A slow start: Use of preventive services among seniors following the Affordable Care Act\u27s enhancement of Medicare benefits in the U.S.

OBJECTIVE: Beginning January 1st, 2011 in the United States the Affordable Care Act enhanced Medicare coverage for preventive services by eliminating patient cost-sharing under Part B and by introducing an Annual Wellness Visit, also free-of-charge. We evaluated the early effects of these reforms on utilization of preventive services. METHOD: We analyzed nationally representative data on 15,044 Medicare seniors from the 2008-2010, and 2012 Medical Expenditure Panel Survey, and examined self-reported cholesterol test, blood pressure check, flu vaccination, endoscopy, fecal occult blood test, prostate specific antigen test, breast examination, and mammography. RESULTS: Enhanced Medicare benefits had no effects on preventive service utilization among Medicare seniors in 2012, including those with traditional Medicare and no other supplemental insurance, who stood to benefit the most from Part B enhancements. CONCLUSION: The muted overall response can be partly attributed to the fact that most seniors already held insurance that fully covered preventive services. While insurance enhancements can sometimes raise utilization, in the case of preventive services there are other fundamental barriers that require attention. Educating and incentivizing physicians about the need to refer/recommend screenings, and enhancing knowledge among seniors about the importance of preventive care are two steps that would likely go a long way towards increasing utilization

Structural and functional analysis of a platelet-activating lysophosphatidylcholine of Trypanosoma cruzi

BACKGROUND: Trypanosoma cruzi is the causative agent of the life-threatening Chagas disease, in which increased platelet aggregation related to myocarditis is observed. Platelet-activating factor (PAF) is a potent intercellular lipid mediator and second messenger that exerts its activity through a PAF-specific receptor (PAFR). Previous data from our group suggested that T. cruzi synthesizes a phospholipid with PAF-like activity. The structure of T. cruzi PAF-like molecule, however, remains elusive. METHODOLOGY/PRINCIPAL FINDINGS: Here, we have purified and structurally characterized the putative T. cruzi PAF-like molecule by electrospray ionization-tandem mass spectrometry (ESI-MS/MS). Our ESI-MS/MS data demonstrated that the T. cruzi PAF-like molecule is actually a lysophosphatidylcholine (LPC), namely sn-1 C18:1(delta 9)-LPC. Similar to PAF, the platelet-aggregating activity of C18:1-LPC was abrogated by the PAFR antagonist, WEB 2086. Other major LPC species, i.e., C16:0-, C18:0-, and C18:2-LPC, were also characterized in all T. cruzi stages. These LPC species, however, failed to induce platelet aggregation. Quantification of T. cruzi LPC species by ESI-MS revealed that intracellular amastigote and trypomastigote forms have much higher levels of C18:1-LPC than epimastigote and metacyclic trypomastigote forms. C18:1-LPC was also found to be secreted by the parasite in extracellular vesicles (EV) and an EV-free fraction. A three-dimensional model of PAFR was constructed and a molecular docking study was performed to predict the interactions between the PAFR model and PAF, and each LPC species. Molecular docking data suggested that, contrary to other LPC species analyzed, C18:1-LPC is predicted to interact with the PAFR model in a fashion similar to PAF. CONCLUSIONS/SIGNIFICANCE: Taken together, our data indicate that T. cruzi synthesizes a bioactive C18:1-LPC, which aggregates platelets via PAFR. We propose that C18:1-LPC might be an important lipid mediator in the progression of Chagas disease and its biosynthesis could eventually be exploited as a potential target for new therapeutic interventions

Proposed molecular structures of the three major LPC species of <i>T. cruzi</i>.

<p>Fragment ions obtained from the MSⁿ analysis of <i>T. cruzi</i> ion species at <i>m/z</i> 530, 528, and 526 are indicated. The tandem fragmentation of C16:0-PAF standard is included as a reference.</p

The content of C18:2-, C18:1-, C18:0- and C16:0-LPC in different life-cycle stages of <i>T. cruzi</i>.

<p>^<i>a</i>The molar relative response factors (MRRF) of C10:0-LPC and LPC standards were used to calculate the amount of each LPC molecular species in Folch lower-phase fractions of <i>T. cruzi</i>.</p><p>^<i>b</i>The number of parasites was determined before lipid extraction by counting live parasites in a hemocytometer. Values are means of three determinations. The standard deviation in all cases was <15%.</p><p>^<i>c</i>Determined by multiplying the number of moles by the Avogadro's constant.</p><p>^<i>d</i>Estimated based on the parasite's length and diameter as determined by scanning electron microscopy, and assuming that each parasite is cylindrical, as previous described <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003077#pntd.0003077-PereiraChioccola1" target="_blank">[103]</a>.</p><p>^<i>e</i>Obtained by dividing the number of LPC molecules per cell by the surface area of each parasite form.</p><p>^<i>f</i>Obtained by dividing the amount of picomoles of LPC per 10⁶ cells of each parasite form (column 1) by the values obtained for Epi.</p><p>^<i>g</i>Not analyzed due to absence or trace amounts of the compound.</p

Activity of C16:0-PAF and different <i>T. cruzi</i> LPC species on the aggregation of rabbit platelets.

