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Background: Thyroid-stimulating hormone (TSH) and subsequent free thyroxine (FT4) concentrations outside the reference interval (RI) are used to diagnose thyroid diseases. Most laboratories do not provide age-specific RIs for TSH and FT4 beyond childhood, although TSH concentrations vary with age. Therefore, we aimed to establish TSH and FT4 age-specific RIs throughout life and aimed to determine whether using these RIs would result in reclassification of thyroid disease diagnoses in adults. Methods: This multicenter retrospective cross-sectional study used big data to determine indirect RIs for TSH and FT4. These RIs were determined by TMC and refineR-analysis, respectively, using four different immunoassay platforms (Roche, Abbott, Siemens, and Beckman Coulter). Retrospective data (2008-2022) from 13 Dutch laboratories for general practitioners and local hospitals were used. RIs were evaluated per manufacturer. Age groups were established from 2 to 20 years by 2-year categories and decade categories between 20 and 100 years. Results: We included totally 7.6 million TSH and 2.2 million FT4 requests. TSH upper reference limits (URLs) and FT4 lower reference limits were higher in early childhood and decreased toward adulthood. In adulthood, TSH URLs increased from 60 years in men, and from 50 years in women, while FT4 URLs increased from 70 years onward. Using adult age-specific RIs resulted in a decrease in diagnoses of subclinical and overt hypothyroidism in women above 50 and men above 60 years in our Roche dataset. Conclusion: This study stressed the known importance of using age-specific RIs for TSH and FT4 in children. This study also showed the clinical relevance of using age-specific RIs for TSH in adulthood to reduce diagnoses of subclinical hypothyroidism in older persons. Therefore, implementation of adult TSH age-specific RIs should be strongly considered. Data are less uniform regarding FT4 age-specific RIs and more research should be performed before implementing these in clinical practice.

Background: The WHO manual for basic semen analysis and ISO 23162 describe sperm morphology assessment as a standard part of semen analysis. Older studies showed a correlation between morphology results and (artificial) conception. In more recent studies this relationship was less apparent and there is more emphasis on sperm morphology as a marker for healthy spermatogenesis (and general male health). Meantime, many laboratories ceased morphology assessment, probably due to unfamiliarity with this paradigmatic shift and to technical difficulties in the assessment, like the interpretation of morphological criteria., Objectives: The aim of this study was to identify morphological criteria with high variability in results in the Dutch External Quality Control (EQC) program., Material and Methods: Over the period 2015-2020, a total of 72 photos of sperm cells along with dichotomous propositions based on 14 criteria as defined in WHO5 (2010) were distributed in the Dutch EQC program for semen analysis. The EQC results were evaluated for variability per criterion and for trends in time., Results: Between 2015 and 2020, 40 to 60 laboratories assessed the photos. Criteria with low variability between participants were related to acrosomal vacuoles, excessive residual cytoplasm, and tail metrics. In contrast, head ovality, regularity of head and midpiece contours, and alignment of the major axis of the midpiece and head led to the highest variability in outcomes. In general, there was a slightly positive trend (lower variability) in time, except for the criteria with the highest variability (stable or declining trend)., Discussion and Conclusion: This study indicates that there are (high) variabilities in the interpretation of the morphological criteria, leading to inconsistent outcomes of morphology assessment. The results are discussed from the perspective of imperfections in definitions and examples of the criteria as given in the WHO manuals., (© 2024 The Author(s). Andrology published by Wiley Periodicals LLC on behalf of American Society of Andrology and European Academy of Andrology.)

