Dose-escalation of adalimumab, golimumab or ustekinumab in inflammatory bowel diseases: characterization and implications in real-life clinical practice.

OBJECTIVES: Dose-escalation is a common practice to optimize treatment with subcutaneously administered biologicals in Crohn's disease (CD) and ulcerative colitis (UC). However, limited data is available on the extent of dose-escalation in real-life. Here, we analyzed treatment persistence, dose-escalation, concomitant corticosteroid use, and costs of adalimumab, golimumab, and ustekinumab in inflammatory bowel diseases (IBD). METHODS: This was a nationwide, retrospective, non-interventional registry study. All adult patients who were diagnosed with CD or UC and had purchased adalimumab, golimumab, or ustekinumab from Finnish pharmacies between 2008 and 2018 were included in the study and followed up for 24 months after treatment initiation. RESULTS: A total of 2884 patients were included in the analyses. For adalimumab, treatment persistence was higher for CD patients compared to UC patients both at months 12 (46.2% versus 37.1%; p < .0001) and 24 (26.1% versus 19.7%; p < 0.0001). For golimumab (UC), treatment persistence was 48.3% at month 12 and 28.1% at month 24. The 12-month treatment persistence rate for patients on ustekinumab (CD) was 47.1%. Cumulative doses exceeding the regular dosing according to the summary of product characteristics (SPC), was observed for adalimumab in CD during the first 6 months of treatment (62.9% of the treatment periods), golimumab in the later stages of the UC treatment (52-54% of treatment periods at months 7-24), and ustekinumab during the first 6 months (70.7%). CONCLUSIONS: Based on this study, dose-escalation of subcutaneously administered biologicals is a common clinical practice in IBD. This has implications for treatment costs, use of concomitant medications, and treatment outcomes.

Benefit of measuring vedolizumab concentrations in inflammatory bowel disease patients in a real-world setting.

OBJECTIVES: We set out to determine the reasons for serum vedolizumab (VDZ) trough concentration (TC) measurements in inflammatory bowel disease (IBD) patients and to evaluate treatment modifications after therapeutic drug measurement (TDM). We also evaluated the effect of increased dosing on patients' response to VDZ therapy. METHODS: We performed a retrospective cohort study of IBD patients who received VDZ therapy at Helsinki University Hospital and whose VDZ levels were measured between June 2014 and December 2018. RESULTS: Altogether, 90 patients (32 Crohn's disease and 58 ulcerative colitis) and 141 VDZ TC measurements were included. 24.1% of measurements took place during induction and 75.9% during the maintenance phase. During induction, 64.7% reached the target TC >20µg/ml. During maintenance therapy, 82.2% of VDZ TCs were within or exceeded the suggested target range of 5-15µg/ml. Reasons for TDM were: secondary nonresponse (44.0%), assessment of adequate VDZ TC (25.5%), primary nonresponse (12.8%), adverse events (6.4%), and other (11.3%). No treatment changes occurred after 60.3% of VDZ measurements. Increased dose frequency was used after 25.5% of VDZ measurements and 33.3% of these patients experienced improvement. Altogether, 31 (34.4%) patients discontinued the therapy due to inadequate treatment response. No anti-vedolizumab antibodies were detected. CONCLUSIONS: During the maintenance of VDZ therapy, the majority of VDZ TCs were within the suggested range. Measurement of VDZ TC did not lead to any treatment changes in two-thirds of patients. Dose optimization occurred in a quarter of patients and a third of them benefited from it.

In situ measurement of the electrical potential across the lysosomal membrane using FRET.

The progressive acidification of the endocytic pathway is generated by H(+) pumping of electrogenic vacuolar-type ATPases (V-ATPases) on the endosomal/lysosomal membrane. The determinants of pH during endosome maturation are not completely understood, but the permeability to ions that neutralize the electrogenic effect of the V-ATPase has been proposed to play a central role. If counter-ion conductance becomes limiting, the generation of a large membrane potential would dominate the proton-motive force (pmf), diminishing the pH gradient proportionally. Validation of this notion requires direct measurement of the electrical potential that develops across the endosomal/lysosomal membrane. To date, the measurement of lysosomal membrane potential (ψ(φ) ) in situ has been hampered by the inability to access endosomes by electrophysiological means and the fact that individual organelles cannot be discerned when using potentiometric fluorescent dyes. Here, we describe a noninvasive procedure to estimate ψ(φ) in intact cells, based on fluorescence resonance energy transfer (FRET). At steady state, ψ(φ) averaged 19 mV (lumen positive) and was only partially dissipated by inhibition of the V-ATPase with concanamycin A (CcA). ψ(φ) was considerably increased by alkalinization of the lysosome lumen by NH(4) Cl, implying that at steady state the V-ATPase operates at submaximal rates and that the contribution of ψ(φ) to pmf is relatively small. Our method should enable systematic studies of endosomal/lysosomal potential.

