Impact of methodological choices in comparative effectiveness studies: application in natalizumab versus fingolimod comparison among patients with multiple sclerosis.

BACKGROUND: Natalizumab and fingolimod are used as high-efficacy treatments in relapsing-remitting multiple sclerosis. Several observational studies comparing these two drugs have shown variable results, using different methods to control treatment indication bias and manage censoring. The objective of this empirical study was to elucidate the impact of methods of causal inference on the results of comparative effectiveness studies. METHODS: Data from three observational multiple sclerosis registries (MSBase, the Danish MS Registry and French OFSEP registry) were combined. Four clinical outcomes were studied. Propensity scores were used to match or weigh the compared groups, allowing for estimating average treatment effect for treated or average treatment effect for the entire population. Analyses were conducted both in intention-to-treat and per-protocol frameworks. The impact of the positivity assumption was also assessed. RESULTS: Overall, 5,148 relapsing-remitting multiple sclerosis patients were included. In this well-powered sample, the 95% confidence intervals of the estimates overlapped widely. Propensity scores weighting and propensity scores matching procedures led to consistent results. Some differences were observed between average treatment effect for the entire population and average treatment effect for treated estimates. Intention-to-treat analyses were more conservative than per-protocol analyses. The most pronounced irregularities in outcomes and propensity scores were introduced by violation of the positivity assumption. CONCLUSIONS: This applied study elucidates the influence of methodological decisions on the results of comparative effectiveness studies of treatments for multiple sclerosis. According to our results, there are no material differences between conclusions obtained with propensity scores matching or propensity scores weighting given that a study is sufficiently powered, models are correctly specified and positivity assumption is fulfilled.

Modulation of soleus stretch reflexes during walking in people with chronic incomplete spinal cord injury.

In people with spasticity due to chronic incomplete spinal cord injury (SCI), it has been presumed that the abnormal stretch reflex activity impairs gait. However, locomotor stretch reflexes across all phases of walking have not been investigated in people with SCI. Thus, to understand modulation of stretch reflex excitability during spastic gait, we investigated soleus stretch reflexes across the entire gait cycle in nine neurologically normal participants and nine participants with spasticity due to chronic incomplete SCI (2.5-11 year post-injury). While the participant walked on the treadmill at his/her preferred speed, unexpected ankle dorsiflexion perturbations (6° at 250°/s) were imposed every 4-6 steps. The soleus H-reflex was also examined. In participants without SCI, spinal short-latency "M1", spinal medium latency "M2", and long-latency "M3" were clearly modulated throughout the step cycle; the responses were largest in the mid-stance and almost completely suppressed during the stance-swing transition and swing phases. In participants with SCI, M1 and M2 were abnormally large in the mid-late-swing phase, while M3 modulation was similar to that in participants without SCI. The H-reflex was also large in the mid-late-swing phase. Elicitation of H-reflex and stretch reflexes in the late swing often triggered clonus and affected the soleus activity in the following stance. In individuals without SCI, moderate positive correlation was found between H-reflex and stretch reflex sizes across the step cycle, whereas in participants with SCI, such correlation was weak to non-existing, suggesting that H-reflex investigation would not substitute for stretch reflex investigation in individuals after SCI.

Differential effects of FXR or TGR5 activation in cholangiocarcinoma progression.

