The Importance of Periodical Screening for Primary Hyperparathyroidism in a Pituitary Tumor Cohort in Searching Patients With MEN1 and Its Genetic Profile

ObjectiveMultiple endocrine neoplasia type 1 (MEN1) is a rare genetic syndrome characterized by parathyroid, anterior pituitary, and/or duodenopancreatic neuroendocrine tumors. Studies have indicated that investigating primary hyperparathyroidism (pHPT) with subsequent genetic screening may be an essential tool for the early diagnosis of MEN1 in patients with pituitary tumors (PTs). This study aimed to investigate the presence of pHPT in patients with PTs and, subsequently, to screen for genetic mutations and related tumors in patients with MEN1 syndrome.MethodsThis study included 255 patients with PTs who were assessed for the presence of MEN1 by serum calcium and parathyroid hormone measurements. Mutation screening of the MEN1, CDKN1B, and AIP genes was performed in the index cases showing the MEN1 phenotype.ResultsFive patients with PTs presented a clinical condition compatible with MEN1. These patients had a younger age of onset and a more severe clinical condition. Genetic analysis identified a frameshift mutation in the MEN1 gene in one of the cases with the MEN1 phenotype, but point mutations in CDKN1B and AIP were not detected in any of these patients.ConclusionOur results show that periodic screening for pHPT in patients with PTs may be useful to detect MEN1 syndrome; thus, it is recommended in those patients with both findings a genetic analysis of MEN1 gene and an additional search of related tumors. By contrast, our data suggest that CDKN1B and AIP mutations do not seem to play a relevant role in the pathogenesis of MEN1.     

Expression analysis of irisin during different development stages of skeletal muscle in mice

BackgroundAs a newly discovered muscle factor secreted by skeletal muscle cells, irisin is a polypeptide fragment formed from hydrolysis of fibronectin type Ⅲ domain-containing protein 5 (FNDC5). Irisin can promote beigeing of white adipose tissue (WAT) and regulate glucose and lipid metabolisms. However, the functions of irisin in skeletal muscle development remain largely unknown. In order to characterize the expression of irisin, this study investigated the expression of irisin precursor FNDC5 in myoblasts and skeletal muscles during different developmental stages of SPF mice.ResultsThe Western blot, quantitative real-time PCR (qRT-PCR), and immunofluorescence assay results showed that FNDC5 was expressed in all the developmental stages of myoblasts and gastrocnemius, but its expression differed at different stages. FNDC5 protein exhibited the highest expression in gastrocnemius of sexually mature mice, followed by elderly mice and adolescent mice, and it displayed the lowest expression in pups. Additionally, FNDC5 protein was mainly expressed in cytoplasm, and it had the highest expression in primary myoblasts, followed by the myotubes with the lowest expression in C2C12 myogenic cells.ConclusionsOverall, FNDC5 was mainly expressed in cytoplasm and extracellular matrix with different expression levels at different developmental stages of skeletal muscle cells and tissues in mice. This study will provide new strategies for promoting skeletal muscle development and treating muscle- and metabolism-related disease by using irisin.     

Fucoidan exerts antidepressant-like effects in mice via regulating the stability of surface AMPARs

The inflammatory hypothesis is one of the most important mechanisms of depression. Fucoidan is a bioactive sulfated polysaccharide abundant in brown seaweeds with anti-inflammatory activity. However, the antidepressant effects of fucoidan on chronic stress-induced depressive-like behaviors have not been well elucidated. Here, we used two different depressive-like mouse models, lipopolysaccharide (LPS) and chronic restraint stress (CRS) models, to explore the detailed molecular mechanism underlying its antidepressant-like effects in C57BL/6J mice by combining multiple behavioral, molecular and immunofluorescence experiments. Adenovirus-mediated overexpression of caspase-1 and pharmacological inhibitors were also used to clarify the antidepressant mechanisms of fucoidan. We found that acute administration of fucoidan did not produce antidepressant effects in the tail suspension test (TST) and forced swim test (FST). Interestingly, chronic fucoidan administration not only dose-dependently reduced stress-induced depressive-like behaviors in the TST, FST, sucrose preference test (SPT), and novelty-suppressed feeding test (NSFT), but also alleviated the downregulation of brain-derived neurotrophic factor (BDNF)-dependent synaptic plasticity via inhibiting caspase-1-mediated inflammation in the hippocampus of mice. Moreover, fucoidan significantly ameliorated behavioral and synaptic plasticity abnormalities in the overexpression of caspase-1 in the hippocampus of mice. Furthermore, blocking BDNF abolished the antidepressant-like effects of fucoidan in mice. Therefore, our findings clearly indicate that fucoidan provides a potential supplementary noninvasive treatment for depression by inhibition of hippocampal inflammation.     

