Natural Histories and Disease Complications in a Cohort of 151 Children With Gastric or Duodenal Eosinophilia.

INTRODUCTION: Eosinophilic gastritis (EoG) and duodenitis (EoD) are rare conditions that are poorly understood. Our aim was to describe the natural history of children with varying degrees of gastric or duodenal eosinophilia with respect to disease complications and histologic and endoscopic longitudinal trajectories. METHODS: The electronic medical record at a tertiary children's hospital was queried to identify patients with EoG, EoD, or EoG + EoD who were cared for between January 2010 and 2022. Multiple logistic regression was performed to explore associations between baseline features and persistence/recurrence of eosinophilia or complications remote from diagnosis. RESULTS: We identified 151 patients: 92 with EoG, 24 with EoD, 12 with EoG + EoD, and 23 with tissue eosinophilia but did not meet histologic criteria for EoG or EoD (low grade). The average age at diagnosis was 10.6 years, and average follow-up was 5.8 years. Twenty-five percent of patients with EoG or EoD had persistence/recurrence of eosinophilia; this was associated with increases in the EoG Endoscopic Reference Score (adjusted odds ratio [aOR] 1.34, confidence interval [CI] 1.03-1.74) on diagnostic endoscopy. Eighteen percent suffered from disease complications, and development of late complications was associated with presenting with a complication (aOR 9.63, CI 1.09-85.20), severity of duodenal endoscopic abnormalities (aOR 8.74, CI 1.67-45.60), and increases in the EoG Endoscopic Reference Score (aOR 1.70, CI 1.11-2.63). DISCUSSION: Patients with gastric and duodenal eosinophilia should be followed closely to monitor for recurrence and complications, especially those presenting with endoscopic abnormalities or complications.

Engineering Cancer Antigen-Specific T Cells to Overcome the Immunosuppressive Effects of TGF-ß.

Adoptive T cell therapy with T cells expressing affinity-enhanced TCRs has shown promising results in phase 1/2 clinical trials for solid and hematological tumors. However, depth and durability of responses to adoptive T cell therapy can suffer from an inhibitory tumor microenvironment. A common immune-suppressive agent is TGF-ß, which is secreted by tumor cells and cells recruited to the tumor. We investigated whether human T cells could be engineered to be resistant to inhibition by TGF-ß. Truncating the intracellular signaling domain from TGF-ß receptor (TGFßR) II produces a dominant-negative receptor (dnTGFßRII) that dimerizes with endogenous TGFßRI to form a receptor that can bind TGF-ß but cannot signal. We previously generated specific peptide enhanced affinity receptor TCRs recognizing the HLA-A*02-restricted peptides New York esophageal squamous cell carcinoma 1 (NY-ESO-1)157-165/l-Ag family member-1A (TCR: GSK3377794, formerly NY-ESO-1c259) and melanoma Ag gene A10254-262 (TCR: ADP-A2M10, formerly melanoma Ag gene A10c796). In this article, we show that exogenous TGF-ß inhibited in vitro proliferation and effector functions of human T cells expressing these first-generation high-affinity TCRs, whereas inhibition was reduced or abolished in the case of second-generation TCRs coexpressed with dnTGFßRII (e.g., GSK3845097). TGF-ß isoforms and a panel of TGF-ß-associated genes are overexpressed in a range of cancer indications in which NY-ESO-1 is commonly expressed, particularly in synovial sarcoma. As an example, immunohistochemistry/RNAscope identified TGF-ß-positive cells close to T cells in tumor nests and stroma, which had low frequencies of cells expressing IFN-γ in a non-small cell lung cancer setting. Coexpression of dnTGFßRII may therefore improve the efficacy of TCR-transduced T cells.

Utilization and impact of esophageal string testing in children with eosinophilic esophagitis: A 1 year experience.

