High-protein meals require 30% additional insulin to prevent delayed postprandial hyperglycaemia.

AIM: To determine the amount of additional insulin required for a high-protein meal to prevent postprandial hyperglycaemia in individuals with type 1 diabetes using insulin pump therapy. METHODS: In this randomized cross-over study, 26 participants aged 8-40 years, HbA1c < 65 mmol/mol (8.1%), received a 50 g protein, 30 g carbohydrate, low-fat (< 1 g) breakfast drink over five consecutive days at home. A standard insulin dose (100%) was compared with additional doses of 115, 130, 145 and 160% for the protein, in randomized order. Doses were commenced 15-min pre-drink and delivered over 3 h using a combination bolus with 65% of the standard dose given up front. Postprandial glycaemia was assessed by 4 h of continuous glucose monitoring. RESULTS: The 100% dosing resulted in postprandial hyperglycaemia. From 120 min, ≥ 130% doses resulted in significantly lower postprandial glycaemic excursions compared with 100% (P < 0.05). A 130% dose produced a mean (sd) glycaemic excursion that was 4.69 (2.42) mmol/l lower than control, returning to baseline by 4 h (P < 0.001). From 120 min, there was a significant increase in the risk of hypoglycaemia compared with control for 145% [odds ratio (OR) 25.4, 95% confidence interval (CI) 5.5-206; P < 0.001) and 160% (OR 103, 95% CI 19.2-993; P < 0.001). Some 81% (n = 21) of participants experienced hypoglycaemia following a 160% dose, whereas 58% (n = 15) experienced hypoglycaemia following a 145% dose. There were no hypoglycaemic events reported with 130%. CONCLUSIONS: The addition of 30% more insulin to a standard dose for a high-protein meal, delivered using a combination bolus, improves postprandial glycaemia without increasing the risk of hypoglycaemia.

Brain pseudotumour secondary to Behçet's disease.

Neuro-Behçet's disease (NBD) is a serious manifestation of Behçet's disease (BD) and can affect either the central or peripheral nervous systems, or both. It occurs in 10-50% of patients with BD. We report on a patient with an unusual intraparenchymal lesion, initially thought to be a brain tumour. Histological examination revealed vasculitis consistent with BD. Clinicians should include NBD as a differential diagnosis when considering an isolated inflammatory intracranial lesion.

Associations between HbA1c and continuous glucose monitoring-derived glycaemic variables.

AIMS: To identify clinically useful associations between HbA1c levels and various continuous glucose monitoring-derived metrics. METHODS: We retrospectively analysed end-of-study HbA1c levels and >2 weeks of continuous glucose monitoring data collected from 530 adults with Type 1 diabetes or insulin-requiring Type 2 diabetes during four randomized trials. Each trial lasted ≥24 weeks and provided central laboratory end-of-study HbA1c levels and continuous glucose monitoring data from the preceding 3 months. Participants were assigned to groups based on either HbA1c levels or continuous glucose monitoring-derived glucose values. RESULTS: HbA1c was strongly correlated with mean glucose value (r=0.80), time spent with glucose values in the 3.9-10.0 mmol/l range (time in range; r=-0.75) and percentage of glucose values >13.9 mmol/l (r=0.72), but was weakly correlated with the percentage of glucose values <3.9 mmol/l (r=-0.39) or <3.0 mmol/l (r=-0.21). The median percentage of glucose values <3.0 mmol/l was <1.2% (<20 min/day) for all HbA1c -based groups, but the median percentage of values >13.9 mmol/l varied from 2.5% (0.6 h/day) to 27.8% (6.7 h/day) in the lowest and highest HbA1c groups, respectively. More than 90% of participants with either <2% of glucose values >13.9 mmol/l, mean glucose <7.8 mmol/l, or time in range >80% had HbA1c levels ≤53 mmol/mol (≤7.0%). For participants with HbA1c ≥64 mmol/mol (≥8.0%), the median time in range was 44%, with 90% of participants having a time in range of <59%. CONCLUSIONS: The associations shown in the present study suggest that continuous glucose monitoring-derived metrics may help guide diabetes therapy intensification efforts in an HbA1c -independent manner.

