Investigations into the concentrations and metabolite profiles of doping agents and antidepressants in human seminal fluid using liquid chromatography-mass spectrometry.

Exogenous substances, including drugs and chemicals, can transfer into human seminal fluid and influence male fertility and reproduction. In addition, substances relevant in the context of sports drug testing programs, can be transferred into the urine of a female athlete (after unprotected sexual intercourse) and trigger a so-called Adverse Analytical Finding. Here, the question arises as to whether it is possible to distinguish analytically between intentional doping offences and unintentional contamination of urine by seminal fluid. To this end, 480 seminal fluids from non-athletes were analysed to identify concentration ranges and metabolite profiles of therapeutic drugs that are also classified as doping agents. Therefore, a screening procedure was developed using liquid chromatography connected to a triple quadrupole mass spectrometer, and suspect samples (i.e. samples indicating the presence of relevant compounds) were further subjected to liquid chromatography-high-resolution accurate mass (tandem) mass spectrometry. The screening method yielded 90 findings (including aromatase inhibitors, selective estrogen receptor modulators, diuretics, stimulants, glucocorticoids, beta-blockers, antidepressants, and the non-approved PPARδ agonist GW1516) in a total of 81 samples, with 91 % of these suspected cases being verified by the confirmation method. Besides the intact drug, phase-I and -II metabolites were also occasionally observed in the seminal fluid. This study demonstrated that various drugs including those categorized as doping agents partition into seminal fluid. Monitoring substances and metabolites may contribute to a better understanding of the distribution and metabolism of exogenous substances in seminal fluid that may be responsible for the impairment of male fertility. Significance Statement This study demonstrates that doping agents as well as clinically relevant substances are transferred/eliminated into seminal fluid to a substantial extent and that knowledge about drug levels (and potential consequences for the male fertility and female exposure) is limited. The herein generated new dataset provides new insights into an important and yet little explored area of drug deposition and elimination, and hereby a basis for the assessment of contamination cases by seminal fluid in sports drug testing.

Triple Blockade of Oncogenic RAS Signaling Using KRAS and MEK Inhibitors in Combination with Irradiation in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest of human malignancies and carries an exceptionally poor prognosis. It is mostly driven by multiple oncogenic alterations, with the highest mutation frequency being observed in the KRAS gene, which is a key oncogenic driver of tumorogenesis and malignant progression in PDAC. However, KRAS remained undruggable for decades until the emergence of G12C mutation specific KRAS inhibitors. Despite this development, this therapeutic approach to target KRAS directly is not routinely used for PDAC patients, with the reasons being the rare presence of G12C mutation in PDAC with only 1-2% of occurring cases, modest therapeutic efficacy, activation of compensatory pathways leading to cell resistance, and absence of effective KRASG12D or pan-KRAS inhibitors. Additionally, indirect approaches to targeting KRAS through upstream and downstream regulators or effectors were also found to be either ineffective or known to cause major toxicities. For this reason, new and more effective treatment strategies that combine different therapeutic modalities aiming at achieving synergism and minimizing intrinsic or adaptive resistance mechanisms are required. In the current work presented here, pancreatic cancer cell lines with oncogenic KRAS G12C, G12D, or wild-type KRAS were treated with specific KRAS or SOS1/2 inhibitors, and therapeutic synergisms with concomitant MEK inhibition and irradiation were systematically evaluated by means of cell viability, 2D-clonogenic, 3D-anchorage independent soft agar, and bioluminescent ATP assays. Underlying pathophysiological mechanisms were examined by using Western blot analyses, apoptosis assay, and RAS activation assay.

Sunlight exposure exerts immunomodulatory effects to reduce multiple sclerosis severity.

