Efficient measurement of opsonising antibodies to Plasmodium falciparum merozoites

Background: Antibodies targeting merozoites are important in protection from malaria. Therefore, merozoite surface proteins are attractive vaccine candidates. There is a need for robust functional assays to investigate mechanisms of acquired immunity and vaccine efficacy. To date, the study of merozoite phagocytosis has been confounded by the complexity and variability of in vitro assays. Methodology/Principal findings: We have developed a new flow cytometry-based merozoite phagocytosis assay. An optimized merozoite preparation technique produced high yields of merozoites separated from haemozoin. Phagocytosis by the undifferentiated THP-1 monocytic cell line was mediated only by Fc Receptors, and was therefore ideal for studying opsonising antibody responses. The assay showed robust phagocytosis with highly diluted immune sera and strong inter-assay correlation. The assay effectively measured differences in opsonisation-dependent phagocytosis among individuals. Conclusions/Significance: This highly reproducible assay has potential applications in assessing the role of opsonic phagocytosis in naturally acquired immunity and vaccine trials

IgG antibodies to synthetic GPI are biomarkers of immune-status to both Plasmodium falciparum and Plasmodium vivax malaria in young children

BACKGROUND: Further reduction in malaria prevalence and its eventual elimination would be greatly facilitated by the development of biomarkers of exposure and/or acquired immunity to malaria, as well as the deployment of effective vaccines against Plasmodium falciparum and Plasmodium vivax. A better understanding of the acquisition of immunity in naturally-exposed populations is essential for the identification of antigens useful as biomarkers, as well as to inform rational vaccine development. METHODS: ELISA was used to measure total IgG to a synthetic form of glycosylphosphatidylinositol from P. falciparum (PfGPI) in a cohort of 1-3 years old Papua New Guinea children with well-characterized individual differences in exposure to P. falciparum and P. vivax blood-stage infections. The relationship between IgG levels to PfGPI and measures of recent and past exposure to P. falciparum and P. vivax infections was investigated, as well as the association between antibody levels and prospective risk of clinical malaria over 16 months of follow-up. RESULTS: Total IgG levels to PfGPI were low in the young children tested. Antibody levels were higher in the presence of P. falciparum or P. vivax infections, but short-lived. High IgG levels were associated with higher risk of P. falciparum malaria (IRR 1.33-1.66, P = 0.008-0.027), suggesting that they are biomarkers of increased exposure to P. falciparum infections. Given the cross-reactive nature of antibodies to PfGPI, high IgG levels were also associated with reduced risk of P. vivax malaria (IRR 0.65-0.67, P = 0.039-0.044), indicating that these antibodies are also markers of acquired immunity to P. vivax. CONCLUSIONS: This study highlights that in young children, IgG to PfGPI might be a useful marker of immune-status to both P. falciparum and P. vivax infections, and potentially useful to help malaria control programs to identify populations at-risk. Further functional studies are necessary to confirm the potential of PfGPI as a target for vaccine development

Phagocytosis by THP-1 cells is antibody and Fc Receptor dependent.

<p>A) EtBr stained merozoites were incubated with i) non-immune plasma or ii) immune PNG plasma, and were added to THP-1 cells. THP-1 cells were gated by forward and side scatter, and EtBr fluorescence was determined by flow cytometry. A non-immune control sample was used to set the EtBr positive gate. B) THP-1 cells were stained with anti-CD14 antibody and treated with trypan blue (TB) buffer which quenched all surface FITC fluorescence (black:THP-1 cells only, white: CD14-FITC stained THP-1, grey: CD14-FITC stained THP-1 with TB buffer). C) FITC stained merozoites were opsonised with i) non-immune or ii) immune PNG plasma and added to THP-1 cells. FITC fluorescence was measured by flow cytometry before and after quenching, and phagocytosed merozoites were resistant to quenching (black:THP-1 cells only, white: fluorescence with FITC stained merozoites, grey: fluorescence with FITC stained merozoites after TB buffer). D) The % phagocytosis measured for EtBr stained merozoites was equivalent to FITC stained merozoites after quenching. Opsonised EtBr stained and FITC stained merozoites were added to THP-1 cells at 3∶1 and 10∶1 merozoite:THP-1 ratios. E) Phagocytosis is active and Fc Receptor dependent. THP-1 cells were treated with cytochalasin D (CytoD) or blocked with non-specific IgG prior to addition to immune plasma opsonised merozoites in the phagocytosis assay. Each point represents the mean ± standard error. <b>*</b>, <i>p</i><0.05; **, <i>p</i><0.01; ***, <i>p</i><0.005.</p

Efficient merozoite phagocytosis at low antibody concentrations.

<p>A) Increasing numbers of EtBr labeled merozoites were incubated with a pool of immune plasma from PNG children and then added to THP-1 cells. The % phagocytosis was determined by flow cytometry. The ratio of 4∶1 was chosen for subsequent assays, and is indicated by the arrow above. B) Titration of a pool of immune plasma from PNG children for opsonising activity using the 4∶1 merozoite:THP-1 cell ratio. % phagocytosis was determined by flow cytometry. The chosen dilution, 1/30,000, is indicated by an arrow above.</p

Merozoite opsonisation proceeds rapidly at low antibody concentrations.

<p>Merozoites were added to wells containing THP-1 cells simultaneously (white bars), or after 40 mins of preincubation (grey bars), with varying dilutions of an immune plasma pool. % phagocytosis was determined by flow cytometry.</p

Isolated merozoites maintain surface coat integrity.

