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IntroductionExtracellular matrix (ECM) remodelling has been associated with chronic lung diseases. However, information about specific age-associated differences in lung ECM is currently limited. In this study we aimed to identify and localize age-associated ECM differences in human lung using comprehensive transcriptomic, proteomic and immunohistochemical analyses.MethodsOur previously identified age-associated gene expression signature of the lung was re-analysed limiting it to an aging signature based on 270 control patients (37-80 years) and focused on the Matrisome core geneset using geneset enrichment analysis. To validate the age-associated transcriptomic differences on protein level, we compared the age-associated ECM genes (F ResultsComparative analysis of transcriptomic and proteomic data identified 7 ECM proteins with higher expression with age at both gene and protein level: COL1A1, COL6A1, COL6A2, COL14A1, FBLN2, LTBP4 and LUM. With immunohistochemistry we demonstrated higher protein expression with age for COL6A2 in whole tissue, parenchyma, airway wall and blood vessel, for COL14A1 in bronchial epithelium and blood vessel, and for FBLN2 and COL1A1 in lung parenchyma.ConclusionOur study revealed that higher age is associated with lung ECM remodelling, with specific differences occurring in defined regions within the lung. These differences may affect lung structure and physiology with aging and as such may increase susceptibility for developing chronic lung diseases.Key messagesWhat is already known on this topicsummarise the state of scientific knowledge on this subject before you did your study and why this study needed to be done.❖ In animal models, it has been demonstrated that aging alters the composition of the lung ECM, with more deposition of collagen and degradation of elastin. Similar ECM differences have been observed in age-associated chronic lung diseases, including COPD; moreover, we observed in lung tissue that several ECM genes associate differently with age in COPD patients compared to non-COPD controls(1). Detailed knowledge on age-associated changes in specific ECM proteins as well as regional differences within the lung is lacking.What this study addssummarise what we now know as a result of this study that we did not know before.❖ We identified 7 age-associated ECM proteins i.e. COL1A1, COL6A1, COL6A2 COL14A1, FBLN2, LTBP4 and LUM with higher transcript and protein levels in human lung tissue with age. Extensive immunohistochemical analysis revealed significant age-associated differences for 3 of these ECM proteins in specific compartments of the lung, with the most notable differences in the blood vessels and parenchyma.How this study might affect research, practice, or policysummarise the implications of this study.❖ The identification of age-associated differences in specific human lung ECM proteins lays a new foundation for the investigation of ECM differences in age-associated chronic lung diseases. Additionally, examining the function of these age-associated ECM proteins and their cellular interactions in lung injury and repair responses may provide novel insight in mechanisms underlying chronic lung diseases.

Contains fulltext : 291900.pdf (Publisher’s version ) (Open Access) OBJECTIVE: To investigate the cost-effectiveness of urethral bulking polydimethylsiloxane-Urolastic® (PDMS-U) compared with mid-urethral sling (MUS) surgery for stress urinary incontinence (SUI) at 1-year follow-up. DESIGN: Prospective, two-arm cohort study with 2-year follow-up. SETTING: International multicentre. POPULATION: Women with moderate to severe SUI. MAIN OUTCOME MEASURES: Primary outcome was subjective cure (Patient Global Impression of Improvement). SECONDARY OUTCOMES: objective cure (negative cough stress test), Urogenital Distress Inventory (UDI-6), complications and re-interventions. Cost-effectiveness outcomes: total costs, quality-adjusted life year (QALY) using IIQ7-scores (Incontinence Impact Questionnaire) and EQ-5D-5L, incremental cost-effectiveness ratio (ICER) and monetary benefit (adjusted for baseline confounders). RESULTS: In all, 131 PDMS-U and 153 MUS surgery patients were treated. Subjective cure rates for MUS surgery and PDMS-U were, respectively: 101/112 (90%) versus 40/87 (46%), adjusted odds ratio (aOR; for age, body mass index [BMI], severity, type of urinary incontinence and previous SUI procedure) was 4.9. Objective cure rates for MUS surgery and PDMS-U were respectively: 98/109 (90%) versus 58/92 (63%), aOR 5.4. Average total costs for PDMS-U and MUS surgery were €3567 and €6688. ICER for MUS surgery cost €15 598 per IIQ QALY and €37 408 per EQ-5D-5L QALY. With a willingness to pay (WTP) of €25 000, MUS has a 84% chance of being cost-effective using IIQ, whereas PDMS-U has a 99% chance of being cost-effective using EQ-5D-5L. CONCLUSION: MUS surgery is more cost-effective in realising improved disease-specific quality of life (QoL), while PDMS-U is more cost-effective in realising improved generic QoL.

Background and ObjectivesDeterminants of disease activity and prognosis are limited in anti-NMDA receptor (NMDAR) encephalitis. Neurofilament light chains (NfL) are markers of axonal damage and have been identified as valuable biomarkers for neurodegenerative and other neuroinflammatory disorders. We aimed to investigate serum NfL levels in patients with anti-NMDAR encephalitis as a biomarker for disease severity and outcome.MethodsIn this retrospective study, NfL values were measured in all available pretreatment serum and paired CSF samples of the nationwide anti-NMDAR encephalitis cohort. The values were analyzed in duplicate using single-molecule array and compared with measurements in healthy references. Follow-up sera were tested to analyze longitudinal responsiveness, if at least available from 2 time points after diagnosis. Serum NfL levels were compared with data on disease activity (seizures, MRI, and CSF findings), severity (modified Rankin Scale [mRS] score, admission days, and intensive care unit admission), and outcome (mRS score and relapses), using regression analysis.ResultsWe have included 71 patients (75% female; mean age 31.4 years, range 0–85 years) of whom pretreatment serum samples were analyzed. Paired CSF samples were available of 33 patients, follow-up serum samples of 20 patients. Serum NfL levels at diagnosis were higher in patients (mean 19.5 pg/mL, 95% CI 13.7–27.7) than in references (mean 6.4 pg/mL, 95% CI 5.8–7.2,p< 0.0001). We observed a good correlation between serum and CSF NfL values (R= 0.84,p< 0.0001). Serum NfL levels and age correlated in patients (PearsonR= 0.57,p< 0.0001) and references (R= 0.62,p< 0.0001). Increased NfL values were detected in patients post–herpes simplex virus 1 encephalitis (mean 248.8 vs 14.1 pg/mL,p< 0.0001) and in patients with brain MRI lesions (mean 27.3 vs 11.1 pg/mL,p= 0.019). NfL levels did relate to the long-term follow-up (mRS score at 12 months; βNfL= 0.55,p= 0.013), although largely explained by the effect of age on NfL levels and prognosis. In serial samples, NfL values did roughly follow clinical disease activity, albeit with delay.DiscussionIncreased serum NfL levels reflect neuroaxonal damage in anti-NMDAR encephalitis. No relationship was identified with disease severity, whereas the association with outcome was confounded by age. The implied role of sampling timing on NfL levels also limits the applicability of NfL as a prognostic marker.

