Novel PNKP mutations associated with reduced DNA single-strand break repair and severe microcephaly, seizures, and developmental delay.

BACKGROUND: Microcephaly with early-onset seizures (MCSZ) is a neurodevelopmental disorder caused by pathogenic variants in the DNA strand break repair protein, polynucleotide kinase 3'-phosphatase (PNKP). METHODS: We have used whole genome sequencing and Sanger sequencing to identify disease-causing variants, followed by a minigene assay, Western blotting, alkaline comet assay, γH2AX, and ADP-ribose immunofluorescence. RESULTS: Here, we describe a patient with compound heterozygous variants in PNKP, including a missense variant in the DNA phosphatase domain (T323M) and a novel splice acceptor site variant within the DNA kinase domain that we show leads to exon skipping. We show that primary fibroblasts derived from the patient exhibit greatly reduced levels of PNKP protein and reduced rates of DNA single-strand break repair, confirming that the mutated PNKP alleles are dysfunctional. CONCLUSION: The data presented show that the detected compound heterozygous variants result in reduced levels of PNKP protein, which affect the repair of both oxidative and TOP1-induced single-strand breaks, and most likely causes MCSZ in this patient.

Membranous NECTIN-4 Expression Frequently Decreases during Metastatic Spread of Urothelial Carcinoma and Is Associated with Enfortumab Vedotin Resistance.

PURPOSE: The antibody-drug conjugate enfortumab vedotin (EV) releases a cytotoxic agent into tumor cells via binding to the membrane receptor NECTIN-4. EV was recently approved for patients with metastatic urothelial carcinoma (mUC) without prior assessment of the tumor receptor status as ubiquitous NECTIN-4 expression is assumed. Our objective was to determine the prevalence of membranous NECTIN-4 protein expression in primary tumors (PRIM) and patient-matched distant metastases (MET). EXPERIMENTAL DESIGN: Membranous NECTIN-4 protein expression was measured (H-score) by IHC in PRIM and corresponding MET (N = 137) and in a multicenter EV-treated cohort (N = 47). Progression-free survival (PFS) after initiation of EV treatment was assessed for the NECTIN-4-negative/weak (H-score 0-99) versus moderate/strong (H-score 100-300) subgroup. The specificity of the NECTIN-4 IHC staining protocol was validated by establishing CRISPR-Cas9-induced polyclonal NECTIN-4 knockouts. RESULTS: In our cohort, membranous NECTIN-4 expression significantly decreased during metastatic spread (Wilcoxon matched pairs P < 0.001; median H-score = 40; interquartile range, 0-140), with 39.4% of MET lacking membranous NECTIN-4 expression. In our multicenter EV cohort, absence or weak membranous NECTIN-4 expression (34.0% of the cohort) was associated with a significantly shortened PFS on EV (log-rank P < 0.001). CONCLUSIONS: Membranous NECTIN-4 expression is frequently decreased or absent in mUC tissue. Of note, the clinical benefit of EV strongly depends on membranous NECTIN-4 expression. Thus, our results are of highest clinical relevance and argue for a critical reconsideration of the current practice and suggest that the NECTIN-4 receptor status should be determined (ideally in a metastatic/progressive lesion) before initiation of EV. See related commentary by Aggen et al., p. 1377.

Health Promotion as a Motivational Factor in Alpine Cycling.

The present study examines motives for cycling in the alpine region and focuses on the relative importance of health promotion with respect to other motives. Furthermore, the influences of person-specific characteristics on the rank of the motives are examined, and possibilities for advertising bike tourism based on these motives and characteristics are derived. By applying a quantitative approach, a total of 175 cyclists were surveyed using questionnaires on person-specific characteristics, motives, and their relevance for alpine cycling. Data analysis revealed that health promotion is the most important motive for alpine cycling after fun and action as well as nature experience. Further health-related motives such as stress reduction are also perceived as important. The social component, on the other hand, was given the least priority. The results also showed that person-specific characteristics influence the relative importance of motives. For example, elderly persons and people with children perceive the motive of health promotion as the most important. The study shows that the health-promoting effect of alpine cycling is noticed and may be further encouraged. This study demonstrates that alpine cyclists are a heterogeneous group and that health benefits are perceived by various sub-groups therein. Therefore, any marketing for alpine cycling needs to reflect the diversity of cyclists, and approaches need to be adapted according to the respective target group.

