Supervised clustering of peripheral immune cells associated with clinical response to checkpoint inhibitor therapy in patients with advanced melanoma.

Background and purpose: Immune therapy with checkpoint inhibitors (CPIs) is a highly successful therapy in many cancers including metastatic melanoma. Still, many patients do not respond well to therapy and there are no blood-borne biomarkers available to assess the clinical outcome. Materials and methods: To investigate cellular changes after CPI therapy, we carried out flow cytometry-based immune monitoring in a cohort of 90 metastatic melanoma patients before and after CPI therapy using the FlowSOM algorithm. To evaluate associations to the clinical outcome with therapy, we divided the patients based on progression-free survival. Results: We found significant associations with CPI therapy in both peripheral blood mononuclear cell and T-cell subsets, but with the most pronounced effects in the latter. Particularly CD4+ effector memory T-cell subsets were associated with response with a positive correlation between CD27+HLA-DR+CD4+ effector memory T cells in a univariate (odds ratio: 1.07 [95% confidence interval 1.02-1.12]) and multivariate regression model (odds ratio: 1.08 [95% confidence interval 1.03-1.14]). We also found a trend towards stronger accumulation of CD57+CD8+ T cells in non-responding patients. Conclusion: Our results show significant associations between immune monitoring and clinical outcome of therapy that could be evaluated as biomarkers in a clinical setting.

'25-Hydroxyvitamin D, Autoantigenic and Total Antibody Concentrations: Results from a Danish Case-control Study of Newly Diagnosed Patients with Childhood Type 1 Diabetes and their Healthy Siblings'.

B cells have recently entered the stage as an important accessory player in type 1 diabetes (T1D) etiopathogenesis. Experimental studies suggest regulatory functions of vitamin D on B cells. However, only a few human studies, with considerable methodological limitations, have been conducted within this field. Our objective was to investigate whether higher 25-hydroxyvitamin D (25(OH)D) concentrations were inversely associated with ß-cell autoantigens glutamic acid decarboxylase (isoform 65) (GADA) and insulinoma-associated antigen-2A (IA-2A). Further, we also wanted to examine the relationship between 25(OH)D and total antibody concentrations. We randomly selected 500 patients with newly diagnosed T1D and 500 siblings for 25(OH)D, antibody and genetic analysis from the population-based Danish Registry of Childhood and Adolescent Diabetes. The relative change (RC) in the mean concentration of GADA, IA-2A and antibody isotypes by a 10 nmol/l increase in 25(OH)D concentration was modelled by a robust log-normal regression model. We found no association between 25(OH)D and GADA [adjusted RC per 10 nmol/l increase: 1.00; 95% confidence interval (CI): 0.98-1.02] and IA-2A [adjusted RC per 10 nmol/l increase: 0.92; CI: 0.76-1.12]. Further, 25(OH)D was not associated with the total concentration of antibody isotypes [immunoglobulin (Ig)A, IgE, IgG and IgM]. All null findings were unaltered after adjustment for genetic variation in the vitamin D pathway. Physiological concentrations of 25(OH)D are unlikely to have a clinically important effect on antibody concentrations in a paediatric population of newly diagnosed patients with T1D and their healthy siblings.

Regular use of medication for musculoskeletal pain and risk of long-term sickness absence: A prospective cohort study among the general working population.

