High tibial osteotomy in medial compartment osteoarthritis and varus deformity using the Taylor spatial frame: early results

We report the early results of high tibial osteotomy (HTO) in medial compartment osteoarthritis (OA) and varus deformity using the Taylor spatial frame (TSF). Between October 2005 and April 2007, 9 patients with medial compartment OA and varus deformity underwent TSF application and medial opening wedge HTO. Pre- and post-operative Oxford knee scores, SF-12 and visual analogue pain scores were recorded along with radiographic outcomes. Median follow-up was 19 months (range 15–35). Mean age at operation was 49 years (range 37–59). The median time spent in the frame was 18 weeks (range 12–37). The mean preoperative Oxford knee score was 28.7. This improved to a mean of 35.4 post-operatively (P = 0.0142). 6 (67%) patients had a documented pin-site infection. With TKR as an end point, the survival rate of HTOs was 88.9% at a median of 19 months follow-up. This study demonstrates that in selected patients the TSF provides a viable treatment option for performing HTO in medial compartment OA with varus deformity

Observation of the spin-orbit gap in bilayer graphene by one-dimensional ballistic transport

We report on measurements of quantized conductance in gate-defined quantum point contacts in bilayer graphene that allow the observation of subband splittings due to spin-orbit coupling. The size of this splitting can be tuned from 40 to 80 $\mu$eV by the displacement field. We assign this gate-tunable subband-splitting to a gap induced by spin-orbit coupling of Kane-Mele type, enhanced by proximity effects due to the substrate. We show that this spin-orbit coupling gives rise to a complex pattern in low perpendicular magnetic fields, increasing the Zeeman splitting in one valley and suppressing it in the other one. In addition, we observe the existence of a spin-polarized channel of 6 e$^2$/h at high in-plane magnetic field and of signatures of interaction effects at the crossings of spin-split subbands of opposite spins at finite magnetic field.Comment: 5 pages, 4 figures, Supplement 6 figure

Readmission and mortality in malnourished, older, hospitalized adults treated with a specialized oral nutritional supplement: A randomized clinical trial

SummaryBackgroundHospitalized, malnourished older adults have a high risk of readmission and mortality.ObjectiveEvaluation of a high-protein oral nutritional supplement (HP-HMB) containing beta-hydroxy-beta-methylbutyrate on postdischarge outcomes of nonelective readmission and mortality in malnourished, hospitalized older adults.DesignMulticenter, randomized, placebo-controlled, double-blind trial.SettingInpatient and posthospital discharge.PatientsOlder (≥65 years), malnourished (Subjective Global Assessment [SGA] class B or C) adults hospitalized for congestive heart failure, acute myocardial infarction, pneumonia, or chronic obstructive pulmonary disease.InterventionsStandard-of-care plus HP-HMB (n = 328) or a placebo supplement (n = 324), 2 servings/day.MeasurementsPrimary composite endpoint was 90-day postdischarge incidence of death or nonelective readmission. Other endpoints included 30- and 60-day postdischarge incidence of death or readmission, length of stay (LOS), SGA class, body weight, and activities of daily living (ADL).ResultsThe primary composite endpoint was similar between HP-HMB (26.8%) and placebo (31.1%). No between-group differences were observed for 90-day readmission rate, but 90-day mortality was significantly lower with HP-HMB relative to placebo (4.8% vs. 9.7%; relative risk 0.49, 95% confidence interval [CI], 0.27 to 0.90; p = 0.018). The number-needed-to-treat to prevent 1 death was 20.3 (95% CI: 10.9, 121.4). Compared with placebo, HP-HMB resulted in improved odds of better nutritional status (SGA class, OR, 2.04, 95% CI: 1.28, 3.25, p = 0.009) at day 90, and an increase in body weight at day 30 (p = 0.035). LOS and ADL were similar between treatments.LimitationsLimited generalizability; patients represent a selected hospitalized population.ConclusionsAlthough no effects were observed for the primary composite endpoint, compared with placebo HP-HMB decreased mortality and improved indices of nutritional status during the 90-day observation period.Clinical trial registrationwww.ClinicalTrials.gov NCT01626742

Membrane-Bound IL-21 Promotes Sustained Ex Vivo Proliferation of Human Natural Killer Cells

