Compact portable higher harmonic generation microscopy for the real time assessment of unprocessed thyroid tissue.

During thyroid surgery fast and reliable intra-operative pathological feedback has the potential to avoid a two-stage procedure and significantly reduce health care costs in patients undergoing a diagnostic hemithyroidectomy (HT). We explored higher harmonic generation (HHG) microscopy, which combines second harmonic generation (SHG), third harmonic generation (THG), and multiphoton excited autofluorescence (MPEF) for this purpose. With a compact, portable HHG microscope, images of freshly excised healthy tissue, benign nodules (follicular adenoma) and malignant tissue (papillary carcinoma, follicular carcinoma and spindle cell carcinoma) were recorded. The images were generated on unprocessed tissue within minutes and show relevant morphological thyroid structures in good accordance with the histology images. The thyroid follicle architecture, cells, cell nuclei (THG), collagen organization (SHG) and the distribution of thyroglobulin and/or thyroid hormones T3 or T4 (MPEF) could be visualized. We conclude that SHG/THG/MPEF imaging is a promising tool for clinical intraoperative assessment of thyroid tissue.

Facilitators and Barriers of Adherence to Multi-Disease Exacerbation Action Plans in COPD Patients - A Qualitative Study.

Whereas exacerbation action plans to self-manage Chronic Obstructive Pulmonary Disease (COPD) significantly improve health outcomes, patients' adherence to those action plans is often poor. This study aimed to identify facilitators and barriers of adherence to tailored multi-disease exacerbation action plans. We also explored patients' perspectives toward disease management roles. Individual semi-structured interviews were conducted with a sample of COPD patients who completed a Dutch-Australian self-management intervention evaluating tailored exacerbation action plans for COPD and relevant comorbidities. Interviews were thematically analyzed using a deductive approach guided by the Capability, Opportunity and Motivation of Behavior (COM-B) model. In 2016, ten patients (5 Australian; 5 Dutch; 6 men; age 59-83 years) were interviewed at the end of their one-year follow-up. Facilitators of adherence included improved patients' comprehension of disease and treatment, positive feelings about the intervention, improved self-confidence, and professional support. Barriers included difficulties to recognize symptoms, dislike toward daily symptom monitoring, negative feelings about the intervention, negative mood state, and complexity of symptom diaries and action plans. Patients indicated three distinctive perspectives of their own and their healthcare professional's role in their disease management: 1) patients felt mainly responsible; 2) patients felt shared responsibility with their healthcare professional; and 3) patients felt not responsible as they perceived their healthcare professional to be mainly responsible. We successfully used the COM-B model as a guide to identify facilitators and barriers of patients' adherence to multi-disease exacerbation action plans. Improving patients' adherence in future self-management interventions by targeting specific facilitators or barriers should be considered.

Clinical evaluation of a deep-learning-based computer-aided detection system for the detection of pulmonary nodules in a large teaching hospital.

AIM: To evaluate a deep-learning-based computer-aided detection (DL-CAD) software system for pulmonary nodule detection on computed tomography (CT) images and assess its added value in the clinical practice of a large teaching hospital. MATERIALS AND METHODS: A retrospective analysis was performed of 145 chest CT examinations by comparing the output of the DL-CAD software with a reference standard based on the consensus reading of three radiologists. For every nodule in each scan, the location, composition, and maximum diameter in the axial plane were recorded. The subgroup of chest CT examinations (n = 97) without any nodules was used to determine the negative predictive value at the given clinical sensitivity threshold setting. RESULTS: The radiologists found 91 nodules and the CAD system 130 nodules of which 80 were true positive. The measured sensitivity was 88% and the mean false-positive rate was 1.04 false positives/scan. The negative predictive value was 95%. For 23 nodules, there was a size discrepancy of which 19 (83%) were measured smaller by the radiologist. The agreement of nodule composition between the CAD results and the reference standard was 95%. CONCLUSIONS: The present study found a sensitivity of 88% and a false-positive rate of 1.04 false positives/scan, which match the vendor specification. Together with the measured negative predictive value of 95% the system performs very well; however, these rates are still not good enough to replace the radiologist, even for the specific task of nodule detection. Furthermore, a surprisingly high rate of overestimation of nodule size was observed, which can lead to too many follow-up examinations.

