NICE guideline on ME/CFS: robust advice based on a thorough review of the evidence.

In 2021, the National Institute for Health and Care Excellence produced an evidence-based guideline on the diagnosis and management of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), a disabling long-term condition of unknown cause. The guideline provides clear support for people living with ME/CFS, their families and carers, and for clinicians. A recent opinion piece published in the journal suggested that there were anomalies in the processing and interpretation of the evidence when developing the guideline and proposed eight areas where these anomalies were thought to have occurred. We outline how these opinions are based on a misreading or misunderstanding of the guideline process or the guideline, which provides a balanced and reasoned approach to the diagnosis and management of this challenging condition.

P36-A151†Primary human RPE cells as individual starting material for non-viral gene transfer of therapeutically active factors.

PURPOSE: Our aging society leads to an increasing incidence of neurodegenerative diseases. To date, the development of defined therapies has been hampered because the pathological mechanisms are poorly understood. Cell-based additive gene therapies to enhance the expression of protective factors are considered a promising modality for the treatment of neurodegenerative diseases, such as agerelated macular degeneration (AMD). We have developed a method to stably overexpress the genes encoding pigment epithelium-derived factor (PEDF) and brain-derived neurotrophic factor (BDNF) into the genome of primary human retinal pigment epithelial (RPE) cells by electroporation using the Sleeping Beauty (SB) transposon system. BDNF is the most abundant neurotrophin in the central nervous system. PEDF is a multifunctional protein with anti-angiogenic and neurotrophic properties. METHODS: Primary RPE cells were isolated from various human donor eyes and maintained individually in culture. After reaching confluence, RPE cells were trypsinized and co-transfected in suspension with two plasmids encoding SB100X transposase and the transposon carrying a PEDF and BDNF transcription cassette, respectively. The results of transfection were evaluated by different methods including microscopy, immunoblotting, ELISA, and quantitative PCR (qPCR). RESULTS: Seeding of sufficient numbers of primary human RPE cells allows cultivation and growth into an integrated monolayer of pigmented, hexagonally shaped cells, independent of the donor age (65.3 ± 9.94 a, min: 49 a, max: 83 a, n = 12), post-mortem time of isolation (37.3 ± 17.0 h, min: 16 h, max: 68 h), and cultivation time (27.6 ± 14.1 d, min: 13 d, max: 61 d). Successful transfection was demonstrated in experiments performed independently. Applied electrical pulses had no negative effects on cell morphology. Gene expression of PEDF and BDNF was significantly increased compared with non-transfected control cells. Secretion of recombinant PEDF and BDNF proteins was also significantly elevated and remained stable over time. CONCLUSION: The studies using primary human RPE cells are an important step in the development of a cell-based PEDF or BDNF gene therapy that could be applied as an advanced therapy medicinal product to treat AMD or other degenerative retinal diseases.

P41-A155†Flying human corneal tissues for transplantation - a transport network connected by drones.

PURPOSE: Transportation of human corneal tissue for transplantation always needs to be conducted in a timely manner. For this reason, even single corneal tissue samples are frequently transported by cars. This causes higher operational costs, increases the traffic load, and contributes to environmental pollution in general. Because of their small size, it is technically possible to transport corneal tissue transplants by unmanned aerial vehicles (UAV), more commonly referred to as drones. Such way of transportation would be faster, cheaper, environmentally friendly and would reduce the overall traffic load. METHODS: We conducted an interdisciplinary workshop as part of a larger project called EULE (European UAV-based solutions for transportation of medical goods), funded by the German Ministry for Digitalization and Traffic (BMDV). Together with the Cornea Bank based at the RWTH University Hospital in Aachen, Germany and several project partners specialized in drone technology and aerial transportation, we identified the specific requirements of such a concept. RESULTS: Typical transport routes have been identified that correspond to the range of the UAV. Initially, the payload area of the intended flight system was too small. As a result, the transport vessel for corneal tissue had to be downsized to be placed horizontally in the payload area. Also, the packaging material needed to be modified for the same reason. In addition, sensors had to be integrated to monitor the conditions during transport. CONCLUSION: Because of the mentioned modification in the transportation packaging and the lack of clarity on possible side effects of this novel kind of transportation on human corneal tissue, a field study needs to be conducted on corneal samples not intended for transplantation to evaluate the proposed concept. We plan on conducting 20 test flights and compare the condition of corneal tissue samples before and after each flight. Also, paired corneal samples will be transported by a car in a control group. We will begin with the first test flights after acquiring permission to fly on the designated route, expected in first quartal of 2023.