Autoimmune encephalitis (AE) can be classified into antibody-defined subtypes, which can manifest with immunotherapy-responsive movement disorders sometimes mimicking non-inflammatory etiologies. In the elderly, anti-leucin-rich-glioma-inactivated-1 (LGI1) and contactin-associated-protein-like-2 (CASPR2) antibody-associated diseases compose a relevant fraction of AE. Patients with LGI1 autoantibodies are known to present with limbic encephalitis and additionally faciobrachial dystonic seizures (FBDS) may occur. However, the clinical spectrum of CASPR2 autoantibody-associated disorders is more diverse including limbic encephalitis, Morvan`s syndrome, peripheral nerve hyperexcitability syndrome, ataxia, pain and sleep disorders. Reports on unusual, sometimes isolated and immunotherapy-responsive movement disorders in CASPR2-autoantibody associated syndromes have caused substantial concern regarding necessity of autoantibody-testing in patients with movement disorders. Therefore, we aimed to systematically assess their prevalence and manifestation in patients with CASPR2 autoimmunity. This international, retrospective cohort study included patients with CASPR2 autoimmunity from participating expert centers in Europe. Patients with ataxia and/or movement disorders were analyzed in detail using questionnaires and video recordings. We recruited a comparator group with anti-leucin-rich-glioma-inactivated-1 (LGI1) encephalitis from the GENERATE network. Characteristics were compared according to serostatus. We identified 164 patients with CASPR2 autoantibodies. Of these, 149 (90.8%) had only CASPR2- and 15 (9.1%) both CASPR2- and LGI1-autoantibodies. Compared to 105 patients with LGI1 encephalitis, patients with CASPR2 autoantibodies more often had movement disorders and/or ataxia (35.6% versus 3.8%; p

Fetal heart-rate irregularities occur in 1-2% of pregnancies and are usually caused by premature atrial contractions (PAC). Although PAC are considered benign, they may be associated with cardiac defects and tachyarrhythmia. We aimed to determine the incidence of congenital heart defects (CHDs) and complications in fetuses with PAC.This was a systematic review and meta-analysis conducted in accordance with the PRISMA statement for reporting items for systematic reviews and meta-analyses. MEDLINE and EMBASE were searched from 1990 to June 2021 to identify studies on fetuses with PAC. The primary outcome was CHD; secondary outcomes were complications using the endpoints supraventricular tachyarrhythmia (SVT), cardiac failure and intrauterine fetal demise. Meta-analysis of proportions was performed, subdivided into high-risk and low-risk populations based on reason for referral. Pooled incidences with 95% CIs were calculated.Of 2443 unique articles identified, 19 cohort studies including 2260 fetuses were included. The pooled incidence of CHD in fetuses with PAC was 2.8% (95% CI, 1.5-4.1%), when 0.6% is the incidence expected in the general population. The pooled incidence of CHD was 7.2% (95% CI, 3.5-10.9%) in the high-risk population and 0.9% (95% CI, 0.0-2.0%) in the low-risk population. SVT occurred in 1.4% (95% CI, 0.6-3.4%) of fetuses diagnosed with PAC. Cardiac failure was described in 16 fetuses (1.4% (95% CI, 0.5-3.5%)), of which eight were CHD-related. Intrauterine fetal demise occurred in four fetuses (0.9% (95% CI, 0.5-1.7%)) and was related to CHD in two cases.Our findings suggest that the risk of CHD in fetuses with PAC is 4-5 times higher than that in the general population. CHD was present more frequently in the high-risk population. Consequently, an advanced ultrasound examination to diagnose PAC correctly and exclude CHD is recommended. Complications of PAC are rare but can result in fetal demise, thus weekly fetal heart-rate monitoring remains advisable to enable early detection of SVT and to prevent cardiac failure. © 2022 The Authors. Ultrasound in ObstetricsGynecology published by John WileySons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.

Aims/hypothesis The general population is ageing, involving an enhanced incidence of chronic diseases such as type 2 diabetes. With ageing, DNA methylation of FHL2 increases, as well as expression of the four and a half LIM domains 2 (FHL2) protein in human pancreatic islets. We hypothesised that FHL2 is actively involved in glucose metabolism. Methods Publicly available microarray datasets from human pancreatic islets were analysed for FHL2 expression. In FHL2-deficient mice, we studied glucose clearance and insulin secretion. Gene expression analysis and glucose-stimulated insulin secretion (GSIS) were determined in isolated murine FHL2-deficient islets to evaluate insulin-secretory capacity. Moreover, knockdown and overexpression of FHL2 were accomplished in MIN6 cells to delineate the underlying mechanism of FHL2 function. Results Transcriptomics of human pancreatic islets revealed that individuals with elevated levels of HbA1c displayed increased FHL2 expression, which correlated negatively with insulin secretion pathways. In line with this observation, FHL2-deficient mice cleared glucose more efficiently than wild-type littermates through increased plasma insulin levels. Insulin sensitivity was comparable between these genotypes. Interestingly, pancreatic islets isolated from FHL2-deficient mice secreted more insulin in GSIS assays than wild-type mouse islets even though insulin content and islet size was similar. To support this observation, we demonstrated increased expression of the transcription factor crucial in insulin secretion, MAF BZIP transcription factor A (MafA), higher expression of GLUT2 and reduced expression of the adverse factor c-Jun in FHL2-deficient islets. The underlying mechanism of FHL2 was further delineated in MIN6 cells. FHL2-knockdown led to enhanced activation of forkhead box protein O1 (FOXO1) and its downstream genes such as Mafa and Pdx1 (encoding pancreatic and duodenal homeobox 1), as well as increased glucose uptake. On the other hand, FHL2 overexpression in MIN6 cells blocked GSIS, increased the formation of reactive oxygen species and increased c-Jun activity. Conclusions/interpretation Our data demonstrate that FHL2 deficiency improves insulin secretion from beta cells and improves glucose tolerance in mice. Given that FHL2 expression in humans increases with age and that high expression levels of FHL2 are associated with beta cell dysfunction, we propose that enhanced FHL2 expression in elderly individuals contributes to glucose intolerance and the development of type 2 diabetes. Data availability The human islet microarray datasets used are publicly available and can be found on https://www.ncbi.nlm.nih.gov/geo/. Graphical abstract