BACKGROUND AND AIMS: Cholangiocarcinoma (CCA) is an aggressive tumor type affecting cholangiocytes. CCAs frequently arise under certain cholestatic liver conditions. Intrahepatic accumulation of bile acids may facilitate cocarcinogenic effects by triggering an inflammatory response and cholangiocyte proliferation. Here, the role of bile acid receptors FXR and TGR5 in CCA progression was evaluated. METHODS: FXR and TGR5 expression was determined in human CCA tissues and cell lines. An orthotopic model of CCA was established in immunodeficient mice and tumor volume was monitored by magnetic resonance imaging under chronic administration of the specific FXR or TGR5 agonists, obeticholic acid (OCA) or INT-777 (0,03% in chow; Intercept Pharmaceuticals), respectively. Functional effects of FXR or TGR5 activation were evaluated on CCA cells in vitro. RESULTS: FXR was downregulated whereas TGR5 was upregulated in human CCA tissues compared to surrounding normal liver tissue. FXR expression correlated with tumor differentiation and TGR5 correlated with perineural invasion. TGR5 expression was higher in perihilar than in intrahepatic CCAs. In vitro, FXR was downregulated and TGR5 was upregulated in human CCA cells compared to normal human cholangiocytes. OCA halted CCA growth in vivo, whereas INT-777 showed no effect. In vitro, OCA inhibited CCA cell proliferation and migration which was associated with decreased mitochondrial energy metabolism. INT-777, by contrast, stimulated CCA cell proliferation and migration, linked to increased mitochondrial energy metabolism. CONCLUSION: Activation of FXR inhibits, whereas TGR5 activation may promote, CCA progression by regulating proliferation, migration and mitochondrial energy metabolism. Modulation of FXR or TGR5 activities may represent potential therapeutic strategies for CCA.

IUGR decreases cardiomyocyte endowment and alters cardiac metabolism in a sex- and cause-of-IUGR-specific manner.

Intrauterine growth restriction (IUGR) increases the risk of ischemic heart disease in adulthood. Studies in rats suggest cardiac vulnerability is more pronounced in males and in offspring that were exposed to hypoxia in utero. Therefore, we aimed to test the hypotheses that 1) IUGR adolescent males, but not females, have fewer cardiomyocytes and altered expression of cardiometabolic genes compared with controls; and 2) IUGR due to hypoxia has a greater effect on these parameters compared with IUGR due to nutrient restriction. IUGR was induced in guinea pigs by maternal hypoxia (MH; 10% O2, n = 9) or maternal nutrient restriction (MNR; ~30% reduction in food intake, n = 9) in the second half of pregnancy and compared with control ( n = 11). At 120 days of age, postmortem was performed and the left ventricle perfusion fixed for stereological determination of cardiomyocyte number or snap frozen to determine the abundance of cardiometabolic genes and proteins by quantitative RT-PCR and Western blotting, respectively. MH reduced the number of cardiomyocytes in female ( P < 0.05), but not male or MNR, adolescent offspring. Furthermore, IUGR males had decreased expression of genes responsible for fatty acid activation in the sarcoplasm ( FACS) and transport into the mitochondria ( AMPK-a2 and ACC; P < 0.05) and females exposed to MH had increased activation/phosphorylation of AMP-activated protein kinase-α ( P < 0.05). We postulate that the changes in cardiomyocyte endowment and cardiac gene expression observed in the present study are a direct result of in utero programming, as offspring at this age did not suffer from obesity, hypertension, or left ventricular hypertrophy.

Breaking up Prolonged Sitting does not Alter Postprandial Glycemia in Young, Normal-Weight Men and Women.

A randomized, controlled, cross-over study was used to investigate the effects of breaking up prolonged sitting with low intensity physical activity on postprandial blood glucose concentrations in healthy, young, normal-weight adults. 14 men (n=6) and women (n=8) were assigned to 2.5 h of prolonged sitting (CON) and 2.5 h of prolonged sitting with 2-min bouts of walking every 20 min (LIPA). After ingesting a standardized test drink, capillary blood was sampled every 10 min to establish a postprandial blood glucose profile. Based on individual glucose responses, peak blood glucose, time-to-peak glucose, and incremental area under the glucose curve (iAUC) were determined. Paired sample t-tests were used to detect differences between trials. Peak blood glucose (p=0.55) and iAUC (CON: 252 mmol·L-1·2.5 h-1 [163-340]; LIPA: 214 mmol·L-1·2.5 h-1 [146-282]; p=0.45) were not different between trials. Also, time-to-peak glucose was not different between LIPA and CON (p=0.37). Taking advantage of high temporal resolution blood glucose profiles, we showed that breaking up prolonged sitting with low-intensity physical activity does not alter the postprandial blood glucose response in young, healthy, normal-weight adults. Our results indicate that postprandial glycemic control is maintained during prolonged sitting in young, healthy adults.