BACKGROUND: The aim was to determine the prospective association between use of pain medication - due to musculoskeletal pain in the low back, neck/shoulder and hand/wrist - and long-term sickness absence. METHODS: Cox-regression analysis was performed to estimate the prospective association between regular use of pain medication and long-term sickness absence (LTSA; at least 6 consecutive weeks) among 9,544 employees from the general working population (Danish Work Environment Cohort Study 2010) and free from LTSA during 2009-2010. The fully adjusted model was controlled for age, gender, body mass index, smoking, leisure physical activity, job group, physical activity at work, psychosocial work environment, pain intensity, mental health and chronic disease. RESULTS: In 2010, the proportion of regular pain medication users due to musculoskeletal disorders was 20.8%: 13.4% as over-the-counter (i.e. non-prescription) and 7.4% as doctor prescribed. In the fully adjusted model, regular use of over-the-counter [HR 1.44 (95% CI 1.13-1.83)] and doctor prescribed (HR 2.18 (95% CI 1.67-2.86)) pain medication were prospectively associated with LTSA. CONCLUSIONS: Regular use of pain medication due to musculoskeletal pain is prospectively associated with LTSA even when adjusted for pain intensity. This study suggests that use of pain medication can be an important factor to be aware of in the prevention of sickness absence. Thus, regular use of pain medication - and not solely the intensity of pain - can be an early indicator that musculoskeletal pain can lead to serious consequences such as long-term sickness absence. SIGNIFICANCE: Use of medication due to musculoskeletal pain is prospectively associated with long-term sickness absence even when adjusted for pain intensity. Use of pain medication can be a red flag to be aware of in the prevention of sickness absence.

No association between vitamin D levels around time of birth and later risk of developing oligo- and polyarticular juvenile idiopathic arthritis: a Danish case-cohort study.

OBJECTIVES: Basic and epidemiological studies on rheumatic autoimmune diseases have suggested an association between vitamin D levels around time of birth and disease risk. The literature on vitamin D and juvenile idiopathic arthritis (JIA) is scarce. We hypothesized that low levels of 25-hydroxyvitamin D [25(OH)D] around time of birth would be associated with increased risk of oligo- or polyarticular JIA. METHOD: We conducted a case-cohort study of validated cases diagnosed with oligo- and polyarticular JIA (1993-2012) and controls matched on date of birth. Cases and controls were born in the period 1983-2010. Cases were diagnosed using international criteria. The concentration of 25(OH)D was assessed from neonatal dried blood spot (DBS) samples using high-sensitivity liquid chromatography tandem mass spectrometry (LC-MS/MS). Odds ratios (ORs) were calculated using conditional logistic regression and a two-way analysis of variance (ANOVA) was used to test for season and birth year 25(OH)D variations. A total of 300 matched pairs were included in the statistical analyses. RESULTS: No significant association was found between levels of 25(OH)D and JIA risk in the adjusted model [OR (per 25 nmol/L increase) 1.2, 95% confidence interval (CI) 0.9-1.6, p = 0.2]. 25(OH)D levels were found to fluctuate significantly with season (p < 0.0001) and year (p < 0.0001). The median level of 25(OH)D was 34.4 nmol/L in cases and 31.5 nmol/L in controls. CONCLUSIONS: Our study does not support the hypothesis that a window of vulnerability exists around time of birth with regard to 25(OH)D levels and later JIA risk. Further studies should explore whether 25(OH)D levels during early pregnancy or infancy may influence JIA risk.

Nationwide reduction in the frequency of severe hypoglycemia by half.

AIMS: To examine contemporary rates of severe hypoglycemia (SH) and identify the effect of predictors of SH in a pediatric type 1 diabetes population. METHODS: The national diabetes register provided data on children residing in Denmark from 2008 to 2013 in this register-based population study. Robust Poisson regression models were applied. RESULTS: The study population [n = 2,715 (50.9 % boys), mean (SD) age at onset; 8.1 (4.0) years, diabetes duration; 5.6 (4.9) years] comprised 7,390 person-years of data and 561 events of SH. The overall incidence of SH was 7.6 per 100 person-years. The incidence rate peaked with 16.0 per 100 person-years in 2008 reaching a nadir of 4.9 in 2011. Overall, insulin pump reduced the rate of SH with 27 % compared to any pen treatment (P = 0.003). When stratifying pen treatment, premixed insulin increased the rate of SH by 1.9-fold (P = 0.0015) and NPH increased the rate by 1.6-fold (P = 0.003) versus pump treatment, whereas long-acting insulin analogues were comparable with pump treatment (P = 0.1485). We found no association of SH with glycemic control (P > 0.05). CONCLUSIONS: A nationwide halving in rates of severe hypoglycemia was observed during the study period independent of the prevailing average HbA1c level. Changes in diabetes care and successful educational programs may have influenced the lower incidence rate of severe hypoglycemia.