Purpose: Postoperative complications increase mortality, disability and costs. Advanced understanding of the risk factors for postoperative complications is needed to improve surgical outcomes. This paper discusses the rationale and profile of the BIGPROMISE (biomarkers to guide perioperative management and improve outcome in high-risk surgery) cohort, that aims to investigate risk factors, pathophysiology and outcomes related to postoperative complications., Participants: Adult patients undergoing major surgery in two tertiary teaching hospitals. Clinical data and blood samples are collected before surgery, at the end of surgery and on the first, second and third postoperative day. At each time point a panel of cardiovascular, inflammatory, renal, haematological and metabolic biomarkers is assessed. Aliquots of plasma, serum and whole blood of each time point are frozen and stored. Data on severe complications are prospectively collected during 30 days after surgery. Functional status is assessed before surgery and after 120 days using the WHO Disability Assessment Schedule (WHODAS) 2.0. Mortality is followed up until 2 years after surgery., Findings to Date: The first patient was enrolled on 8 October 2021. Currently (1 January 2024) 3086 patients were screened for eligibility, of whom 1750 (57%) provided informed consent for study participation. Median age was 66 years (60; 73), 28% were female, and 68% of all patients were American Society of Anaesthesiologists (ASA) physical status class 3. Most common types of major surgery were cardiac (49%) and gastro-intestinal procedures (26%). The overall incidence of 30-day severe postoperative complications was 16%., Future Plans: By the end of the recruitment phase, expected in 2026, approximately 3000 patients with major surgery will have been enrolled. This cohort allows us to investigate the role of pathophysiological perioperative processes in the cause of postoperative complications, and to discover and develop new biomarkers to improve risk stratification for adverse postoperative outcomes., Trial Registration Number: NCT05199025., Competing Interests: Competing interests: PGN has participated in advisory boards for perioperative use of biomarkers, for which he has received a honorarium by Roche Diagnostics (Rotkreuz, Switzerland). PGN and TCDR have held lectures on perioperative biomarkers for which they have received a honorarium by Roche Diagnostics. OC has received research grants from ImmuneXpress Inc. (Seattle, WA) and Abionic SA (Epalinges, Switzerland) for related work., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Objectives: Correct interpretation of thyroid function tests relies on correct reference intervals (RIs) for thyroid-stimulating hormone (TSH) and free thyroxine (FT4). ISO15189 mandates periodic verification of RIs, but laboratories struggle with cost-effective approaches. We investigated whether indirect methods (utilizing historical laboratory data) could replace the direct approach (utilizing healthy reference individuals) and compared results with manufacturer-provided RIs for TSH and FT4., Methods: We collected historical data (2008-2022) from 13 Dutch laboratories to re-establish RIs by employing indirect methods, TMC (for TSH) and refineR (for FT4). Laboratories used common automated platforms (Roche, Abbott, Beckman or Siemens). Indirect RIs (IRIs) were determined per laboratory per year and clustered per manufacturer (>1.000.000 data points per manufacturer). Direct RIs (DRIs) were established in 125 healthy individuals per platform., Results: TSH IRIs remained robust over the years for all manufacturers. FT4 IRIs proved robust for three manufacturers (Roche, Beckman and Siemens), but the IRI upper reference limit (URL) of Abbott showed a decrease of 2 pmol/L from 2015. Comparison of the IRIs and DRIs for TSH and FT4 showed close agreement using adequate age-stratification. Manufacturer-provided RIs, notably Abbott, Roche and Beckman exhibited inappropriate URLs (overall difference of 0.5-1.0 µIU/mL) for TSH. For FT4, the URLs provided by Roche, Abbott and Siemens were overestimated by 1.5-3.5 pmol/L., Conclusions: These results underscore the importance of RI verification as manufacturer-provided RIs are often incorrect and RIs may not be robust. Indirect methods offer cost-effective alternatives for laboratory-specific or platform-specific verification of RIs., (© 2024 the author(s), published by De Gruyter, Berlin/Boston.)

Background: Acute kidney injury (AKI) is associated with mortality after cardiac surgery. Novel risk factors may improve identification of patients at risk for renal injury. The authors evaluated the association between preoperative biomarkers that reflect cardiac, inflammatory, renal, and metabolic disorders and cardiac surgery-associated AKI (CSA-AKI) in elderly patients., Methods: This was a secondary analysis of the 2-center prospective cohort study "Anesthesia Geriatric Evaluation." Twelve biomarkers were determined preoperatively in 539 patients. Primary outcome was CSA-AKI. The association between biomarkers and CSA-AKI was investigated with multivariable logistic regression analysis. Secondary outcomes were 1-year mortality and patient-reported disability and were assessed with relative risks (RR) between patients with and without CSA-AKI., Results: CSA-AKI occurred in 88 (16.3%) patients and was associated with increased risk of mortality (RR, 6.70 [95% confidence interval {CI}, 3.38-13.30]) and disability (RR, 2.13 [95% CI, 1.53-2.95]). Preoperative concentrations of N-terminal pro B-type natriuretic peptide (NT-proBNP), high-sensitive C-reactive protein (hs-CRP), hemoglobin, and magnesium had the strongest association with CSA-AKI. Identification of patients with CSA-AKI improved when a biomarker panel was used (area under the curve [AUC] 0.75 [95% CI, 0.69-0.80]) compared to when only clinical risk factors were used (European System for Cardiac Operative Risk Evaluation [EuroSCORE II] AUC 0.67 [95% CI, 0.62-0.73])., Conclusions: Preoperative cardiac, inflammatory, renal, and metabolic biomarkers are associated with CSA-AKI and may improve identification of patients at risk., Competing Interests: Conflicts of Interest: See Disclosures at the end of the article., (Copyright © 2021 International Anesthesia Research Society.)

