Participation of Actin on Giardia lamblia Growth and Encystation

BACKGROUND:Microfilaments play a determinant role in different cell processes such as: motility, cell division, phagocytosis and intracellular transport; however, these structures are poorly understood in the parasite Giardia lamblia. METHODOLOGY AND PRINCIPAL FINDINGS:By confocal microscopy using TRITC-phalloidin, we found structured actin distributed in the entire trophozoite, the label stand out at the ventral disc, median body, flagella and around the nuclei. During Giardia encystation, a sequence of morphological changes concurrent to modifications on the distribution of structured actin and in the expression of actin mRNA were observed. To elucidate whether actin participates actively on growth and encystation, cells were treated with Cytochalasin D, Latrunculin A and Jasplakinolide and analyzed by confocal and scanning electron microscopy. All drugs caused a growth reduction (27 to 45%) and changes on the distribution of actin. Besides, 60 to 80% of trophozoites treated with the drugs, exhibited damage at the caudal region, alterations in the flagella and wrinkles-like on the plasma membrane. The drugs also altered the cyst-yield and the morphology, scanning electron microscopy revealed diminished cytokinesis, cysts with damages in the wall and alterations in the size and on the intermembranal space. Furthermore, the drugs caused a significant reduction of the intensity of fluorescence-labeled CWP1 on ESV and on cyst wall, this was coincident with a reduction of CWP1 gene expression (34%). CONCLUSIONS AND SIGNIFICANCE:All our results, indicated an important role of actin in the morphology, growth and encystation and indirectly suggested an actin role in gene expression

Rab11 and Actin Cytoskeleton Participate in Giardia lamblia Encystation, Guiding the Specific Vesicles to the Cyst Wall

The encystation process is crucial for survival and transmission of Giardia lamblia to new hosts. During this process, vesicular trafficking and the cytoskeleton play important roles. In eukaryotic cells, intracellular transport is regulated by proteins, including Rab-GTPases and SNAREs, which regulate vesicle formation along with recognition of and binding to the target membrane. Cytoskeletal structures are also involved in these processes. In this study, we demonstrate the participation of Rab11 in the transport of encystation-specific vesicles (ESVs). Additionally, we demonstrate that disruption of actin microfilaments affects ESVs transport. The modification of actin dynamics was also correlated with a reduction in rab11 and cwp1 expression. Furthermore, down-regulation of rab11 mRNA by a specific hammerhead ribozyme caused nonspecific localization of CWP1. We thus provide new information about the molecular machinery that regulates Giardia lamblia encystation. Given our findings, Rab11 and actin may be useful targets to block Giardia encystation

Design and baseline characteristics of the finerenone in reducing cardiovascular mortality and morbidity in diabetic kidney disease trial

Background: Among people with diabetes, those with kidney disease have exceptionally high rates of cardiovascular (CV) morbidity and mortality and progression of their underlying kidney disease. Finerenone is a novel, nonsteroidal, selective mineralocorticoid receptor antagonist that has shown to reduce albuminuria in type 2 diabetes (T2D) patients with chronic kidney disease (CKD) while revealing only a low risk of hyperkalemia. However, the effect of finerenone on CV and renal outcomes has not yet been investigated in long-term trials. Patients and Methods: The Finerenone in Reducing CV Mortality and Morbidity in Diabetic Kidney Disease (FIGARO-DKD) trial aims to assess the efficacy and safety of finerenone compared to placebo at reducing clinically important CV and renal outcomes in T2D patients with CKD. FIGARO-DKD is a randomized, double-blind, placebo-controlled, parallel-group, event-driven trial running in 47 countries with an expected duration of approximately 6 years. FIGARO-DKD randomized 7,437 patients with an estimated glomerular filtration rate >= 25 mL/min/1.73 m(2) and albuminuria (urinary albumin-to-creatinine ratio >= 30 to <= 5,000 mg/g). The study has at least 90% power to detect a 20% reduction in the risk of the primary outcome (overall two-sided significance level alpha = 0.05), the composite of time to first occurrence of CV death, nonfatal myocardial infarction, nonfatal stroke, or hospitalization for heart failure. Conclusions: FIGARO-DKD will determine whether an optimally treated cohort of T2D patients with CKD at high risk of CV and renal events will experience cardiorenal benefits with the addition of finerenone to their treatment regimen. Trial Registration: EudraCT number: 2015-000950-39; ClinicalTrials.gov identifier: NCT02545049