Multiple sclerosis (MS) disease risk is associated with reduced sun-exposure. This study assessed the relationship between measures of sun exposure (vitamin D [vitD], latitude) and MS severity in the setting of two multicenter cohort studies (nNationMS = 946, nBIONAT = 990). Additionally, effect-modification by medication and photosensitivity-associated MC1R variants was assessed. High serum vitD was associated with a reduced MS severity score (MSSS), reduced risk for relapses, and lower disability accumulation over time. Low latitude was associated with higher vitD, lower MSSS, fewer gadolinium-enhancing lesions, and lower disability accumulation. The association of latitude with disability was lacking in IFN-ß-treated patients. In carriers of MC1R:rs1805008(T), who reported increased sensitivity toward sunlight, lower latitude was associated with higher MRI activity, whereas for noncarriers there was less MRI activity at lower latitudes. In a further exploratory approach, the effect of ultraviolet (UV)-phototherapy on the transcriptome of immune cells of MS patients was assessed using samples from an earlier study. Phototherapy induced a vitD and type I IFN signature that was most apparent in monocytes but that could also be detected in B and T cells. In summary, our study suggests beneficial effects of sun exposure on established MS, as demonstrated by a correlative network between the three factors: Latitude, vitD, and disease severity. However, sun exposure might be detrimental for photosensitive patients. Furthermore, a direct induction of type I IFNs through sun exposure could be another mechanism of UV-mediated immune-modulation in MS.

Human CCR5high effector memory cells perform CNS parenchymal immune surveillance via GZMK-mediated transendothelial diapedesis.

Although the CNS is immune privileged, continuous search for pathogens and tumours by immune cells within the CNS is indispensable. Thus, distinct immune-cell populations also cross the blood-brain barrier independently of inflammation/under homeostatic conditions. It was previously shown that effector memory T cells populate healthy CNS parenchyma in humans and, independently, that CCR5-expressing lymphocytes as well as CCR5 ligands are enriched in the CNS of patients with multiple sclerosis. Apart from the recently described CD8+ CNS tissue-resident memory T cells, we identified a population of CD4+CCR5high effector memory cells as brain parenchyma-surveilling cells. These cells used their high levels of VLA-4 to arrest on scattered VCAM1, their open-conformation LFA-1 to crawl preferentially against the flow in search for sites permissive for extravasation, and their stored granzyme K (GZMK) to induce local ICAM1 aggregation and perform trans-, rather than paracellular diapedesis through unstimulated primary brain microvascular endothelial cells. This study included peripheral blood mononuclear cell samples from 175 healthy donors, 29 patients infected with HIV, with neurological symptoms in terms of cognitive impairment, 73 patients with relapsing-remitting multiple sclerosis in remission, either 1-4 weeks before (n = 29), or 18-60 months after the initiation of natalizumab therapy (n = 44), as well as white matter brain tissue of three patients suffering from epilepsy. We here provide ex vivo evidence that CCR5highGZMK+CD4+ effector memory T cells are involved in CNS immune surveillance during homeostasis, but could also play a role in CNS pathology. Among CD4+ T cells, this subset was found to dominate the CNS of patients without neurological inflammation ex vivo. The reduction in peripheral blood of HIV-positive patients with neurological symptoms correlated to their CD4 count as a measure of disease progression. Their peripheral enrichment in multiple sclerosis patients and specific peripheral entrapment through the CNS infiltration inhibiting drug natalizumab additionally suggests a contribution to CNS autoimmune pathology. Our transcriptome analysis revealed a migratory phenotype sharing many features with tissue-resident memory and Th17.1 cells, most notably the transcription factor eomesodermin. Knowledge on this cell subset should enable future studies to find ways to strengthen the host defence against CNS-resident pathogens and brain tumours or to prevent CNS autoimmunity.

Does the environment influence multiple sclerosis pathogenesis via UVB light and/or induction of vitamin D?

Multiple sclerosis (MS) is a disease of presumed auto-immune origin. Long-standing observations such as the correlation between MS incidence and geographical latitude or the levels of Vitamin D (Vit D) in the serum have implicated the environmental factors UVB radiation and diet in the etiology of the disease. Clinical trials have been conducted and are currently underway to elucidate whether a Vit D enriched diet or treatment with UVB can influence MS incidence, -severity, and -progression, as well as the ideal time point for treatment. This review summarizes the current scientific knowledge to the environmental factors UVB-light and Vit D concerning the clinical aspects of MS in epidemiological studies and clinical trials.

Blockade of MCAM/CD146 impedes CNS infiltration of T cells over the choroid plexus.