Treatment with antibodies directed against programed-cell death ligand 1 (PD-L1) is a novel therapy for patients with gestational trophoblastic disease. Assessment of PD-L1 expression in tumor tissue is commonly used to identify patients who might benefit from anti-PD-L1 treatment. Multiple antibodies are available to detect PD-L1-expressing cells, and percentages of PD-L1-expressing cells in samples of patients with gestational trophoblastic disease indicated by these antibodies differ substantially. This raises the question which PD-L1 antibody best reflects PD-L1 expression to select patients for treatment.Seven commercially available antibodies for PD-L1 staining (E1L3N, 73-10, 22C3, CAL10, SP142, 28-8, SP263) were validated on Chinese hamster ovarian (CHO) cells transfected with PD-L1, PD-L2, wildtype CHO cells and tonsil tissue. Next, four complete hydatidiform moles and four choriocarcinomas were stained. Samples were independently assessed by two pathologists.All seven antibodies showed membranous staining in the PD-L1-transfected CHO cells. E1L3N and 22C3 scored the highest percentages of PD-L1-positive cells (70%-90% and 60%-70%, respectively). E1L3N stained the cytoplasm of non-transfected CHO cells and was excluded from analysis. The remaining six antibodies predominantly stained syncytiotrophoblast cells of both complete hydatidiform moles and choriocarcinomas. The percentage of PD-L1-stained trophoblast cells and staining intensity varied substantially per used PD-L1 antibody and between complete hydatidiform moles and choriocarcinomas. Agreement between pathologists was best with 22C3 (intraclass correlation coefficient 0.94-0.96).Based on staining results of the CHO cells, gestational trophoblastic disease samples and intraclass correlation coefficient, 22C3 seems the most suitable for adequate detection of PD-L1-expressing trophoblast cells. All antibodies detected PD-L1-expressing cells in the gestational trophoblastic disease samples, though with great variability, hampering comparison of results between studies in this rare disease and emphasizing the need for uniformity in detecting PD-L1-expressing cells.

Background: In recent years, an increasing number of people adapt to a vegetarian, pescatarian or flexitarian dietary pattern that reduces the consumption of meat and fish. Although these dietary patterns have a risk for developing vitamin B12 deficiency associated polyneuropathy, it is unknown whether this risk is still increased when vitamin B12 levels are adequate. Objective: To examine whether a vegetarian, pescatarian or flexitarian dietary pattern is associated with an increased risk for idiopathic axonal polyneuropathy. Methods: We conducted a case-control study that included 256 idiopathic axonal polyneuropathy patients with adequate vitamin B12 blood levels and 630 controls. We used questionnaire data to determine the frequency of meat and fish consumption and defined dietary patterns. Results: The vegetarian (no meat or fish consumption) and the pescatarian (fish consumption, no meat consumption) dietary patterns showed no increased risk of axonal polyneuropathy. Frequency-effect analysis and quantity-effect analysis also did not show that a reduction of meat or fish consumption (flexitarian dietary pattern), either small or large, changed the risk of axonal polyneuropathy. Conclusions: We did not find an increased risk for axonal polyneuropathy among people with a vegetarian, pescatarian or flexitarian diet and an adequate vitamin B12 level.

Purpose: To analyse the risk of inguinal lymph node (ILN) metastases in T1G2 penile cancer stratified by lymphovascular invasion (LVI), perineural invasion (PNI) and tumour size. Methods: Retrospective study of men with localised T1G2 penile cancer with non-palpable lymph nodes and no local recurrence during follow-up at six European institutional high-volume centres was performed. ILN involvement was defined as cancer detected during ultrasound-guided fine-needle aspiration cytology, core needle biopsy, dynamic sentinel lymph node biopsy, ILN dissection or inguinal recurrence during follow-up. Uni- and multivariable logistic regression analyses were performed. Results: In the cohort of 554 men with T1G2 penile cancer, from 6 European institutions, ILN metastases were observed in 46/554 men (8%, 95% confidence interval (CI) 6–11%). Men with both, LVI- and PNI- primary cancers had the lowest risk of ILN involvement (6%) whereas men with LVI + or PNI + showed ILN metastases in 22% and 30%. In multivariable regression, men with LVI + or PNI + had higher odds for ILN metastases compared to men with LVI- and PNI- (OR 3.9, 95% CI 1.6–9.0, p value < 0.01) Tumour size was not associated with ILN risk (OR 1.01 95% CI 0.99–1.04, p = 0.17). Conclusion: Approximately, one out of ten men with T1G2 overall and one out of four men with either LVI + or PNI + still have ILN metastases despite being clinically node negative. Therefore, invasive ILN staging should strongly be recommended in T1G2 with LVI + or PNI + but importantly, must be discussed in patients with T1G2 with LVI- or PNI-.

