The Cytokine IL-17A Limits Th17 Pathogenicity via a Negative Feedback Loop Driven by Autocrine Induction of IL-24.

Dysregulated Th17 cell responses underlie multiple inflammatory and autoimmune diseases, including autoimmune uveitis and its animal model, EAU. However, clinical trials targeting IL-17A in uveitis were not successful. Here, we report that Th17 cells were regulated by their own signature cytokine, IL-17A. Loss of IL-17A in autopathogenic Th17 cells did not reduce their pathogenicity and instead elevated their expression of the Th17 cytokines GM-CSF and IL-17F. Mechanistic in vitro studies revealed a Th17 cell-intrinsic autocrine loop triggered by binding of IL-17A to its receptor, leading to activation of the transcription factor NF-κB and induction of IL-24, which repressed the Th17 cytokine program. In vivo, IL-24 treatment ameliorated Th17-induced EAU, whereas silencing of IL-24 in Th17 cells enhanced disease. This regulatory pathway also operated in human Th17 cells. Thus, IL-17A limits pathogenicity of Th17 cells by inducing IL-24. These findings may explain the disappointing therapeutic effect of targeting IL-17A in uveitis.

Noncanonical autophagy promotes the visual cycle.

Phagocytosis and degradation of photoreceptor outer segments (POS) by retinal pigment epithelium (RPE) is fundamental to vision. Autophagy is also responsible for bulk degradation of cellular components, but its role in POS degradation is not well understood. We report that the morning burst of RPE phagocytosis coincided with the enzymatic conversion of autophagy protein LC3 to its lipidated form. LC3 associated with single-membrane phagosomes containing engulfed POS in an Atg5-dependent manner that required Beclin1, but not the autophagy preinitiation complex. The importance of this process was verified in mice with Atg5-deficient RPE cells that showed evidence of disrupted lysosomal processing. These mice also exhibited decreased photoreceptor responses to light stimuli and decreased chromophore levels that were restored with exogenous retinoid supplementation. These results establish that the interplay of phagocytosis and autophagy within the RPE is required for both POS degradation and the maintenance of retinoid levels to support vision.

α-Ketoglutarate links p53 to cell fate during tumour suppression.

The tumour suppressor TP53 is mutated in the majority of human cancers, and in over 70% of pancreatic ductal adenocarcinoma (PDAC)1,2. Wild-type p53 accumulates in response to cellular stress, and regulates gene expression to alter cell fate and prevent tumour development2. Wild-type p53 is also known to modulate cellular metabolic pathways3, although p53-dependent metabolic alterations that constrain cancer progression remain poorly understood. Here we find that p53 remodels cancer-cell metabolism to enforce changes in chromatin and gene expression that favour a premalignant cell fate. Restoring p53 function in cancer cells derived from KRAS-mutant mouse models of PDAC leads to the accumulation of α-ketoglutarate (αKG, also known as 2-oxoglutarate), a metabolite that also serves as an obligate substrate for a subset of chromatin-modifying enzymes. p53 induces transcriptional programs that are characteristic of premalignant differentiation, and this effect can be partially recapitulated by the addition of cell-permeable αKG. Increased levels of the αKG-dependent chromatin modification 5-hydroxymethylcytosine (5hmC) accompany the tumour-cell differentiation that is triggered by p53, whereas decreased 5hmC characterizes the transition from premalignant to de-differentiated malignant lesions that is associated with mutations in Trp53. Enforcing the accumulation of αKG in p53-deficient PDAC cells through the inhibition of oxoglutarate dehydrogenase-an enzyme of the tricarboxylic acid cycle-specifically results in increased 5hmC, tumour-cell differentiation and decreased tumour-cell fitness. Conversely, increasing the intracellular levels of succinate (a competitive inhibitor of αKG-dependent dioxygenases) blunts p53-driven tumour suppression. These data suggest that αKG is an effector of p53-mediated tumour suppression, and that the accumulation of αKG in p53-deficient tumours can drive tumour-cell differentiation and antagonize malignant progression.

A preclinical platform for assessing antitumor effects and systemic toxicities of cancer drug targets.

