TSLP-induced Mechanisms and Potential Therapies for CRLF2 B-cell Acute Lymphoblastic Leukemia

Childhood CRLF2 B-cell Acute Lymphoblastic Leukemia (CRLF2 B-ALL) is a high-risk form of leukemia that is associated with poor patient survival outcomes. CRLF2 B-ALL is five times more prevalent in Hispanic children than others and is associated with a higher rate of relapse, thus contributing significantly to childhood cancer health disparities. This disease occurs due to alterations of the CRLF2 gene, leading to overexpression of the CRLF2 protein- a component of the receptor signaling complex for the cytokine Thymic Stromal LymphoPoietin (TSLP) on the surface of B-ALL cells. TSLP has been shown to induce proliferation of human and mouse B-cell precursors via activation of the JAK-STAT5 and more recently the PI3K/AKT/mTOR pathways. However, the mechanisms by which TSLP-CRLF2 interactions contribute to high-risk leukemia have not been elucidated. Therefore the objective of our studies was to elucidate the TSLP-induced mechanisms that contribute to high-risk CRLF2 B-ALL and to evaluate promising drug candidates to target high-risk B-ALL. Based on literature findings and preliminary data, we hypothesized that TSLP contributes to CRLF2 B-ALL by increasing proliferation and/or survival of leukemia cells via regulation of genes downstream of CRLF2 pathway activation. This hypothesis was tested using experiments developed under the following specific aims:- Aim 1: Optimize the novel hTSLP+/- xenograft model for use in defining the role of TSLP in CRLF2 B-ALL; Aim 2: Identify TSLP-induced cellular and molecular mechanisms that contribute to CRLF2 B-ALL; Aim 3: Assess candidate drugs’ efficacy against high-risk B-ALL. Results from the studies conducted to address the specific aims show that 1) the hTSLP+/hTSLP- mouse model produces detectable levels of hTSLP, which activates CRLF2 and expands normal B-cells and activates the mTOR cell survival pathway in CRLF2 B-ALL cells in vivo; 2) cellular assays of apoptosis along with gene expression data demonstrate that TSLP regulates the expression of genes that promote cell death and survival of CRLF2 B-ALL cells in vivo and in vitro; 3) drug efficacy studies performed on high-risk leukemia cells using several drug candidates demonstrate that the Mcl-1 inhibitor MIM1 and the Novomedix drug series induced cell death in CRLF2 B-ALL and Nalm6 cells respectively. Taken together, the data provide evidence that TSLP regulates cell survival mechanisms in CRLF2 B-ALL cells and provides a rationale for the use of combination therapies that include drugs which target cell survival molecules and/or cellular functions regulated by TSLP

Pediatric High Risk Leukemia — Molecular Insights

Acute leukemia comprises of 31% of all cancers in children making it the most common childhood malignancy. Significant strides have been made in treatment, partly through risk stratification and intensified therapy. A number of subtypes remain at high risk for relapse and poor outcome, despite current therapies. Here we describe risk stratification and molecular diagnosis used to identify high risk leukemias and guide treatment. Specific cytogenetic alterations that contribute to high risk B and T cell acute lymphoblastic leukemia (ALL), as well as infant leukemia are discussed. Particular attention is given to genetic alterations in IKZF1, CRLF2, and JAK, that have been identified by whole genome sequencing and recently associated with Ph-like ALL. Ongoing studies of disease mechanisms and challenges in developing pre-clinical patient-derived xenograft models to evaluate therapies are discussed

Detecting Endometrial Cancer by Blood Spectroscopy: A Diagnostic Cross-Sectional Study

Endometrial cancer is the sixth most common cancer in women, with a rising incidence worldwide. Current approaches for the diagnosis and screening of endometrial cancer are invasive, expensive or of moderate diagnostic accuracy, limiting their clinical utility. There is a need for cost-effective and minimally invasive approaches to facilitate the early detection and timely management of endometrial cancer. We analysed blood plasma samples in a cross-sectional diagnostic accuracy study of women with endometrial cancer (n = 342), its precursor lesion atypical hyperplasia (n = 68) and healthy controls (n = 242, total n = 652) using attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy and machine learning algorithms. We show that blood-based infrared spectroscopy has the potential to detect endometrial cancer with 87% sensitivity and 78% specificity. Its accuracy is highest for Type I endometrial cancer, the most common subtype, and for atypical hyperplasia, with sensitivities of 91% and 100%, and specificities of 81% and 88%, respectively. Our large-cohort study shows that a simple blood test could enable the early detection of endometrial cancer of all stages in symptomatic women and provide the basis of a screening tool in high-risk groups. Such a test has the potential not only to differentially diagnose endometrial cancer but also to detect its precursor lesion atypical hyperplasia—the early recognition of which may allow fertility sparing management and cancer prevention