Late type 1a endoleaks (T1aELs) after endovascular aneurysm repair (EVAR) are hazardous complications which should be avoided. This study investigated the evolution of the shortest apposition length (SAL) post-EVAR and hypothesised that a declining apposition during follow-up may be an indicator of T1aEL development. Patients with a late T1aEL were selected from a consecutive multicentre database. For each T1aEL patient, the preoperative computed tomography angiography (CTA), first postoperative CTA, and pre-endoleak CTA were analysed. T1aEL patients were matched 1:1 to uncomplicated controls, based on endograft type and follow-up duration. Anatomical characteristics and endograft dimensions, including the post-EVAR SAL, were measured. Included were 28 patients with a late T1aEL and 28 matched controls. The SAL decreased from 11.2 mm (5.6–20.6 mm) to 3.9 mm (0.0–11.4 mm) in the T1aEL group (p = 0.006), whereas an increase in SAL was seen in the control group from 21.3 mm (14.1–25.8 mm) to 25.4 mm (19.0–36.2 mm; p = 0.015). On the pre-endoleak CTA, 18 patients (64%) in the T1aEL group had a SAL < 10 mm, and one (4%) patient in the control group had a SAL < 10 mm on the matched CTAs. Moreover, three mechanisms of decreasing sealing zone were identified, which might be used to determine optimal imaging or reintervention strategies. Diminishing SAL < 10 mm is an indicator for T1aEL during follow-up, it is imperative to include apposition analysis during follow-up.

Background: The Agatston score on noncontrast computed tomography (CT) scans is the gold standard for calcium load determination. However, contrast-enhanced CT is commonly used for patients with atherosclerotic cardiovascular diseases (ASCVDs), such as peripheral arterial occlusive disease (PAOD) and abdominal aortic aneurysm (AAA). Currently, there is no validated method to determine calcium load in the aorta and peripheral arteries with a contrast-enhanced CT. This study validated a length-adjusted calcium score (LACS) method for contrast-enhanced CT scans. Method: The LACS (calcium volume in mm3/arterial length in cm) in the abdominal aorta was calculated using four-phase liver CT scans of 30 patients treated between 2017 and 2021 at the University Medical Center Groningen (UMCG) with no aortic disease. Noncontrast CT scans were segmented with a 130 Hounsfield units (HU) threshold, and a patient-specific threshold was used for contrast-enhanced CTs. The LACS was calculated and compared from both segmentations. Secondly, the interobserver variability and the influence of slice thickness (0.75 mm vs. 2.0 mm) was determined. Results: There was a high correlation between the LACS from contrast-enhanced CT scans and the LACS of noncontrast CTs (R2 = 0.98). A correction factor of 1.9 was established to convert the LACS derived from contrast-enhanced CT to noncontrast CT scans. LACS interobserver agreement on contrast-enhanced CT was excellent (1.0, 95% confidence interval = 1.0–1.0). The 0.75 mm CT threshold was 541 (459–625) HU compared with 500 (419–568) HU on 2 mm CTs (p = 0.15). LACS calculated with both thresholds was not significantly different (p = 0.63). Conclusion: The LACS seems to be a robust method for scoring calcium load on contrast-enhanced CT scans in arterial segments with various lengths.

Knowledge about anatomical shape variations in the pelvis is mandatory for selection, fitting, positioning, and fixation in pelvic surgery. The current knowledge on pelvic shape variation mostly relies on point-to-point measurements on 2D X-ray images and computed tomography (CT) slices. Three-dimensional region-specific assessments of pelvic morphology are scarce. Our aim was to develop a statistical shape model of the hemipelvis to assess anatomical shape variations in the hemipelvis. CT scans of 200 patients (100 male and 100 female) were used to obtain segmentations. An iterative closest point algorithm was performed to register these 3D segmentations, so a principal component analysis (PCA) could be performed, and a statistical shape model (SSM) of the hemipelvis was developed. The first 15 principal components (PCs) described 90% of the total shape variation, and the reconstruction ability of this SSM resulted in a root mean square error of 1.58 (95% CI: 1.53–1.63) mm. In summary, an SSM of the hemipelvis was developed, which describes the shape variations in a Caucasian population and is able to reconstruct an aberrant hemipelvis. Principal component analyses demonstrated that, in a general population, anatomical shape variations were mostly related to differences in the size of the pelvis (e.g., PC1 describes 68% of the total shape variation, which is attributed to size). Differences between the male and female pelvis were most pronounced in the iliac wing and pubic rami regions. These regions are often subject to injuries. Future clinical applications of our newly developed SSM may be relevant for SSM-based semi-automatic virtual reconstruction of a fractured hemipelvis as part of preoperative planning. Lastly, for companies, using our SSM might be interesting in order to assess which sizes of pelvic implants should be produced to provide proper-fitting implants for most of the population.

Displacement of the anterior and posterior column complicates decision making for both-column acetabular fractures. We questioned whether pelvic surgeons agree on treatment strategy, and whether the use of virtual 3D reconstructions changes the treatment strategy of choice. A nationwide cross-sectional survey was performed in all pelvic trauma centers in the Netherlands. Twenty surgeons assessed 15 both-column fractures in 2D as well as 3D. Based on conventional imaging, surgical treatment was recommended in 89% of cases, and by adding 3D reconstructions this was 93% (p = 0.09). Surgical approach was recommended as anterior (65%), posterior (8%) or combined (27%) (poor level of agreement, κ = 0.05) based on conventional imaging. The approach changed in 37% (p = 0.006), with most changes between a combined and anterior approach (still poor level of agreement, κ = 0.13) by adding 3D reconstructions. Additionally, surgeons’ level of confidence increased from good in 38% to good in 50% of cases. In conclusion, surgeons do not agree on the treatment strategy for both-column acetabular fractures. Additional information given by 3D reconstructions may change the chosen surgical approach and increase surgeons’ confidence about their treatment decision. Therefore, virtual 3D reconstructions are helpful for assessing both-column fracture patterns and aid in the choice of treatment strategy.

