Simultaneous precise editing of multiple genes in human cells

Abstract. When double-strand breaks are introduced in a genome by CRISPR they are repaired either by non-homologous end joining (NHEJ), which often results i

Commensal-Induced Regulatory T Cells Mediate Protection against Pathogen-Stimulated NF-κB Activation

Host defence against infection requires a range of innate and adaptive immune responses that may lead to tissue damage. Such immune-mediated pathologies can be controlled with appropriate T regulatory (Treg) activity. The aim of the present study was to determine the influence of gut microbiota composition on Treg cellular activity and NF-κB activation associated with infection. Mice consumed the commensal microbe Bifidobacterium infantis 35624 followed by infection with Salmonella typhimurium or injection with LPS. In vivo NF-κB activation was quantified using biophotonic imaging. CD4+CD25+Foxp3+ T cell phenotypes and cytokine levels were assessed using flow cytometry while CD4+ T cells were isolated using magnetic beads for adoptive transfer to naïve animals. In vivo imaging revealed profound inhibition of infection and LPS induced NF-κB activity that preceded a reduction in S. typhimurium numbers and murine sickness behaviour scores in B. infantis–fed mice. In addition, pro-inflammatory cytokine secretion, T cell proliferation, and dendritic cell co-stimulatory molecule expression were significantly reduced. In contrast, CD4+CD25+Foxp3+ T cell numbers were significantly increased in the mucosa and spleen of mice fed B. infantis. Adoptive transfer of CD4+CD25+ T cells transferred the NF-κB inhibitory activity. Consumption of a single commensal micro-organism drives the generation and function of Treg cells which control excessive NF-κB activation in vivo. These cellular interactions provide the basis for a more complete understanding of the commensal-host-pathogen trilogue that contribute to host homeostatic mechanisms underpinning protection against aberrant activation of the innate immune system in response to a translocating pathogen or systemic LPS

EpCAM an immunotherapeutic target for gastrointestinal malignancy: current experience and future challenges

Despite advances in surgery and adjuvant regimes, gastrointestinal malignancy remains a major cause of neoplastic mortality. Immunotherapy is an emerging and now successful treatment modality for numerous cancers that relies on the manipulation of the immune system and its effector functions to eradicate tumour cells. The discovery that the pan-epithelial homotypic cell adhesion molecule EpCAM is differentially expressed on gastrointestinal tumours has made this a viable target for immunotherapy. Clinical trials using naked anti EpCAM antibody, immunoconjugates, anti-idiotypic and dendritic cell vaccines have met variable success. The murine IgG2a Edrecolomab was shown to reduce mortality and morbidity at a level slightly lower than treatment with 5FU and Levamisole when administered to patients with advanced colorectal carcinoma in a large randomised controlled trial. Fully human and trifunctional antibodies that specifically recruit CD3-positive lymphocytes are now being tested clinically in the treatment of minimal residual disease and ascites. Although clinical trials are in their infancy, the future may bring forth an EpCAM mediated approach for the effective activation and harnessing of the immune system to destroy a pathological aberrance that has otherwise largely escaped its attention

Hydroxyurea and sickle cell anemia: effect on quality of life

BACKGROUND: The Multicenter Study of Hydroxyurea (HU) in Sickle Cell Anemia (MSH) previously showed that daily oral HU reduces painful sickle cell (SS) crises by 50% in patients with moderate to severe disease. The morbidity associated with this disease is known to have serious negative impact on the overall quality of life(QOL) of affected individuals. METHODS: The data in this report were collected from the 299 patients enrolled in the MSH. Health quality of llife (HQOL) measures were assessed in the MSH as a secondary endpoint to determine if the clinical benefit of HU could translate into a measurable benefit perceptible to the patients. HQOL was assessed with the Profile of Mood States, the Health Status Short Form 36 (SF-36), including 4-week pain recall, and the Ladder of Life, self-administered twice 2-weeks apart pre-treatment and every 6 months during the two-year, randomized, double-blind, treatment phase. The effects of factors including randomized treatment, age, gender, pre-treatment crises frequency, Hb-F level mean, daily pain from 4-week pre-treatment diaries, and 2-year Hb-F response level (low or high) were investigated. RESULTS: Over two years of treatment, the benefit of HU treatment on QOL, other than pain scales, was limited to those patients taking HU who maintained a high HbF response, compared to those with low HbF response or on placebo. These restricted benefits occurred in social function, pain recall and general health perception. Stratification according to average daily pain prior to treatment showed that responders to HU whose average daily pain score was 5–9 (substantial pain) achieved significant reduction in the tension scale compared to the placebo group and to non-responders. HU had no apparent effect on other QOL measures. CONCLUSION: Treatment of SS with HU improves some aspects of QOL in adult patients who already suffer from moderate-to-severe SS

Solid Tumor-Targeted Infiltrating Cytotoxic T Lymphocytes Retained by a Superantigen Fusion Protein

Successful immune-mediated regression of solid tumors is difficult because of the small number of cytotoxic T lymphocytes (CTLs) that were traffic to the tumor site. Here, the targeting of tumor-specific infiltrating CTLs was dependent on a fusion protein consisting of human epidermal growth factor (EGF) and staphylococcal enterotoxin A (SEA) with the D227A mutation. EGF-SEA strongly restrained the growth of murine solid sarcoma 180 (S180) tumors (control versus EGF-SEA, mean tumor weight: 1.013 versus 0.197 g, difference  = 0.816 g). In mice treated with EGF-SEA, CD4+, CD8+ and SEA-reactive T lymphocytes were enriched around the EGFR expressing tumor cells. The EGF receptors were potentially phosphorylated by EGF-SEA stimulation and the fusion protein promoted T cells to release the tumoricidal cytokines interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). Intratumoral CTLs secreted cytolytic pore-forming perforins and granzyme B proteins near the surface of carcinomas, causing the death of many tumor cells. We additionally show that labeled EGF-SEA was directly targeted to the tumor tissue after intravenous (i.v.) injection. The findings demonstrate that antibody-like EGF-SEA plays an important role in arresting CTLs in the solid tumor site and has therapeutic potential as a tumor-targeting agent