N-acetylcysteine (NAC) in schizophrenia resistant to clozapine: a double blind randomised placebo controlled trial targeting negative symptoms

BACKGROUND: Clozapine is an effective treatment for a proportion of people with schizophrenia (SZ) who are resistant to the beneficial effects of other antipsychotic drugs. However, anything from 40-60&nbsp;% of people on clozapine experience residual symptoms even on adequate doses of the medication, and thus could be considered \u27clozapine resistant\u27. Agents that could work alongside clozapine to improve efficacy whilst not increasing the adverse effect burden are both desired and necessary to improve the lives of individuals with clozapine-resistant SZ. N-Acetylcysteine (NAC) is one such possible agent. Previous research from our research group provided promising pilot data suggesting the efficacy of NAC in this patient population. The aim of the study reported here is to expand this work by conducting a large scale clinical trial of NAC in the treatment of clozapine-resistant SZ. METHODS: This study is an investigator initiated, multi-site, randomised, placebo-controlled trial. It aims to include 168 patients with clozapine-resistant SZ, divided into an intervention group (NAC) and a control group (placebo). Participants in the intervention group will receive 2&nbsp;g daily of NAC. The primary outcome measures will be the negative symptom scores of the Positive and Negative Syndrome Scale (PANSS). Secondary outcome measures will include: changes in quality of life (QoL) as measured by the Lancashire Quality of Life Profile (LQoLP) and cognitive functioning as measured by the total score on the MATRICS. Additionally we will examine peripheral and cortical glutathione (GSH) concentrations as process outcomes. DISCUSSION: This large scale clinical trial will investigate the efficacy of NAC as an adjunctive medication to clozapine. This trial, if successful, will establish a cheap, safe and easy-to-use agent (NAC) as a \u27go to\u27 adjunct in patients that are only partly responsive to clozapine.<br /

Culturally valuable minority crops provide a succession of floral resources for flower visitors in traditional orchard gardens

Agricultural intensification typically has detrimental effects on pollinator communities, but diverse cropping systems that contain sequentially-flowering crops have the potential to benefit pollinators through the provision of additional floral resources. In this study we investigate the importance of cultivated flora for flower visitors in ten agricultural gardens in South Sinai, Egypt. Insect-flower interactions in gardens and unmanaged plots were surveyed across a four-month period in two environmentally distinct years (pre-flood and post-flood). Despite containing an equal abundance and diversity of wild plants as unmanaged habitat, gardens supported a higher abundance and diversity of flower visitors due to the additional presence of cultivated flora. Visitation networks exhibited dramatic intra-annual changes in composition, with cultivated plants becoming increasingly important in later months. Trends were highly conserved across 2 years despite highly contrasting rainfall. Several key crop species were strongly involved in shaping the structure of the networks, the majority of which were herbs with strong cultural significance (fennel, rosemary, mint) and grown incidentally alongside the primary orchard crops. Minority crops are frequently overlooked in agricultural systems due to their low economic value, but we show that they can have a dramatic influence upon the structure of visitation networks, increasing both pollinator abundance and diversity, and emphasising the link between cultural practices and biodiversity conservation

Development and evaluation of low-volume tests to detect and characterize antibodies to SARS-CoV-2

Low-volume antibody assays can be used to track SARS-CoV-2 infection rates in settings where active testing for virus is limited and remote sampling is optimal. We developed 12 ELISAs detecting total or antibody isotypes to SARS-CoV-2 nucleocapsid, spike protein or its receptor binding domain (RBD), 3 anti-RBD isotype specific luciferase immunoprecipitation system (LIPS) assays and a novel Spike-RBD bridging LIPS total-antibody assay. We utilized pre-pandemic (n=984) and confirmed/suspected recent COVID-19 sera taken pre-vaccination rollout in 2020 (n=269). Assays measuring total antibody discriminated best between pre-pandemic and COVID-19 sera and were selected for diagnostic evaluation. In the blind evaluation, two of these assays (Spike Pan ELISA and Spike-RBD Bridging LIPS assay) demonstrated >97% specificity and >92% sensitivity for samples from COVID-19 patients taken >21 days post symptom onset or PCR test. These assays offered better sensitivity for the detection of COVID-19 cases than a commercial assay which requires 100-fold larger serum volumes. This study demonstrates that low-volume in-house antibody assays can provide good diagnostic performance, and highlights the importance of using well-characterized samples and controls for all stages of assay development and evaluation. These cost-effective assays may be particularly useful for seroprevalence studies in low and middle-income countries

