Lysine Residue at Position 22 of the AID Protein Regulates Its Class Switch Activity

Background: Activation induced deaminase (AID) mediates class switch recombination and somatic hypermutation of immunoglobulin (Ig) genes in germinal centre B cells. In order to regulate its specific activity and as a means to keep offtarget mutations low, several mechanisms have evolved, including binding to specific cofactors, phosphorylation and destabilization of nuclear AID protein. Although ubiquitination at lysine residues of AID is recognized as an essential step in initiating degradation of nuclear AID, any functional relevance of lysine modifications has remained elusive. Methodology/Principal Findings: Here, we report functional implications of lysine modifications of the human AID protein by generating a panel of lysine to arginine mutants of AID and assessment of their catalytic class switch activity. We found that only mutation of Lys22 to Arg resulted in a significant reduction of class switching to IgG1 in transfected primary mouse B cells. This decrease in activity was neither reflected in reduced hypermutation of Ig genes in AID-mutant transfected DT40 B cell lines nor recapitulated in bacterial deamination assays, pointing to involvement of post-translational modification of Lys22 for AID activity in B cells. Conclusions/Significance: Our results imply that lysine modification may represent a novel level of AID regulation and tha

Prognostic value of procalcitonin in Legionella pneumonia

The diagnostic reliability and prognostic implications of procalcitonin (PCT) (ng/ml) on admission in patients with community-acquired pneumonia (CAP) due to Legionella pneumophila are unknown. We retrospectively analysed PCT values in 29 patients with microbiologically proven Legionella-CAP admitted to the University Hospital Basel, Switzerland, between 2002 and 2007 and compared them to other markers of infection, namely, C-reactive protein (CRP) (mg/l) and leukocyte count (109/l), and two prognostic severity assessment scores (PSI and CURB65). Laboratory analysis demonstrated that PCT values on admission were >0.1in over 93%, >0.25 in over 86%, and >0.5 in over 82% of patients with Legionella-CAP. Patients with adverse medical outcomes (59%, n = 17) including need for ICU admission (55%, n = 16) and/or inhospital mortality (14%, n = 4) had significantly higher median PCT values on admission (4.27 [IQR 2.46-9.48] vs 0.97 [IQR 0.29-2.44], p = 0.01), while the PSI (124 [IQR 81-147] vs 94 [IQR 75-116], p = 0.19), the CURB65 (2 [IQR 1-2] vs 1 [1-3], p = 0.47), CRP values (282 [IQR 218-343], p = 0.28 vs 201 [IQR 147-279], p = 0.28), and leukocyte counts (12 [IQR 10-21] vs 12 [IQR 9-15], p = 0.58) were similar. In receiver operating curves, PCT concentrations on admission had a higher prognostic accuracy to predict adverse outcomes (AUC 0.78 [95%CI 0.61-96]) as compared to the PSI (0.64 [95%CI 0.43-0.86], p = 0.23), the CURB65 (0.58 [95%CI 0.36-0.79], p = 0.21), CRP (0.61 [95%CI 0.39-0.84], p = 0.19), and leukocyte count (0.57 [95%CI 0.35-0.78], p = 0.12). Kaplan-Meier curves demonstrated that patients with initial PCT values above the optimal cut-off of 1.5 had a significantly higher risk of death and/or ICU admission (log rank p = 0.003) during the hospital stay. In patients with CAP due to Legionella, PCT levels on admission might be an interesting predictor for adverse medical outcome

Imprecision and DNA break repair biased towards incompatible end joining in leukemia