Endocytic trafficking of sphingomyelin depends on its acyl chain length.

To study the principles of endocytic lipid trafficking, we introduced pyrene sphingomyelins (PyrSMs) with varying acyl chain lengths and domain partitioning properties into human fibroblasts or HeLa cells. We found that a long-chain, ordered-domain preferring PyrSM was targeted Hrs and Tsg101 dependently to late endosomal compartments and recycled to the plasma membrane in an NPC1- and cholesterol-dependent manner. A short-chain, disordered domain preferring PyrSM recycled more effectively, by using Hrs-, Tsg101- and NPC1-independent routing that was insensitive to cholesterol loading. Similar chain length-dependent recycling was observed for unlabeled sphingomyelins (SMs). The findings 1) establish acyl chain length as an important determinant in the endocytic trafficking of SMs, 2) implicate ESCRT complex proteins and NPC1 in the endocytic recycling of ordered domain lipids to the plasma membrane, and 3) introduce long-chain PyrSM as the first fluorescent lipid tracing this pathway.

Impact of Anti-Dementia Medication on the Risk of Death and Causes of Death in Alzheimer's Disease.

BACKGROUND: The Finnish population offers many advantages for evaluating the impact of anti-dementia medication on mortality in Alzheimer's disease (AD) due to broad range of individual-level data collected in national health and social care registries and the fact that Finland has one of the highest mortality rates for dementia globally. OBJECTIVE: The aim of this study was to investigate the association of anti-dementia medication with 2-year risk of death and all-cause mortality in patients with AD. METHODS: This was a retrospective, non-interventional registry study based on individual-level data using Finnish national health and social care registries. An incident cohort of 9,204 AD patients (first AD diagnosis in 2012) was formed from a population of 316,470 individuals ≥74 years of age. The main outcome measure was overall 2-year risk of death. Statistical modelling was used to assess mortality (Kaplan-Meier) and adjusted hazard ratios (HR) (Cox proportional hazard model). RESULTS: Early start of anti-dementia medication (treatment started ≤3 months from AD diagnosis) reduced significantly the risk of all-cause death compared to AD patients who had late medication initiation (defined as treatment started >3 months from AD diagnosis/no medication; HR, 0.51; 95% confidence interval (CI), 0.46-0.57). Dementia was the most common recorded cause of death in both groups. CONCLUSION: This study places importance on early diagnosis of AD and subsequent early initiation of drug treatment in decreasing 2-year risk of death.

Phosphatidylserine dynamics in cellular membranes.

Much has been learned about the role of exofacial phosphatidylserine (PS) in apoptosis and blood clotting using annexin V. However, because annexins are impermeant and unable to bind PS at low calcium concentration, they are unsuitable for intracellular use. Thus little is known about the topology and dynamics of PS in the endomembranes of normal cells. We used two new probes-green fluorescent protein (GFP)-LactC2, a genetically encoded fluorescent PS biosensor, and 1-palmitoyl-2-(dipyrrometheneboron difluoride)undecanoyl-sn-glycero-3-phospho-L-serine (TopFluor-PS), a synthetic fluorescent PS analogue-to examine PS distribution and dynamics inside live cells. The mobility of PS was assessed by a combination of advanced optical methods, including single-particle tracking and fluorescence correlation spectroscopy. Our results reveal the existence of a sizable fraction of PS with limited mobility, with cortical actin contributing to the confinement of PS in the plasma membrane. We were also able to measure the dynamics of PS in endomembrane organelles. By targeting GFP-LactC2 to the secretory pathway, we detected the presence of PS in the luminal leaflet of the endoplasmic reticulum. Our data provide new insights into properties of PS inside cells and suggest mechanisms to account for the subcellular distribution and function of this phospholipid.

Amiloride inhibits macropinocytosis by lowering submembranous pH and preventing Rac1 and Cdc42 signaling.

Macropinocytosis is differentiated from other types of endocytosis by its unique susceptibility to inhibitors of Na(+)/H(+) exchange. Yet, the functional relationship between Na(+)/H(+) exchange and macropinosome formation remains obscure. In A431 cells, stimulation by EGF simultaneously activated macropinocytosis and Na(+)/H(+) exchange, elevating cytosolic pH and stimulating Na(+) influx. Remarkably, although inhibition of Na(+)/H(+) exchange by amiloride or HOE-694 obliterated macropinocytosis, neither cytosolic alkalinization nor Na(+) influx were required. Instead, using novel probes of submembranous pH, we detected the accumulation of metabolically generated acid at sites of macropinocytosis, an effect counteracted by Na(+)/H(+) exchange and greatly magnified when amiloride or HOE-694 were present. The acidification observed in the presence of the inhibitors did not alter receptor engagement or phosphorylation, nor did it significantly depress phosphatidylinositol-3-kinase stimulation. However, activation of the GTPases that promote actin remodelling was found to be exquisitely sensitive to the submembranous pH. This sensitivity confers to macropinocytosis its unique susceptibility to inhibitors of Na(+)/H(+) exchange.