Thyroid Immune-Related Adverse Events in Patients with Cancer Treated with anti-PD1/anti-CTLA4 Immune Checkpoint Inhibitor Combination: Clinical Course and Outcomes

ObjectiveThyroid immune-related adverse events (irAEs) have been reported to have prognostic significance among patients with cancer treated with anti-programmed cell death-1 (PD1) and anti-programmed death-ligand 1 monotherapies. We evaluated the clinical course and predictors of thyroid irAEs in relation to outcomes of patients with advanced cancer treated with combination anti-PD1/anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA4).MethodsWe conducted a regional study and identified patients with advanced cancer who received ≥1 cycle of combination anti-PD1/anti-CTLA4 between 2015 and 2019 in Hong Kong. Thyroid function tests (TFTs) were monitored every 3 weeks. Thyroid irAE was defined by ≥2 abnormal TFTs after initiation of combination anti-PD1/anti-CTLA4 in the absence of other causes.ResultsOne hundred and three patients were included (median age: 59 years; 71.8% men). About 45% had prior anti-PD1 exposure. Upon median follow-up of 6.8 months, 17 patients (16.5%) developed thyroid irAEs, where 6 initially presented with thyrotoxicosis (overt, n = 4; subclinical, n = 2) and 11 with hypothyroidism (overt, n = 2; subclinical, n = 9). Eventually, 10 patients (58.8%) required continuous thyroxine replacement. Systemic steroid was not required in all cases. Prior anti-PD1 exposure (odds ratio, 3.67; 95% CI, 1.19–11.4; P = .024) independently predicted thyroid irAEs. Multivariable Cox regression analysis revealed that occurrence of thyroid irAEs was independently associated with better overall survival (adjusted hazard ratio, 0.34; 95% CI, 0.17–0.71; P = .004).ConclusionThyroid irAEs are common in routine clinical practice among patients with advanced cancer treated with anti-PD1/anti-CTLA4 combination and might have potential prognostic significance. Regular TFT monitoring is advised for timely treatment of thyroid irAEs to prevent potential morbidities.     

Regenerating islet-derived protein (Reg)3β plays a crucial role in attenuation of ileitis and colitis in mice

Regenerating islet-derived protein (Reg)3β belongs to a member of the Reg family of proteins and has pleiotropic functions, including antimicrobial activity and tissue repair. However, whether Reg3β plays a protective role in the development of colitis and ileitis has not been fully investigated. We generated transgenic mice expressing a short form of cellular FLICE-inhibitory protein (cFLIPs) that promotes necroptosis, a regulated form of cell death. cFLIPs transgenic (CFLARs Tg) mice develop severe ileitis in utero. Although Reg3β is undetectable in the small intestine of wild-type embryos, its expression is aberrantly elevated in the small intestine of CFLARs Tg embryos. To test whether elevated Reg3β attenuates or exacerbates ileitis in CFLARs Tg mice, we generated a Reg3b^?/? strain. Reg3b^?/? mice grew to adulthood without apparent abnormalities. Deletion of Reg3b in CFLARs Tg mice exacerbated the embryonic lethality of CFLARs Tg mice. Dextran sulfate sodium-induced colitis, characterized by body weight loss and infiltration of neutrophils, was exacerbated in Reg3b^?/? compared to wild-type mice. Moreover, the expression of Interleukin 6, an inflammatory cytokine and Chitinase-like 3, a marker for tissue repair macrophages was elevated in the colon of Reg3b^?/? mice compared to wild-type mice after DSS treatment. Together, these results suggest that attenuation of colitis and ileitis is a result of Reg3β′s real function.     