Heat shock proteins are differentially expressed in brain and spinal cord: implications for multiple sclerosis.

Multiple sclerosis (MS) is a chronic neurodegenerative disease characterized by demyelination, inflammation and neurodegeneration throughout the central nervous system. Although spinal cord pathology is an important factor contributing to disease progression, few studies have examined MS lesions in the spinal cord and how they differ from brain lesions. In this study we have compared brain and spinal cord white (WM) and grey (GM) matter from MS and control tissues, focusing on small heat shock proteins (HSPB) and HSP16.2. Western blotting was used to examine protein levels of HSPB1, HSPB5, HSPB6, HSPB8 and HSP16.2 in brain and spinal cord from MS and age-matched non-neurological controls. Immunohistochemistry was used to examine expression of the HSPs in MS spinal cord lesions and controls. Expression levels were quantified using ImageJ. Western blotting revealed significantly higher levels of HSPB1, HSPB6 and HSPB8 in MS and control spinal cord compared to brain tissues. No differences in HSPB5 and HSP16.2 protein levels were observed, although HSPB5 protein levels were higher in brain WM versus GM. In MS spinal cord lesions, increased HSPB1 and HSPB5 expression was observed in astrocytes, and increased neuronal expression of HSP16.2 was observed in normal-appearing GM and type 1 GM lesions. The high constitutive expression of several HSPBs in spinal cord and increased expression of HSPBs and HSP16.2 in MS illustrate differences between brain and spinal cord in health and upon demyelination. Regional differences in HSP expression may reflect differences in astrocyte cytoskeleton composition and influence inflammation, possibly affecting the effectiveness of pharmacological agents.

The Mozart study: a relation between dynamic hyperinflation and physical activity in patients with chronic obstructive pulmonary disease?

BACKGROUND: Many patients with chronic obstructive pulmonary disease (COPD) experience dyspnoea during exercise, resulting in a reduction of physical activity (PA). Dynamic hyperinflation (DH) is seen as a major cause of dyspnoea in COPD. OBJECTIVE: The objective of the current study was to investigate the relationship between DH, in terms of the amount of DH and the development and recovery rate of DH in patients with COPD, and PA. METHODS: Thirty-five patients with stable COPD were included from an outpatient clinic (14 GOLD II and 21 GOLD III, median age 65). PA was assessed using an accelerometer. Subjects underwent metronome-paced tachypnoea (MPT) to induce DH. To quantify the amount of DH during MPT, a decrease in inspiratory capacity (IC) or a change in IC as percentage of total lung capacity was used. RESULTS: No significant correlations were found between the parameters describing DH and PA. Secondary correlation analyses showed a negative correlation between static hyperinflation (SH) and PA (r = -0·39; P = 0·02). The pattern of breathing during MPT and the test itself showed high interpatient variability. CONCLUSIONS: The absence of a significant correlation between DH and PA is contrary to previous studies. SH did show a correlation with PA. The variety in results and the technical difficulties in execution of the measurements ask for a new, more reliable, method to detect DH and investigate its relation with PA in patients with COPD.

The association of haemophilic arthropathy with Health-Related Quality of Life: a post hoc analysis.