The 1-h esophageal string test (EST) is a minimally invasive test that can be used to monitor eosinophilic esophagitis (EoE) disease activity and guide treatment without endoscopy. We aimed to describe the real-world utilization and impact of EST on the care of children with EoE over the first year this was used at our center. Between 12/1/2022 and 11/30/2023, 39 ESTs were successful in 45 attempts (87% completion rate) in 31 patients. Five patients underwent multiple ESTs. Adverse events during the EST included vomiting. Reasons for failure to complete the EST (13%, n = 6) were patients could not swallow the capsule (n = 5) and vomiting (n = 1). EST was used to assess EoE without the need for endoscopy in 95% (n = 37) of cases. Treatment approach varied based on whether the EST indicated active (38.5%) or inactive (61.5%) EoE. The EST is a well-tolerated minimally invasive disease monitoring tool for patients with EoE.

Influence of oral contrast type and volume on patient experience and quality of luminal distension at MR Enterography in Crohn's disease: an observational study of patients recruited to the METRIC trial.

OBJECTIVES: To compare the distention quality and patient experience of oral mannitol and polyethylene glycol (PEG) for MRE. METHODS: This study is a retrospective, observational study of a subset of patients enrolled in a multicentre, prospective trial evaluating the diagnostic accuracy of MRE for small bowel Crohn's. Overall and segmental MRE small bowel distention, from 105 patients (64 F, mean age 37) was scored from 0 = poor to 4 = excellent by two experienced observers (68 [65%] mannitol and 37 [35%] PEG). Additionally, 130 patients (77 F, mean age 34) completed a questionnaire rating tolerability of various symptoms immediately and 2 days after MRE (85 [65%] receiving mannitol 45 [35%] receiving PEG). Distension was compared between agents and between those ingesting ≤ 1 L or > 1 L of mannitol using the test of proportions. Tolerability grades were collapsed into "very tolerable," "moderately tolerable," and "not tolerable." RESULTS: Per patient distension quality was similar between agents ("excellent" or "good" in 54% [37/68] versus 46% [17/37]) with mannitol and PEG respectively. Jejunal distension was significantly better with mannitol compared to PEG (40% [27/68] versus 14% [5/37] rated as excellent or good respectively). There was no significant difference according to the volume of mannitol ingested. Symptom tolerability was comparable between agents, although fullness following MRE was graded as "very tolerable" in 27% (12/45) of patients ingesting PEG, verses 44% (37/84) ingesting mannitol, difference 17% (95% CI 0.6 to 34%). CONCLUSION: Mannitol-based solutions and PEG generally achieve comparable distension quality and side effect profiles, although jejunal distension is better quality with mannitol. Neither distension quality nor side-effect profile is altered by ingestion of more than 1 L of mannitol. KEY POINTS: • Mannitol-based and PEG-based oral preparation agents generally achieve comparable distension quality for MRE with the exception of the jejunum which is better distended with mannitol. • Mannitol-based and PEG-based oral preparation agents used for MRE have similar side effect profiles. • Neither distension quality nor side-effect profile is altered by ingestion of more than 1 L of mannitol.

Bayesian analysis of a systematic review of early versus late tracheostomy in ICU patients.

BACKGROUND: A recent systematic review and meta-analysis of RCTs of early vs late tracheostomy in mechanically ventilated patients suggest that early tracheostomy reduces the duration of ICU stay and mechanical ventilation, but does not reduce short-term mortality or ventilator-associated pneumonia (VAP). Meta-analysis of randomised trials is typically performed using a frequentist approach, and although reporting confidence intervals, interpretation is usually based on statistical significance. To provide a robust basis for clinical decision-making, we completed the search used from the previous review and analysed the data using Bayesian methods to estimate posterior probabilities of the effect of early tracheostomy on clinical outcomes. METHODS: The search was completed for RCTS comparing early vs late tracheostomy in the databases PubMed, EMBASE, and Cochrane library in June 2022. Effect estimates and 95% confidence intervals were calculated for the outcomes short-term mortality, VAP, duration of ICU stay, and mechanical ventilation. A Bayesian meta-analysis was performed with uninformative priors. Risk ratios (RRs) and standardised mean differences (SMDs) with 95% credible intervals were reported alongside posterior probabilities for any benefit (RR<1; SMD<0), a small benefit (number needed to treat, 200; SMD<-0.5), or modest benefit (number needed to treat, 100; SMD<-1). RESULTS: Nineteen RCTs with 3508 patients were included. Comparing patients with early vs late tracheostomy, the posterior probabilities for any benefit, small benefit, and modest benefit, respectively, were: 99%, 99%, and 99% for short-term mortality; 94%, 78%, and 51% for VAP; 97%, 43%, and 1% for duration of mechanical ventilation; and 97%, 75%, and 27% and for length of ICU stay. CONCLUSIONS: Bayesian meta-analysis suggests a high probability that early tracheostomy compared with delayed tracheostomy has at least some benefit across all clinical outcomes considered.