Distinct roles of adipose triglyceride lipase and hormone-sensitive lipase in the catabolism of triacylglycerol estolides.

Branched esters of palmitic acid and hydroxy stearic acid are antiinflammatory and antidiabetic lipokines that belong to a family of fatty acid (FA) esters of hydroxy fatty acids (HFAs) called FAHFAs. FAHFAs themselves belong to oligomeric FA esters, known as estolides. Glycerol-bound FAHFAs in triacylglycerols (TAGs), named TAG estolides, serve as metabolite reservoir of FAHFAs mobilized by lipases upon demand. Here, we characterized the involvement of two major metabolic lipases, adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL), in TAG estolide and FAHFA degradation. We synthesized a library of 20 TAG estolide isomers with FAHFAs varying in branching position, chain length, saturation grade, and position on the glycerol backbone and developed an in silico mass spectra library of all predicted catabolic intermediates. We found that ATGL alone or coactivated by comparative gene identification-58 efficiently liberated FAHFAs from TAG estolides with a preference for more compact substrates where the estolide branching point is located near the glycerol ester bond. ATGL was further involved in transesterification and remodeling reactions leading to the formation of TAG estolides with alternative acyl compositions. HSL represented a much more potent estolide bond hydrolase for both TAG estolides and free FAHFAs. FAHFA and TAG estolide accumulation in white adipose tissue of mice lacking HSL argued for a functional role of HSL in estolide catabolism in vivo. Our data show that ATGL and HSL participate in the metabolism of estolides and TAG estolides in distinct manners and are likely to affect the lipokine function of FAHFAs.

An ultra-high affinity synthetic nanobody blocks SARS-CoV-2 infection by locking Spike into an inactive conformation

Without an effective prophylactic solution, infections from SARS-CoV-2 continue to rise worldwide with devastating health and economic costs. SARS-CoV-2 gains entry into host cells via an interaction between its Spike protein and the host cell receptor angiotensin converting enzyme 2 (ACE2). Disruption of this interaction confers potent neutralization of viral entry, providing an avenue for vaccine design and for therapeutic antibodies. Here, we develop single-domain antibodies (nanobodies) that potently disrupt the interaction between the SARS-CoV-2 Spike and ACE2. By screening a yeast surface-displayed library of synthetic nanobody sequences, we identified a panel of nanobodies that bind to multiple epitopes on Spike and block ACE2 interaction via two distinct mechanisms. Cryogenic electron microscopy (cryo-EM) revealed that one exceptionally stable nanobody, Nb6, binds Spike in a fully inactive conformation with its receptor binding domains (RBDs) locked into their inaccessible down-state, incapable of binding ACE2. Affinity maturation and structure-guided design of multivalency yielded a trivalent nanobody, mNb6-tri, with femtomolar affinity for SARS-CoV-2 Spike and picomolar neutralization of SARS-CoV-2 infection. mNb6-tri retains stability and function after aerosolization, lyophilization, and heat treatment. These properties may enable aerosol-mediated delivery of this potent neutralizer directly to the airway epithelia, promising to yield a widely deployable, patient-friendly prophylactic and/or early infection therapeutic agent to stem the worst pandemic in a century.

Reproducibility and reliability of central corneal thickness determination in more and less profound corneal edema using ultrasound pachymetry, a Scheimpflug camera and anterior segment OCT.

PURPOSE: The purpose of this study is to determine the influence of different degrees of corneal edema on the reliability and reproducibility of central corneal thickness(CCT) measurements by a Scheimpflug camera (Pentacam), anterior segment optical coherence tomography(AS-OCT) and ultrasound pachymetry(USP). METHODS: Forty-four patients undergoing ophthalmic surgery were included in this prospective study. All measurements were acquired by two investigators. The Pentacam and AS-OCT measurements were performed in randomized order followed by USP. Two measurements were taken by each investigator with each device. CCT was evaluated by using the apex value provided by the Pentacam, the corneal apex cut in the AS-OCT and averaging 2 cycles of 4 measurements for USP. Coefficients of variation (COV) and intraclass correlation coefficients (ICC) were determined. To investigate the reproducibility in different degrees of corneal edema, patients were subdivided into edema more/less than 10% of CCT, ≥/< 600 µm and > 650 µm CCT. RESULTS: No significant differences were recorded for each individual investigator and measuring device. However, overall the devices differed significantly in the < 600 µm group (ANOVA p < 0.04). The reproducibility decreased with higher degrees of corneal edema in particular for investigator 1 and USP measurements. No significant overestimation of corneal thickness by the Pentacam was recorded in higher degrees of corneal edema. CONCLUSION: USP measurements are highly user dependent especially in higher degrees of corneal edema. Nevertheless, all methods were able to reach a high level of agreement in CCT measurement in higher degrees of corneal edema. Interestingly lower degrees of corneal edema revealed the only significant differences in-between the 3 devices.

