Association of apo A-IV 360 (Gln --> His) polymorphism with plasma lipids and lipoproteins: the Framingham Offspring Study

The effect of a common apolipoprotein (apo) A-IV polymorphism (substitution of histidine for glutamine at position 360) on plasma lipid, lipoprotein cholesterol and lipoprotein(a) (Lp(a)) levels, and on low-density lipoprotein (LDL) particle size was examined by genotyping in 2322 Caucasian men and women (mean age: 48.9+/-10.1 years) participating in the Framingham Offspring Study (FOS). The relative frequencies of the apo A-IV-Gln (apo A-IV-1) and the apo A-IV-His (apo A-IV-2) alleles were 0.932 and 0.068, respectively, and were in Hardy-Weinberg equilibrium. No effect of the apo A-IV-2 genotype was observed on plasma triglyceride, total and lipoprotein cholesterol, and LDL particle size in either men or women after adjustment for age and body mass index. To avoid a possible interaction between the apo E genotype and the apo A-IV genotype, subgroup analyses were undertaken in 1,414 male and female subjects with the apo E3/3 genotype. Among women in this group there was a significant effect of the apo A-IV-2 allele on triglyceride levels (p=0.046). This effect was no longer significant after adjustment for age and BMI (p=0.074). No significant allele effect on other lipoprotein levels, including Lp(a), was noted in apo E3/3 men or women. We have also conducted a meta-analysis of our own data and of other studies found in the literature, indicating a significant lowering effect of apo A-IV-2 on plasma triglycerides, but no effects on other parameters. In conclusion, the apo A-IV-2 allele is associated with a modest reduction in plasma triglyceride levels in the general population.     

Membrane metabolism mediated by Sec14 family members influences Arf GTPase activating protein activity for transport from the trans-Golgi

The budding yeast Saccharomyces cerevisiae contains a family of Arf (ADP-ribosylation factor) GTPase activating protein (GAP) proteins with the Gcs1 + Age2 ArfGAP pair providing essential overlapping function for the movement of transport vesicles from the trans-Golgi network. We have generated a temperature-sensitive but stable version of the Gcs1 protein that is impaired only for trans-Golgi transport and find that deleterious effects of this enfeebled Gcs1-4 mutant protein are relieved by increased gene dosage of the gcs1-4 mutant gene itself or by the SFH2 gene (also called CSR1), encoding a phosphatidylinositol transfer protein (PITP). This effect was not seen for the SEC14 gene, encoding the founding member of the yeast PITP protein family, even though the Gcs1 and Age2 ArfGAPs are known to be downstream effectors of Sec14-mediated activity for trans-Golgi transport. Sfh2-mediated suppression of inadequate Gcs1-4 function depended on phospholipase D, whereas inadequate Gcs1-4 activity was relieved by increasing levels of diacylglycerol (DAG). Recombinant Gcs1 protein was found to bind certain phospholipids but not DAG. Our findings favor a model of Gcs1 localization through binding to specific phospholipids and activation of ArfGAP activity by DAG-mediated membrane curvature as the transport vesicle is formed. Thus, ArfGAPs are subject to both temporal and spatial regulation that is facilitated by Sfh2-mediated modulation of the lipid environment.     

The effect of temperature and host age on the infectivity and development of Angiostrongylus vasorum in the slug Arion lusitanicus

Experimental infection with Angiostrongylus vasorum was conducted in Iberian slugs Arion lusitanicus. Initially, different size/age groups of juvenile slugs (small, <0.5 g and medium, 0.5–1 g) were exposed to freshly isolated first-stage parasitic larvae (L1) of A. vasorum. The slugs were subsequently incubated at 5, 10 and 15°C for 6 weeks. Larval development within the slugs differed significantly with temperature. At 15°C, all larvae developed into the third larval stage (L3), at 10°C into the second stage (L2), whereas no development was observed at 5°C. The mean larval burdens were highest in the largest group of slugs and tended to increase with higher temperature. In a second experiment isolated L1 were incubated at 5, 10 and 15°C for 3 and 7 days prior to infection of slugs, which then were kept for 6 weeks at 15°C. The infectivity decreased significantly with the larval storage time and the mean larval burden per slug was lower at higher incubating temperature. However, all established larvae developed into infective L3. Temperature had an effect on the development of the larvae and thus an impact on transmission of the parasite as only L3 are infective to the definitive canid hosts.     