Anticancer drug development campaigns often fail due to an incomplete understanding of the therapeutic index differentiating the efficacy of the agent against the cancer and its on-target toxicities to the host. To address this issue, we established a versatile preclinical platform in which genetically defined cancers are produced using somatic tissue engineering in transgenic mice harboring a doxycycline-inducible short hairpin RNA against the target of interest. In this system, target inhibition is achieved by the addition of doxycycline, enabling simultaneous assessment of efficacy and toxicity in the same animal. As proof of concept, we focused on CDK9­a cancer target whose clinical development has been hampered by compounds with poorly understood target specificity and unacceptable toxicities. We systematically compared phenotypes produced by genetic Cdk9 inhibition to those achieved using a recently developed highly specific small molecule CDK9 inhibitor and found that both perturbations led to robust antitumor responses. Remarkably, nontoxic levels of CDK9 inhibition could achieve significant treatment efficacy, and dose-dependent toxicities produced by prolonged CDK9 suppression were largely reversible upon Cdk9 restoration or drug withdrawal. Overall, these results establish a versatile in vivo target validation platform that can be employed for rapid triaging of therapeutic targets and lend support to efforts aimed at advancing CDK9 inhibitors for cancer therapy.

Histopathology and SARS-CoV-2 Cellular Localization in Eye Tissues of COVID-19 Autopsies.

Ophthalmic manifestations and tissue tropism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been reported in association with coronavirus disease 2019 (COVID-19), but the pathology and cellular localization of SARS-CoV-2 are not well characterized. The objective of this study was to evaluate macroscopic and microscopic changes and investigate cellular localization of SARS-CoV-2 across ocular tissues at autopsy. Ocular tissues were obtained from 25 patients with COVID-19 at autopsy. SARS-CoV-2 nucleocapsid gene RNA was previously quantified by droplet digital PCR from one eye. Herein, contralateral eyes from 21 patients were fixed in formalin and subject to histopathologic examination. Sections of the droplet digital PCR-positive eyes from four other patients were evaluated by in situ hybridization to determine the cellular localization of SARS-CoV-2 spike gene RNA. Histopathologic abnormalities, including cytoid bodies, vascular changes, and retinal edema, with minimal or no inflammation in ocular tissues were observed in all 21 cases evaluated. In situ hybridization localized SARS-CoV-2 RNA to neuronal cells of the retinal inner and outer layers, ganglion cells, corneal epithelia, scleral fibroblasts, and oligodendrocytes of the optic nerve. In conclusion, a range of common histopathologic alterations were identified within ocular tissue, and SARS-CoV-2 RNA was localized to multiple cell types. Further studies will be required to determine whether the alterations observed were caused by SARS-CoV-2 infection, the host immune response, and/or preexisting comorbidities.

Limbic Connectivity Underlies Pain Treatment Response in Small-Fiber Neuropathy.

OBJECTIVE: Small-fiber neuropathy (SFN) is characterized by neuropathic pain due to degeneration of small-diameter nerves in the skin. Given that brain reorganization occurs following chronic neuropathic pain, this study investigated the structural and functional basis of pain-related brain changes after skin nerve degeneration. METHODS: Diffusion-weighted and resting-state functional MRI data were acquired from 53 pathologically confirmed SFN patients, and the structural and functional connectivity of the pain-related network was assessed using network-based statistic (NBS) analysis. RESULTS: Compared with age- and sex-matched controls, the SFN patients exhibited a robust and global reduction of functional connectivity, mainly across the limbic and somatosensory systems. Furthermore, lower functional connectivity was associated with skin nerve degeneration measured by reduced intraepidermal nerve fiber density and better therapeutic response to anti-neuralgia medications, particularly for the connectivity between the insula and the limbic areas including the anterior and middle cingulate cortices. Similar to the patterns of functional connectivity changes, the structural connectivity was robustly reduced among the limbic and somatosensory areas, and the cognition-integration areas including the inferior parietal lobule. There was shared reduction of structural and functional connectivity among the limbic, somatosensory, striatal, and cognition-integration systems: (1) between the middle cingulate cortex and inferior parietal lobule and (2) between the thalamus and putamen. These observations indicate the structural basis underlying altered functional connectivity in SFN. INTERPRETATION: Our findings provide imaging evidence linking structural and functional brain dysconnectivity to sensory deafferentation caused by peripheral nerve degeneration and therapeutic responses for neuropathic pain in SFN. ANN NEUROL 2023;93:655-667.

Effects of Toll-like receptor 1 and 2 agonist Pam3CSK4 on uveal melanocytes and relevant experimental mouse model.