Sensors, transducers, and effectors that regulate cell size and shape.

Cell volume and shape are stringently regulated. This homeostasis requires the cells to sense their size and shape and to convey this information to effectors that will counteract deformations induced by osmotic or mechanical challenges. The sensors, transducers, and effectors of volume change are the subject of this review.

Cholesterol substitution increases the structural heterogeneity of caveolae.

Caveolin-1 binds cholesterol and caveola formation involves caveolin-1 oligomerization and cholesterol association. The role of cholesterol in caveolae has so far been addressed by methods that compromise membrane integrity and abolish caveolar invaginations. To study the importance of sterol specificity for the structure and function of caveolae, we replaced cholesterol in mammalian cells with its immediate precursor desmosterol by inhibiting 24-dehydrocholesterol reductase. Desmosterol could substitute for cholesterol in maintaining cell growth, membrane integrity, and preserving caveolar invaginations. However, in desmosterol cells the affinity of caveolin-1 for sterol and the stability of caveolin oligomers were decreased. Moreover, caveolar invaginations became more heterogeneous in dimensions and in the number of caveolin-1 molecules per caveola. Despite the altered caveolar structure, caveolar ligand uptake was only moderately inhibited. We found that in desmosterol cells, Src kinase phosphorylated Cav1 at Tyr(14) more avidly than in cholesterol cells. Taken the role of Cav1 Tyr(14) phosphorylation in caveolar endocytosis, this may help to preserve caveolar uptake in desmosterol cells. We conclude that a sterol C24 double bond interferes with caveolin-sterol interaction and perturbs caveolar morphology but facilitates Cav1 Src phosphorylation and allows caveolar endocytosis. More generally, substitution of cholesterol by a structurally closely related sterol provides a method to selectively modify membrane protein-sterol affinity, structure and function of cholesterol-dependent domains without compromising membrane integrity.

Significance of sterol structural specificity. Desmosterol cannot replace cholesterol in lipid rafts.

Desmosterol is an immediate precursor of cholesterol in the Bloch pathway of sterol synthesis and an abundant membrane lipid in specific cell types. The significance of the difference between the two sterols, an additional double bond at position C24 in the tail of desmosterol, is not known. Here, we provide evidence that the biophysical and functional characteristics of the two sterols differ and that this is because the double bond at C24 significantly weakens the sterol ordering potential. In model membranes, desmosterol was significantly weaker than cholesterol in promoting the formation or stability of ordered domains, and in mammalian cell membranes, desmosterol associated less avidly than cholesterol with detergent-resistant membranes. Atomic scale molecular dynamics simulations showed that the double bond gives rise to additional stress in the tail, creating a rigid structure between C24 and C27 and favoring tilting of desmosterol distinct from cholesterol. Functional effects of desmosterol in cell membranes were assessed upon acutely exchanging approximately 70% of cholesterol to desmosterol. This led to impaired raft-dependent signaling via the insulin receptor, whereas non-raft-dependent protein secretion was not affected. We suggest that the choice of cholesterol synthesis route may provide a physiological mechanism to modulate raft-dependent functions in cells.

Partitioning of pyrene-labeled phospho- and sphingolipids between ordered and disordered bilayer domains.

Here we have studied how the length of the pyrene-labeled acyl chain (n) of a phosphatidylcholine, sphingomyelin, or galactosylceramide affects the partitioning of these lipids between 1), gel and fluid domains coexisting in bovine brain sphingomyelin (BB-SM) or BB-SM/spin-labeled phosphatidylcholine (PC) bilayers or 2), between liquid-disordered and liquid-ordered domains in BB-SM/spin-labeled PC/cholesterol bilayers. The partitioning behavior was deduced either from modeling of pyrene excimer/monomer ratio versus temperature plots, or from quenching of the pyrene monomer fluorescence by spin-labeled PC. New methods were developed to model excimer formation and pyrene lipid quenching in segregated bilayers. The main result is that partition to either gel or liquid-ordered domains increased significantly with increasing length of the labeled acyl chain, probably because the pyrene moiety attached to a long chain perturbs these ordered domains less. Differences in partitioning were also observed between phosphatidylcholine, sphingomyelin, and galactosylceramide, thus indicating that the lipid backbone and headgroup-specific properties are not severely masked by the pyrene moiety. We conclude that pyrene-labeled lipids could be valuable tools when monitoring domain formation in model and biological membranes as well as when assessing the role of membrane domains in lipid trafficking and sorting.