BACKGROUND: Very late antigen 4 (VLA-4; integrin α4ß1) is critical for transmigration of T helper (TH) 1 cells into the central nervous system (CNS) under inflammatory conditions such as multiple sclerosis (MS). We have previously shown that VLA-4 and melanoma cell adhesion molecule (MCAM) are important for trans-endothelial migration of human TH17 cells in vitro and here investigate their contribution to pathogenic CNS inflammation. METHODS: Antibody blockade of VLA-4 and MCAM is assessed in murine models of CNS inflammation in conjunction with conditional ablation of α4-integrin expression in T cells. Effects of VLA-4 and MCAM blockade on lymphocyte migration are further investigated in the human system via in vitro T cell transmigration assays. RESULTS: Compared to the broad effects of VLA-4 blockade on encephalitogenic T cell migration over endothelial barriers, MCAM blockade impeded encephalitogenic T cell migration in murine models of MS that especially depend on CNS migration across the choroid plexus (CP). In transgenic mice lacking T cell α4-integrin expression (CD4::Itga4-/-), MCAM blockade delayed disease onset. Migration of MCAM-expressing T cells through the CP into the CNS was restricted, where laminin 411 (composed of α4, ß1, γ1 chains), the proposed major ligand of MCAM, is detected in the endothelial basement membranes of murine CP tissue. This finding was translated to the human system; blockade of MCAM with a therapeutic antibody reduced in vitro transmigration of MCAM-expressing T cells across a human fibroblast-derived extracellular matrix layer and a brain-derived endothelial monolayer, both expressing laminin α4. Laminin α4 was further detected in situ in CP endothelial-basement membranes in MS patients' brain tissue. CONCLUSIONS: Our findings suggest that MCAM-laminin 411 interactions facilitate trans-endothelial migration of MCAM-expressing T cells into the CNS, which seems to be highly relevant to migration via the CP and to potential future clinical applications in neuroinflammatory disorders.

CD8(+) T-cell pathogenicity in Rasmussen encephalitis elucidated by large-scale T-cell receptor sequencing.

Rasmussen encephalitis (RE) is a rare paediatric epilepsy with uni-hemispheric inflammation and progressive neurological deficits. To elucidate RE immunopathology, we applied T-cell receptor (TCR) sequencing to blood (n=23), cerebrospinal fluid (n=2) and brain biopsies (n=5) of RE patients, and paediatric controls. RE patients present with peripheral CD8(+) T-cell expansion and its strength correlates with disease severity. In addition, RE is the only paediatric epilepsy with prominent T-cell expansions in the CNS. Consistently, common clones are shared between RE patients, who also share MHC-I alleles. Public RE clones share Vß genes and length of the CDR3. Rituximab/natalizumab/basiliximab treatment does not change TCR diversity, stem cell transplantation replaces the TCR repertoire with minimal overlap between donor and recipient, as observed in individual cases. Our study supports the hypothesis of an antigen-specific attack of peripherally expanded CD8(+) lymphocytes against CNS structures in RE, which might be ameliorated by restricting access to the CNS.

LOOKING BACK ON 5 YEARS OF HORIZON SCANNING IN ONCOLOGY.

OBJECTIVES: The regularly structured adaptation of health technology assessment (HTA) programs is of utmost importance to sustain the relevance of the products for stakeholders and to justify investment of scarce financial resources. This study describes internal adjustments and external measures taken to ensure the Horizon Scanning Programme in Oncology (HSO) is current. METHODS: Formal evaluation methods comprising a survey, a download, an environmental analysis, and a Web site questionnaire were used to evaluate user satisfaction. RESULTS: The evaluation showed that users were satisfied with HSO outputs in terms of timeliness, topics selected, and depth of information provided. Discussion of these findings with an expert panel led to changes such as an improved dissemination strategy and the introduction of an additional output, that is, the publication of a league table of emerging oncology drugs. The rather high level of international usage and the environmental analysis highlighted a considerable overlap in topics assessed and, thus, the potential for international collaboration. As a consequence, thirteen reports were jointly published based on eleven "calls for collaboration." To further facilitate collaboration and the usability of reports for other agencies, HSO reports will be adjusted according to tools developed at a European level. CONCLUSIONS: Evaluation of the impact of HTA programs allows the tailoring of outputs to fit the needs of the target population. However, within a fast developing HTA community, estimates of impact will increasingly be determined by international collaborative efforts. Refined methods and a broader definition of impact are needed to ultimately capture the efficiency of national HTA programs.

PML risk stratification using anti-JCV antibody index and L-selectin.