Systemic levels of CCL2, CCL3, CCL4 and CXCL8 differ according to age, time period and season among children newly diagnosed with type 1 diabetes and their healthy siblings.

The mechanisms by which antigen-specific T cells migrate to the islets of Langerhans in type 1 diabetes (T1D) are largely unknown. Chemokines attract immune cells to sites of inflammation. The aim was to elucidate the role of inflammatory chemokines in T1D at time of diagnosis. From a population-based registry of children diagnosed with T1D from 1997 to 2005, we studied five different inflammatory chemokines (CCL2, CCL3, CCL4, CCL5 and CXCL8). Four hundred and eighty-two cases and 479 sibling frequencies matched on age and sample year distribution were included. Patients showed lower levels of CCL4 compared to siblings, but this result was not significant after correction for multiple testing. CCL2, CCL3, CCL4 and CXCL8 levels were highest in the most recent cohorts (P < 0.01) in both patients and siblings. A significant seasonal variation - for most of the chemokines - was demonstrated with the highest level during the summer period in both patients and siblings. In addition, there was a significant inverse relationship between CCL4 levels and age. When comparing patients and siblings, remarkably few differences were identified, but interestingly chemokine levels varied with age, season and period for the entire study population. Such variations should be taken into account when studying chemokines in paediatric populations.

Minor stroke as singular manifestation of hereditary thrombotic thrombocytopenic purpura in a young man.

The authors describe a case of a 38-year-old male with minor stroke due to exacerbation of hereditary deficiency of ADAMTS 13 resulting in a chronic relapsing form of thrombotic thrombocytopenic purpura (TTP). The clue to the unusual pathogenesis was given by laboratory findings of a mild anaemia and thrombocytopenia. After two days of observation, the patient was treated with plasmapheresis resulting in normalized platelet levels and continued clinical improvement. Subsequent clinical and laboratory investigation verified the diagnosis and the patient was put on regular treatments with plasma substitution.

Effect of intravenous N-acetylcysteine infusion on haemostatic parameters in healthy subjects.

BACKGROUND AND AIMS: N-acetylcysteine is used to treat paracetamol overdose but depresses the activity of plasma coagulation factors II, VII, and X, which are often used to assess liver injury. The aim of this study was to investigate the effect of N-acetylcysteine on haemostasis in normal volunteers. METHODS: Haemostatic parameters in 10 healthy subjects were analysed before and following intravenous infusion of therapeutic doses of N-acetylcysteine, as well as in vitro. RESULTS: N-acetylcysteine induced significant decreases in plasma levels of vitamin K dependent haemostatic proteins in vivo, being maximal at one hour following the start of infusion, with maximal decreases from 1.00 to 0.73 (0.67-0.79) (mean (95% confidence interval)), 0.66 (0.58-0.73), 0.81 (0.73-0.90), 0.64 (0.57-0.70), 0.74 (0.65-0.82), and 0.61 (0.54-0.67) for factor II, VII, IX, and X activities, protein C activity, and free protein S reactivity, respectively. These data suggest that N-acetylcysteine induces protein modifications affecting activity. Five subjects developed an adverse reaction to infusion of N-acetylcysteine and these were associated with a rapid increase in levels of factor VIII and its carrier protein von Willebrand factor (vWf) from 1.0 to 1.85 (1.08-2.62) and 1.77 (0.83-2.71), respectively, which suggests that the allergic reaction induced release of vWf from endothelial cells. N-acetylcysteine did not affect factor VIII or vWf in subjects without adverse reactions, and nor did it affect factor V or antithrombin in any of the subjects. CONCLUSION: Therapeutic doses of N-acetylcysteine cause abnormal haemostatic activity, and this should be taken into account when using haemostatic function tests as an indicator of hepatic injury.