Pam3CSK4 activates Toll-like receptors 2 and 1 (TLR1/2), which recognize mainly molecules from gram-positive pathogens. The effect of Pam3CSK4 on various cytokine and chemokine expression in cultured human uveal melanocytes (UM) has not been studied systematically. The purpose of this study was to investigate the mechanistic expressions of seven cytokines and chemokines of interleukin- (IL-) 6, IL-10, MCP-1 (CCL-2), CXCL-1 (GRO-α), CXCL-8 (IL-8), interferon-gamma (IFN-γ), and tumor necrosis factor-alpha (TNF-α) in UM. These cytokines are reported to be increased in intraocular fluids or tissues of the patients with endophthalmitis and non-infectious uveitis, as well as in various experimental animal uveitic models in the literature. Flow cytometry was used to measure the effects of Pam3CSK4 on the expression of TLR1/2 in UM. ELISA and Real-time PCR analysis were used to estimate the ability of Pam3CSK4 to elevate these cytokines and chemokines levels in conditioned media and cell lysates of UM, respectively. Flow cytometry measured and compared the phosphorylated MAPK pathway and activated NF-κB signals pathway in UM, treated with and without Pam3CSK4. ELISA analysis tested the effect of various signal inhibitors (ERK1/2, JNK1/2, p38 and NF-κB) on Pam3CSK4-induced IL-6 levels in cultured UM. The role of TLR2 in Pam3CSK4-induced acute anterior uveitis in experimental mouse model was tested in TLR2 knockout (TLR2 KO) mice and their wild-type C57Bl/6 controls. Pam3CSK4 increased the expression of TLR1/2 proteins in cultured UM. Pam3CSK4 significantly elevated the IL-6, MCP-1, CXCL-1, CXCL-8 protein, and mRNA levels in cultured UM, but not IL-10, TNF-α, or IFN-γ. Pam3CSK4 activated NF-κB, ERK, JNK, and p38 expression. Pam3CSK4-induced expression of IL-6 was decreased by NF-κB, ERK, INK, and p38 inhibitors; especially the NF-κB inhibitor, which can completely block the IL-6 stimulation. Intravitreal injection of Pam3CSK4 induced acute anterior uveitis in C57Bl/6 mice, this effect was significantly reduced in TLR2 KO mice. TLR1/2 plays an important role against invading pathogens, especially gram-positive bacteria; but an excessive reaction to molecules from gram-positive bacteria may promote non-infectious uveitis. UM can produce IL-6, MCP-1, CXCL-1, and CXCL-8, and are one of the target cells of TNF-α and IFN-γ. TLR-2 inhibitors might have a beneficial effect in the treatment of certain types of uveitis and other ocular inflammatory-related diseases and warrant further investigation.

Microbiota-Dependent Activation of an Autoreactive T Cell Receptor Provokes Autoimmunity in an Immunologically Privileged Site.

Activated retina-specific T cells that have acquired the ability to break through the blood-retinal barrier are thought to be causally involved in autoimmune uveitis, a major cause of human blindness. It is unclear where these autoreactive T cells first become activated, given that their cognate antigens are sequestered within the immune-privileged eye. We demonstrate in a novel mouse model of spontaneous uveitis that activation of retina-specific T cells is dependent on gut commensal microbiota. Retina-specific T cell activation involved signaling through the autoreactive T cell receptor (TCR) in response to non-cognate antigen in the intestine and was independent of the endogenous retinal autoantigen. Our findings not only have implications for the etiology of human uveitis, but also raise the possibility that activation of autoreactive TCRs by commensal microbes might be a more common trigger of autoimmune diseases than is currently appreciated.

Electrodiagnostic subtyping in Guillain-Barré syndrome patients in the International Guillain-Barré Outcome Study.