Long-term effects of a catastrophic insecticide spill on stream invertebrates.

Accidental spills or illegal discharges of pesticides in aquatic ecosystems can lead to exposure levels that strongly exceed authorized pesticide concentrations, causing major impacts on aquatic ecosystems. Such short-term events often remain undetected in regular monitoring programs with infrequent sampling. In early spring 2015, we identified a catastrophic pesticide spill with the insecticide cypermethrin in the Holtemme River, Germany. Based on existing pre-event macroinvertebrate community data, we monitored the effects and recovery of the macroinvertebrate community for more than two years after the spill. Strong short-term effects were apparent for all taxa with the exception of Chironomidae and Tubificidae. Effects could also be observed on the community level as total abundance, taxa number and biomass strongly decreased. Total abundance and taxa number showed a fast recovery. Regarding long-term effects, the total biomass remained substantially below the pre-contamination level (76%) until the end of the study. Also the abundances of three taxa (Gammarus, Leuctra, Limnius Ad.) did not return to levels prior to the spill even after 26 months. This lack of the taxon-specific recovery was likely due to their long generation time and a low migration ability due to a restricted connectivity between the contaminated site and uncontaminated stream sections. These factors proved to be stronger predictors for the recovery than the pesticide tolerance. We revealed that the biological indicators SPEARpesticides and share of Ephemeroptera, Plecoptera and Trichoptera (EPT) are not suitable for the identification of such extreme events, when nearly all taxa are eradicated. Both indicators are functioning only when repeated stressors initiate long-term competitive replacement of sensitive by insensitive taxa. We conclude that pesticide spills can have significant long-term effects on stream macroinvertebrate communities. Regular ecological monitoring is imperative to identify such ecosystem impairments, combined with analytical chemistry methods to identify the potential sources of spills.

Clinical-scale isolation of 'minimally manipulated' cytomegalovirus-specific donor lymphocytes for the treatment of refractory cytomegalovirus disease.

BACKGROUND AIMS: Reactivation of cytomegalovirus (CMV) after hematopoietic stem cell transplantation remains a major cause of morbidity despite improved antiviral drug therapies. Selective restoration of CMV immunity by adoptive transfer of CMV-specific T cells is the only alternative approach that has been shown to be effective and non-toxic. We describe the results of clinical-scale isolations of CMV-specific donor lymphocytes with the use of a major histocompatibility (MHC) class I peptide streptamer-based isolation method that yields minimally manipulated cytotoxic T cells of high purity. METHODS: Enrichment of CMV-specific cytotoxic T lymphocytes (CTLs) was performed by labeling 1 × 10(10) leukocytes from a non-mobilized mononuclear cell (MNC) apheresis with MHC class I streptamers and magnetic beads. Thereafter, positively labeled CMV-specific CTLs were isolated through the use of CliniMACS (magnetic-activated cell sorting), and MHC streptamers were released through the use of d-biotin. The purity of enriched CMV-specific CTLs was determined on the basis of MHC streptamer staining and fluorescence-activated cell sorting. RESULTS: A total of 22 processes were performed with the use of five different MHC class I streptamers. The median frequency of CMV-specific CTLs in the starting apheresis product was 0.41% among CD3+ T cells. The isolation process yielded a total of 7.77 × 10(6) CMV-specific CTLs, with a median purity of 90.2%. Selection reagents were effectively removed from the final cell product; the CMV-specific CTLs displayed excellent viability and cytotoxicity and were stable for at least 72 h at 4°C after MNC collection. CONCLUSIONS: Clinical-scale isolation of "minimally manipulated" CMV-specific donor CTLs through the use of MHC class I streptamers is feasible and yields functional CTLs at clinically relevant dosages.

