Balanced impacts of fitness and drug pressure on the evolution of PfMDR1 polymorphisms in Plasmodium falciparum.

BACKGROUND: Anti-malarial drug resistance may be limited by decreased fitness in resistant parasites. Important contributors to resistance are mutations in the Plasmodium falciparum putative drug transporter PfMDR1. METHODS: Impacts on in vitro fitness of two common PfMDR1 polymorphisms, N86Y, which is associated with sensitivity to multiple drugs, and Y184F, which has no clear impact on drug sensitivity, were evaluated to study associations between resistance mediators and parasite fitness, measured as relative growth in competitive culture experiments. NF10 P. falciparum lines engineered to represent all PfMDR1 N86Y and Y184F haplotypes were co-cultured for 40 days, and the genetic make-up of the cultures was characterized every 4 days by pyrosequencing. The impacts of culture with anti-malarials on the growth of different haplotypes were also assessed. Lastly, the engineering of P. falciparum containing another common polymorphism, PfMDR1 D1246Y, was attempted. RESULTS: Co-culture results were as follows. With wild type (WT) Y184 fixed (N86/Y184 vs. 86Y/Y184), parasites WT and mutant at 86 were at equilibrium. With mutant 184 F fixed (N86/184F vs. 86Y/184F), mutants at 86 overgrew WT. With WT N86 fixed (N86/Y184 vs. N86/184F), WT at 184 overgrew mutants. With mutant 86Y fixed (86Y/Y184 vs. 86Y/184F), WT and mutant at 86 were at equilibrium. Parasites with the double WT were in equilibrium with the double mutant, but 86Y/Y184 overgrew N86/184F. Overall, WT N86/mutant 184F parasites were less fit than parasites with all other haplotypes. Parasites engineered for another mutation, PfMDR1 1246Y, were unstable in culture, with reversion to WT over time. Thus, the N86 WT is stable when accompanied by the Y184 WT, but incurs a fitness cost when accompanied by mutant 184F. Culturing in the presence of chloroquine favored 86Y mutant parasites and in the presence of lumefantrine favored N86 WT parasites; piperaquine had minimal impact. CONCLUSIONS: These results are consistent with those for Ugandan field isolates, suggest reasons for varied haplotypes, and highlight the interplay between drug pressure and fitness that is guiding the evolution of resistance-mediating haplotypes in P. falciparum

Expanded Hematopoietic Progenitor Cells Reselected for High Aldehyde Dehydrogenase Activity Demonstrate Islet Regenerative Functions

Human umbilical cord blood (UCB) hematopoietic progenitor cells (HPC) purified for high aldehyde dehydrogenase activity (ALDHhi) stimulate islet regeneration after transplantation into mice with streptozotocin-induced β cell deletion. However, ALDHhi cells represent a rare progenitor subset and widespread use of UCB ALDHhi cells to stimulate islet regeneration will require progenitor cell expansion without loss of islet regenerative functions. Here we demonstrate that prospectively purified UCB ALDHhi cells expand efficiently under serum-free, xeno-free conditions with minimal growth factor supplementation. Consistent with the concept that ALDH-activity is decreased as progenitor cells differentiate, kinetic analyses over 9 days revealed the frequency of ALDHhi cells diminished as culture time progressed such that total ALDHhi cell number was maximal (increased 3-fold) at day 6. Subsequently, day 6 expanded cells (bulk cells) were sorted after culture to reselect differentiated progeny with low ALDH-activity (ALDHlo subset) from less differentiated progeny with high ALDH-activity (ALDHhi subset). The ALDHhi subset retained primitive cell surface marker coexpression (32.0% ± 7.0% CD34+/CD38- cells, 37.0% ± 6.9% CD34+/CD133+ cells), and demonstrated increased hematopoietic colony forming cell function compared with the ALDHlo subset. Notably, bulk cells or ALDHlo cells did not possess the functional capacity to lower hyperglycemia after transplantation into streptozotocin-treated NOD/SCID mice. However, transplantation of the repurified ALDHhi subset significantly reduced hyperglycemia, improved glucose tolerance, and increased islet-associated cell proliferation and capillary formation. Thus, expansion and delivery of reselected UCB cells that retain high ALDH-activity after short-term culture represents an improved strategy for the development of cellular therapies to enhance islet regeneration in situ

