Safety and efficacy of a biodegradable implant releasing tenofovir alafenamide for vaginal protection in a macaque model.

OBJECTIVES: To advance the initiative of ending the global epidemic, long-lasting HIV protection is needed through sustained release of antiretroviral drugs for months to years. We investigated in macaques the safety and efficacy of biodegradable polycaprolactone implants releasing tenofovir alafenamide for HIV pre-exposure prophylaxis (PrEP). METHODS: Implants were administered subcutaneously in the arm using a contraceptive trocar. Efficacy against vaginal simian-HIV (SHIV) infection was investigated in six pigtailed macaques that received two tenofovir alafenamide implants (0.35 mg/day), one in each arm, for a total release rate of tenofovir alafenamide at 0.7 mg/day. Macaques were exposed to SHIV twice weekly for 6 weeks. Statistical analyses were used to compare outcome with eight untreated controls. Histological assessments were performed on skin biopsies collected near implantation sites. RESULTS: Median (range) tenofovir diphosphate level in PBMCs was 1519 (1068-1898) fmol/106 cells. All macaques with tenofovir alafenamide implants were protected against vaginal SHIV infection. In contrast, 7/8 controls were infected after a median of 4 SHIV exposures (P = 0.0047). Histological assessment of tissues near tenofovir alafenamide implant sites showed inflammation and necrosis in 5/6 animals, which were not evident by visual inspection. CONCLUSIONS: We demonstrated complete protection against vaginal SHIV infection with two implants releasing a total of 0.7 mg of tenofovir alafenamide per day. We also identified tenofovir diphosphate concentrations in PBMCs associated with complete vaginal protection. Consistent with previous findings, we observed adverse local toxicity and necrosis near the tenofovir alafenamide implant site. Improved tenofovir alafenamide implants that are safe and maintain high efficacy have the potential to provide long-lasting protection against vaginal HIV infection.

Wildland Fire Emission Sampling at Fishlake National Forest, Utah Using an Unmanned Aircraft System.

Emissions from a stand replacement prescribed burn were sampled using an unmanned aircraft system (UAS, or "drone") in Fishlake National Forest, Utah, U.S.A. Sixteen flights over three days in June 2019 provided emission factors for a broad range of compounds including carbon monoxide (CO), carbon dioxide (CO2), nitric oxide (NO), nitrogen oxide (NO2), particulate matter < 2.5 microns in diameter (PM2.5), volatile organic compounds (VOCs) including carbonyls, black carbon, and elemental/organic carbon. To our knowledge, this is the first UAS-based emission sampling for a fire of this magnitude, including both slash pile and crown fires resulting in wildfire-like conditions. The burns consisted of drip torch ignitions as well as ground-mobile and aerial helicopter ignitions of large stands comprising over 1,000 ha, allowing for comparison of same-species emission factors burned under different conditions. The use of a UAS for emission sampling minimizes risk to personnel and equipment, allowing flexibility in sampling location and ensuring capture of representative, fresh smoke constituents. PM2.5 emission factors varied 5-fold and, like most pollutants, varied inversely with combustion efficiency resulting in lower emission factors from the slash piles than the crown fires.

Control of collagen gel mechanical properties through manipulation of gelation conditions near the sol-gel transition.

The ability to control the mechanical properties of cell culture environments is known to influence cell morphology, motility, invasion and differentiation. The present work shows that it is possible to control the mechanical properties of collagen gels by manipulating gelation conditions near the sol gel transition. This manipulation is accomplished by performing gelation in two stages at different temperatures. The mechanical properties of the gel are found to be strongly dependent on the duration and temperature of the first stage. In the second stage the system is quickly depleted of free collagen which self assembles into a highly branched network characteristic of gelation at the higher temperature (37 °C). An important aspect of the present work is the use of advanced rheometric techniques to assess the transition point between viscoelastic liquid and viscoelastic solid behaviour which occurs upon establishment of a sample spanning network at the gel point. The gel time at the stage I temperature is found to indicate the minimum time that the gelling collagen sample must spend under stage I conditions before the two stage gelation procedure generates an enhancement of mechanical properties. Further, the Fractional Maxwell Model is found to provide an excellent description of the time-dependent mechanical properties of the mature collagen gels.

Emissions from prescribed burning of agricultural fields in the Pacific Northwest.

