Therapeutic efficacy of posaconazole against isolates of Candida albicans with different susceptibilities to fluconazole in a vaginal model.

A battery of 34 vaginal isolates of Candida albicans was tested against posaconazole (POS) and fluconazole (FLU) to determine their in vitro susceptibilities and to obtain FLU-susceptible and FLU-resistant strains for the murine in vivo studies. FLU-resistant strains were chosen on the basis of their 48-h MICs. The 48-h geometric mean MICs for all isolates tested were 0.016 and 0.656 microg/ml for POS and FLU, respectively. The treatment regimens for the vaginal murine infection model were POS or FLU at 10 or 20 mg/kg of body weight/day and 20 mg/kg twice a day. All regimens with POS were effective in reducing fungal burden of both the fluconazole-susceptible and resistant isolates of C. albicans. All FLU regimens were effective against infection induced by the fluconazole-susceptible strain. While FLU at 10 mg/kg was ineffective against fungal burden of the resistant strain, treatment with FLU at 20 mg/kg once or twice a day was effective against this strain. Both POS and FLU at 20 mg/kg twice a day were able to clear C. albicans from vaginas of mice infected with the fluconazole-susceptible strain. POS displayed a more effective in vivo activity than FLU in the treatment of murine C. albicans vaginitis produced by isolates with different susceptibilities to FLU.     

Gain-of-function gene mutations and venous thromboembolism: distinct roles in different clinical settings

Objective

To calculate the prevalence of common gain of function gene mutations in patients with different clinical manifestations of venous thromboembolism.

Design and setting

Case–control study in two hospitals in Italy.

Participants

387 patients with venous thromboembolism and 286 controls.

Main measures

Factor V (FV) Leiden, factor II (FII) A20210 and JAK2 V617F mutations.

Results

Among patients with deep vein thrombosis in one leg, 23 (20.9%) carried FV Leiden and FII A20210 mutations. Similar figures were observed in patients with cerebral vein thrombosis (CVT; n = 9; 20.0%) and in patients presenting with splanchnic vein thrombosis (SVT; n = 26; 18.7%). A lower prevalence was obtained in patients with retinal vein thrombosis (n = 11; 11.8%). The JAK2 F617 mutant allele was found in 27 (21.1%) patients with SVT, but in none of the patients presenting with a thrombotic event from different districts. 13 of the 27 JAK2 V617F‐positive subjects with SVT were previously known to have a myeloproliferative disease (MPD). Three other patients had a diagnosis of MPD after the occurrence of the thrombotic event.

Conclusion

Carriership of FV Leiden or FII A20210 mutations identifies an at‐risk condition for venous thrombosis in the lower extremities, SVT or CVT. In patients with SVT, screening for the JAK2 V617F mutation may be useful in recognising patients who should be carefully observed for the subsequent development of overt MPD. Thus, genetic tests may play a different role, various clinical manifestations of venous thromboembolism being associated with distinct risk profiles.Venous thrombosis is the third most common cardiovascular affliction after ischaemic heart disease and stroke.¹ It is common in Caucasians, affecting 1 in 1000 individuals every year, and is strongly associated with life‐threatening pulmonary embolism. The pathogenesis of venous thrombosis is multifactorial, involving acquired and genetic factors. In addition to circumstantial predisposing factors (eg, surgery, pregnancy, immobilisation and malignancy), genetic predisposition due to molecular abnormalities of components of the coagulation pathway have been found in subjects who had had thromboembolic disease.² Abnormalities within the gene loci encoding for natural anticoagulants (antithrombin, protein C and protein S) and for fibrinogen have been shown to be rather uncommon risk factors for venous thrombosis.³ In patients of European ancestry, common gain‐of‐function mutations within the gene of the coagulation factor V (FV Leiden mutation) and the factor II (FII) gene (a G→A transition at nucleotide position 20210) have been shown to account for a large number of cases of thromboembolism.^2,3 Recently, the JAK2 V617F mutation, an acquired somatic event occurring in most patients with polycythaemia vera (PV) and in about half of the patients with essential thrombocythaemia (ET) or myelofibrosis (MF),^4,5,6,7,8 has been found in a high proportion of patients with Budd–Chiari syndrome⁹ and in patients without cirrhosis with portal and mesenteric venous thrombosis, a heterogeneous group of disorders.¹⁰Thus, the high frequency of common mutations in patients presenting with venous thromboembolism raised the hypothesis that it can be considered as a feasible and practical approach for the identification of predisposing genetic risk factors. Genetic test results can better inform individuals about their risk of recurrence and appropriated tailored strategies for the prevention of venous thrombosis.However, depending on the different clinical manifestations of venous thromboembolism, the prevalence of inherited coagulation abnormalities varies, suggesting pathogenic differences.^11,12,13 These data support the hypothesis that mechanistic differences are involved in the pathogenesis of thrombosis in different clinical settings. Thus, before considering whether it is advisable to investigate a subject for inherited risk factors, we need to know the prevalence of these risk factors in different clinical settings.We, therefore, calculated the prevalence of inherited thrombophilic risk factors in a large cohort of patients referred for a thrombophilic investigation with different clinical manifestations of venous thromboembolism.     

