A phase II study of allogeneic natural killer cell therapy to treat patients with recurrent ovarian and breast cancer

BackgroundNatural killer (NK) cells derived from patients with cancer exhibit diminished cytotoxicity compared with NK cells from healthy individuals. We evaluated the tumor response and in vivo expansion of allogeneic NK cells in recurrent ovarian and breast cancerMethodsPatients underwent a lymphodepleting preparative regimen: fludarabine 25 mg/m² × 5 doses, cyclophosphamide 60 mg/kg × 2 doses, and, in seven patients, 200 cGy total body irradiation (TBI) to increase host immune suppression. An NK cell product, from a haplo-identical related donor, was incubated overnight in 1000 U/mL interleukin (IL)-2 prior to infusion. Subcutaneous IL-2 (10 MU) was given three times/week × 6 doses after NK cell infusion to promote expansion, defined as detection of ≥100 donor-derived NK cells/μL blood 14 days after infusion, based on molecular chimerism and flow cytometryResultsTwenty (14 ovarian, 6 breast) patients were enrolled. The median age was 52 (range 30–65) years. Mean NK cell dose was 2.16 × 10⁷cells/kg. Donor DNA was detected 7 days after NK cell infusion in 9/13 (69%) patients without TBI and 6/7 (85%) with TBI. T-regulatory cells (Treg) were elevated at day +14 compared with pre-chemotherapy (P = 0.03). Serum IL-15 levels increased after the preparative regimen (P = < 0.001). Patients receiving TBI had delayed hematologic recovery (P = 0.014). One patient who was not evaluable had successful in vivo NK cell expansionConclusionsAdoptive transfer of haplo-identical NK cells after lymphodepleting chemotherapy is associated with transient donor chimerism and may be limited by reconstituting recipient Treg cells. Strategies to augment in vivo NK cell persistence and expansion are needed.     

Combination of Immune and Viral Factors Distinguishes Low-Risk versus High-Risk HIV-1 Disease Progression in HLA-B*5701 Subjects

HLA-B*5701 is the host factor most strongly associated with slow HIV-1 disease progression, although rates can vary within this group. Underlying mechanisms are not fully understood but likely involve both immunological and virological dynamics. The present study investigated HIV-1 in vivo evolution and epitope-specific CD8(+) T cell responses in six HLA-B*5701 patients who had not received antiretroviral treatment, monitored from early infection for up to 7 years. The subjects were classified as high-risk progressors (HRPs) or low-risk progressors (LRPs) based on baseline CD4(+) T cell counts. Dynamics of HIV-1 Gag p24 evolution and multifunctional CD8(+) T cell responses were evaluated by high-resolution phylogenetic analysis and polychromatic flow cytometry, respectively. In all subjects, substitutions occurred more frequently in flanking regions than in HLA-B*5701-restricted epitopes. In LRPs, p24 sequence diversity was significantly lower; sequences exhibited a higher degree of homoplasy and more constrained mutational patterns than HRPs. The HIV-1 intrahost evolutionary rate was also lower in LRPs and followed a strict molecular clock, suggesting neutral genetic drift rather than positive selection. Additionally, polyfunctional CD8(+) T cell responses, particularly to TW10 and QW9 epitopes, were more robust in LRPs, who also showed significantly higher interleukin-2 (IL-2) production in early infection. Overall, the findings indicate that HLA-B*5701 patients with higher CD4 counts at baseline have a lower risk of HIV-1 disease progression because of the interplay between specific HLA-linked immune responses and the rate and mode of viral evolution. The study highlights the power of a multidisciplinary approach, integrating high-resolution evolutionary and immunological data, to understand mechanisms underlying HIV-1 pathogenesis.     

Aberrant Active cis-Regulatory Elements Associated with Downregulation of RET Finger Protein Overcome Chemoresistance in Glioblastoma

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Literature sources for primary environmental science teachers

With school days seemingly getting shorter and packed with more and more obligations, teachers integrate core subjects like science and reading in the hopes of delivering a curriculum that is meaningful and content rich. Using trade books, primary teachers can develop and deliver science content designed to introduce students to proenvironmental behaviors built around the Next Generation Science Earth and Human Activity Standard, Human Impacts on Earth Systems, ESS3-C. The Next Generation Science standards call for integrating literacy skills and science to develop knowledge of scientific topics. This article shares ideas on how primary teachers can use a variety of children’s literature as a foundation to inspire young students to take small steps toward developing proenvironmental behaviors. Under an umbrella of three environmental themes, proenvironmental behavior, trash, and water pollution, the article describes key concepts, vocabulary, and activities designed to develop a mindfulness of proenvironmental behaviors for each selected text. While texts and learning experiences are intentionally focused on young learners (Kindergarten/first grade), they can be adapted and used in all primary grades.     