Synthesis and self-assembly of curcumin-modified amphiphilic polymeric micelles with antibacterial activity.

BACKGROUND: The ubiquitous nature of bacterial biofilms combined with the enhanced resistance towards antimicrobials has led to the development of an increasing number of strategies for biofilm eradication. Such strategies must take into account the existence of extracellular polymeric substances, which obstruct the diffusion of antibiofilm agents and assists in the maintenance of a well-defended microbial community. Within this context, nanoparticles have been studied for their drug delivery efficacy and easily customised surface. Nevertheless, there usually is a requirement for nanocarriers to be used in association with an antimicrobial agent; the intrinsically antimicrobial nanoparticles are most often made of metals or metal oxides, which is not ideal from ecological and biomedical perspectives. Based on this, the use of polymeric micelles as nanocarriers is appealing as they can be easily prepared using biodegradable organic materials. RESULTS: In the present work, micelles comprised of poly(lactic-co-glycolic acid) and dextran are prepared and then functionalised with curcumin. The effect of the functionalisation in the micelle's physical properties was elucidated, and the antibacterial and antibiofilm activities were assessed for the prepared polymeric nanoparticles against Pseudomonas spp. cells and biofilms. It was found that the nanoparticles have good penetration into the biofilms, which resulted in enhanced antibacterial activity of the conjugated micelles when compared to free curcumin. Furthermore, the curcumin-functionalised micelles were efficient at disrupting mature biofilms and demonstrated antibacterial activity towards biofilm-embedded cells. CONCLUSION: Curcumin-functionalised poly(lactic-co-glycolic acid)-dextran micelles are novel nanostructures with an intrinsic antibacterial activity tested against two Pseudomonas spp. strains that have the potential to be further exploited to deliver a secondary bioactive molecule within its core.

Tuning T-Cell Receptor Affinity to Optimize Clinical Risk-Benefit When Targeting Alpha-Fetoprotein-Positive Liver Cancer.

Patients with hepatocellular carcinoma (HCC) have a poor prognosis and limited therapeutic options. Alpha-fetoprotein (AFP) is often expressed at high levels in HCC and is an established clinical biomarker of the disease. Expression of AFP in nonmalignant liver can occur, particularly in a subset of progenitor cells and during chronic inflammation, at levels typically lower than in HCC. This cancer-specific overexpression indicates that AFP may be a promising target for immunotherapy. We verified expression of AFP in normal and diseased tissue and generated an affinity-optimized T-cell receptor (TCR) with specificity to AFP/HLA-A*02+ tumors. Expression of AFP was investigated using database searches, by qPCR, and by immunohistochemistry (IHC) analysis of a panel of human tissue samples, including normal, diseased, and malignant liver. Using in vitro mutagenesis and screening, we generated a TCR that recognizes the HLA-A*02-restricted AFP158-166 peptide, FMNKFIYEI, with an optimum balance of potency and specificity. These properties were confirmed by an extension of the alanine scan (X-scan) and testing TCR-transduced T cells against normal and tumor cells covering a variety of tissues, cell types, and human leukocyte antigen (HLA) alleles. Conclusion: We have used a combination of physicochemical, in silico, and cell biology methods for optimizing a TCR for improved affinity and function, with properties that are expected to allow TCR-transduced T cells to differentiate between antigen levels on nonmalignant and cancer cells. T cells transduced with this TCR constitute the basis for a trial of HCC adoptive T-cell immunotherapy.

