PHRF1 promotes the class switch recombination of IgA in CH12F3-2A cells.

PHRF1 is an E3 ligase that promotes TGF-ß signaling by ubiquitinating a homeodomain repressor TG-interacting factor (TGIF). The suppression of PHRF1 activity by PML-RARα facilitates the progression of acute promyelocytic leukemia (APL). PHRF1 also contributes to non-homologous end-joining in response to DNA damage by linking H3K36me3 and NBS1 with DNA repair machinery. However, its role in class switch recombination (CSR) is not well understood. In this study, we report the importance of PHRF1 in IgA switching in CH12F3-2A cells and CD19-Cre mice. Our studies revealed that Crispr-Cas9 mediated PHRF1 knockout and shRNA-silenced CH12F3-2A cells reduced IgA production, as well as decreased the amounts of PARP1, NELF-A, and NELF-D. The introduction of PARP1 could partially restore IgA production in PHRF1 knockout cells. Intriguingly, IgA, as well as IgG1, IgG2a, and IgG3, switchings were not significantly decreased in PHRF1 deficient splenic B lymphocytes isolated from CD19-Cre mice. The levels of PARP1 and NELF-D were not decreased in PHRF1-depleted primary splenic B cells. Overall, our findings suggest that PHRF1 may modulate IgA switching in CH12F3-2A cells.

A robust platform for expansion and genome editing of primary human natural killer cells.

Genome editing is a powerful technique for delineating complex signaling circuitry and enhancing the functionality of immune cells for immunotherapy. Natural killer (NK) cells are potent immune effectors against cell malignancy, but they are challenging to modify genetically by conventional methods due to the toxicity of DNA when introduced into cells coupled with limited transfection and transduction efficiency. Here, we describe an integrated platform that streamlines feeder-free ex vivo expansion of cryopreserved primary human NK cells and nonviral genome editing by the nucleofection of CRISPR-Cas9 ribonucleoproteins (Cas9 RNPs). The optimized Cas9 nucleofection protocol allows efficient and multiplex gene knockout in NK cells while preserving high cell viability and negligible off-target effects. Cointroduction of a DNA template also enables in-frame gene knock-in of an HA affinity tag and a gfp reporter across multiple loci. This work demonstrates the advantages and flexibility of working with cryopreserved NK cells as potential off-the-shelf engineered therapeutic agents.

Enhanced NK-92 Cytotoxicity by CRISPR Genome Engineering Using Cas9 Ribonucleoproteins.

Natural killer (NK) cells are an attractive cell-type for adoptive immunotherapy, but challenges in preparation of therapeutic primary NK cells restrict patient accessibility to NK cell immunotherapy. NK-92 is a well-characterized human NK cell line that has demonstrated promising anti-cancer activities in clinical trials. Unlimited proliferation of NK-92 cells provides a consistent supply of cells for the administration and development of NK cell immunotherapy. However, the clinical efficacy of NK-92 cells has not reached its full potential due to reduced immune functions as compared to primary NK cells. Improvements of NK-92 functions currently rely on conventional transgene delivery by mRNA, plasmid and viral vector with limited efficiencies. To enable precise genetic modifications, we have established a robust CRISPR genome engineering platform for NK-92 based on the nucleofection of Cas9 ribonucleoprotein. To demonstrate the versatility of the platform, we have performed cell-based screening of Cas9 guide RNA, multiplex gene knockout of activating and inhibitory receptors, knock-in of a fluorescent gene, and promoter insertion to reactivate endogenous CD16 and DNAM-1. The CRISPR-engineered NK-92 demonstrated markedly enhanced cytotoxicity and could mediate antibody-dependent cellular cytotoxicity against hard to kill cancer cell lines. Our genome editing platform is straightforward and robust for both functional studies and therapeutic engineering of NK-92 cells.

The association between blood urea nitrogen to creatinine ratio and mortality in patients with upper gastrointestinal bleeding.