Expansion of Umbilical Cord Blood Aldehyde Dehydrogenase Expressing Cells Generates Myeloid Progenitor Cells that Stimulate Limb Revascularization

Uncompromised by chronic disease-related comorbidities, human umbilical cord blood (UCB) progenitor cells with high aldehyde dehydrogenase activity (ALDHhi cells) stimulate blood vessel regeneration after intra-muscular transplantation. However, implementation of cellular therapies using UCB ALDHhi cells for critical limb ischemia, the most severe form of severe peripheral artery disease, is limited by the rarity (\u3c0.5%) of these cells. Our goal was to generate a clinically-translatable, allogeneic cell population for vessel regenerative therapies, via ex vivo expansion of UCB ALDHhi cells without loss of pro-angiogenic potency. Purified UCB ALDHhi cells were expanded \u3e18-fold over 6-days under serum-free conditions. Consistent with the concept that ALDH-activity is decreased as progenitor cells differentiate, only 15.1% ± 1.3% of progeny maintained high ALDH-activity after culture. However, compared to fresh UCB cells, expansion increased the total number of ALDHhi cells (2.7-fold), CD34+/CD133+ cells (2.8-fold), and hematopoietic colony forming cells (7.7-fold). Remarkably, injection of expanded progeny accelerated recovery of perfusion and improved limb usage in immunodeficient mice with femoral artery ligation-induced limb ischemia. At 7 or 28 days post-transplantation, mice transplanted with expanded ALDHhi cells showed augmented endothelial cell proliferation and increased capillary density compared to controls. Expanded cells maintained pro-angiogenic mRNA expression and secreted angiogenesis-associated growth factors, chemokines, and matrix modifying proteins. Coculture with expanded cells augmented human microvascular endothelial cell survival and tubule formation under serum-starved, growth factor-reduced conditions. Expanded UCB-derived ALDHhi cells represent an alternative to autologous bone marrow as an accessible source of pro-angiogenic hematopoietic progenitor cells for the refinement of vascular regeneration-inductive therapies. Stem Cells Translational Medicine 2017;6:1607–1619

Pion Breather States in QCD

We describe a class of pionic breather solutions (PBS) which appear in the chiral lagrangian description of low-energy QCD. These configurations are long-lived, with lifetimes greater than $10^3$ fm/c, and could arise as remnants of disoriented chiral condensate (DCC) formation at RHIC. We show that the chiral lagrangian equations of motion for a uniformly isospin-polarized domain reduce to those of the sine-gordon model. Consequently, our solutions are directly related to the breather solutions of sine-gordon theory in 3+1 dimensions. We investigate the possibility of PBS formation from multiple domains of DCC, and show that the probability of formation is non-negligible.Comment: 9 pages, 4 figure

Lack of Association of Bone Morphogenetic Protein 2 Gene Haplotypes with Bone Mineral Density, Bone Loss, or Risk of Fractures in Men

Introduction. The association of bone morphogenetic protein 2 (BMP2) with BMD and risk of fracture was suggested by a recent linkage study, but subsequent studies have been contradictory. We report the results of a study of the relationship between BMP2 genotypes and BMD, annual change in BMD, and risk of fracture in male subjects. Materials and Methods. We tested three single-nucleotide polymorphisms (SNPs) across the BMP2 gene, including Ser37Ala SNP, in 342 Caucasian Englishmen, comprising 224 control and 118 osteoporotic subjects. Results. BMP2 SNP1 (Ser37Ala) genotypes were found to have similar low frequency in control subjects and men with osteoporosis. The major informative polymorphism, BMP2 SNP3 (Arg190Ser), showed no statistically significant association with weight, height, BMD, change in BMD at hip or lumbar spine, and risk of fracture. Conclusion. There were no genotypic or haplotypic effects of the BMP2 candidate gene on BMD, change in BMD, or fracture risk identified in this cohort

The independent effects of hypovolemia and pulmonary vasoconstriction on ventricular function and exercise capacity during acclimatisation to 3800 m

