Evaluation of genetic variants associated with the risk of thiopurine-related pancreatitis: a case control study from ENEIDA registry.

BACKGROUND: Risk factors for developing pancreatitis due to thiopurines in patients with inflammatory bowel disease (IBD) are not clearly identified. AIM: Our aim was to evaluate the predictive pharmacogenetic risk of pancreatitis in IBD patients treated with thiopurines. METHODS: We conducted an observational pharmacogenetic study of acute pancreatitis events in a cohort study of IBD patients treated with thiopurines from the prospectively maintained ENEIDA registry biobank of GETECCU. Samples were obtained and the CASR, CEL, CFTR, CDLN2, CTRC, SPINK1, CPA1, and PRSS1 genes, selected based on their known association with pancreatitis, were fully sequenced. RESULTS: Ninety-five cases and 105 controls were enrolled, 57% were women. Median age at pancreatitis diagnosis was 39 years. We identified 81 benign variants (50 in cases and 67 in controls) and a total of 35 distinct rare pathogenic and unknown significance variants (10 in CEL, 21 in CFTR, 1 in CDLN2, and 3 in CPA1). None of the cases or controls carried pancreatitis-predisposing variants within the CASR, CPA1, PRSS1, and SPINK1 genes, nor a pathogenic CFTR mutation. Four different variants of unknown significance were detected in the CDLN and CPA1 genes; one of them was in the CDLN gene in a single patient with pancreatitis, and 3 in the CPA1 gene in 5 controls. After the analysis of the variants detected, no significant differences were observed between cases and controls. CONCLUSION: In patients with IBD, genes known to cause pancreatitis seem not to be involved in thiopurine-related pancreatitis onset.

Comparative Study of the Effectiveness of Vedolizumab Versus Ustekinumab After Anti-TNF Failure in Crohn's Disease (Versus-CD): Data from the ENEIDA Registry.

BACKGROUND: Both vedolizumab and ustekinumab are approved for the management of Crohn's disease [CD]. Data on which one would be the most beneficial option when anti-tumour necrosis factor [anti-TNF] agents fail are limited. AIMS: To compare the durability, effectiveness, and safety of vedolizumab and ustekinumab after anti-TNF failure or intolerance in CD. METHODS: CD patients from the ENEIDA registry who received vedolizumab or ustekinumab after anti-TNF failure or intolerance were included. Durability and effectiveness were evaluated in both the short and the long term. Effectiveness was defined according to the Harvey-Bradshaw index [HBI]. The safety profile was compared between the two treatments. The propensity score was calculated by the inverse probability weighting method to balance confounder factors. RESULTS: A total of 835 patients from 30 centres were included, 207 treated with vedolizumab and 628 with ustekinumab. Dose intensification was performed in 295 patients. Vedolizumab [vs ustekinumab] was associated with a higher risk of treatment discontinuation (hazard ratio [HR] 2.55, 95% confidence interval [CI]: 2.02-3.21), adjusted by corticosteroids at baseline [HR 1.27; 95% CI: 1.00-1.62], moderate-severe activity in HBI [HR 1.79; 95% CI: 1.20-2.48], and high levels of C-reactive protein at baseline [HR 1.06; 95% CI: 1.02-1.10]. The inverse probability weighting method confirmed these results. Clinical response, remission, and corticosteroid-free clinical remission were higher with ustekinumab than with vedolizumab. Both drugs had a low risk of adverse events with no differences between them. CONCLUSION: In CD patients who have failed anti-TNF agents, ustekinumab seems to be superior to vedolizumab in terms of durability and effectiveness in clinical practice. The safety profile is good and similar for both treatments.

A Network of Macrophages Supports Mitochondrial Homeostasis in the Heart.

