Inducible deletion of Ezh2 in CD4+ T cells inhibits kidney T cell infiltration and prevents interstitial nephritis in MRL/lpr lupus-prone mice.

Systemic lupus erythematosus is a remitting relapsing autoimmune disease characterized by autoantibody production and multi-organ involvement. T cell epigenetic dysregulation plays an important role in the pathogenesis of lupus. We have previously demonstrated upregulation of the key epigenetic regulator EZH2 in CD4+ T cells isolated from lupus patients. To further investigate the role of EZH2 in the pathogenesis of lupus, we generated a tamoxifen-inducible CD4+ T cell Ezh2 conditional knockout mouse on the MRL/lpr lupus-prone background. We demonstrate that Ezh2 deletion abrogates lupus-like disease and prevents T cell differentiation. Single-cell analysis suggests impaired T cell function and activation of programed cell death pathways in EZH2-deficient mice. Ezh2 deletion in CD4+ T cells restricts TCR clonal repertoire and prevents kidney-infiltrating effector CD4+ T cell expansion and tubulointerstitial nephritis, which has been linked to end-stage renal disease in patients with lupus nephritis.

Intestinal permeability correlates with disease activity and DNA methylation changes in lupus patients.

OBJECTIVE: Systemic lupus erythematosus (SLE or lupus) is a chronic autoimmune disease that can involve various organ systems. Several studies have suggested that increased intestinal permeability may play a role in the pathogenesis of lupus. The aim of this study was to elucidate the relationship between intestinal permeability, disease activity, and epigenetic changes in lupus patients. METHODS: A total of 25 female lupus patients were included in this study. Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) scores were used as indicator of disease activity. Plasma zonulin levels were measured, using an ELISA, as a marker of intestinal permeability. Genome-wide DNA methylation patterns were assessed in neutrophils for 19 of the lupus patients using the Infinium MethylationEPIC array. Linear regression and Pearson's correlation were used to evaluate the correlation between zonulin concentrations and SLEDAI scores. The relationship between DNA methylation levels and zonulin concentrations was assessed using beta regression, linear regression, and Pearson's correlation, adjusting for age and race. RESULTS: Intestinal permeability positively correlated with disease activity in lupus patients (p-value = 7.60 × 10-3, r = 0.53). DNA methylation levels in 926 CpG sites significantly correlated with intestinal permeability. The highest correlation was identified in LRIG1 (cg14159396, FDR-adjusted p-value = 1.35 × 10-12, adjusted r2 = 0.92), which plays a role in intestinal homeostasis. Gene Ontologies related to cell-cell adhesion were enriched among the genes that were hypomethylated with increased intestinal permeability in lupus. CONCLUSION: Our data suggest a correlation between increased intestinal permeability and disease activity in lupus patients. Further, increased intestinal permeability might be associated with epigenetic changes that could play a role in the pathogenesis of lupus.

Mosquito Salivary Antigens and Their Relationship to Dengue and P. vivax Malaria.

In tropical areas, the simultaneous transmission of multiple vector-borne diseases is common due to ecological factors shared by arthropod vectors. Malaria and dengue virus, transmitted by Anopheles and Aedes mosquitoes, respectively, are among the top vector-borne diseases that cause significant morbidity and mortality in endemic areas. Notably, tropical areas often have suitable conditions for the co-existence of these mosquito species, highlighting the importance of identifying markers that accurately indicate the risk of acquiring each specific disease entity. Aedes are daytime-biting mosquitoes, while Anopheles preferentially bite during the night. These biting patterns raise the possibility of concurrent exposure to bites from both species. This is important because mosquito saliva, deposited in the skin during blood feeding, induces immune responses that modulate pathogen establishment and infection. Previous studies have focused on characterizing such effects on the vector-pathogen interface for an individual pathogen and its mosquito vector. In this study, we evaluated associations between immune responses to salivary proteins from non-dengue and non-malaria vector mosquito species with clinical characteristics of malaria and dengue, respectively. Surprisingly, antibody responses against Anopheles antigens in dengue patients correlated with red blood cell count and hematocrit, while antibody responses against Aedes proteins were associated with platelet count in malaria patients. Our data indicate that concurrent exposure to multiple disease-carrying mosquito vectors and their salivary proteins with differing immunomodulatory properties could influence the transmission, pathogenesis, and clinical presentation of malaria, dengue fever, and other vector-borne illnesses.