BACKGROUND AND PURPOSE: Various electrodiagnostic criteria have been developed in Guillain-Barré syndrome (GBS). Their performance in a broad representation of GBS patients has not been evaluated. Motor conduction data from the International GBS Outcome Study (IGOS) cohort were used to compare two widely used criterion sets and relate these to diagnostic amyotrophic lateral sclerosis criteria. METHODS: From the first 1500 patients in IGOS, nerve conduction studies from 1137 (75.8%) were available for the current study. These patients were classified according to nerve conduction studies criteria proposed by Hadden and Rajabally. RESULTS: Of the 1137 studies, 68.3% (N = 777) were classified identically according to criteria by Hadden and Rajabally: 111 (9.8%) axonal, 366 (32.2%) demyelinating, 195 (17.2%) equivocal, 35 (3.1%) inexcitable and 70 (6.2%) normal. Thus, 360 studies (31.7%) were classified differently. The areas of differences were as follows: 155 studies (13.6%) classified as demyelinating by Hadden and axonal by Rajabally; 122 studies (10.7%) classified as demyelinating by Hadden and equivocal by Rajabally; and 75 studies (6.6%) classified as equivocal by Hadden and axonal by Rajabally. Due to more strictly defined cutoffs fewer patients fulfilled demyelinating criteria by Rajabally than by Hadden, making more patients eligible for axonal or equivocal classification by Rajabally. In 234 (68.6%) axonal studies by Rajabally the revised El Escorial (amyotrophic lateral sclerosis) criteria were fulfilled; in axonal cases by Hadden this was 1.8%. CONCLUSIONS AND DISCUSSION: This study shows that electrodiagnosis in GBS is dependent on the criterion set utilized, both of which are based on expert opinion. Reappraisal of electrodiagnostic subtyping in GBS is warranted.

Patisiran, an RNAi therapeutic for hereditary transthyretin-mediated amyloidosis: Sub-analysis in Taiwanese patients from the APOLLO study.

BACKGROUND AND OBJECTIVES: To examine the efficacy and safety of patisiran, an RNA interference therapeutic, in patients from Taiwan with hereditary transthyretin-mediated (hATTR) amyloidosis with polyneuropathy. METHODS: The APOLLO phase 3 trial included patients from Taiwan who received patisiran 0.3 mg/kg intravenously or placebo once every 3 weeks (q3w) for 18 months (18 M), followed by patisiran 0.3 mg/kg q3w in an ongoing global open-label extension (OLE) study. The primary endpoint was change from baseline in modified Neuropathy Impairment Score +7 (mNIS+7) at 18 M. RESULTS: Eighteen Taiwanese patients were enrolled in APOLLO (patisiran, n = 8; placebo, n = 10; all A97S gene variant) and 14 continued in the global OLE. In this Taiwanese sub-population, beneficial treatment effects at 18 M were observed in mNIS+7 (least squares mean difference in change from baseline [patisiran-placebo], -26.5 points; 95% confidence interval: -45.5, -7.5). Patients who switched from placebo to patisiran demonstrated slowing of polyneuropathy progression at month 12 in the global OLE, while those who received patisiran in APOLLO maintained the beneficial treatment effects. Patisiran had an acceptable safety profile in the Taiwanese sub-population. CONCLUSIONS: This analysis suggests that patisiran is well tolerated and may provide a substantial clinical benefit for Taiwanese patients with hATTR amyloidosis with polyneuropathy. TRIAL REGISTRATION INFORMATION: The studies were registered on the ClinicalTrials.gov. The APOLLO study ClinicalTrials.gov identifier is NCT01960348 (https://clinicaltrials.gov/ct2/show/NCT01960348), with the registration date of October 10, 2013, and the first patient was enrolled on December 13, 2013. For the global OLE, the ClinicalTrials.gov identifier is NCT02510261 (https://clinicaltrials.gov/ct2/show/NCT02510261) with the registration date of July 29, 2015, and the first patient was enrolled on July 13, 2015. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that treatment with patisiran is safe and efficacious in Taiwanese patients with hereditary transthyretin-mediated amyloidosis with polyneuropathy.

The effects of double-filtration plasmapheresis on coagulation profiles and the risk of bleeding.

BACKGROUND/PURPOSE: Double-filtration plasmapheresis (DFPP) can be used to remove circulating pathogenic molecules. By reclaiming filtered albumin, DFPP reduces the need for albumin and plasma replacement. Large proteins, such as fibrinogen, are removed. Our institution adopts a DFPP treatment protocol consisting of active surveillance of coagulation profiles and prophylactic supplementation of blood products containing fibrinogen. This study aims to investigate the effects of consecutive DFPP treatments on serial coagulation profiles and the risk of bleeding under this protocol. METHODS: Serial laboratory data and bleeding events at a single tertiary medical center were prospectively collected. Prophylactic transfusion of cryoprecipitate or fresh frozen plasma (FFP) was instituted if significant coagulopathy or a clinically evident bleeding event was observed. RESULTS: After the first treatment session, plasma fibrinogen levels decreased from 332 ± 106 mg/dL to 96 ± 44 mg/dL in the 37 study patients. In the following sessions, plasma fibrinogen levels were maintained at around 100 mg/dL under prophylactic transfusion. No major bleeding events were recorded, but five (14%) patients experienced minor bleeding. CONCLUSION: DFPP treatment might be performed safely along with active monitoring of coagulation profiles and prophylactic transfusion of cryoprecipitate or FFP.