Lowest numbers of primary CD8(+) T cells can reconstitute protective immunity upon adoptive immunotherapy.

Patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT) are threatened by potentially lethal viral manifestations like cytomegalovirus (CMV) reactivation. Because the success of today's virostatic treatment is limited by side effects and resistance development, adoptive transfer of virus-specific memory T cells derived from the stem cell donor has been proposed as an alternative therapeutic strategy. In this context, dose minimization of adoptively transferred T cells might be warranted for the avoidance of graft-versus-host disease (GVHD), in particular in prophylactic settings after T-cell-depleting allo-HSCT protocols. To establish a lower limit for successful adoptive T-cell therapy, we conducted low-dose CD8(+) T-cell transfers in the well-established murine Listeria monocytogenes (L.m.) infection model. Major histocompatibility complex-Streptamer-enriched antigen-specific CD62L(hi) but not CD62L(lo) CD8(+) memory T cells proliferated, differentiated, and protected against L.m. infections after prophylactic application. Even progenies derived from a single CD62L(hi) L.m.-specific CD8(+) T cell could be protective against bacterial challenge. In analogy, low-dose transfers of Streptamer-enriched human CMV-specific CD8(+) T cells into allo-HSCT recipients led to strong pathogen-specific T-cell expansion in a compassionate-use setting. In summary, low-dose adoptive T-cell transfer (ACT) could be a promising strategy, particularly for prophylactic treatment of infectious complications after allo-HSCT.

Overnight resting of PBMC changes functional signatures of antigen specific T- cell responses: impact for immune monitoring within clinical trials.

Polyfunctional CD4 or CD8 T cells are proposed to represent a correlate of immune control for persistent viruses as well as for vaccine mediated protection against infection. A well-suited methodology to study complex functional phenotypes of antiviral T cells is the combined staining of intracellular cytokines and phenotypic marker expression using polychromatic flow cytometry. In this study we analyzed the effect of an overnight resting period at 37 °C on the quantity and functionality of HIV-1, EBV, CMV, HBV and HCV specific CD4 and CD8 T-cell responses in a cohort of 21 individuals. We quantified total antigen specific T cells by multimer staining and used 10-color intracellular cytokine staining (ICS) to determine IFNγ, TNFα, IL2 and MIP1ß production. After an overnight resting significantly higher numbers of functionally active T cells were detectable by ICS for all tested antigen specificities, whereas the total number of antigen specific T cells determined by multimer staining remained unchanged. Overnight resting shifted the quality of T-cell responses towards polyfunctionality and increased antigen sensitivity of T cells. Our data suggest that the observed effect is mediated by T cells rather than by antigen presenting cells. We conclude that overnight resting of PBMC prior to ex vivo analysis of antiviral T-cell responses represents an efficient method to increase sensitivity of ICS-based methods and has a prominent impact on the functional phenotype of T cells.

MHC multimer-guided and cell culture-independent isolation of functional T cell receptors from single cells facilitates TCR identification for immunotherapy.

Adoptive therapy using T cells redirected to target tumor- or infection-associated antigens is a promising strategy that has curative potential and broad applicability. In order to accelerate the screening process for suitable antigen-specific T cell receptors (TCRs), we developed a new approach circumventing conventional in vitro expansion-based strategies. Direct isolation of paired full-length TCR sequences from non-expanded antigen-specific T cells was achieved by the establishment of a highly sensitive PCR-based T cell receptor single cell analysis method (TCR-SCAN). Using MHC multimer-labeled and single cell-sorted HCMV-specific T cells we demonstrate a high efficacy (approximately 25%) and target specificity of TCR-SCAN receptor identification. In combination with MHC-multimer based pre-enrichment steps, we were able to isolate TCRs specific for the oncogenes Her2/neu and WT1 even from very small populations (original precursor frequencies of down to 0.00005% of CD3(+) T cells) without any cell culture step involved. Genetic re-expression of isolated receptors demonstrates their functionality and target specificity. We believe that this new strategy of TCR identification may provide broad access to specific TCRs for therapeutically relevant T cell epitopes.