Primary explants of the postnatal thymus allow the expansion of clonogenic thymic epithelial cells that constitute thymospheres

Abstract Background Thymic epithelial cells (TECs) are responsible for shaping the repertoires of T cells, where their postnatal regeneration depends on a subset of clonogenic TECs. Despite the implications for regenerative medicine, their cultivation and expansion remain challenging. Primary explant cell culture is a technique that allows the seeding and expansion of difficult-to-culture cells. Here, we report a reliable and simple culture system to obtain functional TECs and thymic interstitial cells (TICs). Methods To establish primary thymic explants, we harvested 1 mm cleaned fragments of thymus from 5-week-old C57/BL6 mice. Tissue fragments of a complete thymic lobe were placed in the center of a Petri dish with 1 mL of DMEM/F-12 medium supplemented with 20% fetal bovine serum (FBS) and 1% penicillin‒streptomycin. To compare, thymic explants were also cultivated by using serum-free DMEM/F-12 medium supplemented with 10% KnockOut™. Results We obtained high numbers of functional clonogenic TECs and TICs from primary thymic explants cultivated with DMEM/F-12 with 20% FBS. These cells exhibited a highly proliferative and migration profile and were able to constitute thymospheres. Furthermore, all the subtypes of medullary TECs were identified in this system. They express functional markers to shape T-cell and type 2 innate lymphoid cells repertoires, such as Aire, IL25, CCL21 and CD80. Finally, we also found that ≥ 70% of lineage negative TICs expressed high amounts of Aire and IL25. Conclusion Thymic explants are an efficient method to obtain functional clonogenic TECs, all mTEC subsets and different TICs Aire+IL25+ with high regenerative capacity

Video Supplementary Figure 7

Thymic epithelial cells (TECs) are responsible for shaping the repertoires of T cells, where their postnatal regeneration depends on a subset of clonogenic TECs. Despite the implications for regenerative medicine, their cultivation and expansion remain challenging. Primary explant cell culture is a technique that allows the seeding and expansion of difficult-to-culture cells. Here, we report a reliable and simple culture system to obtain functional TECs and thymic interstitial cells (TICs).</p

Odanacatib for the treatment of postmenopausal osteoporosis : Results of the LOFT multicentre, randomised, double-blind, placebo-controlled trial and LOFT Extension study