Association between maternal blood lipids during pregnancy and offspring growth trajectories in a predominantly macrosomic cohort: findings from the ROLO longitudinal birth cohort study.

The purpose of this study is to examine associations between maternal lipid profiles in pregnancy and offspring growth trajectories in a largely macrosomic cohort. This is a secondary analysis of the ROLO birth cohort (n = 293), which took place in the National Maternity Hospital, Dublin, Ireland. Infants were mostly macrosomic, with 55% having a birthweight > 4 kg. Maternal mean age was 32.4 years (SD 3.9 years), mean BMI was 26.1 kg/m2 (SD 4.4 kg/m2) and 48% of children born were males. Total cholesterol, high density lipoprotein cholesterol (HDL-cholesterol), low density lipoprotein cholesterol (LDL-cholesterol) and triglycerides were measured from fasting blood samples of mothers at 14 and 28 week gestation. The change in maternal lipid levels from early to late pregnancy was also examined. Offspring abdominal circumference and weight were measured at 20- and 34-week gestation, birth, 6 months, 2 years and 5 years postnatal. Linear spline multilevel models examined associations between maternal blood lipid profiles and offspring growth. We found some weak, significant associations between maternal blood lipids and trajectories of offspring growth. Significant findings were close to the null, providing limited evidence. For instance, 1 mmol/L increase in maternal triglycerides was associated with faster infant weight growth from 20- to 34-week gestation (0.01 kg/week, 95% CI - 0.02, - 0.001) and slower abdominal circumference from 2 to 5 years (0.01 cm/week, 95% CI - 0.02, - 0.001). These findings do not provide evidence of a clinically meaningful effect.    Conclusion: These findings raise questions about the efficacy of interventions targeting maternal blood lipid profiles in pregnancies at risk of macrosomia. New studies on this topic are needed. What is Known: • Maternal fat accumulation during early pregnancy may potentially support fetal growth in the third trimester by providing a reserve of lipids that are broken down and transferred to the infant across the placental barrier. • There are limited studies exploring the impact of maternal lipid profiles on infant and child health using growth trajectories spanning prenatal to postnatal life. What is New: • Maternal blood lipid profiles were not associated with offspring growth trajectories of weight and abdominal circumference during pregnancy up to 5 years of age in a largely macrosomic cohort, as significant findings were close to the null, providing limited evidence for a clinically meaningful relationship. • Strengths of this work include the use of infant growth trajectories that span prenatal to postnatal life and inclusion of analyses of the change of maternal lipid levels from early to late pregnancy and their associations with offspring growth trajectories from 20-week gestation to 5 years of age.

Durable alveolar engraftment of PSC-derived lung epithelial cells into immunocompetent mice.

Durable reconstitution of the distal lung epithelium with pluripotent stem cell (PSC) derivatives, if realized, would represent a promising therapy for diseases that result from alveolar damage. Here, we differentiate murine PSCs into self-renewing lung epithelial progenitors able to engraft into the injured distal lung epithelium of immunocompetent, syngeneic mouse recipients. After transplantation, these progenitors mature in the distal lung, assuming the molecular phenotypes of alveolar type 2 (AT2) and type 1 (AT1) cells. After months in vivo, donor-derived cells retain their mature phenotypes, as characterized by single-cell RNA sequencing (scRNA-seq), histologic profiling, and functional assessment that demonstrates continued capacity of the engrafted cells to proliferate and differentiate. These results indicate durable reconstitution of the distal lung's facultative progenitor and differentiated epithelial cell compartments with PSC-derived cells, thus establishing a novel model for pulmonary cell therapy that can be utilized to better understand the mechanisms and utility of engraftment.

A randomized controlled safety and feasibility trial of floatation-REST in anxious and depressed individuals.