Intervention and Mechanisms of Alanyl-glutamine for Inflammation, Nutrition, and Enteropathy: A Randomized Controlled Trial.

OBJECTIVE: Determine the minimum dosage of alanyl-glutamine (Ala-Gln) required to improve gut integrity and growth in children at risk of environmental enteropathy (EE). METHODS: This was a double-blinded randomized placebo-controlled dose-response trial. We enrolled 140 children residing in a low-income community in Fortaleza, Brazil. Participants were 2 to 60 months old and had weight-for-age (WAZ), height-for-age (HAZ), or weight-for-height (WHZ) z-scores less than -1. We randomized children to 10 days of nutritional supplementation: Ala-Gln at 3 g/day, Ala-Gln at 6 g/day, Ala-Gln at 12 g/day, or an isonitrogenous dose of glycine (Gly) placebo at 12.5 g/day. Our primary outcome was urinary lactulose-mannitol excretion testing. Secondary outcomes were anthropometry, fecal markers of inflammation, urine metabolic profiles, and malabsorption (spot fecal energy). RESULTS: Of 140 children, 103 completed 120 days of follow-up (24% dropout). In the group receiving the highest dose of Ala-Gln, we detected a modest improvement in urinary lactulose excretion from 0.19% on day 1 to 0.17% on day 10 (P = 0.05). We observed significant but transient improvements in WHZ at day 10 in 2 Ala-Gln groups, and in WHZ and WAZ in all Ala-Gln groups at day 30. We detected no effects on fecal inflammatory markers, diarrheal morbidity, or urine metabolic profiles; but did observe modest reductions in fecal energy and fecal lactoferrin in participants receiving Ala-Gln. CONCLUSIONS: Intermediate dose Ala-Gln promotes short-term improvement in gut integrity and ponderal growth in children at risk of EE. Lower doses produced improvements in ponderal growth in the absence of enhanced gut integrity.

Understanding genetic learning needs of people affected by rare disease.

Nearly 350 million people worldwide are affected by a rare disease (RD) and ~80% of RDs have a genetic type, underscoring the need for access to reliable genomics education. Patient assistance in resource development can help ensure content is appropriate. The aim of this study was to define the needs and practical usage of the RD community to inform the scope and content of an online genetic course targeted toward the entire RD ecosystem. A high-level online survey (OS) was disseminated to 586 RD patients and family members/caregivers. A total of 251 individuals responded to the OS. Eight respondents were invited to participate in a follow-up focus group (FG). Nearly 87% of OS respondents have made efforts previously to learn more about genetics and 95.6% indicated a current interest in genetic education. Navigating healthcare systems, information sharing, and advocacy support were driving factors for this desire. Respondents indicated difficulty finding information on gene function, genetic testing, disease pathogenesis, and scientific advances. FG outcomes dove deeper into psychological needs including reducing emotional burden, alleviating fear of the unknown and seeking hope. Research identified high levels of interest in genetic education across all stages of the RD journey. Key themes identified in this study may help guide genetic counselors as they create their own patient and family-facing content.

Population dynamics of a dual Pseudomonas putida-Pseudomonas fluorescens biofilm in a capillary bioreactor.

Most biofilm studies employ single species, yet in nature biofilms exist as mixed cultures, with inevitable effects on growth and development of each species present. To investigate how related species of bacteria interact in biofilms, two Pseudomonas spp., Pseudomonas fluorescens and Pseudomonas putida, were cultured in capillary bioreactors and their growth measured by confocal microscopy and cell counting. When inoculated in pure culture, both bacteria formed healthy biofilms within 72 h with uniform coverage of the surface. However, when the bioreactors were inoculated with both bacteria simultaneously, P. putida was completely dominant after 48 h. Even when the inoculation by P. putida was delayed for 24 h, P. fluorescens was eliminated from the capillary within 48 h. It is proposed that production of the lipopeptide putisolvin by P. putida is the likely reason for the reduction of P. fluorescens. Putisolvin biosynthesis in the dual-species biofilm was confirmed by mass spectrometry.