Cardiomyocytes are subjected to the intense mechanical stress and metabolic demands of the beating heart. It is unclear whether these cells, which are long-lived and rarely renew, manage to preserve homeostasis on their own. While analyzing macrophages lodged within the healthy myocardium, we discovered that they actively took up material, including mitochondria, derived from cardiomyocytes. Cardiomyocytes ejected dysfunctional mitochondria and other cargo in dedicated membranous particles reminiscent of neural exophers, through a process driven by the cardiomyocyte's autophagy machinery that was enhanced during cardiac stress. Depletion of cardiac macrophages or deficiency in the phagocytic receptor Mertk resulted in defective elimination of mitochondria from the myocardial tissue, activation of the inflammasome, impaired autophagy, accumulation of anomalous mitochondria in cardiomyocytes, metabolic alterations, and ventricular dysfunction. Thus, we identify an immune-parenchymal pair in the murine heart that enables transfer of unfit material to preserve metabolic stability and organ function. VIDEO ABSTRACT.

Fgr kinase is required for proinflammatory macrophage activation during diet-induced obesity.

Proinflammatory macrophages are key in the development of obesity. In addition, reactive oxygen species (ROS), which activate the Fgr tyrosine kinase, also contribute to obesity. Here we show that ablation of Fgr impairs proinflammatory macrophage polarization while preventing high-fat diet (HFD)-induced obesity in mice. Systemic ablation of Fgr increases lipolysis and liver fatty acid oxidation, thereby avoiding steatosis. Knockout of Fgr in bone marrow (BM)-derived cells is sufficient to protect against insulin resistance and liver steatosis following HFD feeding, while the transfer of Fgr-expressing BM-derived cells reverts protection from HFD feeding in Fgr-deficient hosts. Scavenging of mitochondrial peroxides is sufficient to prevent Fgr activation in BM-derived cells and HFD-induced obesity. Moreover, Fgr expression is higher in proinflammatory macrophages and correlates with obesity traits in both mice and humans. Thus, our findings reveal the mitochondrial ROS-Fgr kinase as a key regulatory axis in proinflammatory adipose tissue macrophage activation, diet-induced obesity, insulin resistance and liver steatosis.

Cell identity and nucleo-mitochondrial genetic context modulate OXPHOS performance and determine somatic heteroplasmy dynamics.

Heteroplasmy, multiple variants of mitochondrial DNA (mtDNA) in the same cytoplasm, may be naturally generated by mutations but is counteracted by a genetic mtDNA bottleneck during oocyte development. Engineered heteroplasmic mice with nonpathological mtDNA variants reveal a nonrandom tissue-specific mtDNA segregation pattern, with few tissues that do not show segregation. The driving force for this dynamic complex pattern has remained unexplained for decades, challenging our understanding of this fundamental biological problem and hindering clinical planning for inherited diseases. Here, we demonstrate that the nonrandom mtDNA segregation is an intracellular process based on organelle selection. This cell type-specific decision arises jointly from the impact of mtDNA haplotypes on the oxidative phosphorylation (OXPHOS) system and the cell metabolic requirements and is strongly sensitive to the nuclear context and to environmental cues.

Early treatment with anti-tumor necrosis factor agents improves long-term effectiveness in symptomatic stricturing Crohn's disease.

BACKGROUND: There is limited evidence on the effectiveness of biological therapy in stricturing complications in patients with Crohn's disease. AIM: The study aims to determine the effectiveness of anti-tumor necrosis factor (TNF) agents in Crohn's disease complicated with symptomatic strictures. METHODS: In this multicentric and retrospective study, we included adult patients with symptomatic stricturing Crohn's disease receiving their first anti-TNF therapy, with no previous history of biological, endoscopic or surgical therapy. The effectiveness of the anti-TNF agent was defined as a composite outcome combining steroid-free drug persistence with no use of new biologics or immunomodulators, hospital admission, surgery or endoscopic therapy during follow-up. RESULTS: Overall, 262 patients with Crohn's disease were included (53% male; median disease duration, 35 months, 15% active smokers), who received either infliximab (N = 141, 54%) or adalimumab (N = 121, 46%). The treatment was effective in 87% and 73% of patients after 6 and 12 months, respectively, and continued to be effective in 26% after a median follow-up of 40 months (IQR, 19-85). Nonetheless, 15% and 21% of individuals required surgery after 1 and 2 years, respectively, with an overall surgery rate of 32%. Postoperative complications were identified in 15% of patients, with surgical site infection as the most common. Starting anti-TNF therapy in the first 18 months after the diagnosis of Crohn's disease or the identification of stricturing complications was associated with a higher effectiveness (HR 1.62, 95% CI 1.18-2.22; and HR 1.55, 95% CI 1.1-2.23; respectively). Younger age, lower albumin levels, strictures located in the descending colon, concomitant aminosalicylates use or presence of lymphadenopathy were associated with lower effectiveness. CONCLUSIONS: Anti-TNF agents are effective in approximately a quarter of patients with Crohn's disease and symptomatic intestinal strictures, and 68% of patients are free of surgery after a median of 40 months of follow-up. Early treatment and some potential predictors of response were associated with treatment success in this setting.