NK cells have therapeutic potential for a wide variety of human malignancies. However, because NK cells expand poorly in vitro, have limited life spans in vivo, and represent a small fraction of peripheral white blood cells, obtaining sufficient cell numbers is the major obstacle for NK-cell immunotherapy. Genetically-engineered artificial antigen-presenting cells (aAPCs) expressing membrane-bound IL-15 (mbIL15) have been used to propagate clinical-grade NK cells for human trials of adoptive immunotherapy, but ex vivo proliferation has been limited by telomere shortening. We developed K562-based aAPCs with membrane-bound IL-21 (mbIL21) and assessed their ability to support human NK-cell proliferation. In contrast to mbIL15, mbIL21-expressing aAPCs promoted log-phase NK cell expansion without evidence of senescence for up to 6 weeks of culture. By day 21, parallel expansion of NK cells from 22 donors demonstrated a mean 47,967-fold expansion (median 31,747) when co-cultured with aAPCs expressing mbIL21 compared to 825-fold expansion (median 325) with mbIL15. Despite the significant increase in proliferation, mbIL21-expanded NK cells also showed a significant increase in telomere length compared to freshly obtained NK cells, suggesting a possible mechanism for their sustained proliferation. NK cells expanded with mbIL21 were similar in phenotype and cytotoxicity to those expanded with mbIL15, with retained donor KIR repertoires and high expression of NCRs, CD16, and NKG2D, but had superior cytokine secretion. The mbIL21-expanded NK cells showed increased transcription of the activating receptor CD160, but otherwise had remarkably similar mRNA expression profiles of the 96 genes assessed. mbIL21-expanded NK cells had significant cytotoxicity against all tumor cell lines tested, retained responsiveness to inhibitory KIR ligands, and demonstrated enhanced killing via antibody-dependent cell cytotoxicity. Thus, aAPCs expressing mbIL21 promote improved proliferation of human NK cells with longer telomeres and less senescence, supporting their clinical use in propagating NK cells for adoptive immunotherapy

Micro-algae come of age as a platform for recombinant protein production

A complete set of genetic tools is still being developed for the micro-alga Chlamydomonas reinhardtii. Yet even with this incomplete set, this photosynthetic single-celled plant has demonstrated significant promise as a platform for recombinant protein expression. In recent years, techniques have been developed that allow for robust expression of genes from both the nuclear and plastid genome. With these advances, many research groups have examined the pliability of this and other micro-algae as biological machines capable of producing recombinant peptides and proteins. This review describes recent successes in recombinant protein production in Chlamydomonas, including production of complex mammalian therapeutic proteins and monoclonal antibodies at levels sufficient for production at economic parity with existing production platforms. These advances have also shed light on the details of algal protein production at the molecular level, and provide insight into the next steps for optimizing micro-algae as a useful platform for the production of therapeutic and industrially relevant recombinant proteins

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

A Novel Method for Assessment of Natural Killer Cell Cytotoxicity Using Image Cytometry

<div><p>Natural killer (NK) cells belong to the innate arm of the immune system and though activated NK cells can modulate immune responses through the secretion of cytokines, their primary effector function is through target cell lysis. Accordingly, cytotoxicity assays are central to studying NK cell function. The ⁵¹Chromium release assay, is the “gold standard” for cytotoxicity assay, however, due to concerns over toxicity associated with the use and disposal of radioactive compounds there is a significant interest in non-radioactive methods. We have previously used the calcein release assay as a non-radioactive alternative for studying NK cell cytotoxicity. In this study, we show that the calcein release assay varies in its dynamic range for different tumor targets, and that the entrapped calcein could remain unreleased within apoptotic bodies of lysed tumor targets or incompletely released resulting in underestimation of percent specific lysis. To overcome these limitations, we developed a novel cytotoxicity assay using the Cellometer Vision Image Cytometer and compared this method to standard calcein release assay for measuring NK cell cytotoxicity. Using tumor lines K562, 721.221, and Jurkat, we demonstrate here that image cytometry shows significantly higher percent specific lysis of the target cells compared to the standard calcein release assay within the same experimental setup. Image cytometry is able to accurately analyze live target cells by excluding dimmer cells and smaller apoptotic bodies from viable target cell counts. The image cytometry-based cytotoxicity assay is a simple, direct and sensitive method and is an appealing option for routine cytotoxicity assay.</p></div

Percent specific lysis of K562 cell line using protocol optimized for direct image cytometry assay.

<p>(a) Cytotoxicity against K562 was repeated by image cytometry with reduced target cell number (50,000 cells/well), in a final media volume of 100 μl. After the standard 4-hour incubation the cells were directly resuspended in the 100 μl volume and read using image cytometer. (b) The data shows that with reduced target cell density (Low) the cytotoxicity of NK cells against K562 was significantly higher compared to previous assay with higher target cell density (High) of 100,000 cells/well. The statistical analysis was performed using Wilcoxon matched-pairs signed rank, non-parametric, two-tailed t test and <i>p</i> value of <0.05 was considered significant. The data is plotted as median with range.</p

Determination of target cell lysis by image cytometry.

<p>The figure shows images from the image cytometer and analysis of fluorescence data using FCS express, to derive live target cell counts. The number of live target cells in the culture was determined by plotting fluorescence intensity of target cells from each E:T ratio compared to spontaneous control. The live K562 cells are shown in blue circles and the lysed cells and apoptotic bodies are highlighted in red circles in the images. Representative fluorescence histograms used for deriving live target cell counts are shown for each tumor cell lines (K562, 721.221 and Jurkat cells).</p