Nonpermissive Skin Environment Impairs Nerve Regeneration in Diabetes via Sec31a.

OBJECTIVE: Although the microenvironment for peripheral nerve regeneration is permissive, such a mechanism is defective in diabetes, and the molecular mediators remain elusive. [Correction added on May 11, 2022, after first online publication: In the preceding sentence, "is ok" was changed to "is defective".] This study aimed to (1) investigate the relationship between skin innervation and collagen pathology in diabetic neuropathy and to (2) clarify the molecular alterations that occur in response to hyperglycemia and their effects on axon regeneration. METHODS: We addressed this issue using two complementary systems: (1) human skin from patients with diabetic neuropathy and to (2) a coculture model of human dermal fibroblasts (HDFs) with rat dorsal root ganglia neurons in the context of intrinsic neuronal factor and extrinsic microenvironmental collagen and its biosynthetic pathways. RESULTS: In diabetic neuropathy, the skin innervation of intraepidermal nerve fiber density (IENFd), a measure of sensory nerve degeneration, was reduced with similar expression of a growth associated protein 43, a marker of nerve regeneration. In contrast, the content and packing of collagen in the diabetic skin became more rigid than the control skin. Sec31a, a protein that regulates the collagen biosynthetic pathway, was upregulated and inversely correlated with IENFd. In the cell model, activated HDFs exposed to high-glucose medium enhanced the expression of Sec31a and collagen I through the activation of transforming growth factor ß, a profibrotic molecule. Sec31a upregulation impaired neurite outgrowth. This effect was reversed by silencing Sec31a expression and neurite outgrowth was resumed. INTERPRETATION: The current study provides evidence that Sec31a plays a key role in inhibiting nerve regeneration in diabetic neuropathy. ANN NEUROL 2022;91:821-833.

Eplontersen for Hereditary Transthyretin Amyloidosis With Polyneuropathy.

Importance: Transthyretin gene silencing is an emerging treatment strategy for hereditary transthyretin (ATTRv) amyloidosis. Objective: To evaluate eplontersen, an investigational ligand-conjugated antisense oligonucleotide, in ATTRv polyneuropathy. Design, Setting, and Participants: NEURO-TTRansform was an open-label, single-group, phase 3 trial conducted at 40 sites across 15 countries (December 2019-April 2023) in 168 adults with Coutinho stage 1 or 2 ATTRv polyneuropathy, Neuropathy Impairment Score 10-130, and a documented TTR variant. Patients treated with placebo from NEURO-TTR (NCT01737398; March 2013-November 2017), an inotersen trial with similar eligibility criteria and end points, served as a historical placebo ("placebo") group. Interventions: Subcutaneous eplontersen (45 mg every 4 weeks; n = 144); a small reference group received subcutaneous inotersen (300 mg weekly; n = 24); subcutaneous placebo weekly (in NEURO-TTR; n = 60). Main Outcomes and Measures: Primary efficacy end points at week 65/66 were changes from baseline in serum transthyretin concentration, modified Neuropathy Impairment Score +7 (mNIS+7) composite score (scoring range, -22.3 to 346.3; higher scores indicate poorer function), and Norfolk Quality of Life Questionnaire-Diabetic Neuropathy (Norfolk QoL-DN) total score (scoring range, -4 to 136; higher scores indicate poorer quality of life). Analyses of efficacy end points were based on a mixed-effects model with repeated measures adjusted by propensity score weights. Results: Among 144 eplontersen-treated patients (mean age, 53.0 years; 69% male), 136 (94.4%) completed week-66 follow-up; among 60 placebo patients (mean age, 59.5 years; 68% male), 52 (86.7%) completed week-66 follow-up. At week 65, adjusted mean percentage reduction in serum transthyretin was -81.7% with eplontersen and -11.2% with placebo (difference, -70.4% [95% CI, -75.2% to -65.7%]; P < .001). Adjusted mean change from baseline to week 66 was lower (better) with eplontersen vs placebo for mNIS+7 composite score (0.3 vs 25.1; difference, -24.8 [95% CI, -31.0 to -18.6; P < .001) and for Norfolk QoL-DN (-5.5 vs 14.2; difference, -19.7 [95% CI, -25.6 to -13.8]; P < .001). Adverse events by week 66 that led to study drug discontinuation occurred in 6 patients (4%) in the eplontersen group vs 2 (3%) in the placebo group. Through week 66, there were 2 deaths in the eplontersen group consistent with known disease-related sequelae (cardiac arrhythmia; intracerebral hemorrhage); there were no deaths in the placebo group. Conclusions and Relevance: In patients with ATTRv polyneuropathy, the eplontersen treatment group demonstrated changes consistent with significantly lowered serum transthyretin concentration, less neuropathy impairment, and better quality of life compared with a historical placebo. Trial Registration: ClinicalTrials.gov Identifier: NCT04136184; EU Clinical Trials Register: EudraCT 2019-001698-10.