Convolutional neural networks (CNNs) may improve response prediction in diffuse large B-cell lymphoma (DLBCL). The aim of this study was to investigate the feasibility of a CNN using maximum intensity projection (MIP) images from 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET) baseline scans to predict the probability of time-to-progression (TTP) within 2 years and compare it with the International Prognostic Index (IPI), i.e. a clinically used score. 296 DLBCL 18F-FDG PET/CT baseline scans collected from a prospective clinical trial (HOVON-84) were analysed. Cross-validation was performed using coronal and sagittal MIPs. An external dataset (340 DLBCL patients) was used to validate the model. Association between the probabilities, metabolic tumour volume (MTV) and Dmaxbulk was assessed. Probabilities for PET scans with synthetically removed tumors were also assessed. The CNN provided a 2-year TTP prediction with area under the curve (AUC) of 0.74, outperforming the IPI-based model (AUC = 0.68). A moderate association of CNN probabilities with MTV (r = 0.57) and Dmaxbulk (r = 0.52) was observed in the external dataset. Furthermore, high probabilities (> 0.6) of the original MIPs were considerably decreased after removing the tumours (

In the literature on alcohol use biomarkers, there has been debate as to what a valid and/or utilitarian cut off level should be for various research applications. In this manuscript, we assessed the sensitivity and specificity of multiple cutoff values for phosphatidylethanol (PEth) from bloodspots relative to self-report, the Alcohol Use Disorder Identification Test (AUDIT) scores, and another alcohol use biomarker ethyl glucuronide (EtG) from fingernails in a sample of 222 pregnant women in the Western Cape Province of South Africa. Receiver operating characteristic (ROC) curves were used to assess the area under the curve (AUC) and assess PEth cutoff values of ≥2, ≥4, ≥8, ≥14, and ≥20 nanograms per milliliter (ng/ml). The highest AUC value was attained when PEth was compared to an AUDIT score of 1 or more. Depending on the cutoff used to determine alcohol consumption, PEth identified 47%–70% of the individuals as alcohol-consuming while 62.6%–75.2% were identified by self-reported measures, and 35.6% were identified by EtG. In this sample, sensitivity and accuracy were highest at less stringent PEth cutoffs when compared to self-report, AUDIT score of 1 or more, 5 or more, 8 or more, and EtG ≥ 8 picograms per milligram (pg/mg). For research purposes, less stringent cutoffs, such as PEth ≥ 8 ng/ml, may be considered a valid, positive cutoff for identifying women who consume alcohol during pregnancy in this population. A cutoff of PEth ≥ 20 ng/ml may miss individuals who reported consuming alcohol (false negatives).

Background Ophthalmological disorders are common and frequently disabling for people with Parkinson’s disease (PD). However, details on the prevalence, severity and impact of ophthalmological disorders thus far lacking. We aimed to identify PD patients with undetected ophthalmological disorders in a large cross-sectional, observational study. Methods We previously delivered a screening questionnaire to detect ophthalmological symptoms (Visual impairment in PD questionnaire; VIPD-Q) to 848 patients. Here, we report on a subgroup of 102 patients who received complete ophthalmological assessment aimed at identifying clinically relevant ophthalmological diseases, which were classified as either vison-threatening or not. Impact on daily life functioning was measured using the visual functioning-25 questionnaire (VFQ-25) and fall frequency. Results Almost all patients (92%) had one or more clinically relevant ophthalmological disorders. Of those, 77% had a potentially vision-threatening disease, while 34% had a potentially treatable ophthalmological disease which impacted on quality of life. The most prevalent ophthalmological disorders were dry eyes (86%), ocular misalignment (50%) and convergence insufficiency (41%). We found a weak but significant association between clinically relevant ophthalmological diseases and both fall frequency (R2 = 0.15, p = 0.037) and VFQ-25 score (R2 = 0.15, p = 0.02). The VIPD-Q could not correctly identify patients with relevant ophthalmological disorders. Conclusions Surprisingly, in our study sample, many participants manifested previously undetected ophthalmological diseases, most of which threatened vision, impacted on daily life functioning and were amenable to treatment. Screening for these ophthalmological disorders using a questionnaire asking about symptoms seems insufficient. Instead, episodic ophthalmological assessments should be considered for PD patients, aiming to identify vision-threatening yet treatable diseases. Trial registration Dutch Trial Registration, NL7421.

Purpose Arterio-ureteral fistula (AUF) is an uncommon diagnosis, but potentially lethal. Although the number of reports has increased over the past two decades, the true incidence and contemporary urologists’ experience and approach in clinical practice remains unknown. This research is conducted to provide insight in the incidence of AUF in The Netherlands, and the applied diagnostic tests and therapeutic approaches in modern practice. Methods A nationwide cross-sectional questionnaire analysis was performed by sending a survey to all registered Dutch urologists. Data collection included information on experience with patients with AUF; and their medical history, diagnostics, treatment, and follow-up, and were captured in a standardized template by two independent reviewers. Descriptive statistics were used. Results Response rate was 62% and 56 AUFs in 53 patients were reported between 2003 and 2018. The estimated incidence of AUF in The Netherlands in this time period is 3.5 AUFs per year. Hematuria was observed in all patients; 9% intermittent microhematuria, and 91% presenting with, or building up to massive hematuria. For the final diagnosis, angiography was the most efficient modality, confirming diagnosis in 58%. Treatment comprised predominantly endovascular intervention. Conclusion The diagnosis AUF should be considered in patients with persistent intermittent or massive hematuria.