Cost implications for the NHS of using the Alere™ i Influenza A & B near patient test with nasal swabs.

BACKGROUND: Influenza is an acute viral infection of the respiratory tract. A rapid confirmatory diagnosis of influenza is important, since it is highly transmissible and outbreaks of influenza within the hospital setting increase morbidity and mortality. The objective of this study was to evaluate the cost implications, from the perspective of the UK NHS, of using on-label nasal swabs with the Alere™ i Influenza A & B test in a near patient setting. METHODS: A cost consequence model was developed. The time horizon of the model was from hospital admission on suspicion of influenza until the end of treatment (following a diagnosis of influenza or discharge from hospital). Data on the prevalence of influenza and the sensitivity and specificity of the Alere™ i Influenza A & B test came from two prospective observational diagnostic accuracy studies. Costs were obtained from published resources. Uncertainties in the model data were investigated using deterministic, one-way sensitivity analyses. RESULTS: Using the Alere™ i Influenza A & B point of care test with nasal swabs (on label) in NHS medical assessment units and emergency departments could save approximately £242,730 per 1000 adults presenting with influenza-like symptoms. The main cause for this was reduced times to availability of the result compared with the laboratory RT-PCR test. Other key drivers of savings were the cost of isolation, the prevalence of influenza, the specificity of the test, and the availability of isolation resources. CONCLUSIONS: The Alere™ i Influenza A & B point of care test would have greatest impact in hospitals that have extensive delays in the time to receive a result. Sensitivity analyses identified the model parameters which would have greatest effect on the result and confirmed that assumptions were conservative, i.e. did not change key results.

Missed opportunities to diagnose syphilis prior to the development of sight-losing uveitis.

The incidence of syphilis in the UK is rapidly rising. Uveitis (intraocular inflammation) usually occurs in the secondary or later stages of syphilis infection and is sight-threatening. Methods A retrospective analysis of the demographics, presentation, diagnosis, treatment and outcomes of patients with syphilitic uveitis managed in Newcastle from 2005-2016 was carried out. Results Ten males (19 eyes) with syphilitic uveitis had a generally good visual and serological response to penicillin treatment. In eight of the patients there had been a failure to test for syphilis during assessments by various medical practitioners for unexplained symptoms that were attributable to syphilis prior to the eye involvement. Conclusion Uveitis associated with syphilis can be sight-threatening but responds well to treatment. In our case series there were multiple missed opportunities to diagnose syphilis prior to presentation with eye disease, with a general failure of healthcare professionals to take an adequate sexual history.

Optimizing the combination insulin bolus split for a high-fat, high-protein meal in children and adolescents using insulin pump therapy.

AIMS: To determine the optimum combination bolus split to maintain postprandial glycaemia with a high-fat and high-protein meal in young people with Type 1 diabetes. METHODS: A total of 19 young people (mean age 12.9 ± 6.7 years) participated in a randomized, repeated-measures trial comparing postprandial glycaemic control across six study conditions after a high-fat and high-protein meal. A standard bolus and five different combination boluses were delivered over 2 h in the following splits: 70/30 = 70% standard /30% extended bolus; 60/40=60% standard/40% extended bolus; 50/50=50% standard/50% extended bolus; 40/60=40% standard/60% extended bolus; and 30/70=30% standard/70% extended bolus. Insulin dose was determined using the participant's optimized insulin:carbohydrate ratio. Continuous glucose monitoring was used to assess glucose excursions for 6 h after the test meal. RESULTS: Standard bolus and combination boluses 70/30 and 60/40 controlled the glucose excursion up to 120 min. From 240 to 300 min after the meal, the glucose area under the curve was significantly lower for combination bolus 30/70 compared with standard bolus (P=0.004). CONCLUSIONS: High-fat and high-protein meals require a ≥60% insulin:carbohydrate ratio as a standard bolus to control the initial postprandial rise. Additional insulin at an insulin:carbohydrate ratio of up to 70% is needed in the extended bolus for a high fat and protein meal to prevent delayed hyperglycaemia.

Diagnostic accuracy and cost analysis of the Alere™ i Influenza A&B near-patient test using throat swabs.

