Two-Faced Immunity? The Evidence for Antibody Enhancement of Malaria Transmission.

Plasmodium gametocytes can induce an immune response in humans that interferes with the development of sexual-stage parasites in the mosquito gut. Many early studies of the sexual-stage immune response noted that mosquito infection could be enhanced as well as reduced by immune sera. For Plasmodium falciparum, these reports are scarce, and the phenomenon is generally regarded as a methodological artefact. Plasmodium transmission enhancement (TE) remains contentious, but the clinical development of transmission-blocking vaccines based on sexual-stage antigens requires that it is further studied. In this essay, we review the early literature on the sexual-stage immune response and transmission-modulating immunity. We discuss hypotheses for the mechanism of TE, suggest experiments to prove or disprove its existence, and discuss its possible implications

Quantification Method of P2X3 Receptors in Rat DRG Neurons: Western Blotting

Skeletal muscle contractions are known to evoke pressor and cardioaccelerator responses in part by stimulating P2X3 receptors found on the peripheral endings of afferents. In diabetic patients, this pressor response is exaggerated. What is currently not known is whether P2X3 receptors play a role in evoking this exaggerated response. PURPOSE: The purpose of this project was to quantify P2X3 receptors in the L4 and L5 dorsal root ganglia (DRG) neurons in both healthy and type 1 diabetic rats using western blot analysis. METHODS: We injected 50 mg/kg streptozotocin (STZ) or the vehicle (CTL) i.p in fasted female and male Sprague Dawley rats and then waited at least 7 days for the rats to become diabetic. We then performed a laminectomy in the anesthetized rats to expose the spinal cord and roots. Using a dissecting microscope, we removed the L4 and L5 DRG from the spinal column. The DRG are the cell bodies of the peripheral afferents found in the hindlimb musculature. The DRG were placed in HBSS (is this buffer?) and stored at -80°C until analysis. For quantification, samples were lysed and proteins were isolated using the NucleoSpin RNA/Protein Kit (Macherey-Nagel, Bethlehem, PA, USA). A Qubit 3.0 Fluorometer was used to quantify the protein concentration of each sample so that equal protein concentrations could then be loaded onto a Bolt Bis-Tris (4-12%) gel. Following electrophoresis, the proteins were transferred to a membrane before being probed with a rabbit polyclonal P2X3 antibody (Alomone Labs), followed by an anti-rabbit secondary antibody conjugated to alkaline phosphatase (Life Technologies). The membrane was then exposed using a ChemiDoc XRS and the results analyzed using BioRad’s Quantity One imaging software. RESULTS: We were able to detect P2X3 receptor proteins. When compared with a molecular weight ladder, P2X3 receptor proteins were 54kDa, which is similar to the molecular weight of P2X3 receptors quantified in other studies. CONCLUSION: This method of quantifying P2X3 receptors in DRG neurons allows for a comparison between non-diabetic and diabetic rats. Further analyses are required to determine whether the quantity of P2X3 receptors in L4 and L5 DRG neurons is different in diabetic rats compared to non-diabetic rats

Combined DNA extraction and antibody elution from filter papers for the assessment of malaria transmission intensity in epidemiological studies.

BACKGROUND: Informing and evaluating malaria control efforts relies on knowledge of local transmission dynamics. Serological and molecular tools have demonstrated great sensitivity to quantify transmission intensity in low endemic settings where the sensitivity of traditional methods is limited. Filter paper blood spots are commonly used a source of both DNA and antibodies. To enhance the operational practicability of malaria surveys, a method is presented for combined DNA extraction and antibody elution. METHODS: Filter paper blood spots were collected as part of a large cross-sectional survey in the Kenyan highlands. DNA was extracted using a saponin/chelex method. The eluate of the first wash during the DNA extraction process was used for antibody detection and compared with previously validated antibody elution procedures. Antibody elution efficiency was assessed by total IgG ELISA for malaria antigens apical membrane antigen-1 (AMA-1) and merozoite-surface protein-1 (MSP-142). The sensitivity of nested 18S rRNA and cytochrome b PCR assays and the impact of doubling filter paper material for PCR sensitivity were determined. The distribution of cell material and antibodies throughout filter paper blood spots were examined using luminescent and fluorescent reporter assays. RESULTS: Antibody levels measured after the combined antibody/DNA extraction technique were strongly correlated to those measured after standard antibody elution (p < 0.0001). Antibody levels for both AMA-1 and MSP-142 were generally slightly lower (11.3-21.4%) but age-seroprevalence patterns were indistinguishable. The proportion of parasite positive samples ranged from 12.9% to 19.2% in the different PCR assays. Despite strong agreement between outcomes of different PCR assays, none of the assays detected all parasite-positive individuals. For all assays doubling filter paper material for DNA extraction increased sensitivity. The concentration of cell and antibody material was not homogenously distributed throughout blood spots. CONCLUSION: Combined DNA extraction and antibody elution is an operationally attractive approach for high throughput assessment of cumulative malaria exposure and current infection prevalence in endemic settings. Estimates of antibody prevalence are unaffected by the combined extraction and elution procedure. The choice of target gene and the amount and source of filter paper material for DNA extraction can have a marked impact on PCR sensitivity

Detecting Gametocytes: How Sensitive Is Sensible?

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Assessing Plasmodium falciparum transmission in mosquito-feeding assays using quantitative PCR.