Background: People with Parkinson’s disease (PD) benefit from boxing exercise. Adding kicking variations to the boxing may provide additional benefit to improve balance. However, the benefits and adherence to such trainings is unknown. Objective: To explore the feasibility, safety, and benefits on balance of boxing training combined with kicking techniques in comparison to boxing without kicking in PD. Methods: Participants were randomized to group-based boxing training with kicking techniques (BK) or to group-based boxing alone training (BO). Both groups trained for one hour, once a week, for a period of 10 weeks. Participants were assessed at baseline and ten weeks post-intervention for difference in balance, fear of falling, balance confidence, walking ability, and quality of life. Results: Twenty-nine people with PD (median age 64 years; median disease duration 5 years) participated. Both interventions were feasible and acceptable for all participants. No adverse events occurred. Most participants (BK 80%; BO 75%) were satisfied with the training. We found no significant between group difference on either the primary (Mini-BEST) or secondary outcomes. The within group comparison showed that balance improved in both groups after the intervention (BK 22.60 (2.7) to 25.33 (2.64) p = 0.02; BO 23.09 (3.44) to 25.80 (2.39); p = 0.01 on the Mini BEST test). Conclusion: Both types of boxing seem to be feasible and safe. Adding kicking techniques to boxing does not improve balance significantly more than boxing alone. Incorporation of kicking may be a valuable addition to the exercise therapy repertoire.

Contains fulltext : 283535.pdf (Publisher’s version ) (Open Access) Dendritic cells (DCs) bridge the connection between innate and adaptive immunity. DCs present antigens to T cells and stimulate potent cytotoxic T-cell responses. Metabolic reprogramming is critical for DC development and activation; however, metabolic adaptations and regulation in DC subsets remains largely uncharacterized. Here, we mapped metabolomic and lipidomic signatures associated with the activation phenotype of human conventional DC type 1, a DC subset specialized in cross-presentation and therefore of major importance for the stimulation of CD8(+) T cells. Our metabolomics and lipidomic analyses showed that Toll-like receptor (TLR) stimulation altered glycerolipids and amino acids in cDC1. Poly I:C or pRNA stimulation reduced triglycerides and cholesterol esters, as well as various amino acids. Moreover, TLR stimulation reduced expression of glycolysis-regulating genes and did not induce glycolysis. Conversely, cDC1 exhibited increased mitochondrial content and oxidative phosphorylation (OXPHOS) upon TLR3 or TLR7/8 stimulation. Our findings highlight the metabolic adaptations required for cDC1 maturation.

Ocular disorders constitute a major component of the non-motor symptoms of Parkinson’s disease (PD). Blurry vision is commonly associated with PD, but often challenging to interpret. The clinical spectrum of blurred vision is broad, and finding the underlying aetiology can be challenging. An incomplete diagnosis impedes therapeutic successes. We report two persons with PD who both experienced blurry vision, but each with a different underlying pathology that called for specific ophthalmological and neurological treatments. In case 1, the blurry vision was presumably caused by strabismus and convergence insufficiency, while case 2 had blurry vision partly due to palinopsia, a higher order visual processing deficit. Adequate treatment improved vision in both cases. Neurologists should be aware of the different underlying causes of blurred vision, should master the basic therapeutic approaches, and know when to refer a patient to the ophthalmology department.

PDF file, 1359K, Vaccine characteristics of first cycle of all patients. (A) Expression of HLA-ABC, HLA-DR/DP, HLA-DQ, CD80, CD86, CD83, and CCR7 was analyzed by flow cytometry. (B) Tumor antigen expression by DCs 2-4 hrs after electroporation with mRNA encoding gp100 and tyrosinase. Data are shown as percentage of positive DCs used for the first vaccination. The graphs represent mean � SD of all patients.

PDF file - 41K, Clinical protocol and immunization schedule.

Purpose: Regulatory T cells (Tregs) are thought to inhibit antitumor immune responses, and their depletion could therefore have a synergistic effect with therapeutic cancer vaccines. We investigated the impact of three medications on blood Treg frequency in vaccinated cancer patients.Experimental Design: To date, the most specific marker for human Tregs is demethylation in the DNA that encodes the transcription factor FOXP3. Thus, we used a FOXP3 methylation-specific quantitative PCR assay (MS-qPCR) to measure Treg frequencies in the peripheral blood mononuclear cells (PBMCs) of melanoma patients. The patients participated in three clinical trials that combined tumor vaccines with potential Treg-depleting agents: low-dose cyclophosphamide, anti-CD25 monoclonal antibody daclizumab, and the IL-2/diphtheria toxin fusion protein denileukin diftitox.Results: In the nine control patients, blood Treg frequencies varied over time; there was a 46% reduction in one patient. In treated patients, a more than 2-fold decrease in Tregs was observed in one out of 11 patients receiving cyclophosphamide and in four out of 13 receiving daclizumab, but there was no such Treg decrease in any of the six patients who received denileukin diftitox. As a positive control, a more than 2-fold increase in blood Tregs was detected in four out of nine patients who were treated with interleukin-2.Conclusions: We used a MS-qPCR method that detects Tregs but not other activated T lymphocytes; however, none of the Treg-depleting strategies that we tested led, in the majority of patients, to a conservative 50% reduction in blood Tregs. Clin Cancer Res; 17(4); 1–8. ©2010 AACR.

Summary of all analyzed primary tumor slides used in the analysis, using the same format as in Figure 1B of the main manuscript to illustrate the T cell landscapes.

PDF file, 96K, Detection of tumor-specific T cells in SKIL cultures does not require challenge with defined epitopes. Tetramer analysis of SKILs cultured from biopsies of DTH reactions to DCs pulsed with tumor peptides (upper plots) or DCs electroporated with mRNA encoding tumor proteins (lower plots) of patients II-E-09 (A), II-E-03 (B), and II-E-10 (C). This figure shows that sampling DTH biopsy cultures with tetramers yields similar results when the patient is challenged with intradermal injections with DC pulsed with defined epitopes or DC electroporated with mRNA encoding the full tumor antigen.

Supplementary Figures S1-S3 from Cancer Patients Treated with Sunitinib or Sorafenib Have Sufficient Antibody and Cellular Immune Responses to Warrant Influenza Vaccination

Supplementary Figure S1; Supplementary Tables S1-S2.

