Mutation-specific reporter for optimization and enrichment of prime editing.

Prime editing is a versatile genome-editing technique that shows great promise for the generation and repair of patient mutations. However, some genomic sites are difficult to edit and optimal design of prime-editing tools remains elusive. Here we present a fluorescent prime editing and enrichment reporter (fluoPEER), which can be tailored to any genomic target site. This system rapidly and faithfully ranks the efficiency of prime edit guide RNAs (pegRNAs) combined with any prime editor variant. We apply fluoPEER to instruct correction of pathogenic variants in patient cells and find that plasmid editing enriches for genomic editing up to 3-fold compared to conventional enrichment strategies. DNA repair and cell cycle-related genes are enriched in the transcriptome of edited cells. Stalling cells in the G1/S boundary increases prime editing efficiency up to 30%. Together, our results show that fluoPEER can be employed for rapid and efficient correction of patient cells, selection of gene-edited cells, and elucidation of cellular mechanisms needed for successful prime editing.

[Systematic detection of physical child abuse at emergency rooms]. / Screening op kindermishandeling op de SEH.

OBJECTIVE: The aim of our diagnostic accuracy study Child Abuse Inventory at Emergency Rooms (CHAIN-ER) was to establish whether a widely used checklist accurately detects or excludes physical abuse among children presenting to ERs with physical injury. DESIGN: A large multicentre study with a 6-month follow-up in 4 ERs in The Netherlands. METHOD: Participants were 4290 children aged 0-7 years, attending the ER because of physical injury. All children were systematically tested with an easy-to-use child abuse checklist (index test). A national expert panel (reference standard) retrospectively assessed all children with positive screens and a 15% random sample of the children with negative screens for physical abuse, using additional information, namely, an injury history taken by a paediatrician, information provided by the general practitioner, youth doctor and social services by structured questionnaires, and 6-month follow-up information. Our main outcome measure was physical child abuse; secondary outcome measure was injury due to neglect and need for help. RESULTS: 4253/4290 (99%) parents agreed to follow-up. At a prevalence of 0.07% (3/4253) for inflicted injury by expert panel decision, the positive predictive value of the checklist was 0.03 (95% CI 0.006 to 0.085), and the negative predictive value 1.0 (0.994 to 1.0). There was 100% (93 to 100) agreement about inflicted injury in children, with positive screens between the expert panel and child abuse experts. CONCLUSION: Rare cases of inflicted injury among preschool children presenting at ERs for injury are very likely captured by easy-to-use checklists, but at very high false-positive rates. Subsequent assessment by child abuse experts can be safely restricted to children with positive screens at very low risk of missing cases of inflicted injury. Because of the high false positive rate, we do advise careful prior consideration of cost-effectiveness and clinical and societal implications before de novo implementation.

FOXP1 acts through a negative feedback loop to suppress FOXO-induced apoptosis.

Transcriptional activity of Forkhead box transcription factor class O (FOXO) proteins can result in a variety of cellular outcomes depending on cell type and activating stimulus. These transcription factors are negatively regulated by the phosphoinositol 3-kinase (PI3K)-protein kinase B (PKB) signaling pathway, which is thought to have a pivotal role in regulating survival of tumor cells in a variety of cancers. Recently, it has become clear that FOXO proteins can promote resistance to anti-cancer therapeutics, designed to inhibit PI3K-PKB activity, by inducing the expression of proteins that provide feedback at different levels of this pathway. We questioned whether such a feedback mechanism may also exist directly at the level of FOXO-induced transcription. To identify critical modulators of FOXO transcriptional output, we performed gene expression analyses after conditional activation of key components of the PI3K-PKB-FOXO signaling pathway and identified FOXP1 as a direct FOXO transcriptional target. Using chromatin immunoprecipitation followed by next-generation sequencing, we show that FOXP1 binds enhancers that are pre-occupied by FOXO3. By sequencing the transcriptomes of cells in which FOXO is specifically activated in the absence of FOXP1, we demonstrate that FOXP1 can modulate the expression of a specific subset of FOXO target genes, including inhibiting expression of the pro-apoptotic gene BIK. FOXO activation in FOXP1-knockdown cells resulted in increased cell death, demonstrating that FOXP1 prevents FOXO-induced apoptosis. We therefore propose that FOXP1 represents an important modulator of FOXO-induced transcription, promoting cellular survival.