BACKGROUND: The aim of replacement therapy in haemophilia is to improve Health-Related Quality of Life (HRQoL) by preventing bleeding and arthropathy. However, the association of arthropathy with HRQoL is unknown. AIM: To explore the association of haemophilic arthropathy with HRQoL. METHODS: A post hoc analysis on patients with severe/moderate haemophilia with SF36 questionnaire (SF36) and X-rays of ankles, knees and elbows made within 2.5-years. The SF36 scores of 'physical functioning' (SF36-PF, range 0-100, optimum 100) and Utility (SF6D-Utility, range 0-1, optimum 1) and radiological Pettersson scores (PS, range 0-78, optimum 0) were calculated. The association of PS with reduced SF6D-Utility and SF36-PF ( 21 points, the risk of reduced SF6D-Utility was stable (OR 4.16; 95% CI: 2.03-8.51) but SF36-PF continued to decrease: compared to lowest PS, OR for reduced SF36-PF was 5.69 (95% CI: 1.62-20.06) for PS 22-39 and 25.15 (95% CI: 6.53-96.81) for PS 40-78. CONCLUSION: Health-Related Quality of Life only showed a significant deterioration in patients with a Pettersson score of >21 points. This suggests that HRQoL is relatively insensitive to early joint changes.

Methylation of IGF2 regulatory regions to diagnose adrenocortical carcinomas.

Adrenocortical carcinoma (ACC) is a rare malignancy with a poor prognosis. Discrimination of ACCs from adrenocortical adenomas (ACAs) is challenging on both imaging and histopathological grounds. High IGF2 expression is associated with malignancy, but shows large variability. In this study, we investigate whether specific methylation patterns of IGF2 regulatory regions could serve as a valuable biomarker in distinguishing ACCs from ACAs. Pyrosequencing was used to analyse methylation percentages in DMR0, DMR2, imprinting control region (ICR) (consisting of CTCF3 and CTCF6) and the H19 promoter. Expression of IGF2 and H19 mRNA was assessed by real-time quantitative PCR. Analyses were performed in 24 ACCs, 14 ACAs and 11 normal adrenals. Using receiver operating characteristic (ROC) analysis, we evaluated which regions showed the best predictive value for diagnosis of ACC and determined the diagnostic accuracy of these regions. In ACCs, the DMR0, CTCF3, CTCF6 and the H19 promoter were positively correlated with IGF2 mRNA expression (P<0.05). Methylation in the most discriminating regions distinguished ACCs from ACAs with a sensitivity of 96%, specificity of 100% and an area under the curve (AUC) of 0.997±0.005. Our findings were validated in an independent cohort of 9 ACCs and 13 ACAs, resulting in a sensitivity of 89% and a specificity of 92%. Thus, methylation patterns of IGF2 regulatory regions can discriminate ACCs from ACAs with high diagnostic accuracy. This proposed test may become the first objective diagnostic tool to assess malignancy in adrenal tumours and facilitate the choice of therapeutic strategies in this group of patients.

Safety and long-term effects of renal denervation: Rationale and design of the Dutch registry.

BACKGROUND: Percutaneous renal denervation (RDN) has recently been introduced as a treatment for therapy-resistant hypertension. Also, it has been suggested that RDN may be beneficial for other conditions characterised by increased sympathetic nerve activity. There are still many uncertainties with regard to efficacy, safety, predictors for success and long-term effects. To answer these important questions, we initiated a Dutch RDN registry aiming to collect data from all RDN procedures performed in the Netherlands. METHODS: The Dutch RDN registry is an ongoing investigator-initiated, prospective, multicentre cohort study. Twenty-six Dutch hospitals agreed to participate in this registry. All patients who undergo RDN, regardless of the clinical indication or device that is used, will be included. Data are currently being collected on eligibility and screening, treatment and follow-up. RESULTS: Procedures have been performed since August 2010. At present, data from 306 patients have been entered into the database. The main indication for RDN was hypertension (n = 302, 99%). Patients had a mean office blood pressure of 177/100 (±29/16) mmHg with a median use of three (range 0-8) blood pressure lowering drugs. Mean 24-hour blood pressure before RDN was 157/93 (±18/13) mmHg. RDN was performed with different devices, with the Simplicity™ catheter currently used most frequently. CONCLUSION: Here we report on the rationale and design of the Dutch RDN registry. Enrolment in this investigator-initiated study is ongoing. We present baseline characteristics of the first 306 participants.