Cancer is a genetic disease caused by mutations and chromosomal abnormalities that contribute to uncontrolled cell growth. In addition, cancer cells can rapidly respond to conventional and targeted therapies by accumulating novel and often specific genetic lesions leading to acquired drug resistance and relapsing disease. In chronic lymphocytic leukemia (CLL), however, diverse chromosomal aberrations often occur. In many cases, improper repair of DNA double-strand breaks (DSB) is a major source for genomic abnormalities. Therefore, this study examined the repair of DNA DSBs by nonhomologous end joining (NHEJ) in CLL by performing plasmid-based repair assays in primary CLL cells and normal B cells, isolated from patients, as well as TALEN/Cas9–induced chromosomal deletions in the CLL cell line Mec1. It is demonstrated that DNA repair is aberrant in CLL cells, featuring perturbed DNA break structure preference with efficient joining of noncohesive ends and more deletions at repair junctions. In addition, increased microhomology-mediated end joining (MMEJ) of DNA substrates was observed in CLL together with increased expression of MMEJ-specific repair factors. In summary, these data identify major differences in DNA repair efficiency between CLL cells and normal B cells isolated from patients

RNA editing contributes to epitranscriptome diversity in chronic lymphocytic leukemia

RNA editing—primarily conversion of adenosine to inosine (A > I)—is a widespread posttranscriptional mechanism, mediated by Adenosine Deaminases acting on RNA (ADAR) enzymes to alter the RNA sequence of primary transcripts. Hence, in addition to somatic mutations and alternative RNA splicing, RNA editing can be a further source for recoding events. Although RNA editing has been detected in many solid cancers and normal tissue, RNA editing in chronic lymphocytic leukemia (CLL) has not been addressed so far. We determined global RNA editing and recurrent, recoding RNA editing events from matched RNA-sequencing and whole exome sequencing data in CLL samples from 45 untreated patients. RNA editing was verified in a validation cohort of 98 CLL patients and revealed substantially altered RNA editing profiles in CLL compared with normal B cells. We further found that RNA editing patterns were prognostically relevant. Finally, we showed that ADAR knockout decreased steady state viability of MEC1 cells and made them more susceptible to treatment with fludarabine and ibrutinib in vitro. We propose that RNA editing contributes to the pathophysiology of CLL and targeting the RNA editing machinery could be a future strategy to maximize treatment efficacy

TIGIT expressing CD4+T cells represent a tumor-supportive T cell subset in chronic lymphocytic leukemia

While research on T cell exhaustion in context of cancer particularly focuses on CD8C cytotoxic T cells, the role of inhibitory receptors on CD4C T-helper cells have remained largely unexplored. TIGIT is a recently identified inhibitory receptor on T cells and natural killer (NK) cells. In this study, we examined TIGIT expression on T cell subsets from CLL patients. While we did not observe any differences in TIGIT expression in CD8C T cells of healthy controls and CLL cells, we found an enrichment of TIGITC T cells in the CD4C T cell compartment in CLL. Intriguingly, CLL patients with an advanced disease stage displayed elevated numbers of CD4C TIGITC T cells compared to low risk patients. Autologous CLL-T cell co-culture assays revealed that depleting CD4C TIGITC expressing T cells from co-cultures significantly decreased CLL viability. Accordingly, a supportive effect of TIGITCCD4C T cells on CLL cells in vitro could be recapitulated by blocking the interaction of TIGIT with its ligands using TIGIT-Fc molecules, which also impeded the T cell specific production of CLL-prosurvival cytokines. Our data reveal that TIGITCCD4CT cells provide a supportive microenvironment for CLL cells, representing a potential therapeutic target for CLL treatment

A Case of Wernicke's Encephalopathy Following Fluorouracil-based Chemotherapy

The pyrimidine antimetabolite 5-fluorouracil (5-FU) is a chemotherapeutic agent used widely for various tumors. Common side effects of 5-FU are related to its effects on the bone marrow and gastrointestinal epithelium. Neurotoxicity caused by 5-FU is uncommon, although acute and delayed forms have been reported. Wernicke's encephalopathy is an acute, neuropsychiatric syndrome resulting from thiamine deficiency, and has significant morbidity and mortality. Central nervous system neurotoxicity such as Wernicke's encephalopathy following chemotherapy with 5-FU has been reported rarely, although it has been suggested that 5-FU can produce adverse neurological effects by causing thiamine deficiency. We report a patient with Wernicke's encephalopathy, reversible with thiamine therapy, associated with 5-FU-based chemotherapy

Genome Analysis Linking Recent European and African Influenza (H5N1) Viruses

Although linked, these viruses are distinct from earlier outbreak strains