Relevant Infections in Inflammatory Bowel Disease, and Their Relationship With Immunosuppressive Therapy and Their Effects on Disease Mortality.

BACKGROUND AND AIMS: There is controversy as to whether the risk of relevant infection in IBD is related to immunosuppressants or the disease itself. The aims of this study were to evaluate: [1] the life-long prevalence and types of relevant infections in patients with IBD related to immunosuppressive treatment, and [2] the relationship of both infection and patient comorbidity to mortality. METHODS: Observational multicentre retrospective study of IBD patients that presented a relevant infection. For each case, four periods of infection exposure were analysed: P1: pre-IBD diagnosis, P2: from IBD diagnosis to immunosuppressant initiation, P3: during immunosuppressant therapy, and P4: after treatment withdrawal. RESULTS: The life-long prevalence of relevant infection in the total cohort of patients [6914] was 3%, and 5% in immunosuppressed patients [4202]. 366 relevant infections were found in 212 patients [P1: 9, P2: 17, P3: 334, and P4: 6]. Differences between periods were significant [p < 0.0001]. The most frequent types of infection were respiratory, intestinal and urinary. The most frequent opportunistic infections were tuberculosis [prevalence: 2.6/1000] and herpes zoster [prevalence: 3.9/1000]. Herpes zoster infection was associated with thiopurines alone or in combination with anti-TNF in 75% of the cases, whereas tuberculosis was associated with anti-TNF in 94% of patients. The overall mortality was 4.2%. Infection-related mortality was 2.8% and it was not influenced by comorbidity. CONCLUSIONS: Relevant infections in IBD patients are rare and appear to be related to immunosuppression. Relevant infection is a major cause of death in IBD.

Effect of Triiodothyroacetic Acid Treatment in Mct8 Deficiency: A Word of Caution.

BACKGROUND: Monocarboxylate transporter 8 (MCT8) is a thyroid hormone-specific cell membrane transporter. Mutations in the MCT8 gene lead to profound psychomotor retardation and abnormal thyroid hormone serum levels with low thyroxine (T4) and high triiodothyronine (T3). Currently, therapeutic options for patients are limited. Triiodothyroacetic acid (TRIAC) has potential therapeutic value. The aim of this study was to evaluate the effects and efficacy of therapeutic doses of TRIAC on Mct8-deficient mice (Mct8KO). METHODS: Wild-type (Wt) and Mct8KO mice were treated with 30 ng TRIAC/g of body weight/day, given in drinking water, from postnatal day 21 to 30. TRIAC, T4 and T3 levels in plasma, as well as T3 and TRIAC content in the cerebral cortex and striatum were measured by specific radioimmunoassays. The activities of deiodinases 1 and 2 were measured in liver and cortex. The effect of TRIAC treatment in the expression of T3-dependent genes was measured in the heart, cerebral cortex, and striatum. RESULTS: Plasma TRIAC concentration were the same in Wt and Mct8KO animals after treatment. TRIAC treatment greatly decreased plasma T4 in Wt and Mct8KO mice, and reduced T3 to normal levels in the Mct8KO mice. Deiodinase 1 activity and gene expression in the liver increased, while it did not have any effect on the expression of Serca2a in the heart. TRIAC treatment did not induce the expression of T3-dependent genes in the cerebral cortex or striatum, but further decreased expression of Flywch2 in the cortex and Aldh1a1 and Flywch2 in the striatum. Direct measurements of TRIAC and T3 content in the cortex and striatum revealed a decrease in T3 after treatment with no significant increase in the level of endogenous TRIAC. CONCLUSIONS: Therapeutic doses of TRIAC in Mct8KO mice restored plasma T3 levels but severely decreased T4 levels. TRIAC has a direct effect on deiodinase 1 in the liver and does not have an effect on gene expression in the heart. The increase in the plasma TRIAC levels after treatment is not sufficient to increase TRIAC levels in the brain and to promote the expression of T3-dependent genes in brain cells. Instead, it leads to a state of brain hypothyroidism with reduced T3 content.