Safety and Tolerability of Nebulized Amoxicillin-Clavulanic Acid in Patients with COPD (STONAC 1 and STONAC 2).

The safety and tolerability of nebulized amoxicillin clavulanic acid were determined in patients with stable COPD and during severe exacerbations of COPD. Nine stable COPD patients received doses ranging from 50:10 mg up to 300:60 mg amoxicillin clavulanic acid and eight patients hospitalised for a COPD exacerbation received fixed doses 200/40 mg twice daily. Safety was evaluated by spirometry before and after inhalation. Tolerability was evaluated by questionnaire. Plasma and expectorated sputum samples were assayed for amoxicillin content. Seventeen patients underwent in total 100 nebulizations with amoxicillin clavulanic acid. In this safety and tolerability study no clinically relevant deteriorations in FEV1 were observed. Nebulized amoxicillin clavulanic acid produces sputum concentrations well above the Minimal Inhibiting Concentration of 90% for potential pathogenic micro-organisms, with low concentrations in the central compartment (low systemic exposure). Based on spirometry and reported side effects, inhalation of nebulized amoxicillin clavulanic acid seems to be safe and well tolerated, both in stable patients with COPD as in those experiencing a severe exacerbation. Levels of amoxicillin were adequate.

What drives MRI-measured cortical atrophy in multiple sclerosis?

BACKGROUND: Cortical atrophy, assessed with magnetic resonance imaging (MRI), is an important outcome measure in multiple sclerosis (MS) studies. However, the underlying histopathology of cortical volume measures is unknown. OBJECTIVE: We investigated the histopathological substrate of MRI-measured cortical volume in MS using combined post-mortem imaging and histopathology. METHODS: MS brain donors underwent post-mortem whole-brain in-situ MRI imaging. After MRI, tissue blocks were systematically sampled from the superior and inferior frontal gyrus, anterior cingulate gyrus, inferior parietal lobule, and superior temporal gyrus. Histopathological markers included neuronal, axonal, synapse, astrocyte, dendrite, myelin, and oligodendrocyte densities. Matched cortical volumes from the aforementioned anatomical regions were measured on the MRI, and used as outcomes in a nested prediction model. RESULTS: Forty-five tissue blocks were sampled from 11 MS brain donors. Mean age at death was 68±12 years, post-mortem interval 4±1 hours, and disease duration 35±15 years. MRI-measured regional cortical volumes varied depending on anatomical region. Neuronal density, neuronal size, and axonal density were significant predictors of GM volume. CONCLUSIONS: In patients with long-standing disease, neuronal and axonal pathology are the predominant pathological substrates of MRI-measured cortical volume in chronic MS.

Detection of calcifications in retinoblastoma using gradient-echo MR imaging sequences: comparative study between in vivo MR imaging and ex vivo high-resolution CT.