Tankyrase inhibition sensitizes melanoma to PD-1 immune checkpoint blockade in syngeneic mouse models

publishedVersio

A phase I trial of the trifunctional anti Her2 × anti CD3 antibody ertumaxomab in patients with advanced solid tumors

Background: Ertumaxomab (ertu) is a bispecific, trifunctional antibody targeting Her2/neu, CD3 and the Fcγ-receptors I, IIa, and III forming a tri-cell complex between tumor cell, T cell and accessory cells. Methods: Patients (pts) with Her2/neu (1+/SISH positive, 2+ and 3+) expressing tumors progressing after standard therapy were treated to investigate safety, tolerability and preliminary efficacy. In this study, ertu was applied i.v. in 2 cycles following a predefined dose escalating scheme. Each cycle consisted of five ascending doses (10–500 μg) applied weekly within 28 days with a 21 day treatment-free interval. If 2 pts experienced a dose limiting toxicity (DLT) at a given dose level, the maximum tolerated dose (MTD) had been exceeded. Results: Fourteen heavily pretreated pts (e.g. breast, rectal, gastric cancer) were enrolled in the four main cohorts. Three (21 %) pts had to be replaced. Two serious adverse events (SAE) with possible relation to the investigational drug were seen, both fully reversible. A DLT was not detected. Consequently, the MTD could not be determined. All adverse events (AE) were transient and completely reversible. Most frequent AEs were fatigue (14/14), pain (13/14), cephalgia (12/14), chills (11/14), nausea (8/14), fever (7/14), emesis (7/14) and diarrhea (5/14). Single doses up to 300 μg were well tolerated (total dose up to 800 μg per cycle). We observed one partial remission and two disease stabilizations after first treatment cycle. Conclusions: Single doses up to 300 μg could be safely administered in an escalating dose scheme. Immunological responses and clinical activity warrant further evaluation in patients with Her2 over expressing tumors. Trial registration EudraCT number: 2011-003201-14; ClinicalTrials.gov identifier: NCT0156941

The James Webb Space Telescope

The James Webb Space Telescope (JWST) is a large (6.6m), cold (50K), infrared-optimized space observatory that will be launched early in the next decade. The observatory will have four instruments: a near-infrared camera, a near-infrared multi-object spectrograph, and a tunable filter imager will cover the wavelength range, 0.6 to 5.0 microns, while the mid-infrared instrument will do both imaging and spectroscopy from 5.0 to 29 microns. The JWST science goals are divided into four themes. The End of the Dark Ages: First Light and Reionization theme seeks to identify the first luminous sources to form and to determine the ionization history of the early universe. The Assembly of Galaxies theme seeks to determine how galaxies and the dark matter, gas, stars, metals, morphological structures, and active nuclei within them evolved from the epoch of reionization to the present day. The Birth of Stars and Protoplanetary Systems theme seeks to unravel the birth and early evolution of stars, from infall on to dust-enshrouded protostars to the genesis of planetary systems. The Planetary Systems and the Origins of Life theme seeks to determine the physical and chemical properties of planetary systems including our own, and investigate the potential for the origins of life in those systems. To enable these observations, JWST consists of a telescope, an instrument package, a spacecraft and a sunshield. The telescope consists of 18 beryllium segments, some of which are deployed. The segments will be brought into optical alignment on-orbit through a process of periodic wavefront sensing and control. The JWST operations plan is based on that used for previous space observatories, and the majority of JWST observing time will be allocated to the international astronomical community through annual peer-reviewed proposal opportunities.Comment: 96 pages, including 48 figures and 15 tables, accepted by Space Science Review

Best practices in heterotrophic high-cell-density microalgal processes: achievements, potential and possible limitations

Microalgae of numerous heterotrophic genera (obligate or facultative) exhibit considerable metabolic versatility and flexibility but are currently underexploited in the biotechnological manufacturing of known plant-derived compounds, novel high-value biomolecules or enriched biomass. Highly efficient production of microalgal biomass without the need for light is now feasible in inexpensive, well-defined mineral medium, typically supplemented with glucose. Cell densities of more than 100 g l−1 cell dry weight have been achieved with Chlorella, Crypthecodinium and Galdieria species while controlling the addition of organic sources of carbon and energy in fedbatch mode. The ability of microalgae to adapt their metabolism to varying culture conditions provides opportunities to modify, control and thereby maximise the formation of targeted compounds with non-recombinant microalgae. This review outlines the critical aspects of cultivation technology and current best practices in the heterotrophic high-cell-density cultivation of microalgae. The primary topics include (1) the characteristics of microalgae that make them suitable for heterotrophic cultivation, (2) the appropriate chemical composition of mineral growth media, (3) the different strategies for fedbatch cultivations and (4) the principles behind the customisation of biomass composition. The review confirms that, although fundamental knowledge is now available, the development of efficient, economically feasible large-scale bioprocesses remains an obstacle to the commercialisation of this promising technology