Introduction-The human multi-drug resistance gene (MDR1, ABCB1) codes for P-glycoprotein (P-gp), an important membrane-bound efflux transporter known to confer anti-cancer drug resistance as well as affect the pharmacokinetics of many drugs and xenobiotics. A number of single nucleotide polymorphisms (SNPs) have been identified throughout the ABCB1 gene which may have an effect on P-gp expression levels and function. Haplotype as well as genotype analysis of SNPs is becoming increasingly important in identifying genetic variants underlying susceptibility to human disease. Three SNPs, 1236C>T, 2677G>T, and 3435C>T have been repeatedly shown to predict changes in the function of P-gp. The frequencies with which these polymorphisms exist in a population have also been shown to be ethnically related.

BackgroundThe prognosis of patients with recurrent/refractory acute myelogenous leukemia (AML) remains poor and cell-based immunotherapies hold promise to improve outcomes. Natural Killer (NK) cells can elicit an antileukemic response via a repertoire of activating receptors that bind AML surface ligands. NK-cell adoptive transfer is safe but thus far has shown limited anti-AML efficacy. Here, we aimed to overcome this limitation by engineering NK cells to express chimeric antigen receptors (CARs) to boost their anti-AML activity and interleukin (IL)-15 to enhance their persistence.MethodsWe characterized in detail NK-cell populations expressing a panel of AML (CD123)-specific CARs and/or IL-15 in vitro and in AML xenograft models.ResultsCARs with 2B4.ζ or 4-1BB.ζ signaling domains demonstrated greater cell surface expression and endowed NK cells with improved anti-AML activity in vitro. Initial in vivo testing revealed that only 2B4.ζ Chimeric Antigen Receptor (CAR)-NK cells had improved anti-AML activity in comparison to untransduced (UTD) and 4-1BB.ζ CAR-NK cells. However, the benefit was transient due to limited CAR-NK-cell persistence. Transgenic expression of secretory interleukin (sIL)-15 in 2B4.ζ CAR and UTD NK cells improved their effector function in the setting of chronic antigen simulation in vitro. Multiparameter flow analysis after chronic antigen exposure identified the expansion of unique NK-cell subsets. 2B4.ζ/sIL-15 CAR and sIL-15 NK cells maintained an overall activated NK-cell phenotype. This was confirmed by transcriptomic analysis, which revealed a highly proliferative and activated signature in these NK-cell groups. In vivo, 2B4.ζ/sIL-15 CAR-NK cells had potent anti-AML activity in one model, while 2B4.ζ/sIL-15 CAR and sIL-15 NK cells induced lethal toxicity in a second model.ConclusionTransgenic expression of CD123-CARs and sIL-15 enabled NK cells to function in the setting of chronic antigen exposure but was associated with systemic toxicities. Thus, our study provides the impetus to explore inducible and controllable expression systems to provide cytokine signals to AML-specific CAR-NK cells before embarking on early-phase clinical testing.

Unlike most intracellular pathogens that gain access into host cells through endocytic pathways, Toxoplasma gondii initiates infection at the cell surface by active penetration through a moving junction and subsequent formation of a parasitophorous vacuole. Here, we describe a noncanonical pathway for T. gondii infection of macrophages, in which parasites are initially internalized through phagocytosis, and then actively invade from within a phagosomal compartment to form a parasitophorous vacuole. This phagosome to vacuole invasion (PTVI) pathway may represent an intermediary link between the endocytic and the penetrative routes for host cell entry by intracellular pathogens. The PTVI pathway is preferentially used by avirulent strains of T. gondii and confers an infectious advantage over virulent strains for macrophage tropism.virulence | Trojan horse | apicomplexa | phagocytes P hagocytosis is one of the most ancient defense mechanisms for the host to destroy invasive pathogens. However, most intracellular pathogens exploit this very pathway for internalization and survival in phagocytes (1). A notable exception to this paradigm is the infection pathway used by apicomplexan parasites-exemplified by Toxoplasma gondii. The current consensus model of T. gondii infection suggests that the parasite actively invades host cells by forming a moving junction (MJ) at the host cell surface (2). Penetration of T. gondii through this junction is largely driven by its own actin motor complex (3). The parasitophorous vacuoles formed by this pathway are nonfusogenic with the host endocytic system, thus evading lysosome-mediated destruction (4-6). However, recent reports including the findings that host F-actin participates in entry by T. gondii (7), and that the parasite is still able to infect cells, albeit much less efficiently, even without some key components of the invasion machinery (8), suggest the existence of alternative infection pathways for Toxoplasma. The active penetration model was defined largely by using nonphagocytic host cells and hypervirulent strains of the parasite. Unlike their virulent counterparts, the interaction of avirulent Toxoplasma strains with macrophage and dendritic cell results in heightened innate cytokine and chemokine production (9) and the development of a "hypermotile" host cellular phenotype (10), which promotes the control of acute infection and mediates dissemination into sites of parasite latency (11,12). Here, we investigated whether avirulent parasites interact with phagocytic host cells in a fundamentally different way from the outset. We found that the avirulent Toxoplasma strains infect macrophages initially via phagocytosis and subsequent active penetration from within the phagosome to form a parasitophorous vacuole. This hybrid invasion pathway may represent an intermediary link between the endocytic and the penetrative routes for host cell entry by intracellular pathogens. ResultsTo investigate whether avirulent Toxoplasma (PTG, type II strain) uses the active penetration p...