BACKGROUND: Clinical diagnostic sensitivity alone is inadequate in the diagnosis of influenza. Polymerase chain reaction (PCR) testing is sensitive but the inherent delays in result availability potentially prolong time to isolation and treatment. Until recently no near-patient test (NPT) has demonstrated adequate sensitivity for routine clinical use. AIM: To evaluate diagnostic accuracy, time to result availability, clinical impact, and cost consequences of Alere™ i Influenza A&B NPT (Alere Inc., Waltham, MA, USA) using off-label throat swabs. METHODS: Prospective, multi-centre [four UK National Health Service (NHS) hospitals], diagnostic accuracy cohort study with cost modelling. Throat swab samples from suspected influenza patients were tested for influenza using the reference standard of PCR; a second throat swab was tested using NPT. FINDINGS: A total of 827 participants were recruited; 589 were suitable for analysis: sensitivity was 75.8% [95% confidence interval (CI): 67.0-84.6]; specificity was 96.8% (95% CI: 95.2-98.3). Sensitivity varied between Sheffield (Northern General Hospital: 82.1%; Royal Hallamshire Hospital: 83.3%) and other sites (Doncaster Royal Infirmary: 71.4%; Newcastle's Royal Victoria Infirmary: 50.0%) whereas specificity was high (92-100%). Positive predictive value (PPV) was 81.2% (95% CI: 72.9-89.5) with negative predictive value 95.6% (95% CI: 93.9-97.4) with observed prevalence of 15.4%. Median time to result for PCR was 1.1 days (on-site laboratories) and 5.2 days (remote laboratories). Isolation findings: 75% influenza positive not isolated; 69% of isolated participants did not have influenza. For a cohort of 1000 participants, annual estimated non-diagnostic cost savings with NPT are £215,040. CONCLUSION: This first prospective study of the Alere i NPT using throat swabs demonstrates high specificity, high PPV during seasonal epidemics, and rapid result availability which could lead to substantial cost savings.

Does efavirenz replacement improve neurological function in treated HIV infection?

OBJECTIVES: The contribution of specific antiretroviral drugs to cognitive function in HIV-infected people remains poorly understood. Efavirenz (EFV) may plausibly cause cognitive impairment. The objective of this study was therefore to determine whether chronic EFV therapy is a modifier of neurocognitive and neurometabolic function in the setting of suppressive highly active antiretroviral therapy. METHODS: We performed an open-label phase IV controlled trial. Adult subjects who were stable on suppressive EFV therapy for at least 6 months were switched to ritonavir-boosted lopinavir (LPV/r) with no change in the nucleoside reverse transcriptase inhibitor (NRTI) backbone. The following parameters were assessed before and 10 weeks after therapy switch: cognitive function (by CogState® computerized battery); brain metabolites (by proton magnetic resonance spectroscopy); brain activity [by attentional processing task-based functional magnetic resonance imaging]; and sleep quantity and quality [by sleep diary, Pittsburgh Sleep Quality Index (PSQI) and Epworth Sleepiness Scale]. RESULTS: Sixteen subjects completed the study. Despite most subjects (81%) self-reporting memory problems at baseline, cognitive function, brain metabolites, and brain activity showed no change at 10 weeks after switch. Sleep quality improved on switch off EFV [mean PSQI (standard deviation): EFV, 8.5 (6.5); LPV/r, 5.8 (5.5); mean difference -0.4; 95% confidence interval -6.0 to -0.7]. CONCLUSIONS: This is the first study to assess the effects of chronic EFV therapy on neurological function in a controlled setting. We conclude that EFV withdrawal is unlikely to result in significant modification of neurocognitive function in otherwise stable HIV-infected people.

Obesity in the HIV-infected population in Northeast England: a particular issue in Black-African women.