Bronchial arterial devascularization. An experimental study in pigs.

OBJECTIVE: The lungs have dual blood supply: The bronchial and the pulmonary circulation. The importance of bronchial circulation is disputed. The purpose of this study was to establish an experimental model to examine the importance of the bronchial artery. DESIGN: Comparative porcine experimental study. The surgical technique was evaluated in group A (n = 9). Group B (n = 8) underwent left bronchial arterial devascularization. In group C (n = 9) the left principal bronchus was devascularized, transsected, and reanastomosed. In groups B and C bronchial mucosal blood flow was studied with laser-Doppler velocimetry. Devascularization was controlled by angiography at section, and specimens were examined with conventional histology and scanning electron microscopy. The right bronchus served as control. RESULTS: In group B devascularization caused considerable, yet insignificant reduction in bronchial mucosal blood flow index (p = 0.1282) postoperatively, and after 1 week (p = 0.0678), insignificant histologic (p > 0.2) changes, and no scanning electron microscopy differences. In group C devascularization with bronchial transsection caused significant reduction in mucosal blood flow index (p = 0.0277) postoperatively and after 1 week (p = 0.0277), significant histologic changes (p = 0.0277), and insignificant (p = 0.069) changes in scanning electron microscopy. CONCLUSION: Bronchial arterial devascularization with transsection caused significant physiologic and morphologic changes, and a model with bronchial devascularization should include transsection.

Symptomatic type 1 protein C deficiency caused by a de novo Ser270Leu mutation in the catalytic domain.

Heterozygosity for a C8524T transition in the protein C gene converting Ser270(TCG) to Leu(TTG) in the protease domain was identified in a family with venous thrombosis. The mutation was associated with parallel reduction in plasma levels of protein C anticoagulant activity and protein C antigen, which is consistent with a type 1 deficiency. Transient expression of mutant protein C cDNA in human kidney 293 cells and analysis of protein C antigen in culture media and cell lysates showed that the secretion of mutant protein compared with wild-type protein was reduced by at least 97% while the intracellular content of mutant and wild-type protein was similar. Northern blot analysis of total mRNA from transfected cells showed no reduction of the mutant protein C mRNA compared with wild-type protein C mRNA. Collectively, these results indicate that the Ser270Leu mutation in the affected family caused the plasma protein C deficiency and are consistent with a disease mechanism that involves synthesis of mutant protein followed by intracellular degradation before its secretion into the extracellular space. The mutation was not present in the parents of the proband, suggesting a de novo mutation. Non-paternity was excluded after the analysis of three intragenic protein C polymorphisms and six dinucleotide repeat allele sets located in five different chromosomes.

Characterization and structural impact of five novel PROS1 mutations in eleven protein S-deficient families.

Heterozygozity for four novel missense mutations (W108C, W342R. E349K and L485S) and one novel 4 bp deletion (ACdelAAAG affecting codons 632-633) was identified in PROS1 of unrelated thrombosis prone Danish families with protein S type I or III deficiency. The 4 bp deletion results in a frameshift leading to replacement of the coding sequence for the 3 C-terminal amino acids by an abnormal extended sequence that codes for 9 amino acids. The E349K substitution was found in 7 families. Haplotype analysis using 7 microsatellite markers flanking PROS1 was consistent with a common founder for this mutation. The mutations reported here are most likely the cause of the protein S deficiency. Firstly, the four missense mutations cosegregate with the abnormal plasma protein S phenotype and lead to the loss of highly conserved amino acids. Secondly, computer analysis of structural models of protein S predicts that the substitutions could affect proper protein folding and/or stability. Analysis of platelet mRNA from subjects with the W108C, E349K, L485S mutation or the 4 bp deletion showed that mutated mRNA was expressed in significant amounts suggesting that mutated molecules are synthesized. Our results are compatible with defective protein folding/unstable molecules, impaired secretion and intracellular degradation of mutated protein, which appear to be the major molecular disease mechanisms for missense mutations and certain other mutations found in genetic disorders.

