Functional assessment for clinical use of serum-free adapted NK-92 cells

Natural killer (NK) cells stand out as promising candidates for cellular immunotherapy due to their capacity to kill malignant cells. However, the therapeutic use of NK cells is often dependent on cell expansion and activation with considerable amounts of serum and exogenous cytokines. We aimed to develop an expansion protocol for NK-92 cells in an effort to generate a cost-efficient, xeno-free, clinical grade manufactured master cell line for therapeutic applications. By making functional assays with NK-92 cells cultured under serum-free conditions (NK-92(SF)) and comparing to serum-supplemented NK-92 cells (NK-92(S)) we did not observe significant alterations in the viability, proliferation, receptor expression levels, or in perforin and granzyme levels. Interestingly, even though NK-92(SF) cells displayed decreased degranulation and cytotoxicity against tumor cells in vitro, the degranulation capacity was recovered after overnight incubation with 20% serum in the medium. Moreover, lentiviral vector-based genetic modification efficiency of NK-92(SF) cells was comparable with NK-92(S) cells. The application of similar strategies can be useful in reducing the costs of manufacturing cells for clinical use and can help us understand and implement strategies towards chemically defined expansion and genetic modification protocols

Are non-responders in a quitline evaluation more likely to be smokers?

BACKGROUND: In evaluation of smoking cessation programs including surveys and clinical trials the tradition has been to treat non-responders as smokers. The aim of this paper is to assess smoking behaviour of non-responders in an evaluation of the Swedish national tobacco cessation quitline a nation-wide, free of charge service. METHODS: A telephone interview survey with a sample of people not participating in the original follow-up. The study population comprised callers to the Swedish quitline who had consented to participate in a 12 month follow-up but had failed to respond. A sample of 84 (18% of all non-responders) was included. The main outcome measures were self-reported smoking behaviour at the time of the interview and at the time of the routine follow-up. Also, reasons for not responding to the original follow-up questionnaire were assessed. For statistical comparison between groups we used Fischer's exact test, odds ratios (OR) and 95% confidence intervals (CI) on proportions and OR. RESULTS: Thirty-nine percent reported to have been smoke-free at the time they received the original questionnaire compared with 31% of responders in the original study population. The two most common reasons stated for not having returned the original questionnaire was claiming that they had returned it (35%) and that they had not received the questionnaire (20%). Non-responders were somewhat younger and were to a higher degree smoke-free when they first called the quitline. CONCLUSION: Treating non-responders as smokers in smoking cessation research may underestimate the true effect of cessation treatment

Divergent β-hairpins determine double-strand versus single-strand substrate recognition of human AlkB-homologues 2 and 3

Human AlkB homologues ABH2 and ABH3 repair 1-methyladenine and 3-methylcytosine in DNA/RNA by oxidative demethylation. The enzymes have similar overall folds and active sites, but are functionally divergent. ABH2 efficiently demethylates both single- and double-stranded (ds) DNA, whereas ABH3 has a strong preference for single-stranded DNA and RNA. We find that divergent F1 β-hairpins in proximity of the active sites of ABH2 and ABH3 are central for substrate specificities. Swapping F1 hairpins between the enzymes resulted in hybrid proteins resembling the donor proteins. Surprisingly, mutation of the intercalating residue F102 had little effect on activity, while the double mutant V101A/F102A was catalytically impaired. These residues form part of an important hydrophobic network only present in ABH2. In this functionally important network, F124 stacks with the flipped out base while L157 apparently functions as a buffer stop to position the lesion in the catalytic pocket for repair. F1 in ABH3 contains charged and polar residues preventing use of dsDNA substrate. Thus, E123 in ABH3 corresponds to F102 in ABH2 and the E123F-variant gained capacity to repair dsDNA with no loss in single strand repair capacity. In conclusion, divergent sequences outside of the active site determine substrate specificities of ABH2 and ABH3

Intestinal Obstruction Syndromes in Cystic Fibrosis: Meconium Ileus, Distal Intestinal Obstruction Syndrome, and Constipation

Meconium ileus at birth, distal intestinal obstruction syndrome (DIOS), and constipation are an interrelated group of intestinal obstruction syndromes with a variable severity of obstruction that occurs in cystic fibrosis patients. Long-term follow-up studies show that today meconium ileus is not a risk factor for impaired nutritional status, pulmonary function, or survival. DIOS and constipation are frequently seen in cystic fibrosis patients, especially later in life; genetic, dietary, and other associations have been explored. Diagnosis of DIOS is based on suggestive symptoms, with a right lower quadrant mass confirmed on abdominal radiography, whereas symptoms of constipation are milder and of longer standing. In DIOS, early aggressive laxative treatment with oral laxatives (polyethylene glycol) or intestinal lavage with balanced osmotic electrolyte solution and rehydration is required, which now makes the need for surgical interventions rare. Constipation can generally be well controlled with polyethylene glycol maintenance treatment

