Inhibition and reversal of a TGF-ß1 induced myofibroblast phenotype by adipose tissue-derived paracrine factors.

BACKGROUND: Hypertrophic scarring results from myofibroblast differentiation and persistence during wound healing. Currently no effective treatment for hypertrophic scarring exists however, autologous fat grafting has been shown to improve scar elasticity, appearance, and function. The aim of this study was to understand how paracrine factors from adipose tissues and adipose-derived stromal cells (ADSC) affect fibroblast to myofibroblast differentiation. METHODS: The transforming growth factor-ß1 (TGF-ß1) induced model of myofibroblast differentiation was used to test the effect of conditioned media from adipose tissue, ADSC or lipid on the proportion of fibroblasts and myofibroblasts. RESULTS: Adipose tissue conditioned media inhibited the differentiation of fibroblasts to myofibroblasts but this inhibition was not observed following treatment with ADSC or lipid conditioned media. Hepatocyte growth factor (HGF) was readily detected in the conditioned medium from adipose tissue but not ADSC. Cells treated with HGF, or fortinib to block HGF, demonstrated that HGF was not responsible for the inhibition of myofibroblast differentiation. Conditioned media from adipose tissue was shown to reduce the proportion of myofibroblasts when added to fibroblasts previously treated with TGF-ß1, however, conditioned media treatment was unable to significantly reduce the proportion of myofibroblasts in cell populations isolated from scar tissue. CONCLUSIONS: Cultured ADSC or adipocytes have been the focus of most studies, however, this work highlights the importance of considering whole adipose tissue to further our understanding of fat grafting. This study supports the use of autologous fat grafts for scar treatment and highlights the need for further investigation to determine the mechanism.

Message in a bubble: the translational potential of extracellular vesicles.

Extracellular vesicles (EVs) are small, membrane-enclosed vesicles released by cells into the extracellular milieu. They are found in all body fluids and contain a variety of functional cargo including DNA, RNA, proteins, glycoproteins and lipids, able to provoke phenotypic responses in cells, both locally and at distant sites. They are implicated in a wide array of physiological and pathological processes and hence have attracted considerable attention in recent years as potential therapeutic targets, drug delivery vehicles and biomarkers of disease. In this review we summarise the major functions of EVs in health and disease and discuss their translational potential, highlighting opportunities of - and challenges to - capitalising on our rapidly increasing understanding of EV biology for patient benefit.

Genetic services survey-experience of people with rare diseases and their families accessing genetic services in the Irish Republic.

Irish Health Service objectives state that patients with rare diseases should have timely access to genomic diagnostics with appropriate pre and post-test counselling. However, waiting times for clinical genetics outpatient appointments, during the study period, were up to two years as staffing levels remain low. A targeted public online survey was conducted in January 2022 to capture the experiences of Rare Disease families trying to access genetic testing and clinical genetic clinics in the Irish Republic. Irish patients experience significant waiting times to access clinical genetic services and self-report anxiety and stress, related to delayed access to diagnosis, clarity around recurrence risk and follow-up management. This negatively impacts personal decisions around family planning, education and employment and has a significant impact on family members seeking clarity on their own risk. Mainstream genetic testing activity is significant. Families report concern over the competency of health care professionals arranging and delivering genetic results and delays in accessing clinical genetics expertise to take them through the clinical implications. Timely access to clinical genetics expertise is important to ensure families with rare diseases have an appropriate understanding of the medical and reproductive implications of a genetic diagnosis and access to relevant care pathways. A national framework to develop competency in genomic literacy for health-care professionals including a national genetic test directory may be beneficial. Clinical genetics teams require ongoing support and investment to ensure the delivery of a safe and effective service for Irish families with rare diseases.

Instruments to assess appropriateness of hip and knee arthroplasty: a systematic review.

OBJECTIVE: To assess criteria and psychometric properties of instruments for assessing appropriateness of elective joint arthroplasty (JA) for adults with primary hip and knee osteoarthritis (OA). METHODS: A systematic review guided by Cochrane methods and PRISMA guidelines. Studies were searched in five databases. Eligible articles include all study designs developing, testing, and/or using an instrument to assess JA appropriateness. Two independent reviewers screened and extracted data. Instruments were compared with Hawker et al. JA consensus criteria. Psychometric properties of instruments were described and appraised guided by Fitzpatrick's and COSMIN approaches. RESULTS: Of 55 instruments included, none met all Hawker et al. JA consensus criteria. Criteria the most met were pain (n = 50), function (n = 49), quality of life (n = 33), and radiography (n = 24). Criteria the least met were clinical evidence of OA (n = 18), expectations (n = 15), readiness for surgery (n = 11), conservative treatments (n = 8), and patient/surgeon agree benefits outweigh risks (n = 0). Instrument by Arden et al. met the most criteria (6 of 9). The most tested psychometric properties were appropriateness (n = 55), face/content validity (n = 55), predictive validity (n = 29), construct validity and feasibility (n = 24). The least tested psychometric properties were intra-rater reliability (n = 3), internal consistency (n = 5), and inter-rater reliability (n = 13). Instruments by Gutacker et al. and Osborne et al. met the most psychometric properties (4 of 10). CONCLUSION: Most instruments included traditional criteria for assessing JA appropriateness but did not include a trial of conservative treatments or shared decision-making elements. There was limited evidence on psychometric properties.