[Acquired warfarin resistance in a patient with mechanical heart valve]. / Erhvervet warfarinresistens hos en patient med mekanisk mitralklap.

We present a case report of a 37-year-old man with a mechanical mitral valve, who, presumably because of malabsorption, developed resistance to oral anticoagulant therapy. The patient was then treated with a replacement of the mechanical valve to biological valves, thereby eliminating the need for anticoagulant therapy. Our case report summarises the diagnostic approach to elucidate apparent resistance to anticoagulant therapy.

[Treatment of respiratory syncytial virus infections in immunocompromised hematological patients]. / Behandling af respiratorisk syncytialvirus-infektioner hos immunkompromitterede haematologiske patienter.

Respiratory syncytial virus (RSV) accounts for considerable morbidity among infants and elderly, that are otherwise immunocompetent. Immunocompromised haematological patients--in particular bone marrow transplant (BMT) recipients with leukopenia--are at high risk for severe, usually fatal, RSV-pneumonia. No randomized, placebo-controlled studies of aerosolized ribavirin for BMT patients with RSV-pneumonia have been conducted, or can be anticipated. We summarize existing case reports providing circumstantial evidence in favour of an active diagnostic and early therapeutic approach to these patients.

Assay of functional plasminogen in rat plasma applicable to experimental studies of thrombolysis.

An improved sensitive, specific, precise and accurate assay of plasminogen in rat plasma was developed. It is performed in 96-well microtiter plates and can be completed within one hour. The assay is based on activation of plasminogen by human urokinase-type plasminogen activator (uPA) and simultaneous measurement of generated plasmin with the specific plasmin substrate H-D-Val-Phe-Lys-4-nitroanilide (S-2390), using purified native rat plasminogen for calibration. The concentration of S-2390 in the final reaction mixture during the whole reaction period is much greater than the Km value (approximately 20 microM) for rat plasmin-cleavage of S-2390 ensuring that hydrolysis of substrate follows zero order kinetics and that the substrate produces a 20-35 fold decrease in rate of inhibition of plasmin by its target inhibitors in plasma. Analogous to the human system the target plasma inhibitors of rat plasmin are shown to be plasmin inhibitor and alpha-macroglobulins. Tranexamic acid (0.8 mM) is incorporated in the reaction mixture resulting in a 19-fold increase in the rate of plasminogen activation and presumably an about 50-fold decrease in the rate of inhibition of generated plasmin by plasmin inhibitor. The assay is suitable for accurate measurement of plasminogen in samples obtained from animals containing pharmacological concentrations of uPA or tissue-type plasminogen activator (tPA) in their plasma when in vitro plasminogen activation is blocked at pH 5 by collecting blood in acidic anticoagulant. Judged from in vitro experiments formation of catalytic active plasmin-alpha-macroglobulin complexes during massive activation of plasminogen in vivo does not interfere with the assay.

A novel missense mutation in the human plasmin inhibitor (alpha2-antiplasmin) gene associated with a bleeding tendency.

