Assessment of approved drugs and developmental drug candidates for rare cystic fibrosis (CF)-causing variants of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) requires abundant ...material from relevant models.
Isogenic cell lines harboring CFTR variants in the native genomic context were created through the development and utilization of a footprint-less, CRISPR/Cas9 gene editing pipeline in 16HBE14o- immortalized bronchial epithelial cells.
Isogenic, homozygous cell lines for three CFTR variants (F508del and the two most common CF-causing nonsense variants, G542X and W1282X) were established and characterized. The F508del model recapitulates the known molecular pathology and pharmacology. The two models of nonsense variants (G542X and W1282X) are sensitive to Nonsense Mediated mRNA Decay (NMD) and responsive to reference compounds that inhibit NMD and promote ribosomal readthrough.
We present a versatile, efficient gene editing pipeline that can be used to create CFTR variants in the native genomic context and the utilization of this pipeline to create homozygous cell models for the CF-causing variants F508del, G542X, and W1282X. The resulting cell lines provide a virtually unlimited source of material with specific pathogenic mutations that can be used in a variety of assays, including functional assays.
•A gene editing pipeline was developed to model CFTR variants in the genomic context.•The model of F508del/M470 responds to known pharmacological agents.•The G542X and W1282X models recapitulate non-sense mediated mRNA decay.•The G542X and W1282X models are sensitive to small molecule induced read-through.
Dysregulation of Notch signaling has been implicated in the development of many different types of cancer. Notch inhibitors are being tested in the clinic, but in most cases gastrointestinal and ...other toxicities have limited the dosage and, therefore, the effectiveness of these therapies. Herein, we describe the generation of a monoclonal antibody against the ligand-binding domain of the Notch1 receptor that specifically blocks ligand-induced activation. This antibody, 23814, recognizes both human and murine Notch1 with similar affinity, enabling examination of the effects on both tumor and host tissue in preclinical models. 23814 blocked Notch1 function in vivo, inhibited functional angiogenesis, and inhibited tumor growth without causing gastrointestinal toxicity. The lack of toxicity allowed for combination of 23814 and the VEGFR inhibitor tivozanib, resulting in significant growth inhibition of several VEGFR inhibitor-resistant tumor models. Analysis of the gene expression profiles of an extensive collection of murine breast tumors enabled the successful prediction of which tumors were most likely to respond to the combination of 23814 and tivozanib. Therefore, the use of a specific Notch1 antibody that does not induce significant toxicity may allow combination treatment with angiogenesis inhibitors or other targeted agents to achieve enhanced therapeutic benefit.
In mammals, the heat shock proteins (HSP) gp96 and hsp70 elicit potent specific MHC class I-restricted CD8(+) T cell (CTL) response to exogenous peptides they chaperone. We show in this study that in ...the adult frog Xenopus, a species whose common ancestors with mammals date back 300 million years, both hsp70 and gp96 generate an adaptive specific cellular immune response against chaperoned minor histocompatibility antigenic peptides that effects an accelerated rejection of minor histocompatibility-locus disparate skin grafts in vivo and an MHC-specific CD8(+) cytotoxic T cell response in vitro. In naturally class I-deficient but immunocompetent Xenopus larvae, gp96 also generates an antitumor immune response that is independent of chaperoned peptides (i.e., gp96 purified from normal tissue also generates a significant antitumor response); this suggests a prominent contribution of an innate type of response in the absence of MHC class I Ags.
The purpose of this study was to analyze data retrospectively from our use of weekly subcutaneous recombinant human erythropoietin (rHuEPO) in predialysis and peritoneal dialysis patients with ...anemia.
All anemic patients with progressive renal failure (12 predialysis and seven home peritoneal dialysis) in whom subcutaneous rHuEPO therapy was begun at, or was reduced to, a weekly dose were studied retrospectively. Patients were not selected for, nor excluded from, these observations for any other reason. Hematocrit and endogenous creatinine clearance were monitored regularly, and no other new treatment for anemia was given except oral iron. Iron-deficiency anemia was considered improbable because of normal red blood cell mean corpuscular volume. Unfortunately, iron parameters were not monitored.