Socio-demographic, lifestyle and health characteristics among snus users and dual tobacco users in Stockholm County, Sweden

<p>Abstract</p> <p>Background</p> <p>Socio-demographic and lifestyle characteristics of snus users have not been systematically described. Such knowledge is pivotal for tobacco control efforts and for the assessment of health effects of snus use.</p> <p>Methods</p> <p>A cross-sectional study was conducted, based on the Stockholm Public Health Survey, including a population-based sample of 34,707 men and women aged 18-84 years. We examined how socio-demographic, lifestyle and health-related characteristics were associated with the prevalence of current daily snus use, smoking and dual tobacco use. Logistic regression was used to calculate odds ratios of prevalence (ORs) and 95% confidence intervals (CIs).</p> <p>Results</p> <p>Low educational level (OR = 1.60, CI = 1.41-1.81 and OR = 1.49, CI = 1.17-1.89, for men and women respectively), as well as occupational class and low income were associated with snus use. Some unfavourable lifestyle characteristics, including risky alcohol consumption (males: OR = 1.81, CI = 1.63-2.02; females: OR = 1.79, CI = 1.45-2.20), binge drinking and low consumption of fruit and vegetables were also associated with snus use. In contrast, physical inactivity and overweight/obesity were not, nor was perceived health. The prevalence of smoking followed steeper gradients for social as well as lifestyle characteristics. Overweight and obese men were however less often smokers. Perceived poor general health and psychological distress were highly related to smoking. Social disadvantage, as well as unhealthy lifestyle and self-reported poor health were strongly associated with dual use. There were limited differences between men and women.</p> <p>Conclusions</p> <p>The social, lifestyle and health profiles of exclusive snus users in Stockholm County are less favourable than those of non-users of tobacco, but more advantageous than those of exclusive smokers. This knowledge should guide tobacco control measures as well as the interpretation of health risks linked to snus use.</p

PCNA directs type 2 RNase H activity on DNA replication and repair substrates

Ribonuclease H2 is the major nuclear enzyme degrading cellular RNA/DNA hybrids in eukaryotes and the sole nuclease known to be able to hydrolyze ribonucleotides misincorporated during genomic replication. Mutation in RNASEH2 causes Aicardi–Goutières syndrome, an auto-inflammatory disorder that may arise from nucleic acid byproducts generated during DNA replication. Here, we report the crystal structures of Archaeoglobus fulgidus RNase HII in complex with PCNA, and human PCNA bound to a C-terminal peptide of RNASEH2B. In the archaeal structure, three binding modes are observed as the enzyme rotates about a flexible hinge while anchored to PCNA by its PIP-box motif. PCNA binding promotes RNase HII activity in a hinge-dependent manner. It enhances both cleavage of ribonucleotides misincorporated in DNA duplexes, and the comprehensive hydrolysis of RNA primers formed during Okazaki fragment maturation. In addition, PCNA imposes strand specificity on enzyme function, and by localizing RNase H2 and not RNase H1 to nuclear replication foci in vivo it ensures that RNase H2 is the dominant RNase H activity during nuclear replication. Our findings provide insights into how type 2 RNase H activity is directed during genome replication and repair, and suggest a mechanism by which RNase H2 may suppress generation of immunostimulatory nucleic acids

PCNA dependent cellular activities tolerate dramatic perturbations in PCNA client interactions

Proliferating cell nuclear antigen (PCNA) is an essential cofactor for DNA replication and repair, recruiting multiple proteins to their sites of action. We examined the effects of the PCNA(S228I) mutation that causes PCNA-associated DNA repair disorder (PARD). Cells from individuals affected by PARD are sensitive to the PCNA inhibitors T3 and T2AA, showing that the S228I mutation has consequences for undamaged cells. Analysis of the binding between PCNA and PCNA-interacting proteins (PIPs) shows that the S228I change dramatically impairs the majority of these interactions, including that of Cdt1, DNMT1, PolD3(p66) and PolD4(p12). In contrast p21 largely retains the ability to bind PCNA(S228I). This property is conferred by the p21 PIP box sequence itself, which is both necessary and sufficient for PCNA(S228I) binding. Ubiquitination of PCNA is unaffected by the S228I change, which indirectly alters the structure of the inter-domain connecting loop. Despite the dramatic in vitro effects of the PARD mutation on PIP-degron binding, there are only minor alterations to the stability of p21 and Cdt1 in cells from affected individuals. Overall our data suggests that reduced affinity of PCNA(S228I) for specific clients causes subtle cellular defects in undamaged cells which likely contribute to the etiology of PARD