On-Table Catheter-Based Neurosalvage Performed by Cardiologists for Embolic Complication during an Endovascular Procedure.

Objectives: To report our experience and clinical results of neurosalvage techniques, performed by interventional cardiologists without moving the patient, to manage cerebral thromboembolic complications. Background: Iatrogenic emboli may be released during an endovascular procedure, causing permanent neurological complications and catastrophic outcomes. Methods: Between July 2013 and December 2017, a total of eight patients suffered from embolic complications during endovascular procedures (two radiofrequency catheter ablation, five coronary angiogram/angioplasty, and one subclavian artery angioplasty). Catheter-based neurosalvage was attempted by experienced interventional cardiologists promptly in the same catheterization room. Results: The embolized locations were the M1 segment of the middle cerebral artery in four patients, the M2/M3 segments in three, and the basilar artery in one. Access to the supra-aortic vessels was achieved. Local intra-arterial thrombolysis was given in five patients (63%) and balloon angioplasty in three (38%). Intra-arterial thrombectomy with a stent retriever was attempted in three patients but failed in one. A combination of different techniques was used in three patients (38%). Final thrombolysis in cerebral infarction grade 3 flow was achieved in seven patients (88%). Favorable clinical outcomes at 1-month follow-up (modified Rankin scale of 0-2) were observed in seven patients (88%), and none of the patients had died at 12 months. Conclusions: Our experience demonstrated that acute embolic complications during an endovascular procedure can be salvaged by interventional cardiologists with acceptable angiographic and clinical results.

Efficacy of Tafamidis in Patients with Ala97Ser Hereditary Transthyretin Cardiac Amyloidosis: A Six-Month Follow-Up Study.

Background: Hereditary transthyretin amyloid cardiomyopathy (ATTR-CM) is a progressive and fatal disease. A97S (p.Ala117Ser) is the most common transthyretin genetic mutation in Taiwan. Tafamidis is a transthyretin stabilizer, and it has been shown to improve outcomes. However, its effect on A97S ATTR-CM subtypes remains unknown. Objectives: This study aimed to investigate the efficacy of tafamidis in patients with hereditary A97S ATTR-CM after 6 months of treatment. Methods: We retrospectively analyzed ATTR-CM patients who received tafamidis (61 mg/day) treatment at National Taiwan University Hospital. Functional status, biochemistry and echocardiography were measured at baseline and after 6 months of tafamidis treatment. The outcome measure was to compare the N-terminal pro-brain natriuretic peptide (NT-proBNP) level at baseline and after 6 months of tafamidis treatment. Results: Twenty patients were enrolled in this study. Their mean age was 63.0 ± 5.8 years and 75% were men. The baseline left ventricular (LV) mass index was 200.9 ± 63.9 g/m2, and the baseline LV ejection fraction was 58.9 ± 13.5%. After 6 months of treatment, the log NT-proBNP level significantly improved from 2.9 ± 0.6 to 2.7 ± 0.5 (p = 0.036). Subgroup analysis showed that the LV posterior wall thickness and left atrial diameter were significantly higher in the patients with improved NT-proBNP, suggesting the benefits of tafamidis for ATTR-CM patients with severe cardiac involvement. Conclusions: The patients with hereditary A97S ATTR-CM in this study had decreased levels of NT-proBNP after 6 months of tafamidis treatment, and this reduction was especially pronounced in those with more severe cardiac involvement.