The hematocrit increased 4 to 9 percentage points in 4 to 13 weeks in all but two patients who were initially treated with weekly doses, and a hematocrit of 31% was achieved in these patients within 6 to 12 weeks. The mean effective dose to accomplish this was 150 U/kg. All but three patients could be maintained on weekly doses at a hematocrit of 31% or higher. The mean effective dose was 75 U/kg.
It is concluded that subcutaneous rHuEPO administered weekly can correct the anemia of predialysis and peritoneal dialysis patients. Weekly dosing is more convenient for patients and may be less costly for Medicare providers.
Growing evidence suggests that some immune responses are mediated not only by conventional and distinct NK cells and CTL, but also by T cell subsets expressing NK receptors and NK cell‐associated ...molecules. Consistent with our previously published finding that the mAb 1F8 identifies non‐T/non‐B cells in Xenopus that effect NK‐like killing in vitro, we now report that in vivo treatment with this mAb impairs rejection of transplanted MHC class I‐negative tumor cells. However, we also find that the NK cell‐associated molecule recognized by mAb 1F8 is expressed by a minor population of CD8+ T cells, in which fully rearranged TCRβ mRNA of at least three different V families can be identified, by contrast, 1F8+/CD8– (NK) cells lack such TCRβ message. Additionally, the expression of the NK cell‐associated molecule can be induced in vitro by a transient submitogenic stimulation of naïve CD8+ T cells with PMA and ionomycin. Such induced expression of 1F8 also occurs in alloantigen‐activated CTL and is coincident with a down‐regulation of MHC‐specific cytotoxicity. Taken together, these new data suggest that regulation of CD8+ T cell activity involving NK cell‐associated molecules is a general and evolutionarily ancient phenomenon.
The genes encoding FcRgamma and TCRzeta homologs were identified using a bioinformatic approach in the amphibian Xenopus laevis. Deduced amino acid sequence of Xenopus TCRzeta is highly similar to ...the mammalian and avian counterparts, whereas that of FcRgamma differs by the presence of an additional ITAM-like motif. The presence of the negatively charged residue in the transmembrane regions of both subunits suggests their ability to serve as signal transducing modules in complex with activating receptors. The short extracellular regions contain characteristic cysteine residues responsible for dimerization in the mammalian subunits. According to Southern blot analysis, Xenopus laevis may possess two non-allelic genes for each subunit. Northern blots revealed FcRgamma transcripts of two sizes differentially expressed in thymus, spleen, intestine, liver and kidney. TCRzeta mRNA was predominantly expressed in the thymus and spleen. These data indicate that the amphibian immune system employs activating receptor complexes arranged in a mammalian-like way.
Research on nonprofit advocacy has grown in recent years, and many nonprofit organizations have expanded and refined their efforts to influence public policies in ways they believe will benefit ...society. Despite the growing body of literature on nonprofit advocacy, there is substantial room for development on questions related to public perceptions of nonprofit advocacy activities. Utilizing an experimental design, we examine the ways in which the involvement of a nonprofit organization in the policy process can shift public opinion regarding a specific policy proposal. We also explore how these perceptions vary when we introduce political conflict that questions the effectiveness of the proposed policy. We find that in the absence of political controversy, the involvement of nonprofits in the policy process can significantly increase positive perceptions, relative to the control condition in which there is no mention on nonprofit involvement. However, we also find that the ways in which nonprofit involvement could boost support for a policy proposal may not hold when there is conflict over the policy in question.
ABSTRACT Background Social factors can play an important role in the development and maintenance of psychosis. Clarifying this relationship is vital for advancing theoretical understanding and ...development of targeted interventions. Psychosis is increasingly researched with an experience sampling methodology (ESM), which provides an ecologically valid approach, that reduces recall biases. Studies examining momentary associations between social factors and psychosis have not yet been summarised. Method We identified 29 ESM studies investigating associations between social factors and positive psychotic experiences through a pre‐registered systematic search of the published literature. Results Being alone did not predict increase in psychosis; however, appraisals and feelings associated with being alone such as feeling socially disconnected, lonely and unwanted did. Being with familiar company was found to reduce psychosis experiences but feeling stressed by the current company increased psychosis. Conclusions While issues with sample size and generalisability mean these results should be interpreted with caution, some putative conclusions can be made. Individuals with psychosis or emerging symptoms should be offered interventions that improve social networks such as peer support, community participation and engagement skills training. These individuals may also benefit from virtual reality or compassion‐based interventions which aim to dampen perceived social threat. Moreover, digital interventions which monitor changes in social variables that predict relapse in symptoms would allow early intervention to prevent mental health crises.