Quantitative interactome analysis with chemical cross-linking and mass spectrometry

Structural plasticity and dynamic protein–protein interactions are critical determinants of protein function within living systems. Quantitative chemical cross-linking with mass spectrometry (qXL-MS) is an emerging technology able to provide information on changes in protein conformations and interactions. Importantly, qXL-MS is applicable to complex biological systems, including living cells and tissues, thereby providing insights into proteins within their native environments. Here, we present an overview of recent technological developments and applications involving qXL-MS, including design and synthesis of isotope-labeled cross-linkers, development of new liquid chromatography–MS methodologies, and computational developments enabling interpretation of the data.     

Elucidation of interleukin-19 as a therapeutic target for breast cancer by computational analysis and experimental validation

Interleukin 19 (IL-19) is a cytokine produced by monocytes and belongs to the family of IL-10. The IL-19 protein stimulates fibronectin (FN) expression and assembly, metastasis, and cell division in breast cancer (BC) cells. IL-19, which is connected to breast pathogenesis and has an autocrine action in BC cells, is a key predictor of prognosis for many tumour forms, including breast cancer. Augmented IL-19 expression has been related to poorer clinical outcomes for patients with BC and directly enhances proliferation and migration while also serving as a microenvironment for tumour formation. The main aim of our study was to examine the expression profile, functional role, and prognostic significance of interleukin-19 in BC pathogenesis and also to find out the molecular mechanism of IL-19 in BC. In this work, we used the various computational approach and tools, to evaluate the expression profile and prognostic implication of IL-19 in BC and discover the role of IL-19 in BC pathogenesis. IL-19 was shown to be highly upregulated in BC as compared to other interleukins. Also, its levels were highly overexpressed in liminal BC patients, mostly in 3rd stage groups under the age group of 21–40 years. IL-19 levels were increased in BC and elevated expression of IL-19 was examined to have worse overall survival (OS). The KEGG analysis and gene ontology of IL-19 depict that IL-19 is significantly augmented in cytokine activity and receptor-ligand activity and also in the JAK-STAT signaling pathway. Moreover, IL-19 showed a high correlation with IL20RA, as later is involved with the JAK-STAT signaling pathway. The in-vivo and in-vitro studies have also reflected that upregulation of IL-19 enhances tumor development and affects clinical outcomes in BC patients through several pathways including the JAK TAT signalling pathway. Overall, our study indicates that IL-19 increases tumour growth and that inhibiting it in addition to standard treatments will greatly improve BC patient’s therapeutic responses.     

10,12-Conjugated linoleic acid supplementation improves HDL composition and function in mice

Obesity is associated with inflammation, insulin resistance, and type 2 diabetes, which are major risk factors for CVD. One dietary component of ruminant animal foods, 10,12-conjugated linoleic acid (10,12 CLA), has been shown to promote weight loss in humans. Previous work has shown that 10,12 CLA is atheroprotective in mice by a mechanism that may be distinct from its weight loss effects, but this exact mechanism is unclear. To investigate this, we evaluated HDL composition and function in obese LDL receptor (Ldlr^?/?) mice that were losing weight because of 10,12 CLA supplementation or caloric restriction (CR; weight-matched control group) and in an obese control group consuming a high-fat high-sucrose diet. We show that 10,12 CLA-HDL exerted a stronger anti-inflammatory effect than CR- or high-fat high-sucrose-HDL in cultured adipocytes. Furthermore, the 10,12 CLA-HDL particle (HDL-P) concentration was higher, attributed to more medium- and large-sized HDL-Ps. Passive cholesterol efflux capacity of 10,12 CLA-HDL was elevated, as was expression of HDL receptor scavenger receptor class B type 1 in the aortic arch. Murine macrophages treated with 10,12 CLA in vitro exhibited increased expression of cholesterol transporters Abca1 and Abcg1, suggesting increased cholesterol efflux potential of these cells. Finally, proteomics analysis revealed elevated Apoa1 content in 10,12 CLA-HDL-Ps, consistent with a higher particle concentration, and particles were also enriched with alpha-1-antitrypsin, an emerging anti-inflammatory and antiatherosclerotic HDL-associated protein. We conclude that 10,12 CLA may therefore exert its atheroprotective effects by increasing HDL-P concentration, HDL anti-inflammatory potential, and promoting beneficial effects on cholesterol efflux.     