Changes in Cardiorespiratory Responses and Kinematics With Hippotherapy in Youth With and Without Cerebral Palsy.

PURPOSE: The purpose of this study was to characterize pelvic displacement and cardiorespiratory responses to simulated horseback riding and walking in youth with cerebral palsy and to compare responses to youth without cerebral palsy before and after 8 weeks of hippotherapy. METHOD: Eight youth with cerebral palsy (Mage = 10 ± 4 years; Mheight = 137 ± 24 cm; Mweight = 32 ± 16 kg) and 8 youth without cerebral palsy (Mage = 11 ± 2 years; Mheight = 149 ± 14 cm; Mweight = 48 ± 15 kg) underwent a hippotherapy intervention. Participants completed simulated horseback riding at an intensity approximating a fast walk (0.65 Hz) and walked on a treadmill (1 mph, 0% grade) before and after hippotherapy. Pelvic displacement along the anterior-posterior, vertical, and medial-lateral axes, heart rate, oxygen consumption, ventilation, and blood pressure were measured at rest and during steady-state exercise in both exercise modes. RESULTS: Kinematics and cardiorespiratory responses were similar between the 2 groups during simulated horseback riding (p > .05 for all) before the intervention. Significantly greater cardiorespiratory responses were observed in the youth with cerebral palsy compared with the group without cerebral palsy while walking before and after the intervention (p <  .05, effect sizes 26% to 237% greater). Eight weeks of hippotherapy did not alter responses, but anecdotal improvements in gait, balance, posture, and range of motion were observed in those with cerebral palsy. CONCLUSION: These results contribute to our understanding regarding the efficacy of hippotherapy as an intervention to improve functional abilities in those with cerebral palsy.

Candesartan cilexetil: an update.

INTRODUCTION: Candesartan cilexetil is one of the most often-used first-line drugs regarding the management of arterial hypertension. Moreover, this drug has proven its effectiveness in chronic heart failure and exerts beneficial effects in diabetes, stroke, dementia and atrial fibrillation. AREAS COVERED: This review focuses on the use of candesartan cilexetil in Phase II and Phase III trials and their implications for clinical usage in the treatment of arterial hypertension and heart failure. The usage of candesartan cilexetil in patients with stroke, migraine or atrial fibrillation, hypertrophy obstructive or nonobstructive cardiomyopathy and diabetic or non diabetic renal diseases will be discussed. Relevant publications were identified by an extensive Medline search. EXPERT OPINION: Candesartan cilexetil is a highly effective ARB for the treatment of arterial hypertension and heart failure. Also, recent trials have suggested beneficial effects in diabetic patients, patients with nondiabetic renal diseases, stroke, migraine and atrial fibrillation.

Long-term prognosis of the transient left ventricular dysfunction syndrome (Tako-Tsubo cardiomyopathy): focus on malignancies.

BACKGROUND: The pathophysiology and long-term prognosis of the transient left ventricular dysfunction syndrome (LVDS, Tako-Tsubo cardiomyopathy) is largely unknown. AIMS: To investigate the prevalence of malignancies and long-term mortality in patients with LVDS. METHODS AND RESULTS: Fifty patients with LVDS (47 females and 3 men, age 70+/-10 years) and 50 age- and gender-matched control patients with acute anterior myocardial infarction (MI) were evaluated. Nine patients (18%) with LVDS and 3 patients (6%) with MI had a previous history of malignancy at the time of the index event. On follow-up (2.9+/-1.6 years), 7 malignancies were newly diagnosed in the LVDS cohort whereas no new case of malignancy was found in the control group (p=0.01, odds ratio 16.95, 95% confidence interval [CI] 1.93-304.60). Overall mortality during follow-up did not differ significantly between both groups (hazard ratio 1.44 for death in LVDS patients, 95% CI 0.52-3.95, p=0.49); however, of those patients who died, cardiac deaths were more frequent in patients with MI (100% versus 11% in patients with LVDS, p<0.001). CONCLUSIONS: Our data suggest an association of LVDS with malignancies, potentially as a result of paraneoplastic phenomena. Long-term prognosis of patients with LVDS is no better than in patients with acute MI.