Background: Reduced Environmental Stimulation Therapy via floatation (floatation-REST) is a behavioral intervention designed to attenuate exteroceptive sensory input to the nervous system. Pilot studies in anxious and depressed individuals demonstrated that single sessions of floatation-REST are safe, well-tolerated, and associated with acute anxiolysis. However, there is not sufficient evidence of the feasibility of floatation-REST as a repeated intervention. Methods: We randomized 75 individuals with anxiety and depression to six sessions of floatation-REST in different formats (pool-REST or pool-REST preferred) or an active comparator (chair-REST). Feasibility was assessed via adherence rate to the assigned intervention, tolerability via duration of REST utilization and overall study dropout rate, and safety via incidence of serious or non-serious adverse events. Results: Six-session adherence was 85% for pool-REST, 89% for pool-REST preferred, and 74% for chair-REST. Dropout rates did not differ significantly between the treatment conditions. Mean session durations were consistently above 50 minutes, and when allowed to choose the duration and frequency, participants opted to float for an average of 75 minutes. There were no serious adverse events associated with any intervention. Positive experiences were endorsed more commonly than negative ones and were also rated at higher levels of intensity. Conclusions: Taken together, six sessions of floatation-REST appear feasible, well-tolerated, and safe in anxious and depressed individuals. Floatation-REST induces positively-valenced experiences with few negative effects. Larger randomized controlled trials evaluating markers of clinical efficacy are warranted.Clinical Trial Registration Identifier: NCT03899090.

Sex, gender, and sexuality in paleopathology: Select current developments and pathways forward.

OBJECTIVE: Overall, paleopathology trails behind related fields in exploring sex, gender, and sexuality in past societies. Here, we interrogatively synthesize scholarship on topics not included in similar reviews, focusing on sex estimation methods, and considering the social determinants of health; trauma; reproduction and family; and childhood - to highlight novel, social epidemiology- and social theory-informed frameworks and interpretive devices. CONCLUSIONS: Many paleopathological interpretations focus on sex-gender differences relative to health, with increasing use of intersectionality. Others consistently project present-day ideologies about sex, gender, and sexuality (e.g., binary sex-gender systems) into paleopathological interpretations (i.e., presentism). SIGNIFICANCE: Paleopathologists have an ethical imperative to generate scholarship that contributes to social justice initiatives focused on dismantling structural inequalities, especially relative to sex, gender, and sexuality (e.g., homophobia), such as through denaturalizing presentist binary systems. They also have a responsibility towards greater inclusivity relative to researcher identity and diversification of method and theory. LIMITATIONS: In addition to material limitations complicating reconstructions of sex, gender, and sexuality relative to health and disease in the past, this review wasn't comprehensive. The review was also limited by the relative paucity of paleopathological work on these topics. SUGGESTIONS FOR FUTURE RESEARCH: The outlook for paleopathological research on sex, gender, and sexuality is, however, positive; paleopathology is well situated to tackle these aspects of social identity. Future work should consider critical, self-reflective movement away from presentism; more robust contextualization; and further engagement with social theory and social epidemiology theory and approaches, including the Developmental Origins of Health and Disease (DOHaD), social determinants of health, and intersectionality.

Pandemic-Era WIC Participation in Wilmington, Delaware: Participants' Experiences and Challenges.

Special Supplemental Nutrition Program for Women, Infants, and Children (WIC) participants faced unprecedented challenges during the coronavirus disease 2019 (COVID-19) pandemic including financial concerns, a national infant formula shortage, and rising food costs. To mitigate these challenges, the United States Department of Agriculture implemented WIC program waivers and flexibilities aiming to simplify program operations (e.g., remote appointments and food package substitutions). However, little is known about WIC participants' perceptions of these changes and their impact on in-store benefit redemption. As such, this study aimed to characterize how pandemic-related events impacted Delaware WIC participants' shopping experiences and program perceptions. The authors conducted semi-structured interviews with 51 WIC participants in Wilmington, Delaware. Survey measures included demographic questions, the Hunger Vital Sign, and open-ended questions regarding WIC program participation experiences during the pandemic. Data were analyzed using a hybrid inductive and deductive coding approach. The results demonstrate that WIC participants benefitted from the pandemic program's flexibilities. However, they continued to experience burdensome shopping trips as well as concerns about their ability to feed their families due to infant formula shortages and inflation. These findings indicate the importance of extending existing WIC flexibilities and providing continued support for both participants and WIC-authorized retailors.

Encouraging Healthier Food and Beverage Purchasing and Consumption: A Review of Interventions within Grocery Retail Settings.