Identification of a Czc-like operon of the periodontal pathobiont Porphyromonas gingivalis involved in metal ion efflux.

OBJECTIVE: The study aimed to investigate the role of the PGN2012 gene of the periodontitis contributing pathobiont Porphyromonas gingivalis. PGN2012 is a homolgue of TolC and is a gene our group previously showed was overexpressed in hyperinvasive cells. METHODS: The study used a combination of bioinformatics, knockout mutagenesis, growth experiments, biofilm assays and human cell invation assays to investigate PGN2012 function. RESULTS: Bioinformatics identified that PGN2012 is part of one of four TolC containing gene loci in P. gingivalis that we predicted may encode a metal resistance RND family tripartite pump, similar to those present in other Gram-negative bacteria, but which are not well understood in anaerobic bacteria. A ΔPGN2012 deletion displayed slightly reduced growth in liquid culture but did not effect biofilm formation or human cell invasion. When metal ions were included in the medium the mutant displayed significantly increased sensitivity to the divalent metal ions Zn2+ (500 µM), Co2+ (2 mM), and Cd2+(0.1 mM) but not Cu2+. CONCLUSIONS: We propose to rename the PGN2012-2014 genes czcCBA, which we suggest plays a role in intracellular stress resistance where zinc is often employed by host cells in antibacterial defence with implications for chronic infection in humans.

A novel bioassay to detect Nociceptin/Orphanin FQ release from single human polymorphonuclear cells.

Nociceptin/Orphanin FQ (N/OFQ) is the endogenous opioid agonist for the N/OFQ receptor or NOP. This receptor system is involved in pain processing but also has a role in immune regulation. Indeed, polymorphonuclear cells (PMNs) express mRNA for N/OFQ precursor and are a potential source for circulating N/OFQ. Current measurements are based on ELISA and RIA techniques. In this study we have designed a bioassay to measure N/OFQ release from single PMNs. Chinese Hamster Ovary (CHO) cells transfected with the human (h) NOP receptor and Gαiq5 chimera force receptor coupling in biosensor cells to increase intracellular Ca2+; this can be measured with FLUO-4 dye. If isolated PMNs from healthy human volunteers are layered next to CHOhNOPGαiq5 biosensor cells then stimulated with the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP) we hypothesise that released N/OFQ will activate the biosensor. PMNs also release ATP and CHO cells express purinergic receptors coupled to elevated Ca2+. In a system where these receptors (P2Y1, P2Y2 and P2X7) are blocked with high concentrations of PPADS and oATP, PMN stimulation with fMLP increases Ca2+ in PMNs then shortly afterwards the biosensor cells. Our data therfore reports detection of single cell N/OFQ release from immune cells. This was absent when cells were preincubated with the selective NOP antagonist; SB-612111. Collectively this is the first description of single cell N/OFQ release. We will deploy this assay with further purified individual cell types and use this to further study the role of the N/OFQ-NOP system in disease; in particular sepsis where there is strong evidence for increased levels of N/OFQ worsening outcome.

Association between allergen component sensitisation and clinical allergic disease in children.

BACKGROUND: Allergen component sensitisation testing is becoming increasingly important in the diagnosis of peanut allergy. The aim of the present study was to evaluate the relationship between sensitisation and symptoms of allergic disease in children by testing a large panel of inhalants, food allergens, and allergen components. METHODS: For 287 children visiting our laboratory for allergy testing, symptoms of allergic disease were recorded by standardised validated questionnaires. Specific IgE to 11 whole allergens was assessed by ImmunoCAP, and to 112 allergen components by ISAC ImmunoCAP assay. We used latent class analysis (LCA) to distinguish clinical phenotypes. RESULTS: Inhalant and food allergen sensitisation was common, irrespective of the children's allergic symptom type. Less than 10% of the variance in symptom scores was explained by variations in the number of allergens (components) that the child was sensitised to. In LCA, 135 children (50.2%) had mild allergy, with few symptoms and sensitisation to no or few allergens, 74 children (27.5%) had more symptoms and sensitisation to inhalant allergens (respiratory allergy) and 60 children (22.3%) showed polysensitisation to a median of six allergens and had more severe symptoms of different organ systems. Adding allergen component test results to LCA failed to result in identifiable classes of allergic disease in children. CONCLUSIONS: In this group of children with allergic symptoms, referred for allergy testing by their physician, broad screening for allergen component sensitisation did not contribute to distinguishing phenotypes of allergic disease.