Calibration and field application of a molecularly imprinted membrane-passive sampler for the sampling of indicator polychlorinated biphenyls in selected aquatic environments of South Africa.

A passive sampling device, based on molecularly imprinted membranes (MIM), was fabricated and optimised for sampling polychlorinated biphenyls (PCBs) in aquatic ecosystems. The newly-developed passive sampler was subjected to in-situ calibration studies to determine PCB sampling rates under various conditions of water turbulence and temperature. This was carried out by exposing the passive samplers to water spiked with PCBs in a continuous-flow exposure setup. The samplers were preloaded with known concentrations of performance reference compounds (PRCs) prior to exposure. Sampling rates of seven indicator PCBs' congeners (PCBs 28, 52, 101, 118, 138, 153 and 180) ranged between 15.3 and 95.6 L/d for the different environmental conditions investigated. To determine the field suitability, the samplers were preloaded with PRCs and deployed for 10 days at the Roodeplaat and Hartbeespoort Dams, in South Africa. Water samples were taken at the end of the deployment period to compare the spot-and-grab samples to the developed samplers. PCBs 28, 101 and 138 were detected in the samplers deployed at Hartbeespoort Dam. The samplers deployed at the Roodeplaat Dam had quantifiable amounts of PCBs 28, 52, 101, 138 and 180 (0.047-0.828 ng mL-1 d-1). The sampler enhanced the detectability of PCB 52 and PCB 180, which were not detected in water samples. The field suitability trials indicated that the developed sampler could successfully be used for PCB monitoring. The sampler enhanced the detection of PCBs that would otherwise be too low to detect in samples collected through the traditional spot-and-grab sampling technique.

Polycyclic aromatic hydrocarbons (PAHs) in sediments from a typical urban impacted river: application of a comprehensive risk assessment.

Soweto and Lenasia, the most densely populated area of South Africa, is simultaneously a thriving metropolis, with a fair share of people still living in squalor conditions directly dependant on the natural resources. Because of industrialisation the populace and environment in this urban area are exposed to various pollutants. The aquatic environment was selected as a proxy to study the effect of industrial pollution in this area. The concentrations, source identification, and various environmental risks of polycyclic aromatic hydrocarbons (PAHs) were determined in sediments of the upper reaches of the Klip River. Composite sediment samples collected in low-flow conditions in 2013 and 2014 ranged from 270-5400 ng/g. The PAHs in this aquatic ecosystem were dominated by 4-ring congeners and could be attributed to combustion of organic fuels by chemical mass balance. Heavy traffic and industrial complexes in the northern part of the study area were responsible for the PAH fingerprints. Probable adverse effects such as toxicity to benthic biota were proven after comparison with international sediment quality guidelines (SQG) both survey years. Toxic equivalence quotients (TEQs) calculated for the sediments using fish potency factors (FPFs) were up to 30 times greater than the Canadian guideline for dioxin-like compounds, indicating high probability of carcinogenic effect to fish mediated through the aryl-hydrocarbon receptor. Finally, sediments in the area posed moderate to high ecological risk, which corroborates the other toxicity assessments. The advantage of investigating multiple risk endpoints, is the comprehensive results obtained that allows for a more realistic representation of the study area. Consequently more aspects are kept into account that results in better conclusions.

Induction of the Lytic Cycle Sensitizes Epstein-Barr Virus-Infected B Cells to NK Cell Killing That Is Counteracted by Virus-Mediated NK Cell Evasion Mechanisms in the Late Lytic Cycle.