Toll-like receptor 2 and 6 agonist fibroblast-stimulating lipopeptide increases expression and secretion of CXCL1 and CXCL2 by uveal melanocytes.

Fibroblast-stimulating lipopeptide (FSL-1) can activate Toll-like receptor 2 and 6 (TLR2/6), which recognize relevant molecules from gram-positive pathogens, fungus, and mycoplasma, and elevates the expression of CXCL1 and CXCL2, neutrophil chemoattractants, in certain types of cells. This effect has not previously been reported in the uveal melanocytes (UM). This study was designed to test the hypothesis that FSL-1 can induce the expression and secretion of CXCL1 and CXCL2 via activation of TLR2/6 in cultured human UM and producing an acute non-infectious uveitis reaction in the mouse. Flow cytometry and fluorescent immunostaining were used to measure the effect of FSL-1 on the expression of TLR2/6 in UM. Real time PCR and ELISA analysis were used to assess the ability of FSL-1 to elevate CXCL1/CXCL2 levels in cell lysates and conditioned media of UM, respectively. Flow cytometry measured phosphorylated MAPK and activated NF-κB signals in UM, with and without FSL-1 treatment. ELISA analysis tested the impact of various signal inhibitors (NF-κB, p38 MAPK, JNK1/2 and ERK1/2) and TLR2/6 antagonists on FSL-1-induced CXCL1/CXCL2 levels in cultured UM. The effects of neutralizing antibodies to TLR2 on FSL-1-induced mouse uveitis were tested in an experimental animal model. FSL-1 induced the expression of TLR2/6 proteins in cultured UM. FSL-1 significantly elevated the CXCL1 and CXCL2 proteins and mRNA levels in cultured UM time- and dose-dependently. FSL-1 mainly activated NF-κB, JNK, and expression of TLR2. FSL-1-induced expression of CXCL1 and CXCL2 was blocked by NF-κB, JNK, ERK inhibitors and TLR2 antagonists. Intravitreal injection of FSL-1 induced acute non-infectious mouse uveitis, which was significantly reduced in severity by a TLR2 antagonist. These results suggest that UM may play a role in the immune reaction, which targets invading pathogens, especially gram-positive bacteria. On the other hand, an excessive reaction to molecules from gram-positive bacteria may promote an inflammatory state of non-infectious uveitis.

Maladaptive motor cortical excitability and connectivity in polyneuropathy with neuropathic pain.

BACKGROUND AND PURPOSE: Sensory symptoms, especially neuropathic pain, are common in polyneuropathy. Conventional diagnostic tools can evaluate structural or functional impairment of nerves but cannot reveal mechanisms of neuropathic pain. Changes in the brain after polyneuropathy may play roles in the genesis of neuropathic pain. METHODS: This cross-sectional study investigated changes of cortical excitability within left primary motor cortex (M1) by measuring resting motor thresholds, short-interval intracortical inhibition (SICI), intracortical facilitation (ICF), and afferent inhibition between polyneuropathy patients and controls, and investigated the correlates of these parameters with neuropathic pain and M1 structural and functional connectivity assessed by diffusion tractography imaging and functional magnetic resonance imaging. RESULTS: Thirty-three painful and 15 nonpainful neuropathic patients and 21 controls were enrolled. There were no differences in intraepidermal nerve fiber density, nerve conduction studies, thermal thresholds, or autonomic functional tests between patients with and without neuropathic pain. Compared to controls, neuropathic patients exhibited similar resting motor thresholds or afferent inhibition, but attenuated SICI and augmented ICF, especially in painful patients. Changes of intracortical excitability in neuropathic patients were correlated with intensities of neuropathic pain, and different presentations of SICI and ICF were noted between patients with and without thermal paresthesia. Additionally, short-latency afferent inhibition at an interstimulus interval of 20 ms was associated with structural connectivity of left M1 with brain areas associated with pain perception. CONCLUSIONS: Maladaptive cortical excitability with altered structural connectivity in left M1 developed after peripheral nerve degeneration and was associated with neuropathic pain and sensory symptoms in polyneuropathy.

Diabetic visceral neuropathy of gastroparesis: Gastric mucosal innervation and clinical significance.