X-linked adrenoleukodystrophy (ALD) is a progressive neurodegenerative disease caused by mutations in ABCD1, the peroxisomal very long-chain fatty acid (VLCFA) transporter. ABCD1 deficiency results in accumulation of saturated VLCFAs. A drug screen using a phenotypic motor assay in a zebrafish ALD model identified chloroquine as the top hit. Chloroquine increased expression of stearoyl-CoA desaturase-1 (scd1), the enzyme mediating fatty acid saturation status, suggesting that a shift toward monounsaturated fatty acids relieved toxicity. In human ALD fibroblasts, chloroquine also increased SCD1 levels and reduced saturated VLCFAs. Conversely, pharmacological inhibition of SCD1 expression led to an increase in saturated VLCFAs, and CRISPR knockout of scd1 in zebrafish mimicked the motor phenotype of ALD zebrafish. Importantly, saturated VLCFAs caused ER stress in ALD fibroblasts, whereas monounsaturated VLCFA did not. In parallel, we used liver X receptor (LXR) agonists to increase SCD1 expression, causing a shift from saturated toward monounsaturated VLCFA and normalizing phospholipid profiles. Finally, Abcd1-/y mice receiving LXR agonist in their diet had VLCFA reductions in ALD-relevant tissues. These results suggest that metabolic rerouting of saturated to monounsaturated VLCFAs may alleviate lipid toxicity, a strategy that may be beneficial in ALD and other peroxisomal diseases in which VLCFAs play a key role.

Background: Cardiac and inflammatory biomarkers have been associated with adverse outcome after major abdominal surgery. This study investigated the effect of remote ischaemic preconditioning (RIPC) on perioperative concentrations of high-sensitive cardiac troponin (hs-cTn) T and interleukin (IL) 6., Methods: Adult patients scheduled for elective pancreatic surgery between March 2017 and February 2019 were randomized to either three cycles of upper-limb ischaemia and reperfusion (each 5 min) or a sham procedure before surgery. The primary endpoint was the maximum postoperative hs-cTnT concentration within 48 h after surgery. Secondary endpoints were postoperative myocardial injury (PMI), defined as an absolute increase of hs-cTnT of at least 14 ng/l above baseline concentration, maximum concentration of IL-6 within 48 h after surgery and postoperative complications within 30 days of surgery., Results: Of 99 eligible patients, 46 underwent RIPC and 46 a sham procedure. RIPC did not reduce the maximum hs-cTnT concentration after surgery (12.6 ng/l RIPC, 16.6 ng/l controls, P = 0.225), nor did it lessen the incidence of PMI (15/45 RIPC, 18/45 controls, P = 0.375). The maximum postoperative IL-6 concentration was 265 pg/ml after RIPC versus 385 pg/ml in controls (P = 0.108). Postoperative complications occurred in 23 RIPC and 24 control patients respectively., Conclusions: Remote ischaemic preconditioning did not reduce the maximum postoperative hs-cTnT concentration. Postoperative myocardial injury, IL-6 concentrations and postoperative complications were similar between RIPC patients and controls., Trial Registration: Clinicaltrials.gov identifier NCT03460938., (© The Author(s) 2021. Published by Oxford University Press on behalf of BJS Society Ltd.)

Mutations in the gene encoding the peroxisomal ATP-binding cassette transporter ( ABCD1 ) cause elevations in very long-chain fatty acids (VLCFAs) and the neurodegenerative disease adrenoleukodystrophy (ALD). In most adults, this manifests as the spinal cord axonopathy adrenomyeloneuropathy (AMN). A challenge in virus-based gene therapy in AMN is how to achieve functional gene correction to the entire spinal cord while minimizing leakage into the systemic circulation, which could contribute to toxicity. In the present study, we used an osmotic pump to deliver adeno-associated viral (AAV) vector into the lumbar cerebrospinal fluid space in mice. We report that slow intrathecal delivery of recombinant AAV serotype 9 (rAAV9) achieves efficient gene transfer across the spinal cord and dorsal root ganglia as demonstrated with two different transgenes, GFP and ABCD1 . In the Abcd1 -/- mouse, gene correction after continuous rAAV9-CBA-h ABCD1 delivery led to a 20% decrease in VLCFA levels in spinal cord compared with controls. The major cell types transduced were astrocytes, vascular endothelial cells, and neurons. Importantly, rAAV9 delivered intrathecally by osmotic pump, in contrast to bolus injection, reduced systemic leakage into peripheral organs, particularly liver and heart tissue.

Peroxisomes are present in virtually every eukaryotic cell type with the exception of the mature erythrocyte. In higher eukaryotes, one of the main functions of peroxisomes is lipid metabolism by means of beta-oxidation of very long-chain fatty acids (VLCFA; ≥22 carbon atoms). A dysfunction in peroxisomal VLCFA beta-oxidation results in elevated VLCFA levels in cells, tissue, and plasma. Here, we describe a straightforward and sensitive method to measure peroxisomal beta-oxidation capacity in living cells using stable-isotope labeled docosanoic acid (D 3 -C22:0).