Cerebral cortex hyperthyroidism of newborn mct8-deficient mice transiently suppressed by lat2 inactivation.

Thyroid hormone entry into cells is facilitated by transmembrane transporters. Mutations of the specific thyroid hormone transporter, MCT8 (Monocarboxylate Transporter 8, SLC16A2) cause an X-linked syndrome of profound neurological impairment and altered thyroid function known as the Allan-Herndon-Dudley syndrome. MCT8 deficiency presumably results in failure of thyroid hormone to reach the neural target cells in adequate amounts to sustain normal brain development. However during the perinatal period the absence of Mct8 in mice induces a state of cerebral cortex hyperthyroidism, indicating increased brain access and/or retention of thyroid hormone. The contribution of other transporters to thyroid hormone metabolism and action, especially in the context of MCT8 deficiency is not clear. We have analyzed the role of the heterodimeric aminoacid transporter Lat2 (Slc7a8), in the presence or absence of Mct8, on thyroid hormone concentrations and on expression of thyroid hormone-dependent cerebral cortex genes. To this end we generated Lat2-/-, and Mct8-/yLat2-/- mice, to compare with wild type and Mct8-/y mice during postnatal development. As described previously the single Mct8 KO neonates had a transient increase of 3,5,3'-triiodothyronine concentration and expression of thyroid hormone target genes in the cerebral cortex. Strikingly the absence of Lat2 in the double Mct8Lat2 KO prevented the effect of Mct8 inactivation in newborns. The Lat2 effect was not observed from postnatal day 5 onwards. On postnatal day 21 the Mct8 KO displayed the typical pattern of thyroid hormone concentrations in plasma, decreased cortex 3,5,3'-triiodothyronine concentration and Hr expression, and concomitant Lat2 inactivation produced little to no modifications. As Lat2 is expressed in neurons and in the choroid plexus, the results support a role for Lat2 in the supply of thyroid hormone to the cerebral cortex during early postnatal development.

Deiodinase activities in thyroids and tissues of iodine-deficient female rats.

Severe iodine deficiency is characterized by goiter, preferential synthesis, and secretion of T(3) in thyroids, hypothyroxinemia in plasma and tissues, normal or low plasma T(3), and slightly increased plasma TSH. We studied changes in deiodinase activities and mRNA in several tissues of rats maintained on low-iodine diets (LIDs) or LIDs supplemented with iodine (LID+I). T(4) and T(3) concentrations decreased in plasma, tissues, and thyroids of LID rats, and T(4) decreased more than T(3) (50%). The highest type 1 iodothyronine deiodinase (D1) activities were found in the thyroid, kidney, and the liver; pituitary, lung, and ovary had lower D1 activities; but the lowest levels were found in the heart and skeletal muscle. D1 activity decreased in all tissues of LID rats (10-40% of LID+I rats), except for ovary and thyroids, which D1 activity increased 2.5-fold. Maximal type 2 iodothyronine deiodinase (D2) activities were found in thyroid, brown adipose tissue, and pituitary, increasing 6.5-fold in thyroids of LID rats and about 20-fold in the whole gland. D2 always increased in response to LID, and maximal increases were found in the cerebral cortex (19-fold), thyroid, brown adipose tissue, and pituitary (6-fold). Lower D2 activities were found in the ovary, heart, and adrenal gland, which increased in LID. Type 3 iodothyronine deiodinase activity was undetectable. Thyroidal Dio1 and Dio2 mRNA increased in the LID rats, and Dio1 decreased in the lung, with no changes in mRNA expression in other tissues. Our data indicate that LID induces changes in deiodinase activities, especially in the thyroid, to counteract the low T(4) synthesis and secretion, contributing to maintain the local T(3) concentrations in the tissues with D2 activity.