MOP and NOP receptor interaction: Studies with a dual expression system and bivalent peptide ligands.

Opioids targeting mu;µ (MOP) receptors produce analgesia in the peri-operative period and palliative care. They also produce side effects including respiratory depression, tolerance/dependence and addiction. The N/OFQ opioid receptor (NOP) also produces analgesia but is devoid of the major MOP side effects. Evidence exists for MOP-NOP interaction and mixed MOP-NOP ligands produce analgesia with reduced side effects. We have generated a HEKMOP/NOP human expression system and used bivalent MOP-NOP and fluorescent ligands to (i) probe for receptor interaction and (ii) consequences of that interaction. We used HEKMOP/NOP cells and two bivalent ligands; Dermorphin-N/OFQ (MOP agonist-NOP agonist; DeNO) and Dermorphin-UFP101 (MOP agonist-NOP antagonist; De101). We have determined receptor binding profiles, GTPγ[35S] binding, cAMP formation and ERK1/2 activation. We have also probed MOP and NOP receptor interactions in HEK cells and hippocampal neurones using the novel MOP fluorescent ligand, DermorphinATTO488 and the NOP fluorescent ligand N/OFQATTO594. In HEKMOP/NOP MOP ligands displaced NOP binding and NOP ligands displaced MOP binding. Using fluorescent probes in HEKMOP/NOP cells we demonstrated MOP-NOP probe overlap and a FRET signal indicating co-localisation. MOP-NOP were also co-localised in hippocampal tissue. In GTPγ[35S] and cAMP assays NOP stimulation shifted the response to MOP rightwards. At ERK1/2 the response to bivalent ligands generally peaked later. We provide evidence for MOP-NOP interaction in recombinant and native tissue. NOP activation reduces responsiveness of MOP activation; this was shown with conventional and bivalent ligands.

The validity of Dutch health claims data for identifying patients with chronic kidney disease: a hospital-based study in the Netherlands.

BACKGROUND: Health claims data may be an efficient and easily accessible source to study chronic kidney disease (CKD) prevalence in a nationwide population. Our aim was to study Dutch claims data for their ability to identify CKD patients in different subgroups. METHODS: From a laboratory database, we selected 24 895 adults with at least one creatinine measurement in 2014 ordered at an outpatient clinic. Of these, 15 805 had ≥2 creatinine measurements at least 3 months apart and could be assessed for the chronicity criterion. We estimated the validity of a claim-based diagnosis of CKD and advanced CKD. The estimated glomerular filtration rate (eGFR)-based definitions for CKD (eGFR < 60 mL/min/1.73 m2) and advanced CKD (eGFR < 30 mL/min/1.73 m2) satisfying and not satisfying the chronicity criterion served as reference group. Analyses were stratified by age and sex. RESULTS: In general, sensitivity of claims data was highest in the population with the chronicity criterion as reference group. Sensitivity was higher in advanced CKD patients than in CKD patients {51% [95% confidence interval (CI) 47-56%] versus 27% [95% CI 25-28%]}. Furthermore, sensitivity was higher in young versus elderly patients. In patients with advanced CKD, sensitivity was 72% (95% CI 62-83%) for patients aged 20-59 years and 43% (95% CI 38-49%) in patients ≥75 years. The specificity of CKD and advanced CKD was ≥99%. Positive predictive values ranged from 72% to 99% and negative predictive values ranged from 40% to 100%. CONCLUSION: When using health claims data for the estimation of CKD prevalence, it is important to take into account the characteristics of the population at hand. The younger the subjects and the more advanced the stage of CKD the higher the sensitivity of such data. Understanding which patients are selected using health claims data is crucial for a correct interpretation of study results.

Interpretation of Folate Results in Hemolytic Plasma Samples: A Practical Approach.