X-linked adrenoleukodystrophy (ALD) is a severe neurodegenerative disorder caused by the accumulation of very long-chain fatty acids (VLCFA) due to mutations in the ABCD1 gene. The phenotypic spectrum ranges from a fatal cerebral demyelinating disease in childhood (cerebral ALD) to a progressive myelopathy without cerebral involvement in adulthood (adrenomyeloneuropathy). Because ABCD1 mutations have no predictive value with respect to clinical outcome a role for modifier genes was postulated. We report that the CYP4F2 polymorphism rs2108622 increases the risk of developing cerebral ALD in Caucasian patients. The rs2108622 polymorphism (c.1297G>A) results in an amino acid substitution valine for methionine at position 433 (p.V433M). Using cellular models of VLCFA accumulation, we show that p.V433M decreases the conversion of VLCFA into very long-chain dicarboxylic acids by ω-oxidation, a potential escape route for the deficient peroxisomal β-oxidation of VLCFA in ALD. Although p.V433M does not affect the catalytic activity of CYP4F2 it reduces CYP4F2 protein levels markedly. These findings open perspectives for therapeutic interventions in a disease with currently limited treatment options., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Objective: To assess the association of systemic inflammation and outcome after major abdominal surgery., Background: Major abdominal surgery carries a high postoperative morbidity and mortality rate. Studies suggest that inflammation is associated with unfavorable outcome., Methods: Levels of C-reactive protein (CRP), interleukin-6 (IL-6), and tumor necrosis factor-α and the systemic inflammatory response syndrome (SIRS) were assessed in 137 patients undergoing major abdominal surgery. Blood samples were drawn on days 0, 1, 3, and 7, and SIRS was scored during 48 hours after surgery. Primary outcome was a composite of mortality, pneumonia, sepsis, anastomotic dehiscence, wound infection, noncardiac respiratory failure, atrial fibrillation, congestive heart failure, myocardial infarction, and reoperation within 30 days of surgery., Results: An IL-6 level more than 432 pg/mL on day 1 was associated with an increased risk of complications (adjusted odds ratio: 3.3; 95% confidence interval [CI]: 1.3-8.5) and a longer median length of hospital stay (7 vs 12 days, P < 0.001). As a single test, an IL-6 cut-off level of 432 pg/mL on day 1 yielded a specificity of 70% and a sensitivity of 64% for the prediction of complications (area under the curve: 0.67; 95% CI: 0.56-0.77). Levels of CRP started to discriminate from day 3 onward with a specificity of 87% and a sensitivity of 58% for a cut-off level of 203 mg/L (AUC: 0.73; 95% CI: 0.63-0.83)., Conclusions: A high IL-6 level on day 1 is associated with postoperative complications. Levels of IL-6 help distinguish between patients at low and high risk for complications before changes in levels of CRP.

X-linked adrenoleukodystrophy (ALD), a progressive neurodegenerative disease, is caused by mutations in ABCD1 and characterized by very-long-chain fatty acids (VLCFA) accumulation. In male patients, an increased plasma VLCFA levels in combination with a pathogenic mutation in ABCD1 confirms the diagnosis. Recent studies have shown that many women with ALD also develop myelopathy. Correct diagnosis is important for management including genetic counseling. Diagnosis in women can only be confirmed when VLCFA levels are elevated or when a known pathogenic ABCD1 mutation is identified. However, in 15-20% of women with ALD VLCFA plasma levels are not elevated. Demonstration that a novel sequence variant is pathogenic can be a challenge when VLCFA levels are in the normal range. Here we report two women with a clinical presentation compatible with ALD, an ABCD1 variation (p.Arg17His and p.Ser358Pro) of unknown significance, but with normal VLCFA levels. We developed a diagnostic test that is based on generating clonal cell lines that express only one of the two alleles. Subsequent biochemical studies enabled us to show that the two sequence variants were not pathogenic, thereby excluding the diagnosis ALD in these women. We conclude that the clonal approach is an important addition to the existing diagnostic array., (Copyright © 2016 Elsevier Inc. All rights reserved.)

X-linked adrenoleukodystrophy (ALD), a progressive neurodegenerative disease, is caused by mutations in ABCD1 and characterized by very-long-chain fatty acids (VLCFA) accumulation. Virtually all males develop progressive myelopathy (AMN). A subset of patients, however, develops a fatal cerebral demyelinating disease (cerebral ALD). Hematopoietic stem cell transplantation is curative for cerebral ALD provided the procedure is performed in an early stage of the disease. Unfortunately, this narrow therapeutic window is often missed. Therefore, an increasing number of newborn screening programs are including ALD. To identify new biomarkers for ALD, we developed an Abcd1 knockout mouse with enhanced VLCFA synthesis either ubiquitous or restricted to oligodendrocytes. Biochemical analysis revealed VLCFA accumulation in different lipid classes and acylcarnitines. Both C26:0-lysoPC and C26:0-carnitine were highly elevated in brain, spinal cord, but also in bloodspots. We extended the analysis to patients and confirmed that C26:0-carnitine is also elevated in bloodspots from ALD patients. We anticipate that validation of C26:0-carnitine for the diagnosis of ALD in newborn bloodspots may lead to a faster inclusion of ALD in newborn screening programs in countries that already screen for other inborn errors of metabolism.