A Pilot Study of Youth With Type 1 Diabetes Initiating Use of a Hybrid Closed-Loop System While Receiving a Behavioral Economics Intervention

ObjectiveMany youth do not use the hybrid closed-loop system for type 1 diabetes effectively. This study evaluated the impact of financial incentives for diabetes-related tasks on use of the 670G hybrid closed-loop system and on glycemia.MethodsAt auto mode initiation and for 16 weeks thereafter, participants received a flat rate for wearing and calibrating the sensor ($1/day), administering at least 3 mealtime insulin boluses per day ($1/day), and uploading ($5/week). Weekly bonuses were given for maintaining at least 70% of the time in auto mode, which were increased for persistent auto mode use from $3/week to a maximum of $13/week. If a participant failed to maintain auto mode for a week, the rewards were reset to baseline. Data from 17 participants aged 15.9 years ± 2.5 years (baseline hemoglobin A1c [HbA1c] 8.6% ± 1.1%) were collected at 6, 12, and 16 weeks. The reinforcers were withdrawn at 16 weeks, with a follow-up assessment at 24 weeks.ResultsWith reinforcers, the participants administered an average of at least 3 mealtime insulin boluses per day and wore the sensor over 70% of the time. However, auto mode use waned. HbA1c levels decreased by 0.5% after 6 weeks, and this improvement was maintained at 12 and 16 weeks (P < .05). Upon withdrawal of reinforcers, HbA1c levels increased back to baseline at 24 weeks.ConclusionCompensation for diabetes-related tasks was associated with lower HbA1c levels, consistent administration of mealtime insulin boluses, and sustained sensor use. These results support the potential of financial rewards for improving outcomes in youth with type 1 diabetes.     

Sulfatide with ceramide composed of phytosphingosine (t18:0) and 2-hydroxy FAs in renal intercalated cells

Diverse molecular species of sulfatide with differences in FA lengths, unsaturation degrees, and hydroxylation statuses are expressed in the kidneys. However, the physiological functions of specific sulfatide species in the kidneys are unclear. Here, we evaluated the distribution of specific sulfatide species in the kidneys and their physiological functions. Electron microscopic analysis of kidneys of Cst-deficient mice lacking sulfatide showed vacuolar accumulation in the cytoplasm of intercalated cells in the collecting duct, whereas the proximal and distal tubules were unchanged. Immunohistochemical analysis revealed that vacuolar H⁺-ATPase-positive vesicles were accumulated in intercalated cells in sulfatide-deficient kidneys. Seventeen sulfatide species were detected in the murine kidney by iMScope MALDI-MS analysis. The distribution of the specific sulfatide species was classified into four patterns. Although most sulfatide species were highly expressed in the outer medullary layer, two unique sulfatide species of m/z 896.6 (predicted ceramide structure: t18:0-C22:0h) and m/z 924.6 (predicted ceramide structure: t18:0-C24:0h) were dispersed along the collecting duct, implying expression in intercalated cells. In addition, the intercalated cell-enriched fraction was purified by fluorescence-activated cell sorting using the anti-vacuolar H⁺-ATPase subunit 6V0A4, which predominantly contained sulfatide species (m/z 896.6 and 924.6). The Degs2 and Fa2h genes, which are responsible for ceramide hydroxylation, were expressed in the purified intercalated cells. These results suggested that sulfatide molecular species with ceramide composed of phytosphingosine (t18:0) and 2-hydroxy FAs, which were characteristically expressed in intercalated cells, were involved in the excretion of NH₃ and protons into the urine.     