BACKGROUND: Natalizumab treatment is associated with progressive multifocal leukoencephalopathy (PML) development. Treatment duration, prior immunosuppressant use, and JCV serostatus are currently used for risk stratification, but PML incidence stays high. Anti-JCV antibody index and L-selectin (CD62L) have been proposed as additional risk stratification parameters. OBJECTIVE: This study aimed at verifying and integrating both parameters into one algorithm for risk stratification. METHODS: Multicentric, international cohorts of natalizumab-treated MS patients were assessed for JCV index (1921 control patients and nine pre-PML patients) and CD62L (1410 control patients and 17 pre-PML patients). RESULTS: CD62L values correlate with JCV serostatus, as well as JCV index values. Low CD62L in natalizumab-treated patients was confirmed and validated as a biomarker for PML risk with the risk factor "CD62L low" increasing a patient's relative risk 55-fold (p < 0.0001). Validation efforts established 86% sensitivity/91% specificity for CD62L and 100% sensitivity/59% specificity for JCV index as predictors of PML. Using both parameters identified 1.9% of natalizumab-treated patients in the reference center as the risk group. CONCLUSIONS: Both JCV index and CD62L have merit for risk stratification and share a potential biological relationship with implications for general PML etiology. A risk algorithm incorporating both biomarkers could strongly reduce PML incidence.

The two-pore domain K2 P channel TASK2 drives human NK-cell proliferation and cytolytic function.

Natural killer (NK) cells are a subset of cytotoxic lymphocytes that recognize and kill tumor- and virus-infected cells without prior stimulation. Killing of target cells is a multistep process including adhesion to target cells, formation of an immunological synapse, and polarization and release of cytolytic granules. The role of distinct potassium channels in this orchestrated process is still poorly understood. The current study reveals that in addition to the voltage-gated KV 1.3 and the calcium-activated KCa 3.1 channels, human NK cells also express the two-pore domain K2 P channel TASK2 (TWIK-related acid-sensitive potassium channel). Expression of Task2 varies among NK-cell subsets and depends on their differentiation and activation state. Despite its different expression in TASK2(high) CD56(bright) CD16(-) and TASK2(low) CD56(dim) CD16(+) NK cells, TASK2 is involved in cytokine-induced proliferation and cytolytic function of both subsets. TASK2 is crucial for leukocyte functional antigen (LFA-1) mediated adhesion of both resting and cytokine-activated NK cells to target cells, an early step in killing of target cells. With regard to the following mechanism, TASK2 plays a role in release of cytotoxic granules by resting, but not IL-15-induced NK cells. Taken together, our data exhibit two-pore potassium channels as important players in NK-cell activation and effector function.

VLA-4 blockade promotes differential routes into human CNS involving PSGL-1 rolling of T cells and MCAM-adhesion of TH17 cells.

The focus of this study is the characterization of human T cell blood-brain barrier migration and corresponding molecular trafficking signatures. We examined peripheral blood and cerebrospinal fluid immune cells from patients under long-term anti-very late antigen-4 (VLA-4)/natalizumab therapy (LTNT) and from CNS specimens. LTNT patients' cerebrospinal fluid T cells exhibited healthy central-/effector-memory ratios, but lacked CD49d and showed enhanced myeloma cell adhesion molecule (MCAM) expression. LTNT led to an increase of PSGL-1 expression on peripheral T cells. Although vascular cell adhesion molecule-1 (VLA-4 receptor) was expressed at all CNS barriers, P-selectin (PSGL-1-receptor) was mainly detected at the choroid plexus. Accordingly, in vitro experiments under physiological flow conditions using primary human endothelial cells and LTNT patients' T cells showed increased PSGL-1-mediated rolling and residual adhesion, even under VLA-4 blockade. Adhesion of MCAM(+)/TH17 cells was not affected by VLA-4 blocking alone, but was abrogated when both VLA-4 and MCAM were inhibited. Consistent with these data, MCAM(+) cells were detected in white matter lesions, and in gray matter of multiple sclerosis patients. Our data indicate that lymphocyte trafficking into the CNS under VLA-4 blockade can occur by using the alternative adhesion molecules, PSGL-1 and MCAM, the latter representing an exclusive pathway for TH17 cells to migrate over the blood-brain barrier.

Ultraviolet B light attenuates the systemic immune response in central nervous system autoimmunity.