In vitro glycolysis poses a problem during diabetes screening, especially in remote laboratories. Point-of-care analysis (POC) may provide an alternative. We compared POC, routine and STAT analysis and a feasible protocol during glucose tolerance test (GTT) for pregnancy diabetes (GDM) screening. In the routine protocol, heparin tubes were used and turn-around-time (TAT) was unsupervised. In the STAT protocol, tubes were processed immediately. The feasible protocol comprised of citrated tubes with a TAT of 1 hour. Outcome was defined as glucose concentration and clinical diagnosis. Glucose measured by POC was higher compared to routine analysis at t = 0 (0.25 mM) and t = 120 (1.17 mM) resulting in 17% more GDM diagnoses. Compared to STAT analysis, POC glucose was also higher, although less pronounced (0.06 and 0.9 mM at t = 0 and t = 120 minutes, respectively) and misclassification was only 2%. Glucose levels and clinical diagnosis were similar using the feasible protocol and STAT analysis (0.03 mM and -0.07 mM at t = 0 and t = 120, 100% identical diagnoses). POC is an viable alternative for STAT glucose analysis in GDM screening (sensitivity: 100%, specificity: 98%). A feasible protocol (citrated phlebotomy tubes with a TAT of 60 minutes) resulted in 100% identical outcome and provides the best alternative.

Background: Postoperative non-cardiac complication rates are as high as 11-28% after high-risk abdominal procedures. Emerging evidence indicates that postoperative cardiac troponin T elevations are associated with adverse outcome in non-cardiac surgery. The aim of this study was to determine the relationship between postoperative high-sensitive cardiac troponin T elevations and non-cardiac complications in patients after major abdominal surgery., Methods: This prospective observational single-centre cohort study included patients at risk for coronary artery disease undergoing elective major abdominal surgery. Cardiac troponin was measured before surgery and at day 1, 3, and 7. Multivariable logistic regression analysis was performed to examine the adjusted association for different cut-off concentrations of postoperative myocardial injury and non-cardiac outcome., Results: In 203 patients, 690 high-sensitive cardiac troponin T measurements were performed. Fifty-three patients (26%) had a non-cardiac complication within 30 days after surgery. Hospital mortality was 4% (8/203). An increase in cardiac troponin T concentration ≥100% compared with baseline was a superior independent predictor of non-cardiac postoperative clinical complications (adjusted odds ratio 4.3, 95% confidence interval 1.8-10.1, P<0.001) and was associated with increased length of stay (9 days, 95% confidence interval 7-11 vs 7 days, 95% confidence interval 6-8, P=0.004) and increased hospital mortality (12 vs 2%, P=0.028)., Conclusions: A postoperative high-sensitive cardiac troponin T increase ≥100% is a strong predictor of non-cardiac 30 day complications, increased hospital stay and hospital mortality in patients undergoing major abdominal surgery., Clinicaltrialsgov Identifier: NCT02150486., (© The Author 2015. Published by Oxford University Press on behalf of the British Journal of Anaesthesia. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

X-linked adrenoleukodystrophy (X-ALD) is a neurometabolic disease that is caused by mutations in the ABCD1 gene. ABCD1 protein deficiency impairs peroxisomal very long-chain fatty acid (VLCFA) degradation resulting in increased cytosolic VLCFA-CoA levels, which are further elongated by the VLCFA-specific elongase, ELOVL1. In adulthood, X-ALD most commonly manifests as a gradually progressive myelopathy (adrenomyeloneuropathy; AMN) without any curative or disease modifying treatments. We recently showed that bezafibrate reduces VLCFA accumulation in X-ALD fibroblasts by inhibiting ELOVL1. Although, in a clinical trial, bezafibrate was unable to lower VLCFA levels in plasma or lymphocytes in X-ALD patients, inhibition of ELOVL1 remains an attractive therapeutic option. In this study, we investigated the kinetic characteristics of ELOVL1 using X-ALD fibroblasts and microsomal fractions from ELOVL1 over-expressing HEK293 cell lines and analyzed the inhibition kinetics of a series of fibrates. Our data show that the CoA esters of bezafibrate and gemfibrozil reduce chain elongation by specifically inhibiting ELOVL1. These fibrates can therefore serve as lead compounds for the development of more potent and more specific inhibitors for ELOVL1., (Copyright © 2014 Elsevier B.V. All rights reserved.)