A novel link between Campylobacter jejuni bacteriophage defence, virulence and Guillain-Barré syndrome.

Guillain-Barré syndrome (GBS) is a post-infectious disease in which the human peripheral nervous system is affected after infection by specific pathogenic bacteria, including Campylobacter jejuni. GBS is suggested to be provoked by molecular mimicry between sialylated lipooligosaccharide (LOS) structures on the cell envelope of these bacteria and ganglioside epitopes on the human peripheral nerves, resulting in autoimmune-driven nerve destruction. Earlier, the C. jejuni sialyltransferase (Cst-II) was found to be linked to GBS and demonstrated to be involved in the biosynthesis of the ganglioside-like LOS structures. Apart from a role in pathogenicity, we report here that Cst-II-generated ganglioside-like LOS structures confer efficient bacteriophage resistance in C. jejuni. By bioinformatic analysis, it is revealed that the presence of sialyltransferases in C. jejuni and other potential GBS-related pathogens correlated significantly with the apparent degeneration of an alternative anti-virus system: type II Clusters of Regularly Interspaced Short Palindromic Repeat and associated genes (CRISPR-Cas). Molecular analysis of the C. jejuni CRISPR-Cas system confirmed the bioinformatic investigation. CRISPR degeneration and mutations in the cas genes cas2, cas1 and csn1 were found to correlate with Cst-II sialyltransferase presence (p < 0.0001). Remarkably, type II CRISPR-Cas systems are mainly found in mammalian pathogens. To study the potential involvement of this system in pathogenicity, we inactivated the type II CRISPR-Cas marker gene csn1, which effectively reduced virulence in primarily cst-II-positive C. jejuni isolates. Our findings indicate a novel link between viral defence, virulence and GBS in a pathogenic bacterium.

NKT cells in mucosal immunity.

The gastrointestinal tract allows the residence of an almost enumerable number of bacteria. To maintain homeostasis, the mucosal immune system must remain tolerant to the commensal microbiota and eradicate pathogenic bacteria. Aberrant interactions between the mucosal immune cells and the microbiota have been implicated in the pathogenesis of inflammatory disorders, such as inflammatory bowel disease (IBD). In this review, we discuss the role of natural killer T cells (NKT cells) in intestinal immunology. NKT cells are a subset of non-conventional T cells recognizing endogenous and/or exogenous glycolipid antigens when presented by the major histocompatibility complex (MHC) class I-like antigen-presenting molecules CD1d and MR1. Upon T-cell receptor (TCR) engagement, NKT cells can rapidly produce various cytokines that have important roles in mucosal immunity. Our understanding of NKT-cell-mediated pathways including the identification of specific antigens is expanding. This knowledge will facilitate the development of NKT cell-based interventions and immune therapies for human intestinal diseases.

Cyclooxygenase-2 in mucosal DC mediates induction of regulatory T cells in the intestine through suppression of IL-4.

Oral intake of protein leads to tolerance through the induction of regulatory T cells (Tr cells) in mesenteric lymph nodes (MLNs). Here we show that the inhibition of cyclooxygenase-2 (COX-2) in vivo suppressed oral tolerance and was associated with enhanced differentiation of interleukin (IL)-4-producing T cells and reduced Foxp3(+) Tr-cell differentiation in MLN. As a result, the functional suppressive capacity of these differentiated mucosal T cells was lost. IL-4 was causally related to loss of tolerance as treatment of mice with anti-IL-4 antibodies during COX-2 inhibition restored tolerance. Dendritic cells (DCs) in the MLN differentially expressed COX-2 and reductionist experiments revealed that selective inhibition of the enzyme in these cells inhibited Foxp3(+) Tr-cell differentiation in vitro. Importantly, the inhibition of COX-2 in MLN-DC caused increased GATA-3 expression and enhanced IL-4 release by T cells, which was directly related to impaired Tr-cell differentiation. These data provide crucial insights into the mechanisms driving de novo Tr-cell induction and tolerance in the intestine.

Early onset IBD: what's the difference?

In this paper we describe an array of differences between paediatric and adult inflammatory bowel diseases. Specifically, patient specifics such as genetics, disease location, immune responses and drug responsiveness are addressed. Given the distinct disease phenotype in children, it seems warranted that early onset inflammatory bowel diseases will be denoted as a specific disease entity.