Exogenous interleukin‐33 targets myeloid‐derived suppressor cells and generates periphery‐induced Foxp3+ regulatory T cells in skin‐transplanted mice

Interleukin-33 (IL-33) has been a focus of study because of its variety of functions shaping CD4⁺ T-cell biology. In the present work, we evaluated the modulatory effect of IL-33 on suppressor cells in an in vivo transplantation model. C57BL/6 wild-type mice were grafted with syngeneic or allogeneic skin transplants and treated with exogenous IL-33 daily. After 10 days of treatment, we analysed draining lymph node cellularity and found in allogeneic animals an increment in myeloid-derived suppressor cells, which co-express MHC-II, and become enriched upon IL-33 treatment. In line with this observation, inducible nitric oxide synthase and arginase 1 expression were also increased in allogeneic animals upon IL-33 administration. In addition, IL-33 treatment up-regulated the number of Foxp3⁺ regulatory T (Treg) cells in the allogeneic group, complementing the healthier integrity of the allografts and the increased allograft survival. Moreover, we demonstrate that IL-33 promotes CD4⁺ T-cell expansion and conversion of CD4⁺ Foxp3⁻ T cells into CD4⁺ Foxp3⁺ Treg cells in the periphery. Lastly, the cytokine pattern of ex vivo-stimulated draining lymph nodes indicates that IL-33 dampens interferon-γ and IL-17 production, stimulating IL-10 secretion. Altogether, our work complements previous studies on the immune-modulatory activity of IL-33, showing that this cytokine affects myeloid-derived suppressor cells at the cell number and gene expression levels. More importantly, our research demonstrates for the first time that IL-33 allows for in vivo Foxp3⁺ Treg cell conversion and favours an anti-inflammatory or tolerogenic state by skewing cytokine production. Therefore, our data suggest a potential use of IL-33 to prevent allograft rejection, bringing new therapeutics to the transplantation field.     