Prescribed burns of winter wheat stubble and Kentucky bluegrass fields in northern Idaho and eastern Washington states (U.S.A.) were sampled using ground-, aerostat-, airplane-, and laboratory-based measurement platforms to determine emission factors, compare methods, and provide a current and comprehensive set of emissions data for air quality models, climate models, and emission inventories. Batch measurements of PM2.5, volatile organic compounds (VOCs), polycyclic aromatic hydrocarbons (PAHs), and polychlorinated dibenzodioxins/dibenzofurans (PCDDs/PCDFs), and continuous measurements of black carbon (BC), particle mass by size, CO, CO2, CH4, and aerosol characteristics were taken at ground level, on an aerostat-lofted instrument package, and from an airplane. Biomass samples gathered from the field were burned in a laboratory combustion facility for comparison with these ground and aerial field measurements. Emission factors for PM2.5, organic carbon (OC), CH4, and CO measured in the field study platforms were typically higher than those measured in the laboratory combustion facility. Field data for Kentucky bluegrass suggest that biomass residue loading is directly proportional to the PM2.5 emission factor; no such relationship was found with the limited wheat data. CO2 and BC emissions were higher in laboratory burn tests than in the field, reflecting greater carbon oxidation and flaming combustion conditions. These distinctions between field and laboratory results can be explained by measurements of the modified combustion efficiency (MCE). Higher MCEs were recorded in the laboratory burns than from the airplane platform. These MCE/emission factor trends are supported by 1-2 min grab samples from the ground and aerostat platforms. Emission factors measured here are similar to other studies measuring comparable fuels, pollutants, and combustion conditions. The size distribution of refractory BC (rBC) was single modal with a log-normal shape, which was consistent among fuel types when normalized by total rBC mass. The field and laboratory measurements of the Angstrom exponent (α) and single scattering albedo (ω) exhibit a strong decreasing trend with increasing MCEs in the range of 0.9-0.99. Field measurements of α and ω were consistently higher than laboratory burns, which is likely due to less complete combustion. When VOC emissions are compared with MCE, the results are consistent for both fuel types: emission factors increase as MCE decreases.

Evaluation of the Sherlock 3CG Tip Confirmation System on peripherally inserted central catheter malposition rates.

Peripherally inserted central catheters are often positioned blindly in the central circulation, and this may result in high malposition rates, especially in critically ill patients. Recently, a new technology has been introduced (Sherlock 3CG Tip Positioning System) that uses an electro-magnetic system to guide positioning in the superior vena cava, and then intra-cavity ECG to guide positioning at the cavo-atrial junction. In this observational study, we investigated how the Sherlock 3CG Tip Positioning System would affect peripherally inserted central catheter malposition rates, defined using a post-insertion chest radiograph, in critically ill patients. A total of 239 catheters positioned using the Sherlock 3CG Tip Positioning System were analysed. When an adequate position was defined as low superior vena cava or cavo-atrial junction, 134 catheters (56.1%; 95% CI 50-62%) were malpositioned. When an adequate position was defined as mid/low superior vena cava, cavo-atrial junction or high right atrium (≤ 2 cm from cavo-atrial junction), 49 (20.5%; 95% CI 16-26%) catheters were malpositioned. These malposition rates are significantly lower than our own historical data, which used a 'blind' anthropometric technique to guide peripherally inserted central catheter insertion.

Brillouin light scattering in niobium doped lead zirconate single crystal.

Brillouin light scattering experiments were performed for lead zirconate single crystals doped with niobium. Special attention was paid to the elastic mode softening near phase transition temperatures. The results are compared with data obtained by Raman light scattering experiments. We observed that the interaction between acoustic and optic modes is responsible for symmetry breaking far above TC, leading to polar regions' appearance. No changes in the acoustic mode frequency and its damping are observed at TC, where ε(T) exhibits a maximum value. The absence of these changes and the central peak observed in Raman experiments suggest that the phase transition at TC is mainly of the order-disorder type. The origin of other phase transitions is discussed as well.

Comparative Pharmacokinetics and Local Tolerance of Tenofovir Alafenamide (TAF) From Subcutaneous Implant in Rabbits, Dogs, and Macaques.