Myasthenia gravis and risks for comorbidity.

Myasthenia gravis (MG) is an autoimmune disorder leading to skeletal muscle weakness and fatigability. MG subgroups are defined according to pathogenetic autoantibody (against acetylcholine receptor, muscle-specific tyrosine kinase or lipoprotein receptor-related protein 4), thymus pathology and clinical manifestations. MG patients have an increased risk for concordant autoimmune disease, in particular with early onset MG. Most common comorbidities are thyroid disease, systemic lupus erythematosus and rheumatoid arthritis. Cardiomyositis and subclinical heart dysfunction have been described in patients with thymoma MG and late onset MG but represent no major threat. A thymic lymphoepithelioma implies an increased risk for another cancer. Autoimmune MG represents no distinct cancer risk factor, although lymphomas and a few other cancer types have been reported with slightly increased frequency. Severe MG-related muscle weakness means a risk for respiratory failure and respiratory tract infection. Drug MG treatment can lead to side-effects. Thymectomy is regarded as a safe procedure both short and long term. Non-MG-related comorbidity represents a diagnostic and therapeutic challenge, especially in elderly patients. Diagnostic accuracy and optimal follow-up is necessary to identify and treat all types of coexisting disease in MG.

Total drug treatment and comorbidity in myasthenia gravis: a population-based cohort study.

BACKGROUND AND PURPOSE: Comorbidity in myasthenia gravis (MG) is important for diagnosis, treatment and prognosis. Disease complexity was assessed by examining total drug treatment, immune therapy and comorbidity in a complete national MG cohort. METHODS: All recipients of the MG-specific drug pyridostigmine 2004-2010 registered in the compulsory Norwegian Prescription Database who met the inclusion criteria were included. The pyridostigmine group was compared with the general Norwegian population. RESULTS: Myasthenia gravis patients received co-medication more often than the controls for nearly all groups of medication, including insulins (95% confidence interval 2.0-3.7), thyroid therapy (1.7-2.5), antidepressants (1.3-1.7), anti-infectives (1.2-1.4), lipid-modifying agents (1.1-1.4) and immunomodulating agents (6.8-8.8). CONCLUSIONS: Myasthenia gravis patients are more often treated with non-MG prescription drugs than controls, reflecting frequent co-medication and comorbidity.

Improving efficiency in stereology: a study applying the proportionator and the autodisector on virtual slides.

Cell counting in stereology is time-consuming. The proportionator is a new stereological sampling method combining automatic image analysis and non-uniform sampling. The autodisector on virtual slides combines automatic generation of disector pairs with the use of digital images. The aim of the study was to investigate the time efficiency of the proportionator and the autodisector on virtual slides compared with traditional methods in a practical application, namely the estimation of osteoclast numbers in paws from mice with experimental arthritis and control mice. Tissue slides were scanned in a digital slide scanner and the autodisector was applied on the obtained virtual tissue slides. Every slide was partitioned into fields of view, and cells were counted in all of them. Based on the original exhaustive data set comprising 100% of fields of view and covering the total section area, a proportionator sampling and a systematic, uniform random sampling were simulated. We found that the proportionator was 50% to 90% more time efficient than systematic, uniform random sampling. The time efficiency of the autodisector on virtual slides was 60% to 100% better than the disector on tissue slides. We conclude that both the proportionator and the autodisector on virtual slides may improve efficiency of cell counting in stereology.

The semi-automatic nucleator.