X-linked adrenoleukodystrophy is the most common peroxisomal disorder. The disease is caused by mutations in the ABCD1 gene that encodes the peroxisomal transporter of very long-chain fatty acids. A defect in the ABCD1 protein results in elevated levels of very long-chain fatty acids in plasma and tissues. The clinical spectrum in males with X-linked adrenoleukodystrophy has been well described and ranges from isolated adrenocortical insufficiency and slowly progressive myelopathy to devastating cerebral demyelination. As in many X-linked diseases, it was assumed that female carriers remain asymptomatic and only a few studies addressed the phenotype of X-linked adrenoleukodystrophy carriers. These studies, however, provided no information on the prevalence of neurological symptoms in the entire population of X-linked adrenoleukodystrophy carriers, since data were acquired in small groups and may be biased towards women with symptoms. Our primary goal was to investigate the symptoms and their frequency in X-linked adrenoleukodystrophy carriers. The secondary goal was to determine if the X-inactivation pattern of the ABCD1 gene was associated with symptomatic status. We included 46 X-linked adrenoleukodystrophy carriers in a prospective cross-sectional cohort study. Our data show that X-linked adrenoleukodystrophy carriers develop signs and symptoms of myelopathy (29/46, 63%) and/or peripheral neuropathy (26/46, 57%). Especially striking was the occurrence of faecal incontinence (13/46, 28%). The frequency of symptomatic women increased sharply with age (from 18% in women <40 years to 88% in women >60 years of age). Virtually all (44/45, 98%) X-linked adrenoleukodystrophy carriers had increased very long-chain fatty acids in plasma and/or fibroblasts, and/or decreased very long-chain fatty acids beta-oxidation in fibroblasts. We did not find an association between the X-inactivation pattern and symptomatic status. We conclude that X-linked adrenoleukodystrophy carriers develop an adrenomyeloneuropathy-like phenotype and there is a strong association between symptomatic status and age. X-linked adrenoleukodystrophy should be considered in the differential diagnosis in women with chronic myelopathy and/or peripheral neuropathy (especially with early faecal incontinence). ABCD1 mutation analysis deserves a place in diagnostic protocols for chronic non-compressive myelopathy.

Insulin can be measured by immunochemical methods using polyclonal or monoclonal antibodies. Monoclonal antibodies are specific in the detection of pure human insulin, and may show little to no cross reactivity with pro-insulin or recombinant insulin. Polyclonal antibodies, however, do show such cross reactivity. Most medical laboratories use commercial (monoclonal) methods to measure insulin 75% of which are not capable of detecting pro-insulin or exogenous insulin. This pitfall in diagnostics may lead to prolonged uncertainty for both patient and physician, which we illustrate with two patients. The first patient was a 45-year-old woman with DM type 1 who for years suffered from hypoglycaemic attacks. Factitious hypoglycaemia went undiagnosed because our monoclonal assay did not detect the overdose insulin analogues. The second patient was a 47-year-old woman with recurrent hypoglycaemic attacks. An insulinoma, which produced pro-insulin, was only detected after using polyclonal insulin and specific pro-insulin assays.

X-linked adrenoleukodystrophy (X-ALD) is caused by mutations in the ABCD1 gene encoding ALDP, an ATP-binding-cassette (ABC) transporter located in the peroxisomal membrane. ALDP deficiency results in impaired peroxisomal β-oxidation and the subsequent accumulation of very long-chain fatty acids (VLCFA; > C22:0) in plasma and tissues. VLCFA are primarily derived from endogenous synthesis by ELOVL1. Therefore inhibiting this enzyme might constitute a feasible therapeutic approach. In this paper we demonstrate that bezafibrate, a PPAR pan agonist used for the treatment of patients with hyperlipidaemia reduces VLCFA levels in X-ALD fibroblasts. Surprisingly, the VLCFA-lowering effect was independent of PPAR activation and not caused by the increase in either mitochondrial or peroxisomal fatty acid β-oxidation capacity. In fact, our results show that bezafibrate reduces VLCFA synthesis by decreasing the synthesis of C26:0 through a direct inhibition of fatty acid elongation activity. Taken together, our data indicate bezafibrate as a potential pharmacotherapeutic treatment for X-ALD. A clinical trial is currently ongoing to evaluate the effect in patients with X-ALD.