Statins inhibit T-acute lymphoblastic leukemia cell adhesion and migration through Rap1b

Statins are known to inhibit signaling of Ras superfamily GTPases and reduce T cell adhesion to ICAM-1. Here, we address the hypothesis that statins affect T cell adhesion and migration by modulating the function of specific GTPases. Statins inhibit the synthesis of mevalonic acid, which is required for farnesyl and geranylgeranyl isoprenoid synthesis. Ras superfamily GTPases are post-translationally isoprenylated to facilitate their anchorage to membranes, where they function to stimulate signal transduction processes. We demonstrate that 1 μM statin inhibits the adhesion, migration, and chemotaxis of the T-ALL cell line CCRF-CEM and TEM of CCRF-CEM and PEER T-ALL cells, but higher statin concentrations are needed to inhibit adhesion of primary T cells. Similar effects are observed following treatment with GGTI-298 or RNA interference-mediated knockdown of Rap1b but not Rap1a, Rac1, Rac2, RhoA, or Cdc42. Statins also alter Rap1 activity and Rap1b localization. Rap1 levels are higher in primary T cells than T-ALL cells, which could explain their reduced sensitivity to statins. These results demonstrate for the first time that the closely related Rap1a and Rap1b isoforms have different functions and suggest that statins or Rap1b depletion could be used to reduce tissue invasion in T-ALL.     

The use of home remedies and complementary health approaches in endometriosis

Research question

Conventional treatments are often associated with adverse effects and endometriosis pain symptoms may reoccur despite treatment. Consequently, many women use complementary health approaches (CHA) and home remedies (HR) to relieve their pain. The aim of this study was to examine the frequency and the subjectively perceived efficacy of CHA/HR use by women affected by endometriosis.

High-throughput genetic screening using matrix-assisted laser desorption/ionization mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has become a powerful and widespread analytical tool in all fields of life science. Compared with other techniques, its high accuracy and sensitivity makes it a superior method, especially for the analysis of nucleic acids. Recent problems in the analysis of nucleic acids by MALDI-TOF MS can be solved using an automated MALDI-compatible sample-preparation system. Together with the reliable minisequencing assay, high-throughput genotyping of single nucleotide polymorphisms by MALDI-TOF MS is able to become a routine method in research, clinical genetics and diagnostics.     