Heterozygosity for a G --> A mutation converting Val384(GTG) to Met(ATG) associated with plasmin inhibitor (alpha2-antiplasmin) deficiency was identified in three family members with bleeding tendency. The proband had traumatic breast haematoma and per-/postoperative bleeds. An affected daughter required a blood transfusion after a normal delivery and a son had prolonged bleeding after tooth extraction. The plasma plasmin inhibitor activities of the affected family members were reduced to 49-66% of normal. The antigenic concentrations determined by electroimmunoassay were reduced to 57-68% of normal. Crossed immunoelectrophoresis of plasma from the proband showed a normal pattern. The amino acid Val384 is located eight residues C-terminal (P8') of the P1 residue (Arg376) in the reactive site. The P8' residues of bovine and mouse plasmin inhibitor are both Val. Among other serpins the P8' residue is unconserved. The mutation was not present in the non-affected family member or 30 blood donors. In addition to the Val384Met mutation two new polymorphisms Ala-26(GCG)/Val(GTG) and Arg407(AGG)/Lys(AAG) and one previously reported polymorphism Arg6(CGG)/Trp(TGG) were identified in the plasmin inhibitor gene of the family. The allelic frequencies among 30 blood donors with normal values of plasma plasmin inhibitor (functional) were 0.84/0.16 for C/T in codon -26, 0.81/0.19 for C/T in codon 6 and 0.83/0.17 for G/A in codon 407.

LDL receptor mutations and ApoB mutations are not risk factors for ischemic cerebrovascular disease of the young, but lipids and lipoproteins are.

BACKGROUND: The genetic background for ischemic cerebrovascular disease of the young and the role of lipids and lipoproteins as risk factors are not clear. METHODS: We determined five LDL receptor mutations (Trp23Stop, Trp66Gly, Trp556Ser, 313+1G --> A, 1846-1G --> A) and three apolipoprotein B mutations (Arg3500Gln, Arg3500Trp, Arg3531Cys), and other risk factors for ischemic cerebrovascular disease in 80 patients (36 women, 44 men) with onset of disease before the age of 50 years compared with 3366 individuals from a general population sample within the same age range. RESULTS: None of the patients were carriers of mutations in the LDL receptor (Trp23Stop, Trp66Gly, Trp556Ser, 313+1G --> A, 1846 - 1G --> A) or the apolipoprotein B gene (Arg3500Gln, Arg3500Trp, Arg3531Cys) associated with hypercholesterolemia. However, on univariate analysis as well as on logistic regression analysis allowing for age and gender, plasma cholesterol (OR 1.4; P < 0.0005), HDL-cholesterol (OR 0.4; P < 0.005), diabetes (OR 5.8; P < 0.0001), and hypertension (OR 3.9; P < 0.001) were significant predictors of ischemic cerebrovascular disease. CONCLUSIONS: The five most common LDL receptor mutations in Danish patients with familial hypercholesterolemia and three mutations in the apolipoprotein B gene did not predispose to ischemic cerebrovascular disease of the young. However, cholesterol and HDL-cholesterol are important risk factors for ischemic cerebrovascular disease of the young in the present study. The elevation in cholesterol could in some patients be due to rare LDL receptor mutations not tested for, and could in other patients be multifactorial in origin.

Trigeminal neuralgia: sudden and long-term remission with transcutaneous electrical nerve stimulation.

OBJECTIVE: To discuss a case of trigeminal neuralgia that responded to an accidental high-intensity discharge of electrical current delivered by a transcutaneous electrical nerve stimulator (TENS). CLINICAL FEATURES: A 36-yr-old man suffering from a 5-month history of worsening paroxysmal pain of the left facial and temporal regions was referred to a neurologist by his family physician. A clinical diagnosis of trigeminal neuralgia was made; before committing to pharmaceutical treatment, however, the patient sought chiropractic consultation. INTERVENTION AND OUTCOME: A trial of self-applied TENS was recommended for pain control. Initial application to patient tolerance provided transient pain relief until an accidental, intense discharge resulted in immediate remission of symptoms, lasting now for three years. CONCLUSION: As an initial treatment of choice, TENS can be a safe and effective therapy for trigeminal neuralgia. The unique effect of this accidental application leads us to speculate that diffuse noxious inhibitory controls may have been the pain inhibitory pathway responsible for the resolution of symptoms in this case. Although firm conclusions are difficult to draw from one incident, using TENS at an intense, noxious level may improve its therapeutic efficacy by decreasing treatment time and frequency and eliciting long-lasting effects. This case suggests the need for further investigation of TENS in the treatment of trigeminal neuralgia and related pain syndromes.