BACKGROUND AND PURPOSE: Intratumoral calcifications are very important in the diagnosis of retinoblastoma. Although CT is considered superior in detecting calcification, its ionizing radiation, especially in patients with hereditary retinoblastoma, should be avoided. The purpose of our study was to validate T2*WI for the detection of calcification in retinoblastoma with ex vivo CT as the criterion standard. MATERIALS AND METHODS: Twenty-two consecutive patients with retinoblastoma (mean age, 21 months; range, 1-71 months) with enucleation as primary treatment were imaged at 1.5T by using a dedicated surface coil. Signal-intensity voids indicating calcification on T2*WI were compared with ex vivo high-resolution CT, and correlation was scored by 2 independent observers as poor, good, or excellent. Other parameters included the shape and location of the signal-intensity voids. In 5 tumors, susceptibility-weighted images were evaluated. RESULTS: All calcifications visible on high-resolution CT could be matched with signal-intensity voids on T2*WI, and correlation was scored as excellent in 17 (77%) and good in 5 (23%) eyes. In total, 93% (25/27) of the signal-intensity voids inside the tumor correlated with calcifications compared with none (0/8) of the signal-intensity voids outside the tumor. Areas of nodular signal-intensity voids correlated with calcifications in 92% (24/26), and linear signal-intensity voids correlated with hemorrhage in 67% (6/9) of cases. The correlation of signal-intensity voids on SWI was better in 4 of 5 tumors compared with T2*WI. CONCLUSIONS: Signal-intensity voids on in vivo T2*WI correlate well with calcifications on ex vivo high-resolution CT in retinoblastoma. Gradient-echo sequences may be helpful in the differential diagnosis of retinoblastoma. The combination of funduscopy, sonography, and high-resolution MR imaging with gradient-echo sequences should become the standard diagnostic approach for retinoblastoma.

CNS myelin induces regulatory functions of DC-SIGN-expressing, antigen-presenting cells via cognate interaction with MOG.

Myelin oligodendrocyte glycoprotein (MOG), a constituent of central nervous system myelin, is an important autoantigen in the neuroinflammatory disease multiple sclerosis (MS). However, its function remains unknown. Here, we show that, in healthy human myelin, MOG is decorated with fucosylated N-glycans that support recognition by the C-type lectin receptor (CLR) DC-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) on microglia and DCs. The interaction of MOG with DC-SIGN in the context of simultaneous TLR4 activation resulted in enhanced IL-10 secretion and decreased T cell proliferation in a DC-SIGN-, glycosylation-, and Raf1-dependent manner. Exposure of oligodendrocytes to proinflammatory factors resulted in the down-regulation of fucosyltransferase expression, reflected by altered glycosylation at the MS lesion site. Indeed, removal of fucose on myelin reduced DC-SIGN-dependent homeostatic control, and resulted in inflammasome activation, increased T cell proliferation, and differentiation toward a Th17-prone phenotype. These data demonstrate a new role for myelin glycosylation in the control of immune homeostasis in the healthy human brain through the MOG-DC-SIGN homeostatic regulatory axis, which is comprised by inflammatory insults that affect glycosylation. This phenomenon should be considered as a basis to restore immune tolerance in MS.

Implementation of a multi-institutional diffuse intrinsic pontine glioma autopsy protocol and characterization of a primary cell culture.

AIMS: Diffuse intrinsic pontine glioma (DIPG) is a fatal paediatric malignancy. Tumour resection is not possible without serious morbidity and biopsies are rarely performed. The resulting lack of primary DIPG material has made preclinical research practically impossible and has hindered the development of new therapies for this disease. The aim of the current study was to address the lack of primary DIPG material and preclinical models by developing a multi-institutional autopsy protocol. METHODS: An autopsy protocol was implemented in the Netherlands to obtain tumour material within a brief post mortem interval. A team of neuropathologists and researchers was available at any time to perform the autopsy and process the material harvested. Whole brain autopsy was performed and primary DIPG material and healthy tissue were collected from all affected brain areas. Finally, the study included systematic evaluation by parents. RESULTS: Five autopsies were performed. The mean time interval between death and time of autopsy was 3 h (range 2-4). All tumours were graded as glioblastoma. None of the parents regretted their choice to participate, and they all derived comfort in donating tissue of their child in the hope to help future DIPG patients. In addition, we developed and characterized one of the first DIPG cell cultures from post mortem material. CONCLUSION: Here we show that obtaining post mortem DIPG tumour tissue for research purposes is feasible with short delay, and that the autopsy procedure is satisfying for participating parents and can be suitable for the development of preclinical DIPG models.

APOE1 mutation in a patient with type III hyperlipoproteinaemia: detailed genetic analysis required.