<p>Platelet aggregation assays were performed as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003077#s2" target="_blank">Materials and Methods</a>, using synthetic 16:0-PAF and C16:0-, C18:0-, C18:1-LPC, and purified C18:2-LPC. Control platelets or platelets pre-treated for 30 min with 10 µM WEB 2086 were assayed in the absence or presence of 1 µM C16:0-PAF or the LPC species at 10 µM (C16:0-LPC, C18:0-LPC, C18:1-LPC, and C18:2-LPC) and 100 µM (C18:1-LPC). Each lipid was tested in duplicate as indicated by black and blue curves in each graph.</p

Full ESI-LIT-MS spectra of <i>T. cruzi</i> phospholipids.

<p>(<b>A</b>) MS1 spectra of lipids obtained in the Folch lower phase prior to fractionation. Lipid samples from all <i>T. cruzi</i> stages were diluted in methanol containing 5 mM LiOH and analyzed by direct infusion in an LTQXL ESI-LIT-MS (positive-ion mode, MS+). Note that the region of spectrum corresponding to LPAF, LPC, and PAF species in Epi, Meta, and TCT has been magnified for better visualization. (<b>B</b>) MS1 spectra of phospholipids obtained by SPE followed by POROS R1 fractionation. Lipids eluted in 25% n-propanol were diluted in methanol containing 5 mM LiOH and analyzed as above. Since the same initial total number of cells (5×10⁸) was used for lipid fractionation from each parasite stage, all spectra were normalized. Magnification of the MS range where PAF and LPC species would be found is indicated (insets). Epi, epimastigote; Meta, metacyclic trypomastigote; ICA, intracellular amastigote; TCT, tissue culture-derived trypomastigote. <i>m/z</i>, mass to charge ratio.</p

Distances between the nitrogen, phosphorous, and oxygen atoms in functional groups of C16:0-PAF and each LPC species.

<p>^<i>a</i>This refers to the nitrogen of the amino group, the phosphorous of the phosphate group, and the oxygen at <i>sn</i>-1, linking the glycerol backbone to the carbonyl group of the acyl chain.</p

Hydrogen bonds between different lysophospholipid ligands and PAFR.

<p>Hydrogen bonds are represented by blue interrupted lines. (<b>A</b>) C16:0-PAF; (<b>B</b>) C16:0-LPC; (<b>C</b>) C18:0-LPC; (<b>D</b>) C18:1-LPC; and (<b>E</b>) C18:2-LPC. Nitrogen atoms are shown in blue, oxygen in red, and ligand carbon chains are filled with the specific color for each ligand: C16:0-PAF, cyan; C16:0-LPC, yellow; C18:0-LPC, orange; C18:1-LPC, green; C18:2-LPC, pink.</p

Structural representation of the PAFR model and its interaction with PAF and LPC species.

<p>(<b>A</b>) Side view of PAFR model. Each TM (alpha-helix) is indicated in a different color: TM1, blue; TM2, red; TM3, dark gray; TM4, orange; TM5, yellow; TM6, purple; TM7, green. The helix 8 (H8) at the end of C-terminus region is indicated (brown). (<b>B–E</b>) Comparison of binding modes of PAF and each LPC species to PAFR. (<b>B</b>) C16:0-PAF (cyan) and C16:0-LPC (yellow); (<b>C</b>) C16:0-PAF (cyan) and C18:0-LPC (orange); (<b>D</b>) C16:0-PAF (cyan) and C18:1-LPC (green); and (<b>E</b>) C16:0-PAF (cyan) and C18:2-LPC (pink). Transmembrane (TM) regions are represented as white rods.</p

Schematic representation of the methodology used for the enrichment and analysis of the putative <i>T. cruzi</i> PAF-like phospholipid.

<p>The total lipid content of the pellets from <i>T. cruzi</i> epimastigote (Epi), metacyclic trypomastigote (Meta), tissue culture-derived trypomastigote (TCT), or intracellular amastigote (ICA) forms was extracted with organic solvents followed by Foch's partition. Folch lower-phase samples were fractionated by SPE in a silica gel (60 Å) column. The different lipid classes were eluted with chloroform (neutral lipids), acetone (glycolipids), and methanol (phospholipids). The latter was further fractionated by perfusion chromatography using POROS R1 50 mini-columns, eluted with a 0%–50% <i>n</i>-propanol gradient. All fractions were diluted in methanol containing 5 mM LiOH and analyzed by MSⁿ. The inset depicts the <i>T. cruzi</i> life cycle with the four stages used in this study.</p