The nucleator is a well-established manual stereological method of estimating mean cell volume from observations on random cell transects through reference points of the cells. In this paper, we present an automated version of the nucleator that uses automatic segmentation of the boundaries of the cell transects. An expert supervises the process. If the segmentation is judged to be satisfactory, an estimate of the cell volume is calculated automatically on the basis of the whole cell transect. In the remaining cases, the expert intervenes and uses the classical nucleator. The resulting estimator is called the semi-automatic nucleator. In this paper, we study the statistical properties of the semi-automatic nucleator. Formulae for the bias and mean square error are derived. The semi-automatic nucleator may have a small bias but will still in most cases be more efficient than the classical nucleator. Procedures for estimating bias and mean square error from a pilot study are provided. The application of the semi-automatic nucleator is illustrated in a study of somatostatin positive inhibitory interneurons which were genetically labelled with green fluorescent protein (GFP). The cells were sampled with an optical disector. The centre of mass in a central cell transect was used as reference point. It is found in this study that the number of cells needed for obtaining, for instance, a 5% precision of the estimate of mean cell volume is 150 and 189 for the semi-automatic and the classical nucleator, respectively. Taking into account that the time spent analysing one cell is shorter for the semi-automatic nucleator than for the classical nucleator, the semi-automatic nucleator is superior to the classical nucleator.

Myasthenia gravis requiring pyridostigmine treatment in a national population cohort.

BACKGROUND: Pyridostigmine is the first drug of choice for patients with myasthenia gravis (MG). The drug is not prescribed regularly to any other patient groups. We aimed to determine the prevalence, incidence and gender-specific characteristics of patients with MG needing drug treatment in a well-defined population cohort. METHODS: Data were retrieved from the Norwegian Prescription Database (NorPD) 2004-2007, containing information on all dispensed drugs in Norway. The study population comprised 677 recipients of pyridostigmine who met the following inclusion criteria (one or more): (i) More than one prescription 1 January 2004-31 December 2007, (ii) prescription from a specialist in neurology, (iii) prescription for MG being specified in NorPD. RESULTS: A total of 435 (64%) women and 242 men were included; female:male ratio 1.8:1. Point prevalence (1 January 2008) of symptomatic MG was 131 per million; 92 for men, 170 for women. Seventy-four new users of pyridostigmine were registered in 2007 (42 women, 32 men), i.e. the incidence rate for 2007 being 16 per million; 14 for men, 18 for women. Mean age of incident cases was 59 years; 64 and 55 years, respectively. Prevalence and incidence were significantly higher in the age group ≥ 50 years than < 50 years (P < 0.001), and highest at 70-79 years. Prevalence and incidence did not differ in the five geographical health regions in Norway. CONCLUSIONS: Reported prevalence and incidence are amongst the highest found in similar studies. This may be explained by optimal case identification, higher incidence of drug requiring MG amongst the elderly, and recurrences of previous MG.

Power variations of wireless communication systems.

The use of wireless digital communication devices like GSM, WCDMA, HSPA, DECT, and WiFi changes the exposure of electromagnetic waves toward the user. Concentrating on the power variations on a slow and fast time scale, these new systems are discussed. Experimental results for both uplink and downlink are included for a sample of systems. The spectrum of the power fluctuations is seen as a convenient and compact way of describing very complex system behavior. The results are of interest for scientific studies of epidemiology and biological effects, and for general electromagnetic compatibility (EMC) aspects.

The interferon regulated ubiquitin-like protein, ISG15, in tumorigenesis: friend or foe?

The interferon-stimulated gene, 15 kDa (ISG15) is an interferon regulated gene that is induced as a primary response to diverse microbial and cell stress stimuli, and encodes the founding member of the ubiquitin-like protein family. ISG15 post-translationally modifies proteins via a pathway parallel to, and partially overlapping with, that of ubiquitin. In addition, ISG15 is released from cells to mediate extracellular cytokine-like activities. Although the biological activities of ISG15 have yet to be fully elucidated, it is clear that ISG15 has the capacity to modulate diverse cellular and physiologic functions. Consistent with this view, alterations in the ISG15 pathway have been identified in human tumors and in tumor cell lines. Here we review evidence of a role for ISG15 as an endogenous tumor suppressor that, when dysregulated in malignant cells, can be subverted to promote oncogenesis.

Role of 2-5A-dependent RNase-L in senescence and longevity.