This review identifies the most promising intervention strategies for promoting the purchase and consumption of healthier items within U.S. grocery retail settings, with a particular focus on those strategies that may be most effective when implemented within SNAP-authorized retail settings. Searches of nine electronic databases, as well as forward and backward searches, yielded 1942 studies. After being screened, 73 peer-reviewed academic articles were identified for inclusion. Of these, 33 analyzed single-component interventions, while 40 assessed multi-component interventions. The following unique intervention types were considered as evaluated in these studies for their ability to increase healthy item purchasing and consumption: (1) nutrition scoring, (2) nutritional messaging, (3) non-nutritional messaging, (4) endcaps and secondary placement, (5) point-of-sale interventions, (6) increased stocking, (7) food tasting and demonstrations, (8) nutrition education, and (9) placement on shelf interventions. Nutritional scoring and nutritional messaging emerged as the most rigorously tested and effective intervention strategies. Other strategies warrant more research attention. Simple intervention strategies, as opposed to complex ones, yield the most successful results and minimize shopper burden. Therefore, these strategies should be reviewed for policy implementation within SNAP-authorized grocery retailers.

Sex differences in training-induced activity of the ubiquitin proteasome system in the dorsal hippocampus and medial prefrontal cortex of male and female mice.

The ubiquitin proteasome system (UPS) is a primary mechanism through which proteins are degraded in cells. UPS activity in the dorsal hippocampus (DH) is necessary for multiple types of memory, including object memory, in male rodents. However, sex differences in DH UPS activation after fear conditioning suggest that other forms of learning may also differentially regulate DH UPS activity in males and females. Here, we examined markers of UPS activity in the synaptic and cytoplasmic fractions of DH and medial prefrontal cortex (mPFC) tissue collected 1 h following object training. In males, training increased phosphorylation of proteasomal subunit Rpt6, 20S proteasome activity, and the amount of PSD-95 in the DH synaptic fraction, as well as proteasome activity in the mPFC synaptic fraction. In females, training did not affect measures of UPS or synaptic activity in the DH synaptic fraction or in either mPFC fraction but increased Rpt6 phosphorylation in the DH cytoplasmic fraction. Overall, training-induced UPS activity was greater in males than in females, greater in the DH than in the mPFC, and greater in synaptic fractions than in cytosol. These data suggest that object training drives sex-specific alterations in UPS activity across brain regions and subcellular compartments important for memory.

WIC Participants' Perceptions of the Cash-Value Benefit Increase during the COVID-19 Pandemic.

Recent changes to the Special Supplemental Nutrition Program for Women, Infants, and Children (WIC) Cash-Value Benefit (CVB), which provides participants with money to spend on fruits and vegetables, have the potential to reduce disparities in healthy food access and food insecurity that were exacerbated by the COVID-19 pandemic. However, few studies have examined how the changes to the CVB allotment that occurred during the pandemic influenced WIC participants' perceptions of the benefit or their fruit and vegetable purchasing and consumption. To address this gap, we conducted semi-structured interviews with 51 WIC participants in Wilmington, Delaware. Survey measures included demographic questions, the Hunger Vital Sign food insecurity screener, and open-ended questions regarding perceptions of the CVB increase and its influence on participants' fruit and vegetable purchasing and consumption. Data were analyzed using a hybrid inductive and deductive coding approach. The results demonstrate that higher CVB allotments increased WIC participants' purchasing and consumption of fruits and vegetables, increased the frequency of their shopping occasions, and enhanced their dietary variety. Our findings also suggest that WIC participants highly value the increased CVB. Consequently, maintaining the increased CVB allotment could improve the nutritional outcomes of low-income mothers, infants, and children participating in WIC.

The impact of waiting time for orthopaedic consultation on pain levels in individuals with osteoarthritis: a systematic review and meta-analysis.

OBJECTIVE: Time spent waiting for access to orthopaedic specialist health services has been suggested to result in increased pain in individuals with osteoarthritis (OA). We assessed whether time spent on an orthopaedic waiting list resulted in a detrimental effect on pain levels in patients with knee or hip OA. METHODS: We searched Ovid MEDLINE, EMBASE and EBSCOhost databases from inception until September 2021. Eligible articles included individuals with OA on an orthopaedic waitlist and not receiving active treatment, and reported pain measures at two or more time points. Random-effects meta-analysis was used to estimate the pooled effect of waiting time on pain levels. Meta-regression was used to determine predictors of effect size. RESULTS: Thirty-three articles were included (n = 2,490 participants, 67 ± 3 years and 62% female). The range of waiting time was 2 weeks to 2 years (20.8 ± 18.8 weeks). There was no significant change in pain over time (effect size = 0.082, 95% CI = -0.009, 0.172), nor was the length of time associated with longitudinal changes in pain over time (ß = 0.004, 95% CI = -0.005, 0.012). Body mass index was a significant predictor of pain (ß = -0.043, 95% CI = -0.079, 0.006), whereas age and sex were not. CONCLUSIONS: Pain remained stable for up to 1 year in patients with OA on an orthopaedic waitlist. Future research is required to understand whether pain increases in patients waiting longer than 1 year.