Neuropsychological speed tests and blood phenylalanine levels in patients with phenylketonuria: a meta-analysis.

Although pathogenesis of phenylketonuria is not completely understood, a low phenylalanine diet is effective to prevent severe neurological impairment, mental retardation and behavioural difficulties. Treatment recommendations heavily rely on neuropsychological research; however, single study results are ambiguous, what is reflected in substantial variation of US, British, German, Dutch and French recommendations for blood phenylalanine concentrations for adolescents and adults. We conducted a meta-analysis estimating the influence of age, phenylalanine level, and type of neuropsychological test on effect sizes (standardized differences between controls and patients) of computer-based speed measurements in phenylketonuric patients. The effect of blood phenylalanine level on effect size was more pronounced in children and adolescents than in adults, with choice reaction time being particularly sensitive for phenylalanine concentrations. Results corroborate all recommendations for children. With the exception of the US and Dutch recommendations, all recommendations for adolescents seem to be too liberal. The same effect size is predicted for adult phenylalanine concentrations between 750 and 1500mumol/L not suggesting a preference for any of the published treatment recommendations for adulthood.

An early sympathetic nervous system influence exacerbates collagen-induced arthritis via CD4+CD25+ cells.

OBJECTIVE: Activation of the sympathetic nervous system (SNS) ameliorates collagen-induced arthritis (CIA) in the late phase of the disease but aggravates it in the presymptomatic phase. The aim of the present study was to determine whether CD4+CD25+ T cells are influenced by the SNS of mice and play a disease-modifying role in the early symptomatic phase of the disease. METHODS: We tested the effects of the SNS on arthritis by transferring CD4+CD25+ T cells from sympathectomized mice immunized with type II collagen and from immunized, SNS-intact animals (controls). We further characterized transferred cells by studying forkhead box P3 (FoxP3) expression, cell proliferation, and cytokine secretion. RESULTS: Using anti-dopamine-beta-hydroxylase antibodies for systemic sympathectomy, we noticed a time-dependent disease amelioration (strongest when sympathectomy was performed 7 days before immunization, with no effect 30 days after immunization). When CD4+CD25+ T cells from immunized and sympathectomized animals were transferred into mice with CIA (on day 32), disease severity was reduced compared with that in controls. However, the number of CD4+CD25+FoxP3+ cells and the FoxP3 expression level in CD4+CD25+ cells were not changed by sympathectomy. In a mixed assay of donor CD4+CD25- and CD4+CD25+ cells, proliferation was reduced when cells from sympathectomized animals were studied. In the same assay, secretion of tumor necrosis factor, interleukin-17 (IL-17), IL-10, and IL-4 (not interferon-gamma) was markedly reduced when cells were taken from sympathectomized animals. Culture of CD4+CD25+ cells with norepinephrine (10(-5)M) for 24 hours before transfer worsened the arthritis. CONCLUSION: The SNS increases disease severity in the early phase of symptomatic CIA by stimulating several proinflammatory aspects of CD4+CD25+ T cells.

Isolation of CD4+CD25+ regulatory T cells for clinical trials.

The adoptive transfer of donor CD4+CD25+ regulatory T cells has been shown to protect from lethal graft-versus-host disease after allogeneic bone marrow transplantation in murine disease models. Efficient isolation strategies that comply with good manufacturing practice (GMP) guidelines are prerequisites for the clinical application of human CD4+CD25+ regulatory T cells. Here we describe the isolation of CD4+CD25+ T cells with regulatory function from standard leukapheresis products by using a 2-step magnetic cell-separation protocol performed under GMP conditions. The generated cell products contained on average 49.5% CD4+CD25high T cells that phenotypically and functionally represented natural CD4+CD25+ regulatory T cells and showed a suppressive activity comparable to that of CD4+CD25+ regulatory T-cell preparations purified by non-GMP-approved fluorescence-activated cell sorting.