UNLABELLED: Epstein-Barr Virus (EBV) persists for the lifetime of the infected host despite eliciting strong immune responses. This persistence requires a fine balance between the host immune system and EBV immune evasion. Accumulating evidence suggests an important role for natural killer (NK) cells in this balance. NK cells can kill EBV-infected cells undergoing lytic replication in vitro, and studies in both humans and mice with reconstituted human immune systems have shown that NK cells can limit EBV replication and prevent infectious mononucleosis. We now show that NK cells, via NKG2D and DNAM-1 interactions, recognize and kill EBV-infected cells undergoing lytic replication and that expression of a single EBV lytic gene, BZLF1, is sufficient to trigger sensitization to NK cell killing. We also present evidence suggesting the possibility of the existence of an as-yet-unidentified DNAM-1 ligand which may be particularly important for killing lytically infected normal B cells. Furthermore, while cells entering the lytic cycle become sensitized to NK cell killing, we observed that cells in the late lytic cycle are highly resistant. We identified expression of the vBcl-2 protein, BHRF1, as one effective mechanism by which EBV mediates this protection. Thus, contrary to the view expressed in some reports, EBV has evolved the ability to evade NK cell responses. IMPORTANCE: This report extends our understanding of the interaction between EBV and host innate responses. It provides the first evidence that the susceptibility to NK cell lysis of EBV-infected B cells undergoing lytic replication is dependent upon the phase of the lytic cycle. Induction of the lytic cycle is associated with acquired sensitization to NK cell killing, while progress through the late lytic cycle is associated with acquired resistance to killing. We provide mechanistic explanations for this novel observation, indicating important roles for the BZLF1 immediate early transactivator, the BHRF1 vBcl-2 homologue, and a novel ligand for the DNAM-1 NK cell receptor.

The Missing Link in Epstein-Barr Virus Immune Evasion: the BDLF3 Gene Induces Ubiquitination and Downregulation of Major Histocompatibility Complex Class I (MHC-I) and MHC-II.

UNLABELLED: The ability of Epstein-Barr virus (EBV) to spread and persist in human populations relies on a balance between host immune responses and EBV immune evasion. CD8(+) cells specific for EBV late lytic cycle antigens show poor recognition of target cells compared to immediate early and early antigen-specific CD8(+) cells. This phenomenon is due in part to the early EBV protein BILF1, whose immunosuppressive activity increases with lytic cycle progression. However, published data suggest the existence of a hitherto unidentified immune evasion protein further enhancing protection against late EBV antigen-specific CD8(+) cells. We have now identified the late lytic BDLF3 gene as the missing link accounting for efficient evasion during the late lytic cycle. Interestingly, BDLF3 also contributes to evasion of CD4(+) cell responses to EBV. We report that BDLF3 downregulates expression of surface major histocompatibility complex (MHC) class I and class II molecules in the absence of any effect upon other surface molecules screened, including CD54 (ICAM-1) and CD71 (transferrin receptor). BDLF3 both enhanced internalization of surface MHC molecules and reduced the rate of their appearance at the cell surface. The reduced expression of surface MHC molecules correlated with functional protection against CD8(+) and CD4(+) T cell recognition. The molecular mechanism was identified as BDLF3-induced ubiquitination of MHC molecules and their subsequent downregulation in a proteasome-dependent manner. IMPORTANCE: Immune evasion is a necessary feature of viruses that establish lifelong persistent infections in the face of strong immune responses. EBV is an important human pathogen whose immune evasion mechanisms are only partly understood. Of the EBV immune evasion mechanisms identified to date, none could explain why CD8(+) T cell responses to late lytic cycle genes are so infrequent and, when present, recognize lytically infected target cells so poorly relative to CD8(+) T cells specific for early lytic cycle antigens. The present work identifies an additional immune evasion protein, BDLF3, that is expressed late in the lytic cycle and impairs CD8(+) T cell recognition by targeting cell surface MHC class I molecules for ubiquitination and proteasome-dependent downregulation. Interestingly, BDLF3 also targets MHC class II molecules to impair CD4(+) T cell recognition. BDLF3 is therefore a rare example of a viral protein that impairs both the MHC class I and class II antigen-presenting pathways.

Cooperation between Epstein-Barr virus immune evasion proteins spreads protection from CD8+ T cell recognition across all three phases of the lytic cycle.