Understanding Impacts of SNAP Fruit and Vegetable Incentive Program at Farmers' Markets: Findings from a 13 State RCT.

Disparities in healthy food access and consumption are a major public health concern. This study reports the findings from a two-year randomized control trial conducted at 77 farmers' markets (FMs) in 13 states and the District of Columbia that sought to understand the impact of fruit and vegetable (FV) incentive vouchers, randomly issued at varied incentive levels to Supplemental Nutrition Assistance Program (SNAP) recipients, for use at FMs. Measures included FV and overall household food purchasing; FV consumption; food insecurity; health status; market expenditure; and demographics. A repeated-measures mixed-effects analysis and the Complier Average Causal Effect (CACE) were used to examine outcomes. Despite 82% reporting food insecurity in the prior year, the findings showed that financial incentives at FMs had statistically significant, positive effects on FV consumption; market expenditures increased with added incentives. SNAP recipients receiving an incentive of USD 0.40 for every USD 1.00 in SNAP spent an average of USD 19.03 per transaction, while those receiving USD 2 for every USD 1 (2:1) spent an average of USD 36.28 per transaction. The data showed that the incentive program at the highest level (2:1) maximally increased SNAP FM expenditure and FV consumption, increasing the latter by 0.31 daily cups among those who used their incentive (CACE model).

Extracellular matrix metalloproteinase-9 (MMP-9) is required in female mice for 17ß-estradiol enhancement of hippocampal memory consolidation.

Hippocampal plasticity and memory are modulated by the potent estrogen 17ß-estradiol (E2). Research on the molecular mechanisms of hippocampal E2 signaling has uncovered multiple intracellular pathways that contribute to these effects, but few have questioned the role that extracellular signaling processes may play in E2 action. Modification of the extracellular matrix (ECM) by proteases like matrix metalloproteinase-9 (MMP-9) is critical for activity-dependent remodeling of synapses, and MMP-9 activity is required for hippocampal learning and memory. Yet little is known about the extent to which E2 regulates MMP-9 in the hippocampus, and the influence this interaction may have on hippocampal memory. Here, we examined the effects of hippocampal MMP-9 activity on E2-induced enhancement of spatial and object recognition memory consolidation. Post-training bilateral infusion of an MMP-9 inhibitor into the dorsal hippocampus of ovariectomized female mice blocked the enhancing effects of E2 on object placement and object recognition memory, supporting a role for MMP-9 in estrogenic regulation of memory consolidation. E2 also rapidly increased the activity of dorsal hippocampal MMP-9 without influencing its protein expression, providing further insight into hippocampal E2/MMP-9 interactions. Together, these results provide the first evidence that E2 regulates MMP-9 to modulate hippocampal memory and highlight the need to further study estrogenic regulation of extracellular modification.

Elamipretide treatment during pregnancy ameliorates the progression of polycystic kidney disease in maternal and neonatal mice with PKD1 mutations.

Pregnancy is proposed to aggravate cyst progression in autosomal dominant polycystic kidney disease (ADPKD) but Tolvaptan, the only FDA-approved drug for adult ADPKD, is not recommended for pregnant ADPKD patients because of potential fetal harm. Since pregnancy itself may increase the risk for ADPKD progression, we investigated the safety and efficacy of Elamipretide, a mitochondrial-protective tetrapeptide. Elamipretide was found to ameliorate the progression of kidney disease in pregnant Pkd1RC/RC mice, in parallel with attenuation of ERK1/2 phosphorylation and improvement of mitochondrial supercomplex formation. Furthermore, Elamipretide was found to pass through the placenta and breast milk and ameliorate aggressive infantile polycystic kidney disease without any observed teratogenic or harmful effect. Elamipretide has an excellent safety profile and is currently tested in multiple phase II and phase III clinical trials. These preclinical studies support a potential clinical trial of Elamipretide for the treatment of ADPKD, particularly for patients that cannot take Tolvaptan.