<p>A) E64-treated schizonts were filtered to release free merozoites and haemozoin crystals, and the filtrate was passed over magnetic columns. Merozoite purification was confirmed by Giemsa-stained smears of i) filtrate, ii) retained haemozoin and iii) purified merozoites. B) Merozites were stained with EtBr and enumerated by flow cytometry. C) Washed Merozoites retained surface proteins MSP-3, MSP-6 and AMA-1 by western blot (S: purified Schizonts; M: filtered merozoites; SM: EtBr stained merozoites). D) Merozoite surface proteins are maintained during merozoite isolation and wash steps as shown by surface localisation of MSP-3 and MSP1₁₉ by immunofluorescence microscopy. Antigens were stained with Alexa-594 and the nucleus with DAPI (panels in order Alexa549; DAPI; brightfield; Alexa549/DAPI; merge).</p

The microtubule inhibitor eribulin demonstrates efficacy in platinum-resistant and refractory high-grade serous ovarian cancer patient-derived xenograft models

Background: Despite initial response to platinum-based chemotherapy and PARP inhibitor therapy (PARPi), nearly all recurrent high-grade serous ovarian cancer (HGSC) will acquire lethal drug resistance; indeed, ~15% of individuals have de novo platinum-refractory disease. Objectives: To determine the potential of anti-microtubule agent (AMA) therapy (paclitaxel, vinorelbine and eribulin) in platinum-resistant or refractory (PRR) HGSC by assessing response in patient-derived xenograft (PDX) models of HGSC. Design and methods: Of 13 PRR HGSC PDX, six were primary PRR, derived from chemotherapy-naïve samples (one was BRCA2 mutant) and seven were from samples obtained following chemotherapy treatment in the clinic (five were mutant for either BRCA1 or BRCA2 ( BRCA1/2) , four with prior PARPi exposure), recapitulating the population of individuals with aggressive treatment-resistant HGSC in the clinic. Molecular analyses and in vivo treatment studies were undertaken. Results: Seven out of thirteen PRR PDX (54%) were sensitive to treatment with the AMA, eribulin (time to progressive disease (PD) ⩾100 days from the start of treatment) and 11 out of 13 PDX (85%) derived significant benefit from eribulin [time to harvest (TTH) for each PDX with p  < 0.002]. In 5 out of 10 platinum-refractory HGSC PDX (50%) and one out of three platinum-resistant PDX (33%), eribulin was more efficacious than was cisplatin, with longer time to PD and significantly extended TTH (each PDX p  < 0.02). Furthermore, four of these models were extremely sensitive to all three AMA tested, maintaining response until the end of the experiment (120d post-treatment start). Despite harbouring secondary BRCA2 mutations, two BRCA2 -mutant PDX models derived from heavily pre-treated individuals were sensitive to AMA. PRR HGSC PDX models showing greater sensitivity to AMA had high proliferative indices and oncogene expression. Two PDX models, both with prior chemotherapy and/or PARPi exposure, were refractory to all AMA, one of which harboured the SLC25A40-ABCB1 fusion, known to upregulate drug efflux via MDR1. Conclusion: The efficacy observed for eribulin in PRR HGSC PDX was similar to that observed for paclitaxel, which transformed ovarian cancer clinical practice. Eribulin is therefore worthy of further consideration in clinical trials, particularly in ovarian carcinoma with early failure of carboplatin/paclitaxel chemotherapy

Targeting homologous recombination deficiency in uterine leiomyosarcoma

Abstract Background Uterine leiomyosarcoma (uLMS) is a rare and aggressive gynaecological malignancy, with individuals with advanced uLMS having a five-year survival of  0.2) but only two samples had a CHORD score > 50%, one of which had a homozygous pathogenic alteration in an HR gene (deletion in BRCA2). A further three samples harboured homozygous HRD alterations (all deletions in BRCA2), detected by WES or panel sequencing, with 5/58 (9%) individuals having HRD uLMS. All five individuals gained access to PARPi therapy. Two of three individuals with mature clinical follow up achieved a complete response or durable partial response (PR) with the subsequent addition of platinum to PARPi upon minor progression during initial PR on PARPi. Corresponding PDX responses were most rapid, complete and sustained with the PARP1-specific PARPi, AZD5305, compared with either olaparib alone or olaparib plus cisplatin, even in a paired sample of a BRCA2-deleted PDX, derived following PARPi therapy in the patient, which had developed PARPi-resistance mutations in PRKDC, encoding DNA-PKcs. Conclusions Our work demonstrates the value of identifying HRD for therapeutic targeting by PARPi and platinum in individuals with the aggressive rare malignancy, uLMS and suggests that individuals with HRD uLMS should be included in trials of PARP1-specific PARPi

Smoothing of initial conditions for high order approximations in option pricing

In this article the Finite Difference method is used to solve the Black Scholes equation. A second order and fourth order accurate scheme is implemented in space and evaluated. The scheme is then tried for different initial conditions. First the discontinuous pay off function of a European Call option is used. Due to the nonsmooth charac- teristics of the chosen initial conditions both schemes show an order of two. Next, the analytical solution to the Black Scholes is used when t=T/2. In this case, with a smooth initial condition, the fourth order scheme shows an order of four. Finally, the initial nonsmooth pay off function is modified by smoothing. Also in this case, the fourth order method shows an order of convergence of four.