Effects of antiplatelet therapy on stroke risk by brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases: subgroup analyses of the RESTART randomised, open-label trial

Background Findings from the RESTART trial suggest that starting antiplatelet therapy might reduce the risk of recurrent symptomatic intracerebral haemorrhage compared with avoiding antiplatelet therapy. Brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases (such as cerebral microbleeds) are associated with greater risks of recurrent intracerebral haemorrhage. We did subgroup analyses of the RESTART trial to explore whether these brain imaging features modify the effects of antiplatelet therapy

PTBP1 Is Required for Embryonic Development before Gastrulation

Polypyrimidine-tract binding protein 1 (PTBP1) is an important cellular regulator of messenger RNAs influencing the alternative splicing profile of a cell as well as its mRNA stability, location and translation. In addition, it is diverted by some viruses to facilitate their replication. Here, we used a novel PTBP1 knockout mouse to analyse the tissue expression pattern of PTBP1 as well as the effect of its complete removal during development. We found evidence of strong PTBP1 expression in embryonic stem cells and throughout embryonic development, especially in the developing brain and spinal cord, the olfactory and auditory systems, the heart, the liver, the kidney, the brown fat and cartilage primordia. This widespread distribution points towards a role of PTBP1 during embryonic development. Homozygous offspring, identified by PCR and immunofluorescence, were able to implant but were arrested or retarded in growth. At day 7.5 of embryonic development (E7.5) the null mutants were about 5x smaller than the control littermates and the gap in body size widened with time. At mid-gestation, all homozygous embryos were resorbed/degraded. No homozygous mice were genotyped at E12 and the age of weaning. Embryos lacking PTBP1 did not display differentiation into the 3 germ layers and cavitation of the epiblast, which are hallmarks of gastrulation. In addition, homozygous mutants displayed malformed ectoplacental cones and yolk sacs, both early supportive structure of the embryo proper. We conclude that PTBP1 is not required for the earliest isovolumetric divisions and differentiation steps of the zygote up to the formation of the blastocyst. However, further post-implantation development requires PTBP1 and stalls in homozygous null animals with a phenotype of dramatically reduced size and aberration in embryonic and extra-embryonic structures

A Scalable System for Production of Functional Pancreatic Progenitors from Human Embryonic Stem Cells

Development of a human embryonic stem cell (hESC)-based therapy for type 1 diabetes will require the translation of proof-of-principle concepts into a scalable, controlled, and regulated cell manufacturing process. We have previously demonstrated that hESC can be directed to differentiate into pancreatic progenitors that mature into functional glucose-responsive, insulin-secreting cells in vivo. In this study we describe hESC expansion and banking methods and a suspension-based differentiation system, which together underpin an integrated scalable manufacturing process for producing pancreatic progenitors. This system has been optimized for the CyT49 cell line. Accordingly, qualified large-scale single-cell master and working cGMP cell banks of CyT49 have been generated to provide a virtually unlimited starting resource for manufacturing. Upon thaw from these banks, we expanded CyT49 for two weeks in an adherent culture format that achieves 50–100 fold expansion per week. Undifferentiated CyT49 were then aggregated into clusters in dynamic rotational suspension culture, followed by differentiation en masse for two weeks with a four-stage protocol. Numerous scaled differentiation runs generated reproducible and defined population compositions highly enriched for pancreatic cell lineages, as shown by examining mRNA expression at each stage of differentiation and flow cytometry of the final population. Islet-like tissue containing glucose-responsive, insulin-secreting cells was generated upon implantation into mice. By four- to five-months post-engraftment, mature neo-pancreatic tissue was sufficient to protect against streptozotocin (STZ)-induced hyperglycemia. In summary, we have developed a tractable manufacturing process for the generation of functional pancreatic progenitors from hESC on a scale amenable to clinical entry

Host candidate gene polymorphisms and clearance of drug-resistant Plasmodium falciparum parasites

Resistance to anti-malarial drugs is a widespread problem for control programmes for this devastating disease. Molecular tests are available for many anti-malarial drugs and are useful tools for the surveillance of drug resistance. However, the correlation of treatment outcome and molecular tests with particular parasite markers is not perfect, due in part to individuals who are able to clear genotypically drug-resistant parasites. This study aimed to identify molecular markers in the human genome that correlate with the clearance of malaria parasites after drug treatment, despite the drug resistance profile of the protozoan as predicted by molecular approaches