BACKGROUND: Evaluating the efficacy of transmission-blocking interventions relies on mosquito-feeding assays, with transmission typically assessed by microscopic identification of oocysts in mosquito midguts; however, microscopy has limited throughput, sensitivity and specificity. Where low prevalence and intensity mosquito infections occur, as observed during controlled human malaria infection studies or natural transmission, a reliable method for detection and quantification of low-level midgut infection is required. Here, a semi-automated, Taqman quantitative PCR (qPCR) assay sufficiently sensitive to detect a single-oocyst midgut infection is described. RESULTS: Extraction of genomic DNA from Anopheles stephensi midguts using a semi-automated extraction process was shown to have equivalent extraction efficiency to manual DNA extraction. An 18S Plasmodium falciparum qPCR assay was adapted for quantitative detection of P. falciparum midgut oocyst infection using synthetic DNA standards. The assay was validated for sensitivity and specificity, and the limit of detection was 0.7 genomes/µL (95% CI 0.4-1.6 genomes/µL). All microscopy-confirmed oocyst infected midgut samples were detected by qPCR, including all single-oocyst positive midguts. The genome number per oocyst was assessed 8-9 days after feeding assay using both qPCR and droplet digital PCR and was 3722 (IQR: 2951-5453) and 3490 (IQR: 2720-4182), respectively. CONCLUSIONS: This semi-automated qPCR method enables accurate detection of low-level P. falciparum oocyst infections in mosquito midguts, and may improve the sensitivity, specificity and throughput of assays used to evaluate candidate transmission-blocking interventions

A Copper(II) Tris-imidazolylphosphine Complex as a Structural and Functional Model of Flavonol 2,4-Dioxygenase

Reaction of tetrakis(acetonitrile)copper(I) perchlorate ([Cu(NCCH3)4][ClO4]), tris-1-ethyl-4-methylimidazolylphosphine (T1Et4MeIP) (1) and 3-hydroxyflavone (flavH) under ambient conditions produces an in-situ generated flavonol bound copper(II) complex, which converts to a stable green complex that formulates to [Cu(T1Et4MeIP)(flav)][ClO4] (2). The crystal structure of 2 was determined by X-ray diffraction and crystallizes in a triclinic system (P1¯ role= presentation style= box-sizing: border-box; margin: 0px; padding: 0px; display: inline-block; line-height: normal; font-size: 14.4px; word-spacing: normal; overflow-wrap: normal; text-wrap: nowrap; float: none; direction: ltr; max-width: none; max-height: none; min-width: 0px; min-height: 0px; border: 0px; position: relative; \u3e1¯) with unit cell dimensions of a = 14.537(15) Å, b = 15.794(14) Å, c = 17.044(17) Å, α = 65.58(3)˚, β = 86.80(5)˚, γ = 73.34(4)˚, V = 3376(6) Å3 and Z = 2. While the five-coordinate copper(II) complex is stable under ambient conditions in the solid state, it undergoes oxidative scission of the flavonol pyrone ring under photolytic (300 nm) and/or thermal (120 °C) conditions in the presence of molecular oxygen. The degradative process produces the corresponding methylated products: methylbenzoate, methyl salicylate and N,N-dimethylbenzamide. In addition, the previously undisclosed single crystal X-ray structure of tris-1-ethyl-4-methylimidazolylphosphine (1), T1Et4MeIP, is also reported

Metric-affine f(R) theories of gravity

General Relativity assumes that spacetime is fully described by the metric alone. An alternative is the so called Palatini formalism where the metric and the connections are taken as independent quantities. The metric-affine theory of gravity has attracted considerable attention recently, since it was shown that within this framework some cosmological models, based on some generalized gravitational actions, can account for the current accelerated expansion of the universe. However we think that metric-affine gravity deserves much more attention than that related to cosmological applications and so we consider here metric-affine gravity theories in which the gravitational action is a general function of the scalar curvature while the matter action is allowed to depend also on the connection which is not {\em a priori} symmetric. This general treatment will allow us to address several open issues such as: the relation between metric-affine $f(R)$ gravity and General Relativity (in vacuum as well as in the presence of matter), the implications of the dependence (or independence) of the matter action on the connections, the origin and role of torsion and the viability of the minimal-coupling principle.Comment: typos corrected, replaced to match published versio

Unravelling the immune signature of Plasmodium falciparum transmission-reducing immunity

Infection with Plasmodium can elicit antibodies that inhibit parasite survival in the mosquito, when they are ingested in an infectious blood meal. Here, we determine the transmission-reducing activity (TRA) of naturally acquired antibodies from 648 malaria-exposed individuals using lab-based mosquito-feeding assays. Transmission inhibition is significantly associated with antibody responses to Pfs48/45, Pfs230, and to 43 novel gametocyte proteins assessed by protein microarray. In field-based mosquito-feeding assays the likelihood and rate of mosquito infection are significantly lower for individuals reactive to Pfs48/45, Pfs230 or to combinations of the novel TRA-associated proteins. We also show that naturally acquired purified antibodies against key transmission-blocking epitopes of Pfs48/45 and Pfs230 are mechanistically involved in TRA, whereas sera depleted of these antibodies retain high-level, complement-independent TRA. Our analysis demonstrates that host antibody responses to gametocyte proteins are associated with reduced malaria transmission efficiency from humans to mosquitoes