Balancing on the crest – Evidence for disruption of the enteric ganglia via inappropriate lineage segregation and consequences for gastrointestinal function

Normal enteric nervous system (ENS) development relies on numerous factors, including appropriate migration, proliferation, differentiation, and maturation of neural crest (NC) derivatives. Incomplete rostral to caudal migration of enteric neural crest-derived progenitors (ENPs) down the gut is at least partially responsible for the absence of enteric ganglia that is a hallmark feature of Hirschsprung disease (HSCR). The thought that ganglia proximal to aganglionosis are normal has guided surgical procedures for HSCR patients. However, chronic gastrointestinal dysfunction suffered by a subset of patients after surgery as well as studies in HSCR mouse models suggest that aberrant NC segregation and differentiation may be occurring in ganglionated regions of the intestine. Studies in mouse models that possess enteric ganglia throughout the length of the intestine (non-HSCR) have also found that certain genetic alterations affect neural crest lineage balance and interestingly many of these mutants also have functional gastrointestinal (GI) defects. It is possible that many GI disorders can be explained in part by imbalances in NC-derived lineages. Here we review studies evaluating ENS defects in HSCR and non-HSCR mouse models, concluding with clinical implications while highlighting areas requiring further study.     

Chasing Jenner's vaccine: revisiting cowpox virus classification

Cowpox virus (CPXV) is described as the source of the first vaccine used to prevent the onset and spread of an infectious disease. It is one of the earliest described members of the genus Orthopoxvirus, which includes the viruses that cause smallpox and monkeypox in humans. Both the historic and current literature describe "cowpox" as a disease with a single etiologic agent. Genotypic data presented herein indicate that CPXV is not a single species, but a composite of several (up to 5) species that can infect cows, humans, and other animals. The practice of naming agents after the host in which the resultant disease manifests obfuscates the true taxonomic relationships of "cowpox" isolates. These data support the elevation of as many as four new species within the traditional "cowpox" group and suggest that both wild and modern vaccine strains of Vaccinia virus are most closely related to CPXV of continental Europe rather than the United Kingdom, the homeland of the vaccine.     

Evidence Against the Involvement of Ionically Bound Cell Wall Proteins in Pea Epicotyl Growth

Ionically bound cell wall proteins were extracted from 7 day old etiolated pea (Pisum sativum L. cv Alaska) epicotyls with 3 molar LiCl. Polyclonal antiserum was raised in rabbits against the cell wall proteins. Growth assays showed that treatment of growing region segments (5-7 millimeters) of peas with either dialyzed serum, serum globulin fraction, affinity purified immunoglobulin, or papain-cleaved antibody fragments had no effect on growth. Immunofluorescence microscopy confirmed antibody binding to cell walls and penetration of the antibodies into the tissues. Western blot analysis, immunoassay results, and affinity chromatography utilizing Sepharose-bound antibodies confirmed recognition of the protein preparation by the antibodies. Experiments employing in vitro extension as a screening measure indicated no effect upon extension by antibodies, by 50 millimolar LiCl perfusion of the apoplast or by 3 molar LiCl extraction. Addition of cell wall protein to protease pretreated segments did not restore extension nor did addition of cell wall protein to untreated segments increase extension. It is concluded that, although evidence suggests that protein is responsible for the process of extension, the class(es) of proteins which are extracted from pea cell walls with 3 molar LiCl are probably not involved in this process.     

A Label-free Selected Reaction Monitoring Workflow Identifies a Subset of Pregnancy Specific Glycoproteins as Potential Predictive Markers of Early-onset Pre-eclampsia