People living with HIV are surviving longer on successful antiretroviral therapy and obesity rates are increasing. We sought to determine the prevalence of being overweight or obese in a regional population of people living with HIV and to explore the demographic and clinical characteristics associated with obesity or being overweight. Data on patients attending three Northeast England clinics were collected including body mass index and demographics. The prevalence of being overweight (body mass index ≥ 25 kg/m2) or obese (body mass index ≥ 30 kg/m2) was determined and compared with regional population data. Associations between being overweight or obese and demographic and other data were further explored using logistic regression models. In 560 patients studied (median age 45 years, 26% Black-African and 69% male), 65% were overweight/obese and 26% obese, which is similar to the local population. However, 83% and 48% of Black-African women were overweight/obese or obese, respectively, with 11% being morbidly obese (body mass index > 40 kg/m2). In the multivariate analyses, the only factors significantly associated with obesity were Black-African race (adjusted odds ratio 2.78, 95% confidence interval 1.60-4.85) and type 2 diabetes (adjusted odds ratio 4.23, 95% confidence interval 1.81-9.91). Levels of obesity and overweight in people living with HIV are now comparable to the levels in the local population of Northeast England; however, the prevalence is significantly higher in Black-African women. Given the additional risk factors for cardiovascular disease inherent in people living with HIV, better strategies to prevent, identify and manage obesity in this population are needed.

Intranasal administration of RSV antigen-expressing MCMV elicits robust tissue-resident effector and effector memory CD8+ T cells in the lung.

Cytomegalovirus vectors are promising delivery vehicles for vaccine strategies that aim to elicit effector CD8+ T cells. To determine how the route of immunization affects CD8+ T-cell responses in the lungs of mice vaccinated with a murine cytomegalovirus vector expressing the respiratory syncytial virus matrix (M) protein, we infected CB6F1 mice via the intranasal or intraperitoneal route and evaluated the M-specific CD8+ T-cell response at early and late time points. We found that intranasal vaccination generated robust and durable tissue-resident effector and effector memory CD8+ T-cell populations that were undetectable after intraperitoneal vaccination. The generation of these antigen-experienced cells by intranasal vaccination resulted in earlier T-cell responses, interferon gamma secretion, and viral clearance after respiratory syncytial virus challenge. Collectively, these findings validate a novel approach to vaccination that emphasizes the route of delivery as a key determinant of immune priming at the site of vulnerability.

Interaction of a dengue virus NS1-derived peptide with the inhibitory receptor KIR3DL1 on natural killer cells.

Killer immunoglobulin-like receptors (KIRs) interact with human leucocyte antigen (HLA) class I ligands and play a key role in the regulation and activation of NK cells. The functional importance of KIR-HLA interactions has been demonstrated for a number of chronic viral infections, but to date only a few studies have been performed in the context of acute self-limited viral infections. During our investigation of CD8(+) T cell responses to a conserved HLA-B57-restricted epitope derived from dengue virus (DENV) non-structural protein-1 (NS1), we observed substantial binding of the tetrameric complex to non-T/non-B lymphocytes in peripheral blood mononuclear cells (PBMC) from a long-standing clinical cohort in Thailand. We confirmed binding of the NS1 tetramer to CD56(dim) NK cells, which are known to express KIRs. Using depletion studies and KIR-transfected cell lines, we demonstrated further that the NS1 tetramer bound the inhibitory receptor KIR3DL1. Phenotypical analysis of PBMC from HLA-B57(+) subjects with acute DENV infection revealed marked activation of NS1 tetramer-binding natural killer (NK) cells around the time of defervescence in subjects with severe dengue disease. Collectively, our findings indicate that subsets of NK cells are activated relatively late in the course of acute DENV illness and reveal a possible role for specific KIR-HLA interactions in the modulation of disease outcomes.

The impact of HLA class I and EBV latency-II antigen-specific CD8(+) T cells on the pathogenesis of EBV(+) Hodgkin lymphoma.