We aimed to determine the isolated and combined contribution of hypovolemia and hypoxic pulmonary vasoconstriction in limiting left ventricular (LV) function and exercise capacity under chronic hypoxemia at high altitude. In a double‐blinded, randomized and placebo‐controlled design, twelve healthy participants underwent echocardiography at rest and during submaximal exercise before completing a maximal test to exhaustion at sea level (SL; 344 m) and after 5–10 days at 3800 m. Plasma volume was normalised to SL values, and hypoxic pulmonary vasoconstriction was reversed by administration of Sildenafil (50 mg) to create four unique experimental conditions that were compared with SL values; high altitude (HA), Plasma Volume Expansion (HA‐PVX), Sildenafil (HA‐SIL) and Plasma Volume Expansion with Sildenafil (HA‐PVX‐SIL). High altitude exposure reduced plasma volume by 11% (P < 0.01) and increased pulmonary artery systolic pressure (19.6 ± 4.3 vs. 26.0 ± 5.4, P < 0.001); these differences were abolished by PVX and SIL respectively. LV end‐diastolic volume (EDV) and stroke volume (SV) were decreased upon ascent to high altitude, but were comparable to sea level in the HA‐PVX. LV EDV and SV were also elevated in the HA‐SIL and HA‐PVX‐SIL trials compared to HA, but to a lesser extent. Neither PVX or SIL had a significant effect on the LV EDV and SV response to exercise, or the maximal oxygen consumption or peak power output. In summary, at 3800 m both hypovolemia and hypoxic pulmonary vasoconstriction contribute to the decrease in LV filling, however, restoring LV filling does not confer an improvement in maximal exercise performance

Field trialling of a pulse airtightness tester in a range of UK homes

A new low pressure ‘quasi-steady’ pulse technique for determining the airtightness of buildings has been developed further and compared with the standard blower-door technique for field-testing a range of typical UK homes. The reported low pressure air pulse unit (APU) has gone through several development stages related to optimizing the algorithm, pressure reference and system construction. The technique, which is compact, portable and easy to use, has been tested alongside the standard blower-door technique to measure the airtightness of a range of typical UK home types. Representative of the UK housing stock, the homes mostly have low levels of airtightness, resulting in poor energy performance, poor indoor air quality and poor thermal comfort. Some of these homes have been targeted for retrofitting and a quick, low cost and simple method for accurately determining their airtightness has clear advantages for correctly predicting the benefits of any improvements. A comparison between the results given by the two techniques is presented and the field trials indicate that the latest version of the pulse technique is reliable for determining building leakage at low pressure. Repeatability of multiple APU tests in the same house is found to be within +/-5% of the mean. A test where the leakage is increased by a known amount shows the APU is able to measure the change more accurately than the blower-door test. The APU also gives convenience in practical applications, due to being more compact and portable, plus it doesn’t need to penetrate the building envelope. The field trials demonstrate the pulse test has the potential to be a feasible alternative to the standard blower-door test

Antibody-drug conjugates plus Janus kinase inhibitors enable MHC-mismatched allogeneic hematopoietic stem cell transplantation

Despite the curative potential of hematopoietic stem cell transplantation (HSCT), conditioning-associated toxicities preclude broader clinical application. Antibody-drug conjugates (ADCs) provide an attractive approach to HSCT conditioning that minimizes toxicity while retaining efficacy. Initial studies of ADC conditioning have largely focused on syngeneic HSCT. However, to treat acute leukemias or induce tolerance for solid organ transplantation, this approach must be expanded to allogeneic HSCT (allo-HSCT). Using murine allo-HSCT models, we show that pharmacologic Janus kinase 1/2 (JAK1/2) inhibition combined with CD45- or cKit-targeted ADCs enables robust multilineage alloengraftment. Strikingly, myeloid lineage donor chimerism exceeding 99% was achievable in fully MHC-mismatched HSCT using this approach. Mechanistic studies using the JAK1/2 inhibitor baricitinib revealed marked impairment of T and NK cell survival, proliferation, and effector function. NK cells were exquisitely sensitive to JAK1/2 inhibition due to interference with IL-15 signaling. Unlike irradiated mice, ADC-conditioned mice did not develop pathogenic graft-versus-host alloreactivity when challenged with mismatched T cells. Finally, the combination of ADCs and baricitinib balanced graft-versus-host disease and graft-versus-leukemia responses in delayed donor lymphocyte infusion models. Our allo-HSCT conditioning strategy exemplifies the promise of immunotherapy to improve the safety of HSCT for treating hematologic diseases