We present the case of a patient with clinical features of familial dysbetalipoproteinaemia (FD) including high levels of total cholesterol, hypertriglyceridaemia and the presence of palmar xanthomas. Whereas genotype analysis identified the APOE3E3 isoform, sequence analysis revealed the presence of one APOE1 allele due to a mutation, p.Lys164Glu, which leads to loss of function of apolipoprotein E (ApoE), a rare cause of dominant FD.

Retinoblastoma: value of dynamic contrast-enhanced MR imaging and correlation with tumor angiogenesis.

BACKGROUND AND PURPOSE: Noninvasive evaluation of retinoblastoma treatment response has become more important due to increased use of eye-sparing treatments. We evaluated the relation between DCE-MR imaging and histopathologic parameters to determine the value of DCE-MR imaging in assessing tumor angiogenesis and prognostic features. MATERIALS AND METHODS: Fifteen consecutive patients with retinoblastoma (mean age, 24 months; range, 2-70 months) undergoing enucleation as the primary treatment (15 eyes) were scanned at 1.5T by using dedicated surface coils. Pretreatment DCE-MR imaging of the most affected eye was evaluated by 2 observers by using curve-pattern analysis, with the first 5 minutes of each curve and the full time-series described as κ(5min) and κ(17min), respectively. Assessed histopathologic and immunologic parameters included optic nerve invasion, choroid invasion, MVD, tumor necrosis, and expression of VEGF and Flt-1. RESULTS: The median value of κ(5min) was 1.28 (range, 0.87-2.07) and correlated positively with MVD (P = .008). The median value of κ(17min) was 1.33 (range, 0.35-3.08) and correlated negatively with tumor necrosis (P = .002). Other histopathologic and immunohistopathologic parameters did not correlate with DCE-MR imaging parameters. Interobserver agreement was 0.53 for κ(5min) and 0.91 for κ(17min). CONCLUSIONS: In retinoblastoma, the early phase of the DCE time curve positively correlates with MVD, while the presence of late enhancement is correlated with necrosis. Thus, the potential for DCE-MR imaging to noninvasively assess tumor angiogenesis and necrosis in retinoblastoma is promising and warrants further investigation.

Postmortem verification of MS cortical lesion detection with 3D DIR.

OBJECTIVE: To assess the sensitivity and specificity of 3D double inversion recovery (DIR) MRI for detecting multiple sclerosis (MS) cortical lesions (CLs) using a direct postmortem MRI to histopathology comparison. METHODS: Single-slab 3D DIR and 3D fluid-attenuated inversion recovery (FLAIR) images of 56 matched fresh brain samples from 14 patients with chronic MS were acquired at 1.5 T. The images of both sequences were prospectively scored for CLs in consensus by 3 experienced raters who were blinded to histopathology and clinical data. Next, CLs were identified histopathologically and were scored again on 3D DIR and 3D FLAIR (retrospective scoring). CLs were classified as intracortical or mixed gray matter (GM)-white matter lesions. Deep GM lesions were also scored. False-positive scores were noted and, from this, specificity was calculated. RESULTS: We found a sensitivity for 3D DIR to detect MS CLs of 18%, which is 1.6-fold higher than 3D FLAIR (improves to 37% with retrospective scoring; 2.0-fold higher than 3D FLAIR). We detected mixed GM-white matter lesions with a sensitivity of 83% using 3D DIR (65% sensitivity for 3D FLAIR), which improved to 96% upon retrospective scoring (91% for 3D FLAIR). For purely intracortical lesions, 3D DIR detected more than 2-fold more than 3D FLAIR (improved to >3-fold upon retrospective scoring). The specificity of 3D DIR to MS CLs was found to be 90%. CONCLUSIONS: In this postmortem verification study, we have shown that 3D DIR is highly pathologically specific, and more sensitive to CLs than 3D FLAIR in MS.