The administration of antiretrovirals (ARVs) for HIV pre-exposure prophylaxis (PrEP) is highly efficacious and may benefit from new long-acting (LA) drug delivery approaches. This paper describes a subcutaneous, reservoir-style implant for the LA delivery of tenofovir alafenamide (TAF) and documents the preclinical assessment of implant safety and pharmacokinetics (PK) in New Zealand White (NZW) rabbits (3 groups of n = 5), beagle dogs (2 groups of n = 6), and rhesus macaques (2 groups of n = 3). Placebo implants were placed in rabbits (n = 10) and dogs (n = 12). Implant parameters, including selection of the TAF form, choice of excipient, and PCL formulation were tuned to achieve targeted concentrations of the active anabolite of TAF, tenofovir diphosphate (TFV-DP), within peripheral blood mononuclear cells (PBMCs) and mucosal tissues relevant to HIV transmission. Sustained concentrations of TFV-DP in PBMCs over 100 fmol/106 cells were achieved in all animal species indicating that the implants effectively delivered TAF for 3-6 months. Unlike placebo implants without TAF, all active implants resulted in local adverse events (AEs) proximal to the implant ranging in severity from mild to moderate and included dermal inflammation and necrosis across all species. Despite these AEs, the implant performed as designed and achieved a constant drug release profile, supporting the continued development of this drug delivery platform.

An engineered Plasmodium falciparum C-terminal 19-kilodalton merozoite surface protein 1 vaccine candidate induces high levels of interferon-gamma production associated with cellular immune responses to specific peptide sequences in Gambian adults naturally exposed to malaria.

The 19-kDa C-terminal region of merozoite surface protein 1 (MSP1(19)), a major blood stage malaria vaccine candidate, is the target of cellular and humoral immune responses in humans naturally infected with Plasmodium falciparum. We have previously described engineered variants of this protein, designed to be better vaccine candidates, but the human immune response to these proteins has not been characterized fully. Here we have investigated the antigenicity of one such variant compared to wild-type MSP1(19)-derived protein and peptides. Gambian adults produced both high T helper type 1 (Th1) [interferon (IFN)-γ] and Th0/Th2 [interleukin (IL)-13 and sCD30] responses to the wild-type MSP1(19) and the modified protein as wells as to peptides derived from both forms. Response to the modified MSP1(19) (with three amino acid substitutions: Glu27Tyr, Leu31Arg and Glu43Leu) relative to the wild-type, included higher IFN-γ production. Interestingly, some peptides evoked different patterns of cytokine responses. Modified peptides induced higher IL-13 production than the wild-type, while the conserved peptides P16 and P19 induced the highest IFN-γ and IL-13 and/or sCD30 release, respectively. We identified P16 as the immunodominant peptide that was recognized by cells from 63% of the study population, and not restricted to any particular human leucocyte antigen D-related (HLA-DR) type. These findings provide new and very useful information for future vaccine development and formulation as well as potential Th1/Th2 immunmodulation using either wild-type or modified protein in combination with their peptides.

Magnetic field driven phase transitions in EuTiO3.

The influence of an external static magnetic field (up to 480 mT) on the structural properties of EuTiO3(ETO) polycrystalline samples was examined by powder XRD at the Elettra synchrotron facilities in the temperature range 100-300 K. While the cubic to tetragonal structural phase transition temperature in this magnetic field range remains almost unaffected, significant lattice effects appear at two characteristic temperatures (∼200 K and ∼250 K), which become more pronounced at a critical threshold field. At ∼200  K a change in the sign of magnetostriction is detected attributed to a modification of the local magnetic properties from intrinsic ferromagnetism to intrinsic antiferromagnetism. These data are a clear indication that strong spin-lattice interactions govern also the high temperature phase of ETO and trigger the appearance of magnetic domain formation and phase transitions.

New candidate vaccines against blood-stage Plasmodium falciparum malaria: prime-boost immunization regimens incorporating human and simian adenoviral vectors and poxviral vectors expressing an optimized antigen based on merozoite surface protein 1.

