Moreover, the increased reactivity to PHE promoted by Gd was endothelium-dependent, reducing NO bioavailability and involving an increased stimulation of angiotensin-converting enzyme and angiotensin II AT1 receptors.

Gadolinium increases the vascular reactivity of rat aortic rings.

Gadolinium (Gd) blocks intra- and extracellular ATP hydrolysis. We determined whether Gd affects vascular reactivity to contractile responses to phenylephrine (PHE) by blocking aortic ectonucleoside triphosphate diphosphohydrolase (E-NTPDase). Wistar rats of both sexes (260-300 g, 23 females, 7 males) were used. Experiments were performed before and after incubation of aortic rings with 3 µM Gd. Concentration-response curves to PHE (0.1 nM to 0.1 mM) were obtained in the presence and absence of endothelium, after incubation with 100 µM L-NAME, 10 µM losartan, or 10 µM enalaprilat. Gd significantly increased the maximum response (control: 72.3 ± 3.5; Gd: 101.3 ± 6.4%) and sensitivity (control: 6.6 ± 0.1; Gd: 10.5 ± 2.8%) to PHE. To investigate the blockade of E-NTDase activity by Gd, we added 1 mM ATP to the bath. ATP reduced smooth muscle tension and Gd increased its relaxing effect (control: -33.5 ± 4.1; Gd: -47.4 ± 4.1%). Endothelial damage abolished the effect of Gd on the contractile responses to PHE (control: 132.6 ± 8.6; Gd: 122.4 ± 7.1%). L-NAME + Gd in the presence of endothelium reduced PHE contractile responses (control/L-NAME: 151.1 ± 28.8; L-NAME + Gd: 67.9 ± 19% AUC). ATP hydrolysis was reduced after Gd administration, which led to ATP accumulation in the nutrient solution and reduced ADP concentration, while adenosine levels remained the same. Incubation with Gd plus losartan and enalaprilat eliminated the pressor effects of Gd. Gd increased vascular reactivity to PHE regardless of the reduction of E-NTPDase activity and adenosine production. Moreover, the increased reactivity to PHE promoted by Gd was endothelium-dependent, reducing NO bioavailability and involving an increased stimulation of angiotensin-converting enzyme and angiotensin II AT1 receptors.

Nucleoside triphosphate diphosphohydrolases role in the pathophysiology of cognitive impairment induced by seizure in early age.

Studies have shown that seizures in young animals lead to later cognitive deficits. There is evidence that long-term potentiation (LTP) and long-term depression (LTD) might contribute to the neural basis for learning and memory mechanism and might be modulated by ATP and/or its dephosphorylated product adenosine produced by a cascade of cell-surface transmembrane enzymes, such as E-NTPDases (ecto-nucleoside triphosphate diphosphohydrolases) and ecto-5'-nucleotidase. Thus, we have investigated if hippocampal ecto-nucleotidase activities are altered at different time periods after one episode of seizure induced by kainic acid (KA) in 7 days old rats. We also have evaluated if 90 day-old rats previously submitted to seizure induced by KA at 7 days of age presented cognitive impairment in Y-maze behavior task. Our results have shown memory impairment of adult rats (Postnatal day 90) previously submitted to one single seizure episode in neonatal period (Postnatal day 7), which is accompanied by an increased ATP hydrolysis in hippocampal synaptosomes. The metabolism of ATP evaluated by HPLC confirmed that ATP hydrolysis was faster in adult rats treated with KA in neonatal period than in controls. Surprisingly, the mRNA and protein levels as seen by PCR and Western blot, respectively, were not altered by the KA administration in early age. Since we have found an augmented hydrolysis of ATP and this nucleotide seems to be important to LTP induction, we could assume that impairment of memory and learning observed in adult rats which have experienced a convulsive episode in postnatal period may be a consequence of the increased ATP hydrolysis. These findings correlate the purinergic signaling to the cognitive deficits induced by neonatal seizures and contribute to a better understanding about the mechanisms of seizure-induced memory dysfunction.

NTPDase and 5' ecto-nucleotidase expression profiles and the pattern of extracellular ATP metabolism in the Walker 256 tumor.