CD8+ T cell responses to Epstein-Barr virus (EBV) lytic cycle expressed antigens display a hierarchy of immunodominance, in which responses to epitopes of immediate-early (IE) and some early (E) antigens are more frequently observed than responses to epitopes of late (L) expressed antigens. It has been proposed that this hierarchy, which correlates with the phase-specific efficiency of antigen presentation, may be due to the influence of viral immune-evasion genes. At least three EBV-encoded genes, BNLF2a, BGLF5 and BILF1, have the potential to inhibit processing and presentation of CD8+ T cell epitopes. Here we examined the relative contribution of these genes to modulation of CD8+ T cell recognition of EBV lytic antigens expressed at different phases of the replication cycle in EBV-transformed B-cells (LCLs) which spontaneously reactivate lytic cycle. Selective shRNA-mediated knockdown of BNLF2a expression led to more efficient recognition of immediate-early (IE)- and early (E)-derived epitopes by CD8+ T cells, while knock down of BILF1 increased recognition of epitopes from E and late (L)-expressed antigens. Contrary to what might have been predicted from previous ectopic expression studies in EBV-negative model cell lines, the shRNA-mediated inhibition of BGLF5 expression in LCLs showed only modest, if any, increase in recognition of epitopes expressed in any phase of lytic cycle. These data indicate that whilst BNLF2a interferes with antigen presentation with diminishing efficiency as lytic cycle progresses (IE>E>>L), interference by BILF1 increases with progression through lytic cycle (IE

CD8+ T cell responses to lytic EBV infection: late antigen specificities as subdominant components of the total response.

EBV elicits primary CD8(+) T cell responses that, by T cell cloning from infectious mononucleosis (IM) patients, appear skewed toward immediate early (IE) and some early (E) lytic cycle proteins, with late (L) proteins rarely targeted. However, L Ag-specific responses have been detected regularly in polyclonal T cell cultures from long-term virus carriers. To resolve this apparent difference between responses to primary and persistent infection, 13 long-term carriers were screened in ex vivo IFN-γ ELISPOT assays using peptides spanning the two IE, six representative E, and seven representative L proteins. This revealed memory CD8 responses to 44 new lytic cycle epitopes that straddle all three protein classes but, in terms of both frequency and size, maintain the IE > E > L hierarchy of immunodominance. Having identified the HLA restriction of 10 (including 7 L) new epitopes using memory CD8(+) T cell clones, we looked in HLA-matched IM patients and found such reactivities but typically at low levels, explaining why they had gone undetected in the original IM clonal screens. Wherever tested, all CD8(+) T cell clones against these novel lytic cycle epitopes recognized lytically infected cells naturally expressing their target Ag. Surprisingly, however, clones against the most frequently recognized L Ag, the BNRF1 tegument protein, also recognized latently infected, growth-transformed cells. We infer that BNRF1 is also a latent Ag that could be targeted in T cell therapy of EBV-driven B-lymphoproliferative disease.

High concentration aqueous sodium fluoride certified reference materials for forensic use certified by complexometric titration.

Sodium fluoride in concentrations of 1 to 2 % is used to prevent the formation of ethanol in blood and urine samples that are to be analysed for ethanol content. The majority of such samples form part of forensic investigations into alleged drunken driving. In South Africa, the laboratory performing the tests is required to prove that the sodium fluoride concentration in the blood samples is above 1 g/100 ml on receipt. This is done by using a fluoride ion-selective electrode calibrated with external aqueous solutions of sodium fluoride. The National Metrology Institute of South Africa (NMISA) prepares sodium fluoride solutions in concentrations from 0.3 to 3.0 g/100 ml. No other certified sodium fluoride reference solutions in these concentrations are available commercially. The sodium fluoride is certified by precipitation of the fluoride as lead chlorofluoride (PbClF) through the addition of a known excess of lead nitrate. The excess lead is back-titrated with ethylenediamine tetraacetic acid (EDTA) using a photometric electrode to detect the endpoint. Aqueous sodium fluoride solutions are prepared and the concentrations verified by the precipitation/back-titration method. This paper shows the application of a classical complexometric method to the certification of reference materials and describes the procedures for the preparation of the sodium fluoride solutions, verification of the concentrations, homogeneity and stability by primary titrimetry. It also briefly covers the calculation of uncertainty, the establishment of traceability and the quality control measures applied to ensure the quality of the certified reference materials (CRMs).

Evaluating time in therapeutic range for hemodialysis patients taking warfarin.