Direct three-dimensional visualization of membrane disruption by amyloid fibrils

Protein misfolding and aggregation cause serious degenerative conditions such as Alzheimer’s, Parkinson, and prion diseases. Damage to membranes is thought to be one of the mechanisms underlying cellular toxicity of a range of amyloid assemblies. Previous studies have indicated that amyloid fibrils can cause membrane leakage and elicit cellular damage, and these effects are enhanced by fragmentation of the fibrils. Here we report direct 3D visualization of membrane damage by specific interactions of a lipid bilayer with amyloid-like fibrils formed in vitro from β₂-microglobulin (β₂m). Using cryoelectron tomography, we demonstrate that fragmented β₂m amyloid fibrils interact strongly with liposomes and cause distortions to the membranes. The normally spherical liposomes form pointed teardrop-like shapes with the fibril ends seen in proximity to the pointed regions on the membranes. Moreover, the tomograms indicated that the fibrils extract lipid from the membranes at these points of distortion by removal or blebbing of the outer membrane leaflet. Tiny (15–25 nm) vesicles, presumably formed from the extracted lipids, were observed to be decorating the fibrils. The findings highlight a potential role of fibrils, and particularly fibril ends, in amyloid pathology, and report a previously undescribed class of lipid–protein interactions in membrane remodelling.The failure of molecular chaperones to prevent the accumulation of misfolded proteins results in protein aggregation and amyloid formation, processes associated with severe human degenerative diseases (1, 2). Despite the attention focused on these problems during the century since these disorders were first identified (3–5) and advances in understanding the structure of the cross-β conformation of amyloid fibrils in atomic detail (6, 7), the basic pathological mechanisms of amyloidosis remain poorly understood and therapeutic intervention is lacking. The identity of the toxic species and the mechanisms of cytotoxicity remain major unsolved problems. In some systems, there is evidence suggesting that prefibrillar oligomers, rather than the fully formed fibrils, are the source of toxicity (8, 9). In these cases, cytotoxicity is thought to result from the formation of specific membrane pores (10, 11) although alternative models including membrane destabilization or membrane thinning have also been proposed (12–15). In other cases, toxicity may reside with the amyloid fibrils themselves. Evidence that toxicity correlates with fibrillar assemblies has been reported for yeast and mammalian prion proteins (16, 17), human lysozyme (18), Huntingtin exon 1, α-synuclein (19), and Amyloid-β (Aβ) (20, 21). Furthermore, Aβ plaques have been shown to form rapidly in vivo and to precede neuropathological changes in a mouse model (22). The end surfaces of fibrils (herein termed “fibril ends”) are unusually reactive entities: they play a key role in catalyzing recruitment and conformational conversion of amyloid-forming proteins (23, 24) and provide the sites for templated elongation of amyloid fibril growth (25, 26). Recently, Xue et al. (27) showed that short fibrils of β₂-microglobulin (β₂m), α-synuclein, and hen lysozyme, each prepared by fragmentation of longer fibrils, cause increased damage to membranes and disruption to cellular function compared with the initial long fibrils. Short and long fibril preparations differ in the number of fibril ends at a given protein concentration. Because these are known to be reactive sites, the above observations suggest a role for fibril ends in amyloid–lipid interaction and possibly in amyloid pathogenesis (23, 24, 27). Fragmented fibrils of all three proteins were also found to induce dye leakage from negatively charged liposomes, the most susceptible of which contain a mixture of the cellular lipids phosphatidylcholine (PC) and phosphatidylglycerol (PG), but liposomes with a variety of compositions were damaged by the fibrils in all cases (27).Here, we use β₂m amyloid fibrils formed in vitro as a model system to investigate the structural basis of membrane damage by amyloid fibrils (27, 28). Previous studies have shown that these fibrils possess a parallel in register cross-β structure (29, 30) assembled into multidomain filaments coiled together, described by cryo-EM (28). These fibrils bind amyloid-specific ligands such as serum amyloid P component with a similar affinity to their ex vivo counterparts (31). Using the conditions under which β₂m amyloid fibrils induce dye release from liposomes (pH 7.4), we examined the effects of both long (∼1,400 nm) and fragmented (∼400 nm) β₂m fibrils (27), as well as various control preparations, on the 3D structures of the liposomes by confocal microscopy, cryo-EM, and tomography. We found pronounced distortions in the liposomes, interruptions to the bilayer structure, and extraction of lipids that were induced by the presence of amyloid fibrils. The most severe distortions were seen in proximity to the fibril ends, which are enriched in the fragmented fibril samples. This type of membrane remodelling appears distinct from the actions of other previously described proteins that induce membrane breakage, as in the action of membrane pore-forming proteins (32). The results suggest a role of fibrils in membrane damage that could contribute to the cellular dysfunction associated with amyloid disease.     

The impact of chronic obstructive pulmonary disease on work loss in the United States

Chronic obstructive pulmonary disease (COPD) is a rapidly growing public health problem in the United States and elsewhere. Although direct costs of COPD are well documented, the impact of COPD and its severity on labor force participation is not well known. Using population-based data from the Third National Health and Nutrition Examination Survey (NHANES III), we determined the adjusted relationship between COPD (and its severity) and labor force participation in the U.S. We used data from 12,436 participants involved in NHANES III; 1,073 of these participants (8.6% of the total) reported COPD. These participants were 3.9% (95% confidence interval, 1.3% to 6.4%) less likely to be in the labor force than those without COPD. Increasing severity of COPD was associated with decreased probability of being in the labor force (p for linear trend = 0.001). Mild, moderate, and severe COPD was associated with a 3.4%, 3.9%, and 14.4% reduction in the labor force participation rate relative to those without COPD. These data suggest that COPD has a considerable adverse impact on work force participation. Based on these data, we estimate that, in 1994, COPD was responsible for work loss of approximately $9.9 billion in the U.S.     