Senescence is a permanent growth arrest that restricts the lifespan of primary cells in culture, and represents an in vitro model for aging. Senescence functions as a tumor suppressor mechanism that can be induced independent of replicative crisis by diverse stress stimuli. RNase-L mediates antiproliferative activities and functions as a tumor suppressor in prostate cancer, therefore, we examined a role for RNase-L in cellular senescence and aging. Ectopic expression of RNase-L induced a senescent morphology, a decrease in DNA synthesis, an increase in senescence-associated beta-galactosidase activity, and accelerated replicative senescence. In contrast, senescence was retarded in RNase-L-null fibroblasts compared with wild-type fibroblasts. Activation of endogenous RNase-L by 2-5A transfection induced distinct senescent and apoptotic responses in parental and Simian virus 40-transformed WI38 fibroblasts, respectively, demonstrating cell type specific differences in the antiproliferative response to RNase-L activation. Replicative senescence is a model for in vivo aging; therefore, genetic disruption of senescence effectors may impact lifespan. RNase-L-/- mice survived 31.7% (P<0.0001) longer than strain-matched RNase-L+/+ mice providing evidence for a physiological role for RNase-L in aging. These findings identify a novel role for RNase-L in senescence that may contribute to its tumor suppressive function and to the enhanced longevity of RNase-L-/- mice.

Priming the cow for mobilization in the periparturient period: effects of supplementing the dry cow with saturated fat or linseed.

High-producing dairy cows experience negative energy balance in early lactation. Dry-cow feeding management will affect the performance and metabolic status of dairy cows in the following early lactation. The present study evaluates dry-cow feeding strategies for priming lipid metabolism in the dairy cow to overcome the metabolic challenges in the following early lactation. Five weeks before expected calving, 27 cows were assigned to 1 of 3 isonitrogenous and isoenergetic dietary treatments: a low-fat control diet (dry-control); a high saturated fat diet (dry-HSF); and a high linseed diet (dry-HUF). The cows were fed the same TMR lactation diet after calving. The treatments were evaluated by performance and metabolic parameters in blood and liver. The cows fed dry-HSF and dry-HUF had significantly greater plasma nonesterified fatty acid concentrations compared with dry-control, and the dry-HUF cows had the greatest C18:3 concentrations in plasma in the prepartum period. Further, the cows fed dry-HSF and dry-HUF diets had a tendency for the greatest capacity for incomplete beta-oxidation of fatty acids in the liver in wk 3 prepartum. The plasma cholesterol concentration was greatest for cows fed dry-HSF in the prepartum period compared with those fed dry-control and dry-HUF. The cows fed dry-HSF had the lowest plasma nonesterified fatty acid and liver fat concentrations in early lactation compared with the cows fed dry-control and dry-HUF. Data in the literature and the present experiment indicate that supplementing dry cows with a saturated fatty acid source is a positive strategy for priming dairy cows for body fat mobilization in the following early lactation.

Continuous lactation in dairy cows: effect on milk production and mammary nutrient supply and extraction.

Reports over the past decade have indicated that normal lactational performance can be achieved in genetically superior and high-producing dairy cows, even when the dry period between 2 lactations is omitted. The hypothesis tested in this experiment was that normal lactogenesis I and metabolic function may be achievable in continuously milked high-yielding dairy cows as a result of the genetic selection for lactation performance and hence longevity of mammary epithelial cells. The milk production and mammary nutrient uptake in response to omission of the dry period for cows with an expected peak milk yield higher than 45 kg/d were studied in 28 Holstein dairy cows managed without bovine somatotropin. Performance and metabolic parameters were followed in late gestation and in the following early lactation. Fourteen cows were milked continuously throughout late gestation, and another 14 dairy cows experienced a 7-wk prepartum dry period. Continuous milking during the prepartum period reduced milk production in the following early lactation period by >20%. The reduced milk production could not be readily ascribed to inefficiency of the mechanisms responsible for nutrient uptake by the lactating mammary epithelial cells, nor to systemic endocrine changes. This suggests that lowered mammary nutrient uptake must have been associated with reduced mammary blood flow, metabolic activity, or both, most likely as a result of disturbed lactogenesis I prepartum or lactogenesis II postpartum triggered by as yet unknown local mechanisms. Milk protein content was elevated by 0.4 percentage units in the continuously milked cows. The underlying reason is unknown, but given the current pricing system for milk, it deserves to be further investigated.