Warfarin is frequently used in the hemodialysis (HD) population for atrial fibrillation (AF) and venous thromboembolism (VTE); however, there is insufficient evidence to support this practice. Given that HD patients have 3 - 10 times the risk for both stroke and bleeding than the general population, anticoagulation in these patients is controversial. Time in therapeutic range (TTR) is accepted as a surrogate outcome of clinical effectiveness and safety of warfarin. The primary objective of this study was to evaluate TTR in an HD population. A 6-year retrospective chart review was performed in 46 HD patients on warfarin (target international normaized ratio (INR)=2-3). One year of patient data was collected, which included weekly INRs, demographics and clinical outcomes. TTR was calculated using the Rosendaal and fraction of INRs in range methods. The mean TTR using the Rosendaal and fraction of INRs in range method was 49.2±14.6% and 44.2±13.5%, respectively. Patients were 3 times more likely to be below target than above it, suggesting they were more often at risk of inadequate efficacy rather than toxicity. There were 9 serious bleeding and 9 thrombotic events; these occurred in patients with a TTR<60%. For the 9 serious bleeding events, the median INR on the day of the event was 2.1 (IQR 1.81-2.75). In conclusion, this HD unit is not meeting the TTR goal established in the literature and patients are often subtherapeutic. Further studies to investigate ways to improve TTR are warranted. Ultimately, a prospective study evaluating the safety and efficacy of warfarin in HD patients is needed.

Production of drug metabolites by immobilised Cunninghamella elegans: from screening to scale up.

Cunninghamella elegans is a fungus that has been used extensively as a microbial model of mammalian drug metabolism, whilst its potential as a biocatalyst for the preparative production of human drug metabolites has been often proposed, little effort has been made to enable this. Here, we describe a workflow for the application of C. elegans for the production of drug metabolites, starting from well-plate screening assays leading to the preparative production of drug metabolites using fungus immobilised either in alginate or as a biofilm. Using 12- and 96-well plates, the simultaneous screening of several drug biotransformations was achieved. To scale up the biotransformation, both modes of immobilisation enabled semi-continuous production of hydroxylated drug metabolites through repeated addition of drug and rejuvenation of the fungus. It was possible to improve the productivity in the biofilm culture for the production of 4'-hydroxydiclofenac from 1 mg/l h to over 4 mg/l h by reducing the incubation time for biotransformation and the number of rejuvenation steps.

Statistical analysis plan for cluster randomised trial to evaluate a community-level complementary food safety and hygiene and nutrition intervention in Mali: the MaaCiwara study.

BACKGROUND: Diarrheal disease is a significant cause of morbidity and mortality in under-fives in many low- and middle-income countries. Changes in food safety, hygiene practices, and nutrition around the weaning period may reduce the risk of disease and improve infant development. The MaaCiwara study aims to evaluate the effectiveness of a community-based educational intervention designed to improve food safety and hygiene behaviours, as well as child nutrition. This update article describes the statistical analysis plan for the MaaCiwara study in detail. METHODS AND DESIGN: The MaaCiwara study is a parallel group, two-arm, superiority cluster randomised controlled trial with baseline measures, involving 120 clusters of rural and urban communities. These clusters are randomised to either receive the community-based behaviour change intervention or to the control group. The study participants will be mother-child pairs, with children aged between 6 and 36 months. Data collection involves a day of observation and interviews with each participating mother-child pair, conducted at baseline, 4 months, and 15 months post-intervention. The primary analysis aims to estimate the effectiveness of the intervention on changes to complementary food safety and preparation behaviours, food and water contamination, and diarrhoea. The primary outcomes will be analysed generalised linear mixed models, at individual level, accounting for clusters and rural/urban status to estimate the difference in outcomes between the intervention and control groups. Secondary outcomes include maternal autonomy, enteric infection, nutrition, child anthropometry, and development scores. In addition, structural equation analysis will be conducted to examine the causal relationships between the different outcomes. TRIAL REGISTRATION: International Standard Randomised Controlled Trial Number (ISRCTN) register: ISRCTN14390796 . Registered on 13 December 2021.