Folate analysis in plasma is affected by hemolysis, which can lead to biased results. However, the degree of hemolysis that is considered acceptable is unclear. We explored the relationship between folate concentration and degree of hemolysis. Heparin plasma samples (N=77, hemolysis index ≤10 µmol/L) were spiked with increasing amounts of corresponding patient-specific hemolysate. Subsequently, the folate concentration and hemolysis index were measured using two Roche Cobas platforms, and their incremental relationship was investigated. The folate concentration ranged from 2.9 to 30.9 nmol/L with a median (interquartile range) of 11.4 (8.6-19.1) nmol/L. The linear relationship between the increments in folate concentration and hemolysis index was approximated by the function y=1.86x+1.56 (R2=0.996), where x represents the laboratory-specific critical difference in folate concentration, which can be calculated from the analytical variation of the employed folate assay(s), and y represents the hemolysis threshold. The hemolysis threshold did not significantly differ between the tertiles of plasma folate concentration (P=0.10). In conclusion, we have provided an evidence-based approach that can be used to reliably interpret folate concentrations in hemolytic samples, independent of the patient's folate status.

[Uveitis and tattoos]. / Uvéites et tatouages.

The complications of tattoos are multiple and known for many years. However, their success explains their exponential multiplication in all countries. This confirmed the development of two new complications: a localized or remote skin sarcoidosis reaction, as well as the development of clinical and histological uveitis. This is in the context of a delayed hypersensitivity reaction, or a granulomatous reaction. Their prevention remains rather poorly known. It requires that each tattoo-carrying subject perform a systemic sarcoidosis check-up as well as an ophthalmological investigation in search of possible uveitis. If confirmed, preventive and therapeutic measures will be carried out in emergencies to avoid blindness. These new complications confirm the extreme severity of the use of uncontrolled inks, real toxic and sensitizing mixtures, especially apparently during extensive colorful tattoos. These particularly aggressive colored inks release multiple substances and nanoparticles into the body, not all of which are measured in the medium and long term.

Reducing Implant Infection in Orthopaedics (RIIiO): Results of a pilot study comparing the influence of forced air and resistive fabric warming technologies on postoperative infections following orthopaedic implant surgery.

BACKGROUND: Active warming during surgery prevents perioperative hypothermia but the effectiveness and postoperative infection rates may differ between warming technologies. AIM: To establish the recruitment and data management strategies needed for a full trial comparing postoperative infection rates associated with forced air warming (FAW) versus resistive fabric warming (RFW) in patients aged >65 years undergoing hemiarthroplasty following fractured neck of femur. METHODS: Participants were randomized 1:1 in permuted blocks to FAW or RFW. Hypothermia was defined as a temperature of <36°C at the end of surgery. Primary outcomes were the number of participants recruited and the number with definitive deep surgical site infections. FINDINGS: A total of 515 participants were randomized at six sites over a period of 18 months. Follow-up was completed for 70.1%. Thirty-seven participants were hypothermic (7.5% in the FAW group; 9.7% in the RFW group). The mean temperatures before anaesthesia and at the end of surgery were similar. For the primary clinical outcome, there were four deep surgical site infections in the FAW group and three in the RFW group. All participants who developed a postoperative infection had antibiotic prophylaxis, a cemented prosthesis, and were operated under laminar airflow; none was hypothermic. There were no serious adverse events related to warming. CONCLUSION: Surgical site infections were identified in both groups. Progression from the pilot to the full trial is possible but will need to take account of the high attrition rate.

Folate reference interval estimation in the Dutch general population.

BACKGROUND: Folate functions as an enzyme co-factor within the one-carbon metabolic pathway, providing key metabolites required for DNA synthesis and methylation. Hence, insufficient intake of folate can negatively affect health. As correct interpretation of folate status is dependent on a well-established reference interval, we set out to perform a new estimation following the restandardization of the Roche folate assay against the international folate standard. MATERIALS AND METHODS: The folate reference interval was estimated using samples obtained from the Dutch population-based Lifelines cohort. The reference interval was estimated using two methods: a nonparametric estimation combined with bootstrap resampling and by fitting the data to a gamma distribution. The lower reference limit was verified in a patient cohort by combined measurement of folate and homocysteine. RESULTS: Dependent on the method used for estimation and in- or exclusion of individuals younger than 21 years of age, the lower reference limit ranged from 6.8 to 7.3 nmol/L and the upper reference limit ranged from 26 to 38.5 nmol/L. Applying a lower reference limit of 7.3 nmol/L resulted in the following percentage of folate deficiencies over a period of 12 months: general practitioner 15.5% (IQR 4.0%), general hospital 12.8% (IQR 5.3%), academic hospital 9.6% (IQR 4.3%). CONCLUSIONS: We estimated the folate reference interval in the Dutch general population which is not affected by a folic acid fortification program and verified the obtained lower reference limit by homocysteine measurements. Based on our results, we propose a folate reference interval independent of age of 7.3-38.5 nmol/L.