Evidence for transcortical reflex pathways in the lower limb of man.

The existence of transcortical reflex pathways in the control of distal arm and hand muscles in man is now widely accepted. Much more controversy exists regarding a possible contribution of such reflexes to the control of leg muscles. It is often assumed that transcortical reflex pathways play no, or only a minor, role in the control of leg muscles. Transcortical reflex pathways according to this view are reserved for the control of the distal upper limb and are seen in close relation to the evolution of the primate hand. Here we review data, which provide evidence that transcortical reflexes do exist for lower limb muscles and may play a significant role in the control of at least some of these muscles. This evidence is based on animal research, recent experiments combining transcranial magnetic stimulation with peripheral electrical and mechanical stimulation in healthy subjects and neurological patients. We propose that afferent activity from muscle and skin may play a role in the regulation of bipedal gait through transcortical pathways.

Effects of diet energy density and milking frequency in early lactation on tumor necrosis factor-alpha responsiveness in dairy cows.

A whole blood stimulation assay (WBA) with Escherichia coli lipopolysaccharide (LPS) and an enzyme-linked immunosorbent assay (ELISA) were established to measure the production of tumor necrosis factor-alpha (TNF-alpha) in bovine plasma. The assays were used to study the effect of time around parturition, and diet energy density, and milking frequency on TNF-alpha responsiveness of dairy cows in early lactation. Forty cows were included in a 2 x 2 factorial block design. One factor was high (H) versus low (L) diet energy density and the other factor was two versus three daily milkings. Blood samples were collected in weeks -3, -1, 2, 3, 5, 9, and 13 around parturition, and investigated for the TNF-alpha production ex vivo and CD14+ monocytes. The TNF-alpha response, CD14+ monocyte number, and CD14 expression level on monocytes were significantly increased in the weeks close to parturition. However, dips of varying sizes were observed for the measured parameters in week 3 after calving. Diet and milking frequency had no effect on the TNF-alpha response ex vivo or CD14 expression level on monocytes, but cows fed diet H had significantly higher numbers of CD14+ monocytes than cows fed diet L. The WBA with LPS was a fast reliable method for repeated measurements of TNF-alpha responsiveness in cattle. Previous findings of increased TNF-alpha responses in periparturient cows were confirmed, whereas diet energy concentration and milking frequency had no effect on the TNF-alpha responsiveness in early lactation.

The effects of dry period versus continuous lactation on metabolic status and performance in periparturient cows.

It has been argued that dairy cows with a high genetic milk production potential can maintain high milk production even with total omission of the dry period. Further, when omitting the dry period, cows are believed to experience fewer metabolic changes during the transition from late gestation to early lactation compared with cows having a traditional dry period. The performance and metabolic response to omission of the dry period for cows with an expected peak milk yield higher than 45 kg/d were studied in 28 Holstein dairy cows. The cows were followed in late gestation and in the subsequent 5 wk of early lactation. Fourteen cows were milked through late gestation (CM) and another 14 dairy cows underwent a 7-wk dry period (DRY). In the early lactation period, the cows had the same dry matter (DM) intake but cows in the CM group had a 22% reduction in milk yield compared with the cows in the DRY group. At calving, the experimental groups had the same average body weight and body condition score and there were no significant differences in body weight and body condition score changes in early lactation. However, the cows in the CM group compared with the cows in the DRY group had a higher plasma concentration of glucose and insulin and a lower plasma concentration of nonesterified fatty acids and beta-hydroxybutyrate in the following 5 wk of early lactation. Furthermore, the cows in the CM group had lower liver triacylglycerol concentration and higher liver glycogen concentration in the following early lactation. It is concluded that, even in dairy cows with an expected peak milk yield above 45 kg/d, omission of the dry period results in a relatively high reduction in milk yield in the following early lactation. Furthermore, these cows are in less metabolic imbalance in the following early lactation.