3D Echo Pilot Study of Geometric Left Ventricular Changes after Acute Myocardial Infarction

Background

Left ventricular remodeling (LVR) after AMI characterizes a factor of poor prognosis. There is little information in the literature on the LVR analyzed with three-dimensional echocardiography (3D ECHO).

Objective

To analyze, with 3D ECHO, the geometric and volumetric modifications of the left ventricle (VE) six months after AMI in patients subjected to percutaneous primary treatment.

Methods

Prospective study with 3D ECHO of 21 subjects (16 men, 56 ± 12 years-old), affected by AMI with ST segment elevation. The morphological and functional analysis (LV) with 3D ECHO (volumes, LVEF, 3D sphericity index) was carried out up to seven days and six months after the AMI. The LVR was considered for increase > 15% of the end diastolic volume of the LV (LVEDV) six months after the AMI, compared to the LVEDV up to seven days from the event.

Results

Eight (38%) patients have presented LVR. Echocardiographic measurements (n = 21 patients): I- up to seven days after the AMI: 1- LVEDV: 92.3 ± 22.3 mL; 2- LVEF: 0.51 ± 0.01; 3- sphericity index: 0.38 ± 0.05; II- after six months: 1- LVEDV: 107.3 ± 26.8 mL; 2- LVEF: 0.59 ± 0.01; 3- sphericity index: 0.31 ± 0.05. Correlation coefficient (r) between the sphericity index up to seven days after the AMI and the LVEDV at six months (n = 8) after the AMI: r: 0.74, p = 0.0007; (r) between the sphericity index six months after the AMI and the LVEDV at six months after the AMI: r: 0.85, p < 0.0001.

Conclusion

In this series, LVR has been observed in 38% of the patients six months after the AMI. The three-dimensional sphericity index has been associated to the occurrence of LVR.     

Vitamin D deficiency in adults: to better understand a new presentation of an old disease

Vitamin D is synthesized in skin through a reaction mediated by sunlight, and it is metabolized to 25-hydroxyvitamin D, in liver, and in 1,25-dihydroxyvitamin D, in kidney. This last reaction has a tight feedback mechanism. 1,25-dihydroxyvitamin D is the active hormone, and its actions are mediated mainly by nuclear receptors. Its major functions are in calcium metabolism and bone mass maintenance. Hypovitaminosis D, as a disease in adult people, manifests itself with hypocalcemia and secondary hyperparathyroidism with subsequent loss of trabecular bone, thinning of cortical bone, and, eventually, a higher risk of fractures. Hypovitaminosis D is a very common condition in Europe, Africa, North America and some South American countries, such as Chile and Argentina. Measurement of serum total 25-hydroxyvitamin D concentration is the gold standard to diagnose vitamin D deficiency. Serum concentrations below 50 nmol/L are associated with an increase in parathyroid hormone concentration, and bone loss. Risk factors for vitamin D deficiency, like poor sunlight exposition, aging skin and factors that interfere with normal vitamin D metabolism, are well established. Oral vitamin D supplementation, an easy and inexpensive treatment, is needed to treat this illness.     

SB-431542, a transforming growth factor beta inhibitor, impairs Trypanosoma cruzi infection in cardiomyocytes and parasite cycle completion

The antiinflammatory cytokine transforming growth factor beta (TGF-beta) plays an important role in Chagas disease, a parasitic infection caused by the protozoan Trypanosoma cruzi. In the present study, we show that SB-431542, an inhibitor of the TGF-beta type I receptor (ALK5), inhibits T. cruzi-induced activation of the TGF-beta pathway in epithelial cells and in cardiomyocytes. Further, we demonstrate that addition of SB-431542 greatly reduces cardiomyocyte invasion by T. cruzi. Finally, SB-431542 treatment significantly reduces the number of parasites per infected cell and trypomastigote differentiation and release. Taken together, these data further confirm the major role of the TGF-beta signaling pathway in both T. cruzi infection and T. cruzi cell cycle completion. Our present data demonstrate that small inhibitors of the TGF-beta signaling pathway might be potential pharmacological tools for the treatment of Chagas disease.