Concurrent helminth infection potently inhibits T cell immunity; however, whether helminths prevent T cell priming or skew clonal recruitment and effector differentiation is not known. Using coinfection with two natural mouse pathogens, Heligsomosoides polygyrus and Toxoplasma gondii to investigate the negative impact of helminthes on the CD8 T cell response, we demonstrate helminth-induced suppression of IL-12-dependent differentiation of KLRG1+ effector CD8 T cells and IFNγ production. Nevertheless, reversal of helminth suppression of the innate IL-12 response of CD8α+ DCs, which occurred in STAT6-deficient mice, was not sufficient to normalize CD8 T cell differentiation. Instead, a combined deficiency in IL-4 and IL-10 was required to reverse the negative effects of helminth coinfection on the CD8 T cell response. Monoclonal T. gondii-specific CD8 T cells adoptively transferred into coinfected mice recapitulated the spectrum of helminth-induced effects on the polyclonal CD8 T response, indicating the lack of requirement for clonal skewing.

PurposeTo compare the accuracy and reliability of a natural language processing (NLP) algorithm with manual coding by radiologists, and the combination of the two methods, for the identification of patients whose computed tomography (CT) reports raised the concern for lung cancer.MethodsAn NLP algorithm was developed using Clinical Text Analysis and Knowledge Extraction System (cTAKES) with the Yale cTAKES Extensions and trained to differentiate between language indicating benign lesions and lesions concerning for lung cancer. A random sample of 450 chest CT reports performed at Veterans Affairs Connecticut Healthcare System between January 2014 and July 2015 was selected. A reference standard was created by the manual review of reports to determine if the text stated that follow-up was needed for concern for cancer. The NLP algorithm was applied to all reports and compared with case identification using the manual coding by the radiologists.ResultsA total of 450 reports representing 428 patients were analyzed. NLP had higher sensitivity and lower specificity than manual coding (77.3% v 51.5% and 72.5% v 82.5%, respectively). NLP and manual coding had similar positive predictive values (88.4% v 88.9%), and NLP had a higher negative predictive value than manual coding (54% v 38.5%). When NLP and manual coding were combined, sensitivity increased to 92.3%, with a decrease in specificity to 62.85%. Combined NLP and manual coding had a positive predictive value of 87.0% and a negative predictive value of 75.2%.ConclusionOur NLP algorithm was more sensitive than manual coding of CT chest reports for the identification of patients who required follow-up for suspicion of lung cancer. The combination of NLP and manual coding is a sensitive way to identify patients who need further workup for lung cancer.

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Peptide nucleic acid (PNA) is known to bind with extraordinarily high affinity and sequence-specificity to complementary nucleic acid sequences and can be used to suppress gene expression. However, effective delivery into cells is a major obstacle to the development of PNA for gene therapy applications. Here, we present a novel method for the in vitro delivery of antigene PNA to cells. By using a nucleocapsid protein derived from Simian virus 40, we have been able to package PNA into pseudovirions, facilitating the delivery of the packaged PNA into cells. We demonstrate that this system can be used effectively to suppress gene expression associated with multidrug resistance in cancer cells, as shown by RT-PCR, flow cytometry, Western blotting, and cell viability under chemotherapy. The combination of PNA with the SV40-based delivery system is a method for suppressing a gene of interest that could be broadly applied to numerous targets.

Summary Although ADAMTS13, the von Willebrand factor-cleaving protease, is expressed in a range of tissues, the physiological significance of tissue-specific ADAMTS13 alternative splicing isoforms have yet to be clarified. Screening a panel of human tissues and cell lines revealed a spliced ADAMTS13 transcript in hepatic stellate cells and a hepatoma cell line that retains the 25th intron. A nonsense codon within the intron truncates the protease, which gains 64 novel amino acids in lieu of both CUB domains. This isoform, while retaining VWF-cleaving capability, accumulates intracellularly and its biological inaccessibility may prevent its participation in regulating hemostasis and other physiologic functions.