In 40% of cases of classical Hodgkin lymphoma (cHL), Epstein-Barr virus (EBV) latency-II antigens [EBV nuclear antigen 1 (EBNA1)/latent membrane protein (LMP)1/LMP2A] are present (EBV(+) cHL) in the malignant cells and antigen presentation is intact. Previous studies have shown consistently that HLA-A*02 is protective in EBV(+) cHL, yet its role in disease pathogenesis is unknown. To explore the basis for this observation, gene expression was assessed in 33 cHL nodes. Interestingly, CD8 and LMP2A expression were correlated strongly and, for a given LMP2A level, CD8 was elevated markedly in HLA-A*02(-) versus HLA-A*02(+) EBV(+) cHL patients, suggesting that LMP2A-specific CD8(+) T cell anti-tumoral immunity may be relatively ineffective in HLA-A*02(-) EBV(+) cHL. To ascertain the impact of HLA class I on EBV latency antigen-specific immunodominance, we used a stepwise functional T cell approach. In newly diagnosed EBV(+) cHL, the magnitude of ex-vivo LMP1/2A-specific CD8(+) T cell responses was elevated in HLA-A*02(+) patients. Furthermore, in a controlled in-vitro assay, LMP2A-specific CD8(+) T cells from healthy HLA-A*02 heterozygotes expanded to a greater extent with HLA-A*02-restricted compared to non-HLA-A*02-restricted cell lines. In an extensive analysis of HLA class I-restricted immunity, immunodominant EBNA3A/3B/3C-specific CD8(+) T cell responses were stimulated by numerous HLA class I molecules, whereas the subdominant LMP1/2A-specific responses were confined largely to HLA-A*02. Our results demonstrate that HLA-A*02 mediates a modest, but none the less stronger, EBV-specific CD8(+) T cell response than non-HLA-A*02 alleles, an effect confined to EBV latency-II antigens. Thus, the protective effect of HLA-A*02 against EBV(+) cHL is not a surrogate association, but reflects the impact of HLA class I on EBV latency-II antigen-specific CD8(+) T cell hierarchies.

Clonotypically similar hybrid αß T cell receptors can exhibit markedly different surface expression, antigen specificity and cross-reactivity.

Emerging data indicate that particular major histocompatibility complex (MHC)-bound antigenic peptides can be recognized by identical or near-identical αß T cell receptors (TCRs) in different individuals. To establish the functional relevance of this phenomenon, we artificially paired α and ß chains from closely related TCRs specific for the human leucocyte antigen (HLA)-B*35:01-restricted HIV-1 negative regulatory factor (Nef)-derived epitope VY8 (VPLRPMTY, residues 74-81). Several hybrid TCRs generated in this manner failed to express at the cell surface, despite near homology with naturally isolated αß chain combinations. Moreover, a substantial proportion of those αß TCRs that did express lost specificity for the index VY8 peptide sequence. One such hybrid αß pair gained neo-variant specificity in the context of the VY8 backbone. Collectively, these data show that clonotypically similar TCRs can display profound differences in surface expression, antigen specificity and cross-reactivity with potential relevance for the control of mutable viruses.

T cell receptor binding affinity governs the functional profile of cancer-specific CD8+ T cells.

Antigen-specific T cell receptor (TCR) gene transfer via patient-derived T cells is an attractive approach to cancer therapy, with the potential to circumvent immune regulatory networks. However, high-affinity tumour-specific TCR clonotypes are typically deleted from the available repertoire during thymic selection because the vast majority of targeted epitopes are derived from autologous proteins. This process places intrinsic constraints on the efficacy of T cell-based cancer vaccines and therapeutic strategies that employ naturally generated tumour-specific TCRs. In this study, we used altered peptide ligands and lentivirus-mediated transduction of affinity-enhanced TCRs selected by phage display to study the functional properties of CD8(+) T cells specific for three different tumour-associated peptide antigens across a range of binding parameters. The key findings were: (i) TCR affinity controls T cell antigen sensitivity and polyfunctionality; (ii) supraphysiological affinity thresholds exist, above which T cell function cannot be improved; and (iii) T cells transduced with very high-affinity TCRs exhibit cross-reactivity with self-derived peptides presented by the restricting human leucocyte antigen. Optimal system-defined affinity windows above the range established for natural tumour-specific TCRs therefore allow the enhancement of T cell effector function without off-target effects. These findings have major implications for the rational design of novel TCR-based biologics underpinned by rigorous preclinical evaluation.

Young women with polycystic ovary syndrome have raised levels of circulating annexin V-positive platelet microparticles.