Unlabelled: X-linked adrenoleukodystrophy (X-ALD) is caused by mutations in the ABCD1 gene and is characterized by impaired beta-oxidation of very-long-chain fatty acids (VLCFA) and subsequent VLCFA accumulation in tissues. In adulthood X-ALD most commonly manifests as a gradually progressive myelopathy, (adrenomyeloneuropathy; AMN) without any curative or disease modifying treatments. We recently showed that bezafibrate (BF), a drug used for the treatment of hyperlipidaemia, reduces VLCFA accumulation in X-ALD fibroblasts by inhibiting ELOVL1, an enzyme involved in the VLCFA synthesis. We therefore designed a proof-of-principal clinical trial to determine whether BF reduces VLCFA levels in plasma and lymphocytes of X-ALD patients. Ten males with AMN were treated with BF for 12 weeks at a dose of 400 mg daily, followed by 12 weeks of 800 mg daily. Every 4 weeks patients were evaluated for side effects and blood samples were taken for analysis. Adherence was good as indicated by a clear reduction in triglycerides. There was no reduction in VLCFA in either plasma or lymphocytes. Plasma levels of BF did not exceed 25 µmol/L. We concluded that BF, at least in the dose given, is unable to lower VLCFA levels in plasma or lymphocytes in X-ALD patients. It is unclear whether this is due to the low levels of BF reached in plasma. Our future work is aimed at the identification of highly-specific inhibitors of ELOVL1 that act at much lower concentrations than BF and are well tolerated. BF appears to have no therapeutic utility in X-ALD., Trial Registration: ClinicalTrials.gov NCT01165060.

X-linked adrenoleukodystrophy (X-ALD) is caused by mutations in the ABCD1 gene encoding the peroxisomal ABC transporter adrenoleukodystrophy protein (ALDP). X-ALD is characterized by the accumulation of very long-chain fatty acids (VLCFA; > or =C24) in plasma and tissues. In this manuscript we provide insight into the pathway underlying the elevated levels of C26:0 in X-ALD. ALDP transports VLCFacyl-CoA across the peroxisomal membrane. A deficiency in ALDP impairs peroxisomal beta-oxidation of VLCFA but also raises cytosolic levels of VLCFacyl-CoA which are substrate for further elongation. We identify ELOVL1 (elongation of very-long-chain-fatty acids) as the single elongase catalysing the synthesis of both saturated VLCFA (C26:0) and mono-unsaturated VLCFA (C26:1). ELOVL1 expression is not increased in X-ALD fibroblasts suggesting that increased levels of C26:0 result from increased substrate availability due to the primary deficiency in ALDP. Importantly, ELOVL1 knockdown reduces elongation of C22:0 to C26:0 and lowers C26:0 levels in X-ALD fibroblasts. Given the likely pathogenic effects of high C26:0 levels, our findings highlight the potential of modulating ELOVL1 activity in the treatment of X-ALD.

Cellular traffic is a central aspect of cell function in health and disease. It is highly dynamic, and can be investigated at increasingly finer temporal and spatial resolution due to new imaging techniques and probes. Manual tracking of these data is labor-intensive and observer-biased and existing automation is only semi-automatic and requires near-perfect object detection and high-contrast images. Here, we describe a novel automated technique for quantifying cellular traffic. Using local intrinsic information from adjacent images in a sequence and a model for object characteristics, our approach detects and tracks multiple objects in living cells via Multiple Hypothesis Tracking and handles several confounds (merge/split, birth/death, and clutters), as reliable as expert observers. By replacing the related component (e.g. using a different appearance model) the method can be easily adapted for quantitative analysis of other biological samples.

Since activated microglia are able to phagocytose damaged cells and subsequently express major histocompatibility complex class II (MHC-II) and co-stimulatory proteins, they are considered to function as antigen presenting cells (APCs) in the central nervous system. The maturation and migratory potential of professional APCs is associated with the expression of chemokine receptor CCR7. We therefore investigated whether the immunological activation of microglia induces CCR7 expression. We here present that activation of cultured microglia by both the innate antigen lipopolysaccharide and protein antigen ovalbumin rapidly induces CCR7 expression, accompanied by increased MHC-II expression. Moreover, it is shown that CCR7 expression in IBA-1 positive cells is induced during the symptom onset and progression of experimental autoimmune encephalomyelitis, a rodent model for multiple sclerosis. These results suggest that microglia express CCR7 under specific inflammatory conditions, corroborating the idea that microglia develop into APCs with migratory potential toward lymphoid chemokines., ((c) 2006 Wiley-Liss, Inc.)

Microglia, the resident inflammatory cells of the CNS, are the only CNS cells that express the fractalkine receptor (CX3CR1). Using three different in vivo models, we show that CX3CR1 deficiency dysregulates microglial responses, resulting in neurotoxicity. Following peripheral lipopolysaccharide injections, Cx3cr1-/- mice showed cell-autonomous microglial neurotoxicity. In a toxic model of Parkinson disease and a transgenic model of amyotrophic lateral sclerosis, Cx3cr1-/- mice showed more extensive neuronal cell loss than Cx3cr1+ littermate controls. Augmenting CX3CR1 signaling may protect against microglial neurotoxicity, whereas CNS penetration by pharmaceutical CX3CR1 antagonists could increase neuronal vulnerability.