In this study, we evaluated the NTPDases and ecto-5'-nucleotidase (CD73) expression profiles and the pattern of adenine nucleotide hydrolysis in rats submitted to the Walker 256 tumor model, 6, 10 and 15 days after the subcutaneous inoculation. Using RT-PCR analysis, we identified mRNA for all of the members of the ecto-nucleoside triphosphate diphosphohydrolase family investigated and a 5'-nucleotidase. By quantitative real-time PCR, Entpd1 (Cd39) and Entpd2 (Cd39L1) and CD73 were identified as the dominant genes expressed by the Walker 256 tumor, at all times studied. Extracellular adenine nucleotide hydrolysis by the Walker 256 tumor was estimated by HPLC analysis. Rapid hydrolysis of extracellular ATP by the tumor cells was observed, leading to the formation of adenosine and inosine in cells obtained from solid tumors at 6 and 10 days after inoculation. Cells obtained from solid tumors at 15 days of growth presented high levels of AMP and presented adenosine as a final product after 90 min of incubation. Results demonstrate that the presence of NTPDases and 5'-nucleotidase enzymes in Walker 256 tumor cells may be important for regulation of the extracellular adenine nucleotides/adenine nucleoside ratio, therefore leading to tumor growth.

Evaluation of the slopes of cognitive impairment and disability in Alzheimer's disease (AD) patients treated with acetylcholinesterase inhibitors (AChEl).

AD is characterized by a widespread cognitive impairment and deficit in functional competency to perform activities of daily living (ADL). The longitudinal reliability of cognitive and functional performance indices and the strength of relationship between patients cognitive impairment and their functional competence are still open issues. The aim of this study has been to evaluate, in a selected sample of patients with AD treated with AChEl, the slopes of cognitive impairment and disability. Among 249 AD patients, according to DSM-IV criteria, with presence/absence of associated vascular lesions (AD+VD), eligible for AChEl treatment, we selected subjects with mild to moderate cognitive impairment, without high comorbidity and severe psychiatric disease, with caregiver who resided with, or had frequent contact with the patient. Patients that changed treatment shifting from one AChEl to another, that didn't tolerate inhibitors (drop-out), and that presented behavioral and psychological symptoms of dementia (BPSD) requiring neuroleptic treatment during the study period were excluded from the final analysis. A sample of 99 subjects (30 males, 69 females; mean age of 79.4+/-5.0 years), completing a 15 months follow-up was considered. Cognitive performance remained stable after 15 months of treatment, but disability increased. No difference was found due to the AChEl compound used. The same hold true for the subgroups with presence/absence of a vascular components, whereas subgroup with mild cognitive performance showed a cognitive decline, parallel to the functional one. Our data underline the efficacy of AChEl in the treatment of AD with presence/absence of vascular component. Nevertheless, the judgement on the level of efficacy of AChEl could be biased by the level of reliability of the indices considered.

Ornithine decarboxylase activity during the development of Anastrepha fraterculus (Diptera, Tephritidae).

Ornithine decarboxylase (ODC) (EC 4.1.1.17) is very important for polyamine biosynthesis, which is required for main biological events. In the present study, ODC activity was measured in samples of Anastrepha fraterculus's egg, larva, pupa body and abdomen, adult body, ovaries, and fat body of young females, and in ovaries of mature flies. The kinetic parameters (Km app and Vmax) for ODC activity were determined for pupa, larva, and young ovary. ODC activity showed fluctuations during A. fraterculus's life development. In its earlier stages, prior to emergence, the egg has high ODC-specific activity probably due to embryogenesis, which is characterized by a high rate of cell division. This enzyme activity is also significantly high in the ovary and fat body of young females possibly related to the increased oogenesis and vitellogenesis. The kinetic parameters (Km app and Vmax) had great variation. Our results using GTP showed that the great variation in kinetic parameters can be accounted for by post-translational modifications.

Thyroid hormone upregulates ecto-5'-nucleotidase/CD73 in C6 rat glioma cells.

Thyroid hormones have profound effects on the central nervous system, such as proliferation, secretion of growth factors and gene expression regulation. Ecto-NTPDases and ecto-5'-nucleotidase can control the extracellular ATP/adenosine levels, which have been described as proliferation factors. Here, we investigated the influence of T(3) on the enzyme cascade which catalyzes interconversion of purine nucleotides in rat C6 glioma cells. Exposure of C6 cells to T(3) caused a dose dependent increase of 30% in the AMP hydrolysis up to 0.25 nM, which was suppressed by actinomycin. No significant alteration was observed on ATP/ADP hydrolysis and T(4) at higher concentrations (10-1000 nM) promoted an increase in AMP hydrolysis that was not dose dependent. T(3) treatment also increased the expression of CD73 mRNA. Besides the importance of the ecto-5'-NT in the cell proliferation and differentiation, its overexpression can enhance extracellular adenosine levels, which could also be an important proliferation signal.