Hormone-sensitive lipase is localized at synapses and is necessary for normal memory functioning in mice

Hormone-sensitive lipase (HSL) is mainly present in adipose tissue where it hydrolyzes diacylglycerol. Although expression of HSL has also been reported in the brain, its presence in different cellular compartments is uncertain, and its role in regulating brain lipid metabolism remains hitherto unexplored. We hypothesized that HSL might play a role in regulating the availability of bioactive lipids necessary for neuronal function and therefore investigated whether dampening HSL activity could lead to brain dysfunction. In mice, we found HSL protein and enzymatic activity throughout the brain, localized within neurons and enriched in synapses. HSL-null mice were then analyzed using a battery of behavioral tests. Relative to wild-type littermates, HSL-null mice showed impaired short-term and long-term memory, yet preserved exploratory behaviors. Molecular analysis of the cortex and hippocampus showed increased expression of genes involved in glucose utilization in the hippocampus, but not cortex, of HSL-null mice compared with controls. Furthermore, lipidomics analyses indicated an impact of HSL deletion on the profile of bioactive lipids, including a decrease in endocannabinoids and eicosanoids that are known to modulate neuronal activity, cerebral blood flow, and inflammation processes. Accordingly, mild increases in the expression of proinflammatory cytokines in HSL mice compared with littermates were suggestive of low-grade inflammation. We conclude that HSL has a homeostatic role in maintaining pools of lipids required for normal brain function. It remains to be tested, however, whether the recruitment of HSL for the synthesis of these lipids occurs during increased neuronal activity or whether HSL participates in neuroinflammatory responses.     

PD-L1 Expression in Normal Endocrine Tissues Is Not Increased Despite High Incidence of PD-1 Inhibitor-Associated Endocrinopathies

ObjectiveTreatment with immune-checkpoint inhibitors often results in endocrine immune-related adverse events (irAEs), affecting the pituitary, thyroid, adrenal, and parathyroid glands and pancreas. The mechanism underlying the endocrine irAEs has not been fully elucidated, and it remains unclear why endocrine organs are so commonly affected. In the present study, we evaluated immunostaining patterns of programmed death-ligand 1 (PD-L1) in normal endocrine tissues to determine whether increased expression may explain the predilection of endocrinopathies in patients treated with programmed cell death-1 inhibitors.MethodsNormal formalin-fixed paraffin-embedded endocrine tissues (pituitary, thyroid, adrenal, pancreas, and parathyroid) were collected from our hospital’s pathology tissue archive. The tissues were assessed for membranous and cytoplasmic PD-L1 immunostaining using the Dako 22C3 pharmDx assay on an automated staining platform.ResultsWe examined 49 endocrine tissues, including 12 thyroid, 5 pancreatic, 17 adrenal, 5 parathyroid, and 10 pituitary samples. Samples with less than 1% membranous PD-L1–positive cells were considered negative, while those with more than 1% of PD-L1 membranous staining were considered positive. Immunostaining result of immune-related cells was also evaluated, considering the cytoplasmic PD-L1–positive cells with the same cutoff of 1%. None of the endocrine tissues demonstrated PD-L1 positivity higher than 1% in the relevant cells.ConclusionWhile our results do not suggest a role of PD-L1 expression in the pathogenesis of endocrine irAEs, they may serve as a basis for future studies further investigating the mechanisms of autoimmune, inflammatory, or malignant endocrine conditions.     

Serum opacity factor rescues fertility among female Scarb1−/− mice by reducing HDL-free cholesterol bioavailability

Human female infertility, 20% of which is idiopathic, is a public health problem for which better diagnostics and therapeutics are needed. A novel cause of infertility emerged from studies of female mice deficient in the HDL receptor gene (Scarb1). These mice are infertile and have high plasma HDL cholesterol (C) concentrations, due to elevated HDL-free cholesterol (FC), which transfers from HDL to all tissues. Previous studies have indicated that oral delivery of probucol, an HDL-lowering drug, to female Scarb1^?/? mice reduces plasma HDL-C concentrations and rescues fertility. Additionally, serum opacity factor (SOF), a bacterial virulence factor, disrupts HDL structure, and bolus SOF injection into mice reduces plasma HDL-C concentrations. Here, we discovered that delivering SOF to female Scarb1^?/? mice with an adeno-associated virus (AAV_SOF) induces constitutive SOF expression, reduces HDL-FC concentrations, and rescues fertility while normalizing ovary morphology. Although AAV_SOF did not alter ovary-FC content, the ovary-mol% FC correlated with plasma HDL-mol% FC in a fertility-dependent way. Therefore, reversing the abnormal plasma microenvironment of high plasma HDL-mol% FC in female Scarb1^-/- mice rescues fertility. These data provide the rationale to search for similar mechanistic links between HDL-mol% FC and infertility and the rescue of fertility in women by reducing plasma HDL-mol% FC.     