Ex vivo localization of the mouse saposin C activation region for acid beta-glucosidase

Saposin C is a biological activator of acid beta-glucosidase (GCase), the lysosomal hydrolase with activity towards glucosylceramide (GC). In addition, saposin C possesses a functional domain that determines the in vitro and ex vivo neuritogenic effects of prosaposin, the precursor of saposins A, B, C, and D. The domains for enzymatic activation and neuritogenic function segregate in vitro, respectively, to the carboxyl- and amino-terminal halves of human and mouse saposin C. A chimeric mouse saposin C(1-8)B(8-28)C(30-80) was created to obliterate the neuritogenic region by substituting amino acids 9-29 of saposin C with amino acids 8-28 of saposin B. This saposin showed normal in vitro enzymatic activation effects toward GCase, but no neuritogenic activity. An altered prosaposin was made to contain the chimeric saposin C region. Expression of this altered or wild-type prosaposin was driven by the PGK-1 promoter as a transgene in prosaposin knock-out mice. In cultured fibroblasts from such mice, expressed saposins localized to the lysosomal compartments. Metabolic lipid labeling using L-[3-(14)C]serine showed retention or clearance of GC in prosaposin deficient or transgene reconstituted cells, respectively. In addition, sulfatide catabolism, that requires saposin B and arylsulfatase, was also normalized in prosaposin KO cells reconstituted with the transgenes. These data show that the transgenic prosaposins were expressed and processed to functional saposins in fibroblasts. These results also show that the enzymatic activation domain is located at carboxyl-terminal half of saposin C and functions only in the context of the general saposin structure.     

The structure of the conus arteriosus of the sturgeon (Acipenser naccarii) heart: II. The myocardium,the subepicardium,and the conus-aorta transition

Sturgeons constitute a family of living "fossil" fish whose heart is related to that of other ancient fish and the elasmobranches. We have undertaken a systematic study of the structure of the sturgeon heart aimed at unraveling the relationship between the heart structure and the adaptive evolutionary changes. In a related paper, data were presented on the conus valves and the subendocardium. Here, the structure of the conus myocardium, the subepicardial tissue, and the conus-aorta transition were studied by conventional light, transmission, and scanning electron microscopy. In addition, actin localization by fluorescent phalloidin was used. The conus myocardium is organized into bundles whose spatial organization changes along the conus length. The variable orientation of the myocardial cell bundles may be effective in emptying the conus lumen during contraction and in preventing reflux of blood. Myocardial cell bundles are separated by loose connective tissue that contains collagen and elastin fibers, vessels, and extremely flat cells separating the cell bundles and enclosing blood vessels and collagen fibers. The ultrastructure of the myocardial cells was found to be very similar to that of other fish groups, suggesting that it is largely conservative. The subepicardium is characterized by the presence of nodular structures that contain lympho-hemopoietic (thymus-like) tissue in the young sturgeons and a large number of lymphocytes after the sturgeons reach sexual maturity. This tissue is likely implicated in the establishment and maintenance of the immune responses. The intrapericardial ventral aorta shows a middle layer of circumferentially oriented cells and internal and external layers with cells oriented longitudinally. Elastin fibers completely surround each smooth muscle cell, and the spaces between the different layers are occupied by randomly arranged collagen bundles. The intrapericardial segment of the ventral aorta is a true transitional segment whose structural characteristics are different from those of both the conus subendocardium and the rest of the ventral aorta.     

Inducible and endothelial nitric oxide synthase expression in human atherogenesis: an immunohistochemical and ultrastructural study

BackgroundNitric oxide has been proven to play an important role in the maintenance of vascular tone and structure. Impairment of nitric oxide production is an early indicator of atherosclerosis, but not much is known about the real mechanisms underlying this phenomenon.MethodsIn the present study, immunocytochemical methods have been used to analyze the patterns of expression of endothelial nitric oxide synthase and inducible nitric oxide synthase proteins in healthy and atherosclerotic human aortae using both confocal laser scanning microscopy and electron microscopy.ResultsInduction of the expression of endothelial nitric oxide synthase and inducible nitric oxide synthase proteins was observed in smooth muscle cells of atherosclerotic human aortae. Altered nitric oxide synthase expression was reported in atheromatous plaques and in apparently normal vascular tissues adjacent to the lesions.ConclusionsOur data confirm and extend previous findings of a direct relationship between dysregulation of nitric oxide pathway and atherosclerosis, suggesting another possible mechanism by which nitric oxide synthase system abnormalities may promote vascular dysfunction during human atherogenesis. Changes in nitric oxide production might be the primary step in the development of atheroma.