Ectonucleotidase activities in Sertoli cells from immature rats.

Sertoli cells have been shown to be targets for extracellular purines such as ATP and adenosine. These purines evoke responses in Sertoli cells through two subtypes of purinoreceptors, P2Y2 and P A1. The signals to purinoreceptors are usually terminated by the action of ectonucleotidases. To demonstrate these enzymatic activities, we cultured rat Sertoli cells for four days and then used them for different assays. ATP, ADP and AMP hydrolysis was estimated by measuring the Pi released using a colorimetric method. Adenosine deaminase activity (EC 3.5.4.4) was determined by HPLC. The cells were not disrupted after 40 min of incubation and the enzymatic activities were considered to be ectocellularly localized. ATP and ADP hydrolysis was markedly increased by the addition of divalent cations to the reaction medium. A competition plot demonstrated that only one enzymatic site is responsible for the hydrolysis of ATP and ADP. This result indicates that the enzyme that acts on the degradation of tri- and diphosphate nucleosides on the surface of Sertoli cells is a true ATP diphosphohydrolase (EC 3.6.1.5) (specific activities of 113 +/- 6 and 21 +/- 2 nmol Pi mg(-1) min(-1) for ATP and ADP, respectively). The ecto-5'-nucleotidase (EC 3.1.3.5) and ectoadenosine deaminase activities (specific activities of 32 +/- 2 nmol Pi mg(-1) min(-1) for AMP and 1.52 +/- 0.13 nmol adenosine mg(-1) min(-1), respectively) were shown to be able to terminate the effects of purines and may be relevant for the physiological control of extracellular levels of nucleotides and nucleosides inside the seminiferous tubules.

Ectonucleotidase activities in Sertoli cells from immature rats

Sertoli cells have been shown to be targets for extracellular purines such as ATP and adenosine. These purines evoke responses in Sertoli cells through two subtypes of purinoreceptors, P2Y2 and P A1. The signals to purinoreceptors are usually terminated by the action of ectonucleotidases. To demonstrate these enzymatic activities, we cultured rat Sertoli cells for four days and then used them for different assays. ATP, ADP and AMP hydrolysis was estimated by measuring the Pi released using a colorimetric method. Adenosine deaminase activity (EC 3.5.4.4) was determined by HPLC. The cells were not disrupted after 40 min of incubation and the enzymatic activities were considered to be ectocellularly localized. ATP and ADP hydrolysis was markedly increased by the addition of divalent cations to the reaction medium. A competition plot demonstrated that only one enzymatic site is responsible for the hydrolysis of ATP and ADP. This result indicates that the enzyme that acts on the degradation of tri- and diphosphate nucleosides on the surface of Sertoli cells is a true ATP diphosphohydrolase (EC 3.6.1.5) (specific activities of 113 + or - 6 and 21 + or - 2 nmol Pi mg-1 min-1 for ATP and ADP, respectively). The ecto-5'-nucleotidase (EC 3.1.3.5) and ectoadenosine deaminase activities (specific activities of 32 + or - 2 nmol Pi mg-1 min-1 for AMP and 1.52 + or - 0.13 nmol adenosine mg-1 min-1, respectively) were shown to be able to terminate the effects of purines and may be relevant for the physiological control of extracellular levels of nucleotides and nucleosides inside the seminiferous tubules

Influence of the biomatrix on the response of Sertoli cells to FSH.

Sertoli cell preparations isolated from 15-day-old Wistar rats were cultured on two different substrates, i.e., plastic and a biomatrix isolated from seminiferous tubules of rat testis. Sertoli cells cultured on a biomatrix acquired a phenotype and morphology more characteristic of in vivo differentiated cells. In order to determine the influence of a biomatrix on the response of Sertoli cells to FSH, on the 7th day of culture, untreated cells, or cells pretreated for 12 h with FSH (1 microgram/ml), were incubated with [U-14C] leucine or [2-3H] mannose. Cells cultured on the biomatrix showed higher [U-14C] leucine and [2-3H] mannose incorporation into proteins and glycoproteins. FSH increased these activities in cells cultured on both substrates, although its stimulating effect was higher on cells cultured on the biomatrix. These results demonstrate that the biomatrix increases protein and glycoprotein synthesis and secretion, and also influences the response of Sertoli cells to FSH.