STUDY QUESTION: Are circulating microparticles (MPs) altered in young women with polycystic ovary syndrome (PCOS)? SUMMARY ANSWER: Women with PCOS have elevated concentrations of circulating platelet-derived MPs, which exhibit increased annexin V binding and altered microRNA (miR) profiles compared with healthy volunteers. WHAT IS KNOWN ALREADY: Some studies have shown that cardiovascular risk is increased in young women with PCOS but the mechanisms by which this occurs are uncertain. Circulating MPs are elevated in patients with cardiovascular disease but the characteristics of MPs in patients with PCOS are unclear. STUDY DESIGN, SIZE, DURATION: Case-control study comprising 17 women with PCOS (mean ± SD; age 31 ± 7 years, BMI 29 ± 6 kg/m(2)) and 18 healthy volunteers (age 31 ± 6 years, BMI 30 ± 6 kg/m(2)). PARTICIPANTS/MATERIALS, SETTING, METHODS: The study was conducted in a University hospital. Nanoparticle tracking analysis (NTA) and flow cytometry (CD41 platelet, CD11b monocyte, CD144 endothelial) were used to determine MP size, concentration, cellular origin and annexin V positivity (reflecting phosphatidylserine exposure). Fatty acid analysis was performed by gas chromatography and MP miR expression profiles were compared by microarray. MAIN RESULTS AND THE ROLE OF CHANCE: PCOS subjects showed increased MP concentrations compared with healthy volunteers (mean ± SD; 11.5 ± 5 × 10(12)/ml versus 10.0 ± 4 × 10(12)/ml, respectively; P = 0.03), which correlated with the homeostasis model of insulin resistance (r = 0.53, P = 0.03). This difference was predominantly seen in MPs whose size was in the small exosomal range (<150 nm in diameter, P< 0.05). PCOS patients showed a greater percentage of annexin V(+) MPs compared with healthy volunteers (84 ± 18 versus 74 ± 24%, respectively, P = 0.05) but the cellular origin of MPs, which were predominantly platelet-derived (PCOS: 99 ± 0.9%; controls: 99 ± 2.5%), did not differ. MP fatty acid concentration and composition was similar between groups but 16 miRs were differentially expressed (P < 0.05). LIMITATIONS, REASON FOR CAUTION: Patients with PCOS were classified by the Rotterdam criteria, which describes a less severe metabolic phenotype than other definitions of the syndrome. Our findings may thus not be generalizable to all patients with PCOS. MicroRNA expression analysis was only undertaken in an exploratory subset of the overall study population hence, validation of our findings in a larger cohort is mandatory. Furthermore, miR levels were unaltered for the highly expressed miRs and it is unclear whether differences in the lowly expressed miRs carries pathological relevance. WIDER IMPLICATIONS OF THE FINDINGS: This study suggests that women with PCOS have an altered MP profile but further studies are needed to confirm this, to explore the mechanisms by which these alterations develop and to establish whether therapies that improve insulin sensitivity are able to reduce circulating MP concentrations. STUDY FUNDING/COMPETING INTERESTS: The study was funded by grants from the Wales Heart Research Institute and Mrs John Nixon Scholarship. The authors have no conflicts of interest to declare.

Comparison of peptide-major histocompatibility complex tetramers and dextramers for the identification of antigen-specific T cells.

Fluorochrome-conjugated peptide-major histocompatibility complex (pMHC) multimers are widely used for flow cytometric visualization of antigen-specific T cells. The most common multimers, streptavidin-biotin-based 'tetramers', can be manufactured readily in the laboratory. Unfortunately, there are large differences between the threshold of T cell receptor (TCR) affinity required to capture pMHC tetramers from solution and that which is required for T cell activation. This disparity means that tetramers sometimes fail to stain antigen-specific T cells within a sample, an issue that is particularly problematic when staining tumour-specific, autoimmune or MHC class II-restricted T cells, which often display TCRs of low affinity for pMHC. Here, we compared optimized staining with tetramers and dextramers (dextran-based multimers), with the latter carrying greater numbers of both pMHC and fluorochrome per molecule. Most notably, we find that: (i) dextramers stain more brightly than tetramers; (ii) dextramers outperform tetramers when TCR-pMHC affinity is low; (iii) dextramers outperform tetramers with pMHC class II reagents where there is an absence of co-receptor stabilization; and (iv) dextramer sensitivity is enhanced further by specific protein kinase inhibition. Dextramers are compatible with current state-of-the-art flow cytometry platforms and will probably find particular utility in the fields of autoimmunity and cancer immunology.