BACKGROUND AND STUDY AIMS: Azotaemia is commonly identified among patients with upper gastrointestinal bleeding (UGIB) due to absorption of blood products in the small bowel. Previous studies have found blood urea nitrogen-to-creatinine (BUN/Cr) ratio to be significantly elevated among patients UGIB bleeding compared to patients with lower GI bleeding. However, no studies have explored the relationship between BUN/Cr ratio and mortality. This study is aimed at investigating how BUN/Cr ratio relates to outcomes for UGIB patients. PATIENTS AND METHODS: This study was conducted prospectively at a university-affiliated teaching hospital with approximate 70,000 annual emergency department (ED) visits. Data from a total of 258 adult UGIB patients were collected between March 1, 2011 and March 1, 2012. Cox regression analysis was used to identify risk factors for 30-day mortality. RESULTS: Malignancy and Rockall score were associated with increased risk of 30-day mortality (Unadjusted hazard ratio (HR): 3.87, 95% CI: 1.59-9.41, p = 0.0029; HR: 1.31, 95% CI: 1.02-1.71, p = 0.0476, respectively). However, BUN/Cr > 30 was associated with lower risk of 30-day mortality (HR: 0.32, 95% CI: 0.11-0.97, p = 0.0441). CONCLUSIONS: A BUN/Cr ratio of >30 was found to be an independent risk factor for mortality and may be useful for pre-endoscopic assessment. Development of future risk scoring systems might warrant consideration of including BUN/Cr ratio as a parameter for estimating risk.

Bacteremia in cirrhotic patients with upper gastrointestinal bleeding.

BACKGROUND/AIMS: Increased risk of bacterial infection is common in cirrhotic patients with upper gastrointestinal bleeding (UGIB). Our study aimed to explore the association of the bacteremia with in-hospital mortality and risk factors of bacteremia in these patients. MATERIALS AND METHODS: In our retrospective cohort study, we collected data for cirrhotic patients with UGIB admitted to our hospital between August 2010 and December 2010. The primary outcome was in-hospital mortality. The secondary outcome was bacteremia. A multivariate logistic regression analysis was performed to determine risk factors for mortality and bacteremia. RESULTS: A total of 202 patients with cirrhosis presenting with UGIB at the emergency department (ED) were enrolled. Bacteremia was associated with a higher mortality rate (adjusted odds ratio [OR]: 9.7; 95% confidence interval [CI]: 1.9-50.6, p=0.007), whereas shock (systolic blood pressure <90 mmHg at ED triage) and bandemia (>0% immature neutrophils of band form) were associated with bacteremia in cirrhotic patients with UGIB (adjusted OR: 5.3; 95% CI: 2.3-12.7, p<0.0001 and adjusted OR: 4.0; 95% CI: 1.6-9.9, p=0.0003, respectively). CONCLUSION: Bacteremia in cirrhotic patients with UGIB is one of the major risk factors leading to in-hospital mortality. On the basis of our findings, prevention of bacteremia in cirrhotic patients with UGIB, especially in those with shock and bandemia, is important; thus, adequate antibiotic treatment is suggested.

Important roles of Vilse in dendritic architecture and synaptic plasticity.

Vilse/Arhgap39 is a Rho GTPase activating protein (RhoGAP) and utilizes its WW domain to regulate Rac/Cdc42-dependent morphogenesis in Drosophila and murine hippocampal neurons. However, the function of Vilse in mammalian dendrite architecture and synaptic plasticity remained unclear. In the present study, we aimed to explore the possible role of Vilse in dendritic structure and synaptic function in the brain. Homozygous knockout of Vilse resulted in premature embryonic lethality in mice. Changes in dendritic complexity and spine density were noticed in hippocampal neurons of Camk2a-Cre mediated forebrain-specific Vilse knockout (VilseΔ/Δ) mice. VilseΔ/Δ mice displayed impaired spatial memory in water maze and Y-maze tests. Electrical stimulation in hippocampal CA1 region revealed that the synaptic transmission and plasticity were defected in VilseΔ/Δ mice. Collectively, our results demonstrate that Vilse is essential for embryonic development and required for spatial memory.