Differential expression patterns of phospholipase D isoforms 1 and 2 in the mammalian brain and retina

Phosphatidic acid is a key signaling molecule heavily implicated in exocytosis due to its protein-binding partners and propensity to induce negative membrane curvature. One phosphatidic acid-producing enzyme, phospholipase D (PLD), has also been implicated in neurotransmission. Unfortunately, due to the unreliability of reagents, there has been confusion in the literature regarding the expression of PLD isoforms in the mammalian brain which has hampered our understanding of their functional roles in neurons. To address this, we generated epitope-tagged PLD1 and PLD2 knockin mice using CRISPR/Cas9. Using these mice, we show that PLD1 and PLD2 are both localized at synapses by adulthood, with PLD2 expression being considerably higher in glial cells and PLD1 expression predominating in neurons. Interestingly, we observed that only PLD1 is expressed in the mouse retina, where it is found in the synaptic plexiform layers. These data provide critical information regarding the localization and potential role of PLDs in the central nervous system.     

Comparison of Clinical Tests for Peripheral Diabetic Neuropathy in a Type 1 Diabetes Cohort

ObjectiveTo examine the performance and agreement of 5 modalities for testing sensory neuropathy against a neurothesiometer among Hispanic patients with type 1 diabetes (T1D) in an outpatient setting.MethodsA cross-sectional study was conducted at a tertiary reference center in Mexico City. Sensitivity, specificity, predictive values, and likelihood ratios were calculated using a VibraTip device, 128 Hz tuning fork, and the Semmes-Weinstein 5.07/10 g monofilament test, Ipswich touch test (IpTT), and pinprick test (PPT). The VPT obtained using a neurothesiometer was used as the standard. Agreement between tests was calculated using kappa coefficients.ResultsOur study included 78 patients (156 examinations), of whom 56.4% were females. The mean age was 38.2 ± 13.0 years, and the mean body mass index was 24.6 ± 4.8 kg/m². The best sensitivity was found for IpTT and VibraTip (89.7% and 79.3%, respectively), while the PPT and IpTT had the highest positive predictive values (94.4% and 92.9%, respectively). The highest kappa coefficients were obtained for the IpTT vs neurothesiometer (kappa coefficient [κ] = 0.893, P < .001), followed by VibraTip vs neurothesiometer (κ = 0.782, P < .001). The VibraTip vs IpTT also had a substantial agreement (κ= 0.713, P < .001).ConclusionOur findings demonstrated that the IpTT had the best diagnostic performance and agreement compared with the standard in this cohort of Hispanic patients with T1D. The IpTT is a useful, simple test for diabetic neuropathy screening. These findings support its inclusion in future guidelines for diabetic foot examination.     

Semaphorin 3A potentiates the profibrotic effects of transforming growth factor-β1 in the cornea

Corneal scarring is a major cause of blindness worldwide with few effective therapeutic options. Finding a treatment would be of tremendous public health benefit, but requires a thorough understanding of the complex interactions that underlie this phenomenon. Here, we tested the hypothesis that the large increase in expression of Semaphorin 3A (SEMA3A) in corneal wounds contributes to the development of stromal fibrosis. We first verified this increased expression in vivo, in a cat model of photorefractive keratectomy-induced corneal wounding. We then examined the impact of adding exogenous SEMA3A to cultured corneal fibroblasts, and assessed how this affected the ability of transforming growth factor-beta1 (TGF-β1) to induce their differentiation into myofibroblasts. Finally, we examined how siRNA knockdown of endogenous SEMA3A affected these same phenomena. We found exogenous SEMA3A to significantly potentiate TGF-β1’s profibrotic effects, with only a minimal contribution from cell-intrinsic SEMA3A. Our results suggest a previously unrecognized interaction between SEMA3A and TGF-β1 in the wounded cornea, and a possible contribution of SEMA3A to the regulation of tissue fibrosis and remodeling in this transparent organ.