Meta-analysis: Inosine triphosphate pyrophosphatase polymorphisms and thiopurine toxicity in the treatment of inflammatory bowel disease.

BACKGROUND: Thiopurines are widely used for the treatment of inflammatory bowel disease, but are associated with the development of side effects. It has been suggested that the enzyme inosine triphosphate pyrophosphatase (ITPA) plays a role in the digestion of thiopurines and that defective activity resulting from polymorphisms in the inosine triphosphate pyrophosphatase encoding genes may be associated with thiopurine-induced side effects. Current studies are controversial regarding this hypothesis. AIM: To perform a meta-analysis and gain more insight into a possible correlation between thiopurine-induced side effects and ITPA polymorphisms. METHODS: We explored Medline for articles on ITPA polymorphisms and thiopurine toxicity. Studies that compared ITPA polymorphism frequencies among thiopurine-tolerant and -intolerant adult inflammatory bowel disease patients were included in this meta-analysis. RESULTS: Nine published studies investigated associations between ITPA polymorphisms and thiopurine toxicity. Six studies (with 751 patients included) met our inclusion criteria and were processed in the meta-analysis. This analysis demonstrates that the ITPA 94C-->A polymorphism, is not significantly associated with any of the studied side effect parameters. CONCLUSIONS: This meta-analysis does not prove a correlation between the development of thiopurine toxicity and the ITPA 94C-->A polymorphism. This implies that there is no clinical relevance to determine ITPA polymorphisms in thiopurine-treated patients.

Antibody targeting of TIRC7 results in significant therapeutic effects on collagen-induced arthritis in mice.

TIRC7 is a cell surface molecule which is expressed in T and B lymphocytes and negatively regulates their function. Anti-TIRC7 specific monoclonal antibody (mAb) inhibited T cell memory response to recall antigens. Up-regulation of TIRC7 on lymphocytes from joint tissue of patients with Rheumatoid Arthritis (RA) and mice with collagen induced arthritis (CIA) suggested TIRC7 as a novel target to promote anti-inflammatory reaction. Anti-TIRC7 mAb administration significantly inhibited the induction and progression of CIA and the anti-collagen IgG1 and IgG2a antibody response. Combination therapy of anti-TIRC7 mAb and soluble TNF-alpha receptor demonstrated an increased inhibitory effect over the single compounds on CIA. The results demonstrate the therapeutic potential of TIRC7 targeting with mAb in diseases associated with exaggerated T and B cell responses.

[Bladder control in 1-4 year old children in the the Eindhoven and Kempen region (The Netherlands) in 1996 and 1966]. / Zindelijkheid voor urine bij 1-4-jarige kinderen in de regio Eindhoven en de Kempen, in 1996 en in 1966.

OBJECTIVE: To determine the age at which children gain bladder control and to compare this with the data from 30 years ago. DESIGN: Questionnaires. METHOD: On the basis of the findings of a 1966 study into toilet training in the Eindhoven and de Kempen region, the Netherlands, a questionnaire was drawn up and distributed via 30 child-health clinics in this region to parents of children aged 12-59 months, during the period 1 March-30 June 1996. The results were compared with those of the earlier study. RESULTS: Data from 1176 children could be evaluated (response rate: 65%). In 1996, the median age for bladder control in boys during the day was 32.6 months and 40.5 months for night-time control. In 1996 boys achieved daytime bladder control 6.7 months earlier and night-time control 7.2 months earlier. In 1996, the median age for girls was 29.7 months for daytime control and 35.4 months for night-time bladder control: in 1966 girls achieved daytime and night-time bladder control 8.2 and 4.8 months earlier, respectively. Factors associated with earlier bladder control were: early age at which parents started toilet training, presence of other children in the family, early age at which the child attended a day-care centre, early age at which the child was able to walk. Other factors such as the presence of a complete family set, parental level of education and professional situation did not show a correlation with the age at which the child achieved bladder control. The type of diaper used was an additional factor for bladder control at all ages but was only statistically significant for 3-year-olds, both during the day and during the night. CONCLUSION: Children in the Eindhoven region achieved daytime and night-time bladder control at a significantly later age than 30 years ago. Various factors such as toilet-training age, day-care attendance, family size and type of diaper played a role in this phenomenon.

Oral antibiotics as a novel therapy for arthritis: evidence for a beneficial effect of intestinal Escherichia coli.