BACKGROUND AND PURPOSE: The pathogenesis of diabetic gastroparesis due to visceral neuropathy involves multidimensional mechanisms with limited exploration of gastric mucosal innervation. This study aimed to examine quantitatively this topic and its relationship with gastroparesis symptoms and gastric emptying in diabetes. METHODS: We prospectively enrolled 22 patients with type 2 diabetes and gastroparesis symptoms and 25 age- and gender-matched healthy controls for comparison. The assessments included: (i) neuropathology with quantification of gastric mucosal innervation density (MID) on endoscopic biopsy; (ii) clinical manifestations based on the Gastroparesis Cardinal Symptom Index (GCSI) questionnaire; and (iii) functional tests of gastric emptying scintigraphy (GES). RESULTS: In patients with diabetes, stomach fullness, bloating and feeling excessively full after meals constituted the most common GCSI symptoms. Seven patients with diabetes (32%) had prolonged gastric emptying patterns. In diabetes, gastric MID was significantly lower in all the regions examined compared with the controls: antrum (294.8 ± 237.0 vs. 644.0 ± 222.0 mm/mm3 ; p < 0.001), body (292.2 ± 239.0 vs. 652.6 ± 260.9 mm/mm3 ; p < 0.001), and fundus (238.0 ± 109.1 vs. 657.2 ± 332.8 mm/mm3 ; p < 0.001). Gastric MID was negatively correlated with gastroparesis symptoms and total scores on the GCSI (p < 0.001). Furthermore, gastric MID in the fundus was negatively correlated with fasting glucose and glycated hemoglobin levels. Gastric emptying variables, including half emptying time and gastric retention, were prolonged in patients with diabetes, and gastric retention at 3 h was correlated with fasting glucose level. CONCLUSION: In diabetes, gastric MID was reduced and GES parameters were prolonged. Both were correlated with gastroparesis symptoms and glycemic control. These findings provide pathology and functional biomarkers for diabetic visceral neuropathy of gastroparesis and underlying pathophysiology.

A combinatorial strategy for treating KRAS-mutant lung cancer.

Therapeutic targeting of KRAS-mutant lung adenocarcinoma represents a major goal of clinical oncology. KRAS itself has proved difficult to inhibit, and the effectiveness of agents that target key KRAS effectors has been thwarted by activation of compensatory or parallel pathways that limit their efficacy as single agents. Here we take a systematic approach towards identifying combination targets for trametinib, a MEK inhibitor approved by the US Food and Drug Administration, which acts downstream of KRAS to suppress signalling through the mitogen-activated protein kinase (MAPK) cascade. Informed by a short-hairpin RNA screen, we show that trametinib provokes a compensatory response involving the fibroblast growth factor receptor 1 (FGFR1) that leads to signalling rebound and adaptive drug resistance. As a consequence, genetic or pharmacological inhibition of FGFR1 in combination with trametinib enhances tumour cell death in vitro and in vivo. This compensatory response shows distinct specificities: it is dominated by FGFR1 in KRAS-mutant lung and pancreatic cancer cells, but is not activated or involves other mechanisms in KRAS wild-type lung and KRAS-mutant colon cancer cells. Importantly, KRAS-mutant lung cancer cells and patients' tumours treated with trametinib show an increase in FRS2 phosphorylation, a biomarker of FGFR activation; this increase is abolished by FGFR1 inhibition and correlates with sensitivity to trametinib and FGFR inhibitor combinations. These results demonstrate that FGFR1 can mediate adaptive resistance to trametinib and validate a combinatorial approach for treating KRAS-mutant lung cancer.

Cis- and trans-binding influences in [NUO·(N2)n].

Uranium nitride-oxide cations [NUO]+ and their complexes with equatorial N2 ligands, [NUO·(N2)n]+ (n = 1-7), were synthesized in the gas phase. Mass-selected infrared photodissociation spectroscopy and quantum chemical calculations confirm [NUO·(N2)5]+ to be a sterically fully coordinated cation, with electronic singlet ground state of 1A1, linear [NUO]+ core, and C5v structure. The presence of short N-U bond distances and high stretching modes, with slightly elongated U-O bond distances and lowered stretching modes, is rationalized by attributing them to cooperative covalent and dative [ǀN≡U≡Oǀ]+ triple bonds. The mutual trans-interaction through flexible electronic U-5f6d7sp valence shell and the linearly increasing perturbation with increase in the number of equatorial dative N2 ligands has also been explained, highlighting the bonding characteristics and distinct features of uranium chemistry.