Arachidonic acid stimulates DNA synthesis in brown preadipocytes through the activation of protein kinase C and MAPK.

Arachidonic acid (AA) is a polyunsaturated fatty acid that stimulates the proliferation of many cellular types. We studied the mitogenic potential of AA in rat brown preadipocytes in culture and the signaling pathways involved. AA is a potent mitogen which induces 4-fold DNA synthesis in brown preadipocytes. The AA mitogenic effect increases by NE addition. AA also increases the mitogenic action of different growth factor combinations. Other unsaturated and saturated fatty acids do not stimulate DNA synthesis to the same extent as AA. We analyzed the role of PKC and MEK/MAPK signaling pathways. PKC inhibition by bisindolilmaleimide I (BIS) abolishes AA and phorbol ester stimulation of DNA synthesis and reduces the mitogenic activity of different growth factors in brown preadipocytes. Brown preadipocytes in culture express PKC α, δ, ε and ζ isoforms. Pretreatment with high doses of the phorbol ester PDBu, induces downregulation of PKCs ε and δ and reproduces the effect of BIS indicating that AA-dependent induction of DNA synthesis requires PKC activity. AA also activates MEK/MAPK pathway and the inhibition of MEK activity inhibits AA stimulation of DNA synthesis and brown adipocyte proliferation. Inhibition of PKC δ by rottlerin abolishes AA-dependent stimulation of DNA synthesis and MAPK activation, whereas PKC ε inhibition does not produce any effect. In conclusion, our results identify AA as a potent mitogen for brown adipocytes and demonstrate the involvement of the PDBu-sensitive PKC δ isoform and MEK/MAPK pathway in AA-induced proliferation of brown adipocytes. Increased proliferative activity might increase the thermogenic capacity of brown fat.

Differences in the response of UCP1 mRNA to hormonal stimulation between rat and mouse primary cultures of brown adipocytes.

UNLABELLED: Uncoupling protein 1 (UCP-1), the specific marker of brown adipose tissue, is transcriptionally activated in response to adrenergic stimuli and thyroid hormones are necessary for its full expression. We describe differences in the regulation of UCP-1 mRNA expression between rat and mouse brown adipocytes in culture, using norepinephrine (NE), triiodothyronine (T3), insulin and retinoic acid (RA). RESULTS: NE and cAMP-elevating agents strongly increase UCP-1 mRNA levels in cultures of mouse adipocytes, but increases are low in those from rat. In rat adipocytes NE poorly increases UCP-1 mRNA expression and T3 markedly increases the adrenergic response of UCP-1, an effect not observed in mouse adipocytes. In the absence of insulin, T3 itself increases UCP-1 mRNA in rat adipocytes and enhances the response to NE, while in mouse adipocytes no effect of T3 is observed. RA by itself stimulates UCP-1 mRNA in mouse adipocytes, but not in those from rat. In rat cultures, RA requires the presence of NE and/or T3. CONCLUSIONS: We find important differences in the hormonal regulation of UCP-1 mRNA expression in cultured preadipocytes depending on the species used as donor; those differences are observed using identical culture conditions and should be considered when doing cultures from these species.

The T3 receptor beta1 isoform regulates UCP1 and D2 deiodinase in rat brown adipocytes.