Whenever neurons in the CNS are injured, microglia become activated. In addition to local activation, microglia remote from the primary lesion site are stimulated. Because this so-called secondary activation of microglia is instrumental for long-term changes after neuronal injury, it is important to understand how microglia activity is controlled. The remote activation of microglia implies that the activating signals are transported along neuronal projections. However, the identity of these signals has not yet been identified. It is shown here that glutamate-treated neurons rapidly express and release the chemokine CCL21. We also provide evidence that neuronal CCL21 is packed in vesicles and transported throughout neuronal processes to reach presynaptic structures. Chemotaxis assays show that functional CCL21 is released from endangered neurons and activate microglia via the chemokine receptor CXCR3. Based on these findings, we suggest that neuronal CCL21 is important in directed neuron-microglia signaling and that this communication could account for the remote activation of microglia, far distant from a primary lesion.

The approximately 50 known chemokines are classified in distinct subfamilies: CXC, CC, CX3C, and C. Although the signaling of chemokines often is promiscuous, signaling events between members of these distinct chemokine classes are hardly observed. The only known exception so far is the murine CC chemokine ligand (CCL)21 (secondary lymphoid tissue chemokine, Exodus-2, 6Ckine), which binds and activates the murine CXC chemokine receptor CXCR3. However, this exception has not been found in humans. In this study, we provide evidence that human CCL21 is a functional ligand for endogenously expressed CXCR3 in human adult microglia. In absence of CCR7 expression, CCL21 induced chemotaxis of human microglia with efficiency similar to the CXCR3 ligands CXC chemokine ligand 9 (monokine induced by IFN-gamma) and CXC chemokine ligand 10 (IFN-gamma-inducible protein-10). Because human CCL21 did not show any effects in CXCR3-transfected HEK293 cells, it is indicated that CXCR3 signaling depends on the cellular background in which the CXCR3 is expressed.

Chemokines in the brain have been recognised as essential elements in neurodegenerative diseases and related neuroinflammation. Recent studies suggest that in addition to the orchestration of chemotaxis of immune cells, chemokines are also involved in neurodevelopment and neurophysiological signalling.

In the present study plastic neural responses to N-methyl-D-aspartate-induced excitotoxic lesions and the neuroprotective effects of the L-type voltage-dependent Ca(2+) channel antagonist nimodipine were investigated in the rat magnocellular nucleus basalis. Assessment of spontaneous behaviour in the elevated plus maze and small open-field paradigms on day 5 and day 14 post-surgery indicated anxiety and persistent hypoactivity of N-methyl-D-aspartate-lesioned rats, as compared with sham-operated controls. Nimodipine administration significantly alleviated the behavioural deficits. Quantitative histochemical analysis of acetylcholinesterase-positive fibre innervation of the somatosensory cortex and determination of the numbers of choline-acetyltransferase-positive proximal fibre branches of cholinergic projection neurons in the magnocellular nucleus basalis demonstrated a severe cholinergic deficit as a consequence of the excitotoxic lesion 14 days post-surgery. Nimodipine pre-treatment significantly attenuated the loss of cortical cholinergic innervation and preserved the functional integrity of cholinergic projection neurons in the magnocellular nucleus basalis. Double-labelling immunocytochemistry demonstrated increased amyloid precursor protein expression in shrinking and presumably apoptotic choline-acetyltransferase-positive neurons, whereas surviving cholinergic nerve cells were devoid of excessive amyloid precursor protein immunoreactivity. Moreover, as a consequence of N-methyl-D-aspartate infusion, rim-like accumulation of amyloid precursor protein-positive astrocytes was visualized in a penumbra-like zone of the excitotoxic injury. Furthermore, abundant sprouting of serotonergic projection fibres invading the damaged magnocellular nucleus basalis subdivision was demonstrated. Pharmacological blockade by the Ca(2+) antagonist nimodipine significantly attenuated both neuronal and glial amyloid precursor protein immunoreactivity and serotonergic fibre sprouting following N-methyl-D-aspartate infusion. The present data characterize plastic endogenous glial and neuronal responses in the magnocellular nucleus basalis model of acute excitotoxic brain damage. The increased amyloid precursor protein expression may indicate effective means of intrinsic neuroprotection, as secreted amyloid precursor protein isoforms are suggested to play a role in neuronal rescue following excitotoxic injury. From a pharmacological point of view, extensive sprouting of serotonergic projections in the damaged magnocellular nucleus basalis may also counteract N-methyl-D-aspartate excitotoxicity via serotonin-induced inhibition of Ca(2+) currents and membrane hyperpolarization. Hence, lesion-induced changes in spontaneous animal behaviour, such as anxiety and novelty-induced hypoactivity, may well be attributed to the considerable re-distribution of serotonergic projections in the basal forebrain. In conclusion, our present data emphasize a role of neuron-glia and neurotransmitter-system interactions in functional recovery after acute excitotoxic brain injury, and the efficacy of L-type Ca(2+) channel blockade by the selective 1,4-dihydropyridine antagonist nimodipine.