Background Odanacatib, a cathepsin K inhibitor, reduces bone resorption while maintaining bone formation. Previous work has shown that odanacatib increases bone mineral density in postmenopausal women with low bone mass. We aimed to investigate the efficacy and safety of odanacatib to reduce fracture risk in postmenopausal women with osteoporosis. Methods The Long-term Odanacatib Fracture Trial (LOFT) was a multicentre, randomised, double-blind, placebo-controlled, event-driven study at 388 outpatient clinics in 40 countries. Eligible participants were women aged at least 65 years who were postmenopausal for 5 years or more, with a femoral neck or total hip bone mineral density T-score between −2·5 and −4·0 if no previous radiographic vertebral fracture, or between −1·5 and −4·0 with a previous vertebral fracture. Women with a previous hip fracture, more than one vertebral fracture, or a T-score of less than −4·0 at the total hip or femoral neck were not eligible unless they were unable or unwilling to use approved osteoporosis treatment. Participants were randomly assigned (1:1) to either oral odanacatib (50 mg once per week) or matching placebo. Randomisation was done using an interactive voice recognition system after stratification for previous radiographic vertebral fracture, and treatment was masked to study participants, investigators and their staff, and sponsor personnel. If the study completed before 5 years of double-blind treatment, consenting participants could enrol in a double-blind extension study (LOFT Extension), continuing their original treatment assignment for up to 5 years from randomisation. Primary endpoints were incidence of vertebral fractures as assessed using radiographs collected at baseline, 6 and 12 months, yearly, and at final study visit in participants for whom evaluable radiograph images were available at baseline and at least one other timepoint, and hip and non-vertebral fractures adjudicated as being a result of osteoporosis as assessed by clinical history and radiograph. Safety was assessed in participants who received at least one dose of study drug. The adjudicated cardiovascular safety endpoints were a composite of cardiovascular death, myocardial infarction, or stroke, and new-onset atrial fibrillation or flutter. Individual cardiovascular endpoints and death were also assessed. LOFT and LOFT Extension are registered with ClinicalTrials.gov (number NCT00529373) and the European Clinical Trials Database (EudraCT number 2007-002693-66). Findings Between Sept 14, 2007, and Nov 17, 2009, we randomly assigned 16 071 evaluable patients to treatment: 8043 to odanacatib and 8028 to placebo. After a median follow-up of 36·5 months (IQR 34·43–40·15) 4297 women assigned to odanacatib and 3960 assigned to placebo enrolled in LOFT Extension (total median follow-up 47·6 months, IQR 35·45–60·06). In LOFT, cumulative incidence of primary outcomes for odanacatib versus placebo were: radiographic vertebral fractures 3·7% (251/6770) versus 7·8% (542/6910), hazard ratio (HR) 0·46, 95% CI 0·40–0·53; hip fractures 0·8% (65/8043) versus 1·6% (125/8028), 0·53, 0·39–0·71; non-vertebral fractures 5·1% (412/8043) versus 6·7% (541/8028), 0·77, 0·68–0·87; all p<0·0001. Combined results from LOFT plus LOFT Extension for cumulative incidence of primary outcomes for odanacatib versus placebo were: radiographic vertebral fractures 4·9% (341/6909) versus 9·6% (675/7011), HR 0·48, 95% CI 0·42–0·55; hip fractures 1·1% (86/8043) versus 2·0% (162/8028), 0·52, 0·40–0·67; non-vertebral fractures 6·4% (512/8043) versus 8·4% (675/8028), 0·74, 0·66–0·83; all p<0·0001. In LOFT, the composite cardiovascular endpoint of cardiovascular death, myocardial infarction, or stroke occurred in 273 (3·4%) of 8043 patients in the odanacatib group versus 245 (3·1%) of 8028 in the placebo group (HR 1·12, 95% CI 0·95–1·34; p=0·18). New-onset atrial fibrillation or flutter occurred in 112 (1·4%) of 8043 patients in the odanacatib group versus 96 (1·2%) of 8028 in the placebo group (HR 1·18, 0·90–1·55; p=0·24). Odanacatib was associated with an increased risk of stroke (1·7% [136/8043] vs 1·3% [104/8028], HR 1·32, 1·02–1·70; p=0·034), but not myocardial infarction (0·7% [60/8043] vs 0·9% [74/8028], HR 0·82, 0·58–1·15; p=0·26). The HR for all-cause mortality was 1·13 (5·0% [401/8043] vs 4·4% [356/8028], 0·98–1·30; p=0·10). When data from LOFT Extension were included, the composite of cardiovascular death, myocardial infarction, or stroke occurred in significantly more patients in the odanacatib group than in the placebo group (401 [5·0%] of 8043 vs 343 [4·3%] of 8028, HR 1·17, 1·02–1·36; p=0·029, as did stroke (2·3% [187/8043] vs 1·7% [137/8028], HR 1·37, 1·10–1·71; p=0·0051). Interpretation Odanacatib reduced the risk of fracture, but was associated with an increased risk of cardiovascular events, specifically stroke, in postmenopausal women with osteoporosis. Based on the overall balance between benefit and risk, the study's sponsor decided that they would no longer pursue development of odanacatib for treatment of osteoporosis