Brown adipose tissue (BAT) thermogenesis increases when uncoupling protein-1 (UCP1) is activated adrenergically and requires T3. In humans, UCP1 activation in BAT seems involved in body weight maintenance. BAT type 2 deiodinase (D2) increases in response to adrenergic agents, producing the T3 required for UCP1 expression. T3 actions are mediated by thyroid hormone nuclear T3 receptors (TR), TRα and TRß. Studies in mice suggest that TRß is required for UCP1 induction, whereas TRα regulates body temperature and adrenergic sensitivity. In the present study, we compare the effects of T3 vs. specific TRß1 and TRα1 agonists [GC-1 and CO23] on the adrenergic induction of UCP1 and D2 in cultured rat brown adipocytes. T3 and GC-1 produced similar increases on UCP1, whereas CO23 increased UCP1 only at high doses (50 nm). GC-1 at low doses (0.2-10 nm) was less potent than T3, increasing the adrenergic stimulation of D2 activity and mRNA. At higher doses, GC-1 further stimulated whereas T3 inhibited D2 activity but not D2 mRNA, suggesting posttranscriptional effects. CO23 had no effect on D2 activity but increased D2 mRNA. T3, GC-1, or CO23 by themselves did not increase UCP1 or D2 mRNA. High T3 doses shortened D2 half-life and increased D2 turnover via proteasome, whereas GC-1 did not change D2 stability. The α1- and α2-adrenergic D2 responses increased using high T3 doses. In summary, T3 increases the adrenergic stimulation of UCP1 and D2 expression mostly via the TRß1 isoform, and in brown adipocytes, D2 is protected from degradation by the action of T3 on TRß1.

Generation and characterization of dickkopf3 mutant mice.

dickkopf (dkk) genes encode a small family of secreted Wnt antagonists, except for dkk3, which is divergent and whose function is poorly understood. Here, we describe the generation and characterization of dkk3 mutant mice. dkk3-deficient mice are viable and fertile. Phenotypic analysis shows no major alterations in organ morphology, physiology, and most clinical chemistry parameters. Since Dkk3 was proposed to function as thyroid hormone binding protein, we have analyzed deiodinase activities, as well as thyroid hormone levels. Mutant mice are euthyroid, and the data do not support a relationship of dkk3 with thyroid hormone metabolism. Altered phenotypes in dkk3 mutant mice were observed in the frequency of NK cells, immunoglobulin M, hemoglobin, and hematocrit levels, as well as lung ventilation. Furthermore, dkk3-deficient mice display hyperactivity.

Iodothyronine levels in the human developing brain: major regulatory roles of iodothyronine deiodinases in different areas.

Thyroid hormones are required for human brain development, but data on local regulation are limited. We describe the ontogenic changes in T(4), T(3), and rT(3) and in the activities of the types I, II, and III iodothyronine deiodinases (D1, D2, and D3) in different brain regions in normal fetuses (13-20 wk postmenstrual age) and premature infants (24-42 wk postmenstrual age). D1 activity was undetectable. The developmental changes in the concentrations of the iodothyronines and D2 and D3 activities showed spatial and temporal specificity but with divergence in the cerebral cortex and cerebellum. T(3) increased in the cortex between 13 and 20 wk to levels higher than adults, unexpected given the low circulating T(3). Considerable D2 activity was found in the cortex, which correlated positively with T(4) (r = 0.65). Cortex D3 activity was very low, as was D3 activity in germinal eminence and choroid plexus. In contrast, cerebellar T(3) was very low and increased only after midgestation. Cerebellum D3 activities were the highest (64 fmol/min.mg) of the regions studied, decreasing after midgestation. Other regions with high D3 activities (midbrain, basal ganglia, brain stem, spinal cord, hippocampus) also had low T(3) until D3 started decreasing after midgestation. D3 was correlated with T(3) (r = -0.682) and rT(3)/T(3) (r = 0.812) and rT(3)/T(4) (r = 0.889). Our data support the hypothesis that T(3) is required by the human cerebral cortex before midgestation, when mother is the only source of T(4). D2 and D3 play important roles in the local bioavailability of T(3). T(3) is produced from T(4) by D2, and D3 protects brain regions from excessive T(3) until differentiation is required.