Who Are You? The Study of Personality in Patients With Anterograde Amnesia.

Little is known about the role of declarative memory in the ongoing perception of one's personality. Seven individuals who developed a rare and severe type of anterograde amnesia following damage to their medial temporal lobes were identified from our neurological patient registry. We examined the stability of their personality ratings on the Big Five Inventory over five retest periods and assessed the accuracy of their ratings via analyses of self-caregiver agreement. The patients portrayed a stable sense of self over the course of 1 year. However, their self-ratings differed from those provided by the caregivers. Intriguingly, these discrepancies diminished when caregivers retrospectively rated the patients' personalities prior to their brain injury, suggesting that patients' perceptions of themselves were stuck in the past. We interpret our findings to indicate that the ability to form new declarative memories is not required for maintaining a stable sense of self but may be important for updating one's sense of self over time.

Bone resorption and dietary calcium in pregnancy-a window to future maternal bone health.

BACKGROUND: Pregnancy is characterized by increased bone turnover and reversible loss of bone mineral density (BMD) to meet fetal calcium demands. The long-term effect of bone turnover and maternal diet in pregnancy on maternal bone is not well established. OBJECTIVE: We aimed to determine if an association exists between [1] bone resorption, [2] dietary calcium, and [3] serum 25-hydroxyvitamin D in pregnancy with maternal BMD 5-year postpartum. DESIGN: This is a prospective, longitudinal study of 107 women recruited to the ROLO low glycemic index dietary intervention trial in pregnancy and followed-up at 13, 28, and 34 weeks' gestation and 5 years' postpartum. At 13 and 28 weeks' gestation, a biomarker of bone resorption, urine cross-linked N-telopeptide of type I collagen (uNTX), was measured. At the 5-year follow-up BMD was measured using dual-energy X-ray absorptiometry. Anthropometry, dietary intakes, and serum 25-hydroxyvitamin D were measured in pregnancy and at 5 years. Multiple linear regression, controlling for confounders, was used for analysis. RESULTS: Mean BMD at 5 years was 1.208 g/cm2. In pregnancy, 24-34% reported dietary calcium intakes <800 mg/day. Vitamin D deficiency (< 30 nmol/L) was observed in 38-41% of women in pregnancy and in 29% of women at the 5-year follow-up. At 13 and 28 weeks' gestation, uNTX levels greater than the median were associated with 0.060 and 0.050 g/cm2 lower BMD 5 years later, respectively. Dietary calcium <800 mg/day in trimester 3 was associated with 0.072 g/cm2 lower BMD 5 years later. Vitamin D deficiency at 5 years, but not in pregnancy, was associated with lower BMD. CONCLUSION: Higher bone resorption and low dietary calcium in pregnancy were associated with lower BMD 5 years later. These findings could enable the identification of women at risk of declining of BMD in later life, but further research is needed. Adequate dietary calcium should be advised in the antenatal setting to promote lifelong maternal bone health.

Group & save sampling in lumbar decompression: A review into current practice.

The risks, benefits and technical aspects of surgery require careful consideration. One element of this is the requirement of postoperative blood transfusion. Patients who undergo elective lumbar decompression are at a low risk of requiring a postoperative transfusion yet undergo multiple preoperative group & save tests. For those who are at a low risk of bleeding, a single group & save sample may be adequate. This review analysed the postoperative blood loss and transfusion rate associated with lumbar decompression surgery without fusion in one institution. A subsequent cost analysis and review of the literature was performed. The aim was to assess whether single group & save sampling, within the context of lumbar decompression, was cost effective and amenable to the patient without impacting patient care. Average blood loss was estimated as a drop in Hb of 12.3g/dl. Six patients (14%) had Hb loss of over 20g/dl. No patients underwent a blood transfusion. Through examination of medical records, we found that 65% of patients (35) were suitable for single group & save sampling, estimating a saving of £2415.95 (53%). Selective group & save testing holds economic potential and safeguards patients from undergoing unnecessary testing. The next step after this review would be a prospective multi-centre study.