Crude diagnostic parameters such as BMI limit recognition of malnutrition in overweight and obese patients. This study applied a robust malnutrition diagnostic measure to investigate whether ...malnutrition impacts clinical outcomes in overweight or obese hip fracture inpatients.
A prospective, consecutive 12-month audit of inpatients admitted to a dedicated hip fracture unit with a BMI of ≥25 for surgical intervention. Univariate and logistic regression analyses were performed to investigate the relationship of demographics (age, gender), comparative measures (type of fracture, Charlson Comorbidity Index (CCI) on admission, time to surgery, type of surgery and anaesthesia, nutrition status) and outcome measures (delirium, time to mobilise post-operatively, length of stay, 12-month mortality). Malnutrition was defined using the International Classification of Diseases, Tenth Revision – Australian Modification protein-energy malnutrition criteria.
127 overweight or obese hip fracture patients for surgical intervention were included in analyses. Patients were predominantly older females (median 81.0, range 48–97 years; 66.9%). Malnutrition prevalence was not infrequent (18.3%) despite the median BMI of 28.3 (range 25.0–63.9). Mortality at 12-months (17.3%) was lower than routinely reported across broader hip fracture populations. Logistic regression modelling demonstrated that malnutrition increased the likelihood of 12-month mortality (OR: 4.47, 95% CI 1.27–15.77; p = 0.020), post-operative delirium (OR: 3.64, 95% CI 1.00 to 13.33; p = 0.051), and delayed post-operative mobility (OR: 3.29, 95% CI 1.05 to 10.31; p = 0.041), in overweight or obese hip fracture patients. Length of stay demonstrated poor association with all predictor measures.
Hip fracture patients who are both overweight or obese, and malnourished, have significantly and substantially worse clinical outcomes than their well-nourished, albeit overweight or obese, counterparts. Comprehensive nutrition assessment measures should be applied to all hip fracture inpatients to ensure appropriate clinical nutrition care is appropriately directed.
Fibrinogen plays an essential role in blood coagulation and inflammation. Circulating fibrinogen levels may be determined based on interindividual differences in DNA methylation at ...cytosine-phosphate-guanine (CpG) sites and vice versa.
To perform an EWAS to examine an association between blood DNA methylation levels and circulating fibrinogen levels to better understand its biological and pathophysiological actions.
We performed an epigenome-wide association study of circulating fibrinogen levels in 18 037 White, Black, American Indian, and Hispanic participants, representing 14 studies from the Cohorts for Heart and Aging Research in Genomic Epidemiology consortium. Circulating leukocyte DNA methylation was measured using the Illumina 450K array in 12 904 participants and using the EPIC array in 5133 participants. In each study, an epigenome-wide association study of fibrinogen was performed using linear mixed models adjusted for potential confounders. Study-specific results were combined using array-specific meta-analysis, followed by cross-replication of epigenome-wide significant associations. We compared models with and without CRP adjustment to examine the role of inflammation.
We identified 208 and 87 significant CpG sites associated with fibrinogen levels from the 450K (p < 1.03 × 10
) and EPIC arrays (p < 5.78 × 10
), respectively. There were 78 associations from the 450K array that replicated in the EPIC array and 26 vice versa. After accounting for overlapping sites, there were 83 replicated CpG sites located in 61 loci, of which only 4 have been previously reported for fibrinogen. The examples of genes located near these CpG sites were SOCS3 and AIM2, which are involved in inflammatory pathways. The associations of all 83 replicated CpG sites were attenuated after CRP adjustment, although many remained significant.
We identified 83 CpG sites associated with circulating fibrinogen levels. These associations are partially driven by inflammatory pathways shared by both fibrinogen and CRP.
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