Hydroxystearic acid effects on CDC2/histone H1 kinase activity in C108 carcinoma cells.

HSA at appropriate concentrations shows cytostatic and/or cytotoxic effects on murine Lewis carcinoma cell line C108. The cytostatic effect is mediated by an arrest in the cell cycle machinery, with accumulation of cells in G2-M. The combination of enzymatic assays, cell cycle kinetics studies and immunoprecipitation shows that HSA causes to a certainty an accumulation of cells in the M phase, while a similar effect in G2 has still to be demonstrated. It also inhibits histone H1 kinase activity up to 95% of that of mitotic cells, having as a direct or indirect target the cdc2 complex.

In vitro effects of hydroxystearic acid on the proliferation of HT29 and I407 cells.

The in vitro effects of hydroxystearic acid on the proliferation of human colon carcinoma cells (HT29) and human embryonic intestine cells (I407) were examined and compared to previous results obtained in murine C108 lung carcinoma cells. The cells were cultured in the presence, or in the absence, of hydroxystearic acid and tested for cell proliferation and viability; the distribution of cells in the cell cycle was evaluated by flow cytometry. Results show that hydroxystearic acid is also an inhibitor of human cell proliferation, and not only of murine C108 cells. Differently from C108 cells, which upon treatment with hydroxystearic acid accumulate in G2-M phases, hydroxystearic acid-treated HT29 cells increase significantly in numbers in G0-G1; I407, embryonic cells used as a control, when treated show only a slight increase in G0-G1.

[Variations of blood flow in pulmonary veins caused by electric stimulation in total atrioventricular block]. / Variazioni del flusso nelle vene polmonari da stimolazione elettrica nel blocco atrioventricolare totale.

It was suggested that Doppler study of pulmonary venous flow (PVF) in combination with mitral flow is useful to evaluate ventricular function. In order to investigate the utility of PVF study in patients with total A-V block treated with pacemaker DDD, we performed transesophageal echocardiography pulsed Doppler of PVF and mitral flow in 12 patients. We measured the following parameters in PVF: peak systolic velocities (S1 and S2), peak diastolic velocity (D) reversal atrial flow diastolic deceleration time. In mitral flow we measured: E wave, A wave and deceleration time of E. We compared our results with the same patients with pacemaker inhibited. In patients with total A-V block with pacemaker inhibited we reported one systolic wave of PVF, a larger D wave with a shortened deceleration time. The PVA seems to disappear. Transmitral flow slowed a higher E wave in patients A-V block with pacemaker inhibited. In patients with total A-V block stimulated by DDD pacemaker we restored the atrioventricular sequentialty; therefore electric stimulation determines the onset of S1 peak and the reduction of S2 peak and the systematic presence of the atrial-pulmonary regurgitant wave.

[Transesophageal echocardiography in the assessment of patients with atrial fibrillation and stroke]. / L'ecocardiografia transesofagea nella valutazione dei pazienti con fibrillazione atriale e stroke.

Cardiac evaluation of stroke or transient ischemic attack (TIA) patients includes transthoracic and transesophageal echocardiography to identify potential cardiac source of embolism. In the present study transesophageal echocardiography was performed in 53 consecutive patients with non valvular atrial fibrillation and a previous embolic stroke. All patients were referred to our department because of an episode of atrial fibrillation. They all underwent computed tomography (CT) scan: 31 patients had a positive scan whereas 22 had a negative scan with a positive history of TIA diagnosed from a neurologist during hospitalization. In the group of 31 patients with a positive scan 9 patients with no history of a previous ischemic attack were found. The transesophageal echocardiography was performed in all the study subjects: 7 patients had a thrombus in the left atrial appendage, 2 patients had a thrombus in the right atrium. Spontaneous echo contrast was reported in 15 patients. Evaluating the interatrial septum we observed a patent foramen ovale in 4 patients, a defect in 2 patients and a fossa ovalis aneurysm in 3 patients. The mitral valve analysis showed a leaflet prolapse in 3 patients. The entire thoracic aorta was imaged in each patient: in 12 an abnormal atherosclerotic plaque was found. Transesophageal echocardiography is an important component of the comprehensive evaluation of potential sources of embolism in patients with ischemic cerebral attack and nonrheumatic atrial fibrillation.