Pre-eclampsia (PE) is a serious complication of pregnancy with potentially life threatening consequences for both mother and baby. Presently there is no test with the required performance to predict which healthy first-time mothers will go on to develop PE. The high specificity, sensitivity, and multiplexed nature of selected reaction monitoring holds great potential as a tool for the verification and validation of putative candidate biomarkersfor disease states. Realization of this potential involves establishing a high throughput, cost effective, reproducible sample preparation workflow. We have developed a semi-automated HPLC-based sample preparation workflow before a label-free selected reaction monitoring approach. This workflow has been applied to the search for novel predictive biomarkers for PE.To discover novel candidate biomarkers for PE, we used isobaric tagging to identify several potential biomarker proteins in plasma obtained at 15 weeks gestation from nulliparous women who later developed PE compared with pregnant women who remained healthy. Such a study generates a number of “candidate” biomarkers that require further testing in larger patient cohorts. As proof-of-principle, two of these proteins were taken forward for verification in a 100 women (58 PE, 42 controls) using label-free SRM. We obtained reproducible protein quantitation across the 100 samples and demonstrated significant changes in protein levels, even with as little as 20% change in protein concentration. The SRM data correlated with a commercial ELISA, suggesting that this is a robust workflow suitable for rapid, affordable, label-free verification of which candidate biomarkers should be taken forward for thorough investigation. A subset of pregnancy-specific glycoproteins (PSGs) had value as novel predictive markers for PE.The identification of clinically relevant plasma biomarkers with diagnostic and/or predictive value continues to challenge the proteomics field. Whereas once the biomarker pipeline was described as a two part discovery and validation process, there is increasing consensus that an intermediate step is required in which the proteins identified in the discovery phase are technically verified in 50 to 200 samples. This verification step identifies false positives from the discovery phase and allows prioritization of proteins to be taken into large-scale clinical validation studies (1). Although commercial ELISA kits may be used in this phase, these are unavailable for many proteins, are expensive, and may lack specificity. In addition, sample requirements may be too high to perform ELISA on all candidates, especially if many proteins are identified as potential markers by low powered, high penetration discovery workflows.Selected reaction monitoring (SRM)¹ mass spectrometry has great potential as an alternative verification method (2–6) as it can be multiplexed, customized, and is highly specific. This potential has not been exploited to date, largely because of technical issues developing a low-cost, reproducible workflow encompassing plasma and serum preparation and LC/MS analysis with the capability to measure protein levels reproducible in hundreds of samples. With traditional stable isotope dilution SRM (SID-SRM), the high cost of accurately quantified, purified stable isotope encoded peptides or proteins may be prohibitive for the verification of multiple peptides from many proteins. Label-free relatively quantitative methods are increasingly popular in discovery proteomics but to a much lesser extent in targeted SRM studies (7, 8).For any SRM method, sample preparation workflows must balance the extent of enrichment and fractionation to enable quantification of lower abundance proteins, against increased technical variability (which is influenced by the number of sample handling steps) and reduced multiplexed potential as a consequence of fractionating peptides from the protein of interest into several distinct fractions. It is also essential that the true technical variation in the workflow is quantitatively evaluated from freezer to MS analysis, rather than just the variation within the LC-SRM part of the experiment. As a paradigm for a label-free SRM assay, we developed our workflow and applied it to the verification of candidate biomarkers that indicate the risk of pre-eclampsia (PE).PE affects 2–8% of pregnancies, and is characterized by hypertension and proteinuria, which may progress to severe maternal complications or death (9). Because delivery of the infant is the only effective intervention, a third of babies are born premature and fetal or newborn mortality is increased three- to 10-fold (10). Its complex etiology involves abnormal placentation, an altered immune response and a sensitized maternal vascular endothelium (11). Prediction of the condition in early pregnancy would allow prevention strategies, such as low dose aspirin, to be targeted to high risk women. In first-time pregnant women, a group particularly at risk, biomarkers continue to fall short of a test that would be useful or cost effective in clinical practice (12–14). Better-performing novel biomarkers are required.The aim of this study was to identify candidate predictive biomarkers for PE and then develop a verification assay using mass spectrometry to determine whether these should be taken forward into more extensive and expensive validation studies. Initial discovery experiments were employed using a pooled sample iTRAQ approach using two different MS platforms to increase plasma proteome coverage. Among the set of proteins discovered, we then developed a label-free SRM assay for relative quantification of CXCL7 (Platelet basic protein; PBP) and members of the Pregnancy specific glycoprotein (PSG) family in a 100-sample set from the international SCreeningfOr Pregnancy Endpoints (SCOPE) study (www.scopestudy.net). Our workflow allowed the specificity and linearity of response for each peptide to be determined, along with true technical variability. Although absolute concentration and LOD/LOQ cannot be calculated using this approach, we aimed to test the hypothesis that a label-free SRM approach could provide a rapid, robust, and efficient screen of candidate plasma biomarkers.     

A phage-encoded nucleoid associated protein compacts both host and phage DNA and derepresses H-NS silencing

Nucleoid Associated Proteins (NAPs) organize the bacterial chromosome within the nucleoid. The interaction of the NAP H-NS with DNA also represses specific host and xenogeneic genes. Previously, we showed that the bacteriophage T4 early protein MotB binds to DNA, co-purifies with H-NS/DNA, and improves phage fitness. Here we demonstrate using atomic force microscopy that MotB compacts the DNA with multiple MotB proteins at the center of the complex. These complexes differ from those observed with H-NS and other NAPs, but resemble those formed by the NAP-like proteins CbpA/Dps and yeast condensin. Fluorescent microscopy indicates that expression of motB in vivo, at levels like that during T4 infection, yields a significantly compacted nucleoid containing MotB and H-NS. motB overexpression dysregulates hundreds of host genes; ∼70% are within the hns regulon. In infected cells overexpressing motB, 33 T4 late genes are expressed early, and the T4 early gene repEB, involved in replication initiation, is up ∼5-fold. We postulate that MotB represents a phage-encoded NAP that aids infection in a previously unrecognized way. We speculate that MotB-induced compaction may generate more room for T4 replication/assembly and/or leads to beneficial global changes in host gene expression, including derepression of much of the hns regulon.