Purpose: The tyrosine kinase inhibitors sorafenib and sunitinib have efficacy in several types of cancer. Recent studies indicate that these agents affect the immune system. The way it affects the immune response to influenza vaccination is unknown. The aim of this study was to elucidate the specific immune response to seasonal flu vaccination in cancer patients treated with sunitinib or sorafenib.Patients and Methods: Sunitinib- or sorafenib-treated cancer patients were vaccinated against seasonal influenza with an inactivated vaccine. Healthy controls and patients with metastatic renal cell cancer (mRCC) without systemic treatment (nontreated mRCC controls) were included for comparison. Antibody responses were measured at baseline, day 8, and day 22 by a standard hemagglutination inhibition assay and cellular T-cell responses at baseline and day 8 by proliferation assay and secretion of cytokines.Results: Forty subjects were enrolled: 16 patients treated with sunitinib, 6 patients with sorafenib, 7 nontreated mRCC controls, and 11 healthy controls. All patients treated with sunitinib and sorafenib developed seroprotection rates comparable with controls. Functional T-cell reactivity was observed in all groups, except for patients treated with sorafenib who showed a decreased proliferation rate and IFN-γ/IL-2 production and increased IL-10 compared with healthy controls.Conclusion: We conclude that influenza vaccination should be recommended to cancer patients treated with sunitinib or sorafenib. Clin Cancer Res; 17(13); 4541–9. ©2011 AACR.

Purpose: Electroporation of dendritic cells (DC) with mRNA encoding tumor-associated antigens (TAA) has multiple advantages compared to peptide loading. We investigated the immunologic and clinical responses to vaccination with mRNA-electroporated DC in stage III and IV melanoma patients.Experimental design: Twenty-six stage III HLA*02:01 melanoma patients scheduled for radical lymph node dissection (stage III) and 19 melanoma patients with irresectable locoregional or distant metastatic disease (referred to as stage IV) were included. Monocyte-derived DC, electroporated with mRNA encoding gp100 and tyrosinase, were pulsed with keyhole limpet hemocyanin and administered intranodally. TAA-specific T-cell responses were monitored in blood and skin-test infiltrating lymphocyte (SKIL) cultures.Results: Comparable numbers of vaccine-induced CD8+ and/or CD4+ TAA-specific T-cell responses were detected in SKIL cultures; 17/26 stage III patients and 11/19 stage IV patients. Strikingly, in this population, TAA-specific CD8+ T cells that recognize multiple epitopes and produce elevated levels of IFNγ upon antigenic challenge in vitro, were significantly more often observed in stage III patients; 15/17 versus 3/11 stage IV patients, P = 0.0033. In stage IV patients, one mixed and one partial response were documented. The presence or absence of IFNγ-producing TAA-specific CD8+ T cells in stage IV patients was associated with marked difference in median overall survival of 24.1 months versus 11.0 months, respectively.Conclusion: Vaccination with mRNA-electroporated DC induces a broad repertoire of IFNγ producing TAA-specific CD8+ and CD4+ T-cell responses, particularly in stage III melanoma patients. Clin Cancer Res; 18(19); 5460–70. ©2012 AACR.

PDF file - 55K, Consort flow chart.

PDF file, 149K, CONSORT flow chart.

PDF file - 62K, This table describes patient details, including disease state and prior treatments.

Supplementary Figure 3. KLH-specific immune responses before and after DC vaccination. (A) KLH-specific T cell proliferation was analyzed before the first vaccination and after each DC vaccination during the first vaccination cycle in peripheral blood of vaccinated metastatic melanoma patients. Per time point each dot represents 1 patient. Proliferative response to KLH is given as proliferation index (proliferation with KLH/proliferation without KLH). The two patients not showing a KLH-specific T cell response are shown in grey. (B) KLH-specific IgG, IgA, and IgM antibodies were quantitatively measured after each vaccination cycle in sera of vaccinated patients. Per isotype each dot represents one patient. Best KLH responses per patient are shown.

Purpose: It is unknown whether the route of administration influences dendritic cell (DC)-based immunotherapy. We compared the effect of intradermal versus intranodal administration of a DC vaccine on induction of immunologic responses in melanoma patients and examined whether concomitant administration of interleukin (IL)-2 increases the efficacy of the DC vaccine.Experimental Design: HLA-A2.1+ melanoma patients scheduled for regional lymph node dissection were vaccinated four times biweekly via intradermal or intranodal injection with 12 × 106 to 17 × 106 mature DCs loaded with tyrosinase and gp100 peptides together with keyhole limpet hemocyanin (KLH). Half of the patients also received low-dose IL-2 (9 MIU daily for 7 days starting 3 days after each vaccination). KLH-specific B- and T-cell responses were monitored in blood. gp100- and tyrosinase-specific T-cell responses were monitored in blood by tetramer analysis and in biopsies from delayed-type hypersensitivity (DTH) skin tests by tetramer and functional analyses with 51Cr release assays or IFNγ release, following coculture with peptide-pulsed T2 cells or gp100- or tyrosinase-expressing tumor cells.Results: In 19 of 43 vaccinated patients, functional tumor antigen–specific T cells could be detected. Although significantly more DCs migrated to adjacent lymph nodes upon intranodal vaccination, this was also highly variable with a complete absence of migration in 7 of 24 intranodally vaccinated patients. Intradermal vaccinations proved superior in inducing functional tumor antigen–specific T cells. Coadministration of IL-2 did not further augment the antigen-specific T-cell response but did result in higher regulatory T-cell frequencies.Conclusion: Intradermal vaccination resulted in superior antitumor T-cell induction when compared with intranodal vaccination. No advantage of additional IL-2 treatment could be shown. Clin Cancer Res; 17(17); 5725–35. ©2011 AACR.

Purpose: Thus far, dendritic cell (DC)-based immunotherapy of cancer was primarily based on in vitro–generated monocyte-derived DCs, which require extensive in vitro manipulation. Here, we report on a clinical study exploiting primary CD1c+ myeloid DCs, naturally circulating in the blood.Experimental Design: Fourteen stage IV melanoma patients, without previous systemic treatment for metastatic disease, received autologous CD1c+ myeloid DCs, activated by only brief (16 hours) ex vivo culture and loaded with tumor-associated antigens of tyrosinase and gp100.Results: Our results show that therapeutic vaccination against melanoma with small amounts (3–10 × 106) of myeloid DCs is feasible and without substantial toxicity. Four of 14 patients showed long-term progression-free survival (12–35 months), which directly correlated with the development of multifunctional CD8+ T-cell responses in three of these patients. In particular, high CD107a expression, indicative for cytolytic activity, and IFNγ as well as TNFα and CCL4 production was observed. Apparently, these T-cell responses are essential to induce tumor regression and promote long-term survival by stalling tumor growth.Conclusions: We show that vaccination of metastatic melanoma patients with primary myeloid DCs is feasible and safe and results in induction of effective antitumor immune responses that coincide with improved progression-free survival. Clin Cancer Res; 22(9); 2155–66. ©2015 AACR.