OBJECTIVE: The intestinal flora is thought to play an important role in regulation of immune responses. We investigated the effects of changing the intestinal flora on the course of adjuvant-induced arthritis (AIA) and on experimental autoimmune encephalomyelitis (EAE) by the use of oral antibiotics. METHODS: Oral treatment with either vancomycin or vancomycin, tobramycin, and colistin was started after AIA and EAE induction. Clinical symptoms of AIA and EAE were monitored, and microbial analysis of ileal samples was performed. RESULTS: Oral vancomycin treatment after disease induction significantly decreased clinical symptoms of AIA. Simultaneously, increased concentrations of Escherichia coli were detected in the distal ileum of vancomycin-treated rats. Ileal concentrations of E coli were inversely related to disease scores in rats with AIA. Coadministration of colistin/tobramycin to prevent the increase in E coli abrogated the beneficial effect of vancomycin on AIA. Vancomycin treatment also reduced the clinical symptoms of EAE. CONCLUSION: We propose oral vancomycin as a novel therapeutic strategy in autoimmune diseases.

Treatment of adjuvant-induced arthritis by oral administration of mycobacterial Hsp65 during disease.

OBJECTIVE: Oral administration of antigen prior to disease induction has been shown to induce peripheral tolerance in several experimental autoimmune diseases. However, the clinical benefit of pretreatment with antigens is limited. The aim of this study was to investigate whether adjuvant-induced arthritis (AIA) could be treated by oral administration of mycobacterial heat-shock protein 65 (Hsp65) during ongoing disease. METHODS: AIA was induced in Lewis rats by immunization with Mycobacterium tuberculosis in Freund's incomplete adjuvant. Oral feeding of Hsp65 in the presence or absence of soybean trypsin inhibitor (SBTI) was started on day 11 after immunization. Arthritis was monitored visually, and joint pathology was examined radiologically. RESULTS: Oral treatment with Hsp65 during ongoing disease significantly reduced the activity of AIA. However, treatment with Hsp65 was only successful when SBTI was coadministered to prevent breakdown of the Hsp65. The beneficial effect of Hsp65/SBTI treatment during AIA was also represented by a clear reduction of articular destruction, as visualized by radiography. Moreover, feeding Hsp65/SBTI resulted in a lower number of both spleen and mesenteric lymph node (MLN) cells expressing the costimulatory molecule CD80 (B7-1). The number of cells expressing CD86 (B7-2) was not altered. Furthermore, MLN cells from AIA animals treated with Hsp65/SBTI contained a lower number of T cells expressing the activation marker CD134 (Ox-40). In addition, treatment with Hsp65/ SBTI was accompanied by an increased proliferative response of spleen cells to the Hsp65 antigen in vitro. Moreover, Hsp65/SBTI-treated rats showed less Hsp65-specific interferon-gamma and increased production of interleukin-10. CONCLUSION: Ongoing AIA activity can be reduced by oral administration of Hsp65 only when protein breakdown in the gastrointestinal tract is inhibited.

Effects of beta-adrenergic blockade on immunologic and cardiovascular changes induced by mental stress.

BACKGROUND: Acute mental stress evokes responses in the cardiovascular and the immune systems. In particular, the subset of natural killer (NK) cells is found to be responsive to mental stress. The role of beta-adrenergic mechanisms in these processes in the subject of this investigation. METHODS AND RESULTS: Healthy male volunteers (n = 31) were subjected to two consecutive mental tasks. Subjects were randomly assigned to a beta-blocker (propranolol 40 mg) or a placebo group. The capsules were ingested 1 hour before the tasks. The tasks evoked sympathetic responses, as indicated by an increase in heart rate and a decrease in the preejection period. These effects were abolished under beta-blockade, indicating that effective beta-blockade was achieved. In the immune system, significant increases were found for the number of NK cells and NK cell activity in the placebo group; these increases were absent in the propranolol group. In addition, an increase in all lymphocyte subsets was observed in subjects who had ingested propranolol. This increase, however, was also observed in subjects who had received propranolol but had not performed the tasks, indicating that these non-subset-specific increases in lymphocytes were a side effect of the beta-blocker. CONCLUSIONS: Mental stress induces activation of the sympathetic nervous system, with concomitant increases in the number of NK cells in the circulation. These changes were inhibited by propranolol, indicating that stress-induced increases in the number and activity of NK cells in the circulation are controlled by a beta-adrenergic mechanism.