Although merozoite surface protein 1 (MSP-1) is a leading candidate vaccine antigen for blood-stage malaria, its efficacy in clinical trials has been limited in part by antigenic polymorphism and potentially by the inability of protein-in-adjuvant vaccines to induce strong cellular immunity. Here we report the design of novel vectored Plasmodium falciparum vaccines capable of overcoming such limitations. We optimized an antigenic insert comprising the four conserved blocks of MSP-1 fused to tandemly arranged sequences that represent both allelic forms of the dimorphic 42-kDa C-terminal region. Inserts were expressed by adenoviral and poxviral vectors and employed in heterologous prime-boost regimens. Simian adenoviral vectors were used in an effort to circumvent preexisting immunity to human adenoviruses. In preclinical studies these vaccines induced potent cellular immune responses and high-titer antibodies directed against MSP-1. The antibodies induced were found to have growth-inhibitory activity against dimorphic allelic families of P. falciparum. These vectored vaccines should allow assessment in humans of the safety and efficacy of inducing strong cellular as well as cross-strain humoral immunity to P. falciparum MSP-1.

Surface antigens of malaria merozoites. A high molecular weight precursor is processed to an 83,000 mol wt form expressed on the surface of Plasmodium falciparum merozoites.

A technique was developed for obtaining high yields of naturally released Plasmodium falciparum merozoites from synchronous cultures of parasitized erythrocytes. The cultured erythrocytes were treated with trypsin to prevent reinvasion (6), and the released merozoites that accumulated extracellularly were harvested by differential centrifugation. The total biosynthetically labeled proteins of schizonts and merozoites, and those immunoprecipitated by human immune serum were analyzed and compared. The surface antigens of free merozoites, labeled by lactoperoxidase-catalyzed iodination, were also described. A monoclonal antibody, specific for a 195,000 mol wt schizont protein, and processing fragments derived from it (3) were used in immunoprecipitation and Western transfer analyses to determine which of the processing fragments are associated with merozoites and which of them are located on the merozoite surface. It was found that processing of the 195,000 mol wt precursor down to an 83,000 mol wt fragment is complete in free merozoites, and that this fragment is expressed as one of the major surface antigens of P. falciparum merozoites.

The three major antigens on the surface of Plasmodium falciparum merozoites are derived from a single high molecular weight precursor.

A 195,000 mol wt Plasmodium falciparum protein and processing fragments derived from it have been purified by monoclonal antibody affinity chromatography. A polyvalent antiserum has been raised against the purified protein and used to identify the terminal processing products associated with the merozoite. Three unique fragments of 83,000, 42,000, and 19,000 mol wt are present and they represent the major surface antigens of P. falciparum merozoites.

Biosynthesis and processing of a Plasmodium falciparum schizont antigen recognized by immune serum and a monoclonal antibody.

Stage-specific protein synthesis by the erythrocytic forms of the malaria parasite Plasmodium falciparum was investigated by pulse labeling synchronous parasite cultures with [35S]methionine at 6-h intervals during a complete 48-h developmental cycle. About 40 labeled parasite proteins could be immunoprecipitated with human immune serum, and most of these were associated with the schizont stage of development. In particular, one schizont protein was a 195,000-mol wt species against which a murine monoclonal antibody was produced. This monoclonal antibody, 89.1 reacted with the parasite membrane in schizonts and also with the surface of free merozoites in the indirect immunofluorescence test. In addition to the 195,000-mol wt protein, antibody 89.1 immunoprecipitated a series of lower-molecular weight polypeptides from extracts of labeled asynchronous P. falciparum parasite cultures. These were shown to be related to the 195,000-mol wt protein by peptide mapping. Pulse-chase labeling of synchronized cultures, and immunoprecipitation with antibody 89.1, showed that specific processing of the 195,000-mol wt polypeptide to the lower-molecular-weight products in concomitant with schizont maturation and merozoite release. It is suggested that this P. falciparum protein may be analogous to a similarly processed 230,000-mol wt protective antigen of the rodent malaria parasite, P. yoelii.

Antibodies inhibit the protease-mediated processing of a malaria merozoite surface protein.