Supplementary Figure legend

Purpose: Anticancer dendritic cell (DC) vaccines require the DCs to relocate to lymph nodes (LN) to trigger immune responses. However, these migration rates are typically very poor. Improving the targeting of ex vivo generated DCs to LNs might increase vaccine efficacy and reduce costs. We investigated DC migration in vivo in humans under different conditions.Experimental Design:HLA-A*02:01 patients with melanoma were vaccinated with mature DCs loaded with tyrosinase and gp100 peptides together with keyhole limpet hemocyanin (NCT00243594). For this study, patients received an additional intradermal vaccination with 111In-labeled mature DCs. The injection site was pretreated with nonloaded, activated DCs, TNFα, or Imiquimod; granulocyte macrophage colony-stimulating factor was coinjected or smaller numbers of DCs were injected. Migration was measured by scintigraphy and compared with an intrapatient control vaccination. In an ex vivo tissue model, we measured CCL21-directed migration of 19F-labeled DCs over a period of up to 12 hours using 19F MRI to supplement our patient data.Results: Pretreatment of the injection site induced local inflammatory reactions but did not improve migration rates. Both in vitro and in vivo, reduction of cell numbers to 5 × 106 or less cells per injection improved migration. Furthermore, scintigraphy is insufficient to study migration of such small numbers of 111In-labeled DCs in vivo.Conclusion: Reduction of cell density, not pretreatment of the injection site, is crucial for improved migration of DCs to LNs in vivo. Clin Cancer Res; 19(6); 1525–33. ©2013 AACR.

Supplementary Figure 1. Schematic overview of the 44 h mDC vaccine preparation cycle and vaccination strategy.

Tumor-infiltrating lymphocytes appear to be a predictor of survival in many cancers, including cutaneous melanoma. We applied automated multispectral imaging to determine whether density and distribution of T cells within primary cutaneous melanoma tissue correlate with survival of metastatic melanoma patients after dendritic cell (DC) vaccination. CD3+ T cell infiltration in primary tumors from 77 metastatic melanoma patients was quantified using the ratio of intratumoral versus peritumoral T-cell densities (I/P ratio). Patients with longer survival after DC vaccination had stronger T-cell infiltration than patients with shorter survival in a discovery cohort of 19 patients (P = 0.000026) and a validation cohort of 39 patients (P = 0.000016). I/P ratio was the strongest predictor of survival in a multivariate analysis including M substage and serum lactate dehydrogenase level. To evaluate I/P ratio as a predictive biomarker, we analyzed 19 chemotherapy-treated patients. Longer survival times of DC-vaccinated compared with chemotherapy-treated patients was observed for high (P = 0.000566), but not low (P = 0.154) I/P ratios. In conclusion, T-cell infiltration into primary melanoma is a strong predictor of survival after DC vaccination in metastatic melanoma patients who, on average, started this therapy several years after primary tumor resection. The infiltration remains predictive even after adjustment for late-stage prognostic markers. Our findings suggest that the I/P ratio is a potential predictive biomarker for treatment selection. Cancer Res; 76(12); 3496–506. ©2016 AACR.

Multispectral imaging and melanin unmixing using automated quantitative analysis of tumor infiltrating lymphocytes by Vectra/InForm platform.

Supplementary Table 2 PDF file - 30K, Patient characteristics and historical controls

The gating strategy applied to isolate and determine the frequency of BDCA1+ DCs, BDCA1+ CD14+ cells, monocytes and MDSCs from PBMCS of melanoma patients or healthy donors by FACS (S1); The distribution of potentially "missed genes" over the RPKM value spectrum in all samples that underwent RNA sequencing (S2); A Venn diagram displaying the overlap between the four gene sets used in GSEA analysis: Mono high, infDC high, MoDC sig and BDCA1+ DC high (S3); The T cell suppressive capacity of BDCA1+ DCs, BDCA1+ CD14+ cells, monocytes and MDSCs (S4); Quantification of immunofluorescence stainings performed on sections of paraffin-embedded resection specimens of healthy and melanoma metastasized lymph nodes or healthy and melanoma metastasized colon (S5); Large view, showing multiple cells, of May-Grunwald/Giemsa-stained healthy donor-derived, FACS-sorted BDCA1+ DCs, BDCA1+CD14+ cells and monocytes (S6); GSEA enrichment plots (S7); BDCA1+ DCs, BDCA1+CD14+ cells and monocytes respond to TLR2 and TLR8 stimulation (S8); IL-12 levels determined in overnight supernatants of BDCA1+ DCs, BDCA1+ CD14+ cells and monocytes cultures following stimulation with pIC and R848 (S9); The T cell polarizing capacity of BDCA1+ DCs, BDCA1+ CD14+ cells and monocytes (S10); The effect of IL-10 on BDCA1+ CD14+ cell-induced T cell proliferation was determined by performing an MLR with allogenic naïve CD4+ T cells in the presence or absence of anit-IL-10 and anti-IL10 receptor or the matching isotype controls (S11); The suppressive capacity of BDCA1+ CD14+ cells is not reversed by PD-L1 blocking (S12).

Supplementary Figure 2 PDF file - 46K, Supplementary Figure 2 Plasmacytoid DCs acquire CCL21-driven chemotactic ability upon activation

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Supplementary Figure 1 PDF file - 34K, Supplementary Figure 1 CliniMACS-based pDC isolation is feasible

PDF file - 34K, KLH specific responses

The detailed composition of all gene sets used in GSEA.