OBJECTIVE: Environmental conditions (eg, latitude) play a critical role in the susceptibility and severity of many autoimmune disorders, including multiple sclerosis (MS). Here, we investigated the mechanisms underlying the beneficial effects of immune regulatory processes induced in the skin by moderate ultraviolet B (UVB) radiation on central nervous system (CNS) autoimmunity. METHODS: Effects of UVB light were analyzed in a murine model of CNS autoimmunity (experimental autoimmune encephalomyelitis). Additionally, patients with relapsing-remitting MS were treated with narrowband UVB phototherapy. Immunomodulatory effects were examined in skin biopsies, serum samples, and immune cells of the peripheral blood. RESULTS: Regulatory T cells (Tregs), which are induced locally in the skin-draining lymph nodes in response to UVB exposure, connect the cutaneous immune response to CNS immunity by migration to the sites of inflammation (blood, spleen, CNS). Here, they attenuate the inflammatory response and ameliorate disease symptoms. Treg-inducing tolerogenic dendritic cells (DCs) were further necessary for induction of this systemic immune regulation by UVB radiation, because ablation of Langerhans cells abolished the UVB-induced phenotype. MS patients treated with UVB phototherapy showed an increase in induced Tregs and tolerogenic DCs accompanied by the downregulation of the T-cell effector cytokine interleukin 21. The treatment further induced elevated serum levels of vitamin D. INTERPRETATION: Local UVB radiation of the skin influences systemic immune reactions and attenuates systemic autoimmunity via the induction of skin-derived tolerogenic DCs and Tregs. Our data could have implications for the understanding or therapeutic modulation of environmental factors that influence immune tolerance.

Molecular, pharmacological, and signaling properties of octopamine receptors from honeybee (Apis mellifera) brain.

G protein-coupled receptors are important regulators of cellular signaling processes. Within the large family of rhodopsin-like receptors, those binding to biogenic amines form a discrete subgroup. Activation of biogenic amine receptors leads to transient changes of intracellular Ca²âº-([Ca²âº](i)) or 3',5'-cyclic adenosine monophosphate ([cAMP](i)) concentrations. Both second messengers modulate cellular signaling processes and thereby contribute to long-lasting behavioral effects in an organism. In vivo pharmacology has helped to reveal the functional effects of different biogenic amines in honeybees. The phenolamine octopamine is an important modulator of behavior. Binding of octopamine to its receptors causes elevation of [Ca²âº](i) or [cAMP](i). To date, only one honeybee octopamine receptor that induces Ca²âº signals has been molecularly and pharmacologically characterized. Here, we examined the pharmacological properties of four additional honeybee octopamine receptors. When heterologously expressed, all receptors induced cAMP production after binding to octopamine with EC50(s) in the nanomolar range. Receptor activity was most efficiently blocked by mianserin, a substance with antidepressant activity in vertebrates. The rank order of inhibitory potency for potential receptor antagonists was very similar on all four honeybee receptors with mianserin >> cyproheptadine > metoclopramide > chlorpromazine > phentolamine. The subroot of octopamine receptors activating adenylyl cyclases is the largest that has so far been characterized in arthropods, and it should now be possible to unravel the contribution of individual receptors to the physiology and behavior of honeybees.

L-selectin is a possible biomarker for individual PML risk in natalizumab-treated MS patients.

OBJECTIVE: To find biomarkers identifying patients at risk for the development of progressive multifocal leukoencephalopathy (PML) during natalizumab treatment. METHODS: Patients were recruited from 10 European and US cohorts. Of 289 patients with multiple sclerosis (MS), 224 had been treated with natalizumab (18-80 months), 21 received other immune-modulatory treatments, and 28 were untreated. We had access to samples from 16 natalizumab PML patients. Eight of these patients had given blood before the diagnosis of PML. We also analyzed non-natalizumab-treated patients who developed PML (n = 10) and age- and sex-matched healthy donors (n = 31). All flow cytometric assessments were done on previously cryopreserved, viable peripheral blood mononuclear cells. RESULTS: The percentage of l-selectin-expressing CD4+ T cells was significantly lower in patients treated long-term with natalizumab (40.2%) when compared with patients not receiving natalizumab treatment (47.2%; p = 0.016) or healthy controls (61.0%; p < 0.0001). An unusually low percentage (9-fold lower; 4.6%) was highly correlated with the risk of developing PML in the patient group with available pre-PML samples when compared with non-PML natalizumab-treated patients (p ≤ 0.0001). Samples were gathered between 4 and 26 months before PML diagnosis. CONCLUSIONS: The cell-based assessment of the percentage of l-selectin-expressing CD4 T cells could provide an urgently needed biomarker for individual PML risk assessment.