When merozoites of the malaria parasite Plasmodium falciparum are released from infected erythrocytes and invade new red cells, a component of a protein complex derived from the merozoite surface protein 1 (MSP-1) precursor undergoes a single proteolytic cleavage known as secondary processing. This releases the complex from the parasite surface, except for a small membrane-bound fragment consisting of two epidermal growth factor (EGF)-like domains, which is the only part of MSP-1 to be carried into invaded erythrocytes. We report that, a group of monoclonal antibodies specific for epitopes within the EGF-like domains, some interfere with secondary processing whereas others do not. Those that most effectively inhibit processing have previously been shown to prevent invasion. Other antibodies, some of which can block this inhibition, not only do not prevent invasion but are carried into the host cell bound to the merozoite surface. These observations unequivocally demonstrate that the binding of antibody to the COOH-terminal region of MSP-1 on the merozoite surface may not be sufficient to prevent erythrocyte invasion, and show that the interaction of different antibodies with adjacent epitopes within the EGF-like domains of MSP-1 can have distinct biochemical effects on the molecule. Inhibition of MSP-1 processing on merozoites may be a mechanism by which protective antibodies interrupt the asexual cycle of the malaria parasite.

Antibodies that inhibit malaria merozoite surface protein-1 processing and erythrocyte invasion are blocked by naturally acquired human antibodies.

Merozoite surface protein-1 (MSP-1) of the human malaria parasite Plasmodium falciparum undergoes at least two endoproteolytic cleavage events during merozoite maturation and release, and erythrocyte invasion. We have previously demonstrated that mAbs which inhibit erythrocyte invasion and are specific for epitopes within a membrane-proximal, COOH-terminal domain of MSP-1 (MSP-119) prevent the critical secondary processing step which occurs on the surface of the extracellular merozoite at around the time of erythrocyte invasion. Certain other anti-MSP-119 mAbs, which themselves inhibit neither erythrocyte invasion nor MSP-1 secondary processing, block the processing-inhibitory activity of the first group of antibodies and are termed blocking antibodies. We have now directly quantitated antibody-mediated inhibition of MSP-1 secondary processing and invasion, and the effects on this of blocking antibodies. We show that blocking antibodies function by competing with the binding of processing-inhibitory antibodies to their epitopes on the merozoite. Polyclonal rabbit antibodies specific for certain MSP-1 sequences outside of MSP-119 also act as blocking antibodies. Most significantly, affinity-purified, naturally acquired human antibodies specific for epitopes within the NH2-terminal 83-kD domain of MSP-1 very effectively block the processing-inhibitory activity of the anti-MSP-119 mAb 12.8. The presence of these blocking antibodies also completely abrogates the inhibitory effect of mAb 12.8 on erythrocyte invasion by the parasite in vitro. Blocking antibodies therefore (a) are part of the human response to malarial infection; (b) can be induced by MSP-1 structures unrelated to the MSP-119 target of processing-inhibitory antibodies; and (c) have the potential to abolish protection mediated by anti-MSP-119 antibodies. Our results suggest that an effective MSP-119-based falciparum malaria vaccine should aim to induce an antibody response that prevents MSP-1 processing on the merozoite surface.

A single fragment of a malaria merozoite surface protein remains on the parasite during red cell invasion and is the target of invasion-inhibiting antibodies.

A complex of polypeptides derived from a precursor is present on the surface of the malaria merozoite. During erythrocyte invasion only a small fragment from this complex is retained on the parasite surface and carried into the newly infected red cell. Antibodies to this fragment will interrupt invasion.

Plasmodium falciparum infection of the placenta impacts on the T helper type 1 (Th1)/Th2 balance of neonatal T cells through CD4(+)CD25(+) forkhead box P3(+) regulatory T cells and interleukin-10.

Placental malaria infection affects the T helper type 1 (Th1)/Th2 balance in neonatal children. We investigated a potential role of regulatory T cells in this balance by comparing T cell responses of cord blood mononuclear cells (CBMC) from parasitized and non-parasitized placenta of Gambian women. CBMC were depleted of CD4(+)CD25(+) forkhead box P3 (FoxP3)(+) regulatory T cells and analysed in vitro for their ability to produce interferon (IFN)-gamma, sCD30 and interleukin (IL)-10 in response to phytohaemagglutinin (PHA), live Plasmodium falciparum, schizont extracts and the recombinant P. falciparum blood stage antigen merozoite surface protein 1 (MSP1(19)). As expected, lower IFN-gamma and higher sCD30 responses were observed for the cells from the parasitized group. In addition, higher IL-10 levels were produced by CBMC from the parasitized group. Depletion of regulatory T cells decreased IL-10 production, which resulted in a restoration of IFN-gamma expression in response to all stimuli. The Th2 marker sCD30 remained significantly higher in the parasitized group in response to malaria protein antigens while similar levels were recovered between both groups in response to live P. falciparum. Similar effects were observed by adding an antibody that blocks IL-10 function. These results suggest that the impact of P. falciparum infection on Th1 differentiation of neonatal T cells can be ascribed to regulatory T cells through production of IL-10.

The carboxy-terminus of merozoite surface protein 1: structure, specific antibodies and immunity to malaria.

Over the last 30 years, evidence has been gathered suggesting that merozoite surface protein 1 (MSP1) is a target of protective immunity against malaria. In a variety of experimental approaches using in vitro methodology, animal models and sero-epidemiological techniques, the importance of antibody against MSP1 has been established but we are still finding out what are the mechanisms involved. Now that clinical trials of MSP1 vaccines are underway and the early results have been disappointing, it is increasingly clear that we need to know more about the mechanisms of immunity, because a better understanding will highlight the limitations of our current assays and identify the improvements required. Understanding the structure of MSP1 will help us design and engineer better antigens that are more effective than the first generation of vaccine candidates. This review is focused on the carboxy-terminus of MSP1.

Signalling in malaria parasites. The MALSIG consortium.

Depending on their developmental stage in the life cycle, malaria parasites develop within or outside host cells, and in extremely diverse contexts such as the vertebrate liver and blood circulation, or the insect midgut and hemocoel. Cellular and molecular mechanisms enabling the parasite to sense and respond to the intra- and the extra-cellular environments are therefore key elements for the proliferation and transmission of Plasmodium, and therefore are, from a public health perspective, strategic targets in the fight against this deadly disease. The MALSIG consortium, which was initiated in February 2009, was designed with the primary objective to integrate research ongoing in Europe and India on i) the properties of Plasmodium signalling molecules, and ii) developmental processes occurring at various points of the parasite life cycle. On one hand, functional studies of individual genes and their products in Plasmodium falciparum (and in the technically more manageable rodent model Plasmodium berghei) are providing information on parasite protein kinases and phosphatases, and of the molecules governing cyclic nucleotide metabolism and calcium signalling. On the other hand, cellular and molecular studies are elucidating key steps of parasite development such as merozoite invasion and egress in blood and liver parasite stages, control of DNA replication in asexual and sexual development, membrane dynamics and trafficking, production of gametocytes in the vertebrate host and further parasite development in the mosquito. This article, which synthetically reviews such signalling molecules and cellular processes, aims to provide a glimpse of the global frame in which the activities of the MALSIG consortium will develop over the next three years.

Methodology for characterizing emissions from small (0.5-2 MTD) batch-fed gasification systems using multiple waste compositions.

A compact, containerized gasification system was characterized for air emissions while burning four waste types. A methodology is presented for developing a standardized test waste composition and demonstrated using three military and one civilian waste types. Batch charges of waste were processed through a gasification chamber, afterburner, and wet scrubber. The 0.5-2 metric ton per day (MTD) system was designed for mobile deployment by the military in forward operations but would be applicable to small scale civilian applications. Emissions data from these types of small capacity, cyclically operated systems are lacking, limiting efforts to compare technologies and their environmental performance. Eight tests were conducted in a 7-day period at the Kilauea Military Camp (KMC) in Hawaii. The pollutants characterized were chosen based on their regulatory and health relevance: particulate matter (PM), mercury (Hg), elemental composition, volatile organic compounds (VOCs), polyaromatic hydrocarbons (PAHs), and polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs). Averaged data from 4-hour runs, including startups and shutdowns, indicated that five of the nine EPA-regulated compounds (lead, cadmium, Hg, sulfur dioxide, and hydrogen chloride) were under the emission limits set for Other Solid Waste Incineration Units (OSWI) while four, PCDD/PCDF, PM, nitrogen oxides, and carbon monoxide, were higher. The procedures through which waste compositions were created and emissions were characterized provide a methodology by which differing waste to energy technologies can be compared on an equivalent basis. This system's emissions compare favorably with alternative disposal methods. PM and PCDD/PCDF emission factors were, respectively, over 39 and 9 times lower from this unit than from published data on burning simulated military waste in an air curtain incinerator and in open burn piles ("burn pits").