Anti-Flavivirus Vaccines: Review of the Present Situation and Perspectives of Subunit Vaccines Produced in Escherichia coli.

This article aims to review the present status of anti-flavivirus subunit vaccines, both those at the experimental stage and those already available for clinical use. Aspects regarding development of vaccines to Yellow Fever virus, (YFV), Dengue virus (DENV), West Nile virus (WNV), Zika virus (ZIKV), and Japanese encephalitis virus (JEV) are highlighted, with particular emphasis on purified recombinant proteins generated in bacterial cells. Currently licensed anti-flavivirus vaccines are based on inactivated, attenuated, or virus-vector vaccines. However, technological advances in the generation of recombinant antigens with preserved structural and immunological determinants reveal new possibilities for the development of recombinant protein-based vaccine formulations for clinical testing. Furthermore, novel proposals for multi-epitope vaccines and the discovery of new adjuvants and delivery systems that enhance and/or modulate immune responses can pave the way for the development of successful subunit vaccines. Nonetheless, advances in this field require high investments that will probably not raise interest from private pharmaceutical companies and, therefore, will require support by international philanthropic organizations and governments of the countries more severely stricken by these viruses.

Autoantigens PLA2R and THSD7A in membranous nephropathy share a common epitope motif in the N-terminal domain.

Patients with membranous nephropathy have autoantibodies against PLA2R (up to 80%), or THSD7A (up to 2%). We previously described the immunodominant epitope within PLA2R but epitopes in THSD7A are still unknown. To find anti-THSD7A sera for this study, we screened 1843 sera from biopsy-proven MN patients by ELISA and identified 22 sera as anti-THSD7A positive representing 1.2% of MN cases. Anti-THSD7A positive sera were further characterized by western blotting and slot blotting on THSD7A protein fragments and peptides. Real time interaction analyses and antibodies off-rate could be reliably determined using bio-layer interferometry. A signature motif in the N-terminal domain of THSD7A (T28mer) with sequence homology to the major PLA2R epitope (P28mer) was identified. B-cell epitope prediction analysis and homology modelling revealed this sequence to be antigenic and surface available suggesting it is accessible for the antibody to bind. All ten selected sera bound to the T28mer confirming this sequence as a dominant epitope in THSD7A. Reactivity to this sequence was lost following kallikrein protease cleavage within the predicted epitope. Importantly, cross-reactivity of both PLA2R and THSD7A autoantibodies was observed at the peptide but not the protein level. We propose that this common motif shared by both autoantigens could be an epitope involved in the initial B-cell triggering event in MN.

Anti-flavivirus vaccines: review of the present situation and perspectives of subunit vaccines produced in escherichia coli

This article aims to review the present status of anti-flavivirus subunit vaccines, both those at the experimental stage and those already available for clinical use. Aspects regarding development of vaccines to Yellow Fever virus, (YFV), Dengue virus (DENV), West Nile virus (WNV), Zika virus (ZIKV), and Japanese encephalitis virus (JEV) are highlighted, with particular emphasis on purified recombinant proteins generated in bacterial cells. Currently licensed anti-flavivirus vaccines are based on inactivated, attenuated, or virus-vector vaccines. However, technological advances in the generation of recombinant antigens with preserved structural and immunological determinants reveal new possibilities for the development of recombinant protein-based vaccine formulations for clinical testing. Furthermore, novel proposals for multi-epitope vaccines and the discovery of new adjuvants and delivery systems that enhance and/or modulate immune responses can pave the way for the development of successful subunit vaccines. Nonetheless, advances in this field require high investments that will probably not raise interest from private pharmaceutical companies and, therefore, will require support by international philanthropic organizations and governments of the countries more severely stricken by these viruses

Persistence and Intra-Host Genetic Evolution of Zika Virus Infection in Symptomatic Adults: A Special View in the Male Reproductive System.

We followed the presence of Zika virus (ZIKV) in four healthy adults (two men and two women), for periods ranging from 78 to 298 days post symptom onset. The patients were evaluated regarding the presence of the virus in different body fluids (blood, saliva, urine and semen), development of immune responses (including antibodies, cytokines and chemokines), and virus genetic variation within samples collected from semen and urine during the infection course. The analysis was focused primarily on the two male patients who shed the virus for up to 158 days after the initial symptoms. ZIKV particles were detected in the spermatozoa cytoplasm and flagella, in immature sperm cells and could also be isolated from semen in cell culture, confirming that the virus is able to preserve integrity and infectivity during replication in the male reproductive system (MRS). Despite the damage caused by ZIKV infection within the MRS, our data showed that ZIKV infection did not result in infertility at least in one of the male patients. This patient was able to conceive a child after the infection. We also detected alterations in the male genital cytokine milieu, which could play an important role in the replication and transmission of the virus which could considerably increase the risk of ZIKV sexual spread. In addition, full genome ZIKV sequences were obtained from several samples (mainly semen), which allowed us to monitor the evolution of the virus within a patient during the infection course. We observed genetic changes over time in consensus sequences and lower frequency intra-host single nucleotide variants (iSNV), that suggested independent compartmentalization of ZIKV populations in the reproductive and urinary systems. Altogether, the present observations confirm the risks associated with the long-term replication and shedding of ZIKV in the MRS and help to elucidate patterns of intra-host genetic evolution during long term replication of the virus.

Persistence and intra-host genetic evolution of zika virus infection in symptomatic adults: A special view in the male reproductive system

We followed the presence of Zika virus (ZIKV) in four healthy adults (two men and two women), for periods ranging from 78 to 298 days post symptom onset. The patients were evaluated regarding the presence of the virus in different body fluids (blood, saliva, urine and semen), development of immune responses (including antibodies, cytokines and chemokines), and virus genetic variation within samples collected from semen and urine during the infection course. The analysis was focused primarily on the two male patients who shed the virus for up to 158 days after the initial symptoms. ZIKV particles were detected in the spermatozoa cytoplasm and flagella, in immature sperm cells and could also be isolated from semen in cell culture, confirming that the virus is able to preserve integrity and infectivity during replication in the male reproductive system (MRS). Despite the damage caused by ZIKV infection within the MRS, our data showed that ZIKV infection did not result in infertility at least in one of the male patients. This patient was able to conceive a child after the infection. We also detected alterations in the male genital cytokine milieu, which could play an important role in the replication and transmission of the virus which could considerably increase the risk of ZIKV sexual spread. In addition, full genome ZIKV sequences were obtained from several samples (mainly semen), which allowed us to monitor the evolution of the virus within a patient during the infection course. We observed genetic changes over time in consensus sequences and lower frequency intra-host single nucleotide variants (iSNV), that suggested independent compartmentalization of ZIKV populations in the reproductive and urinary systems. Altogether, the present observations confirm the risks associated with the long-term replication and shedding of ZIKV in the MRS and help to elucidate patterns of intra-host genetic evolution during long term replication of the virus

Assessing the incidence trend and characteristics of eosinophilic esophagitis in children in Olmsted County, Minnesota.

Previous studies reported increased eosinophilic esophagitis (EoE) incidence in children. It is unclear whether this reported increased EoE incidence is true or due to increased recognition and diagnostic endoscopy among children. A population-based study that evaluated EoE incidence in OC, Minnesota, from 1976 to 2005 concluded that EoE incidence increased significantly over the past three 5-year intervals (from 0.35 [range: 0-0.87] per 100,000 person-years for 1991-1995 to 9.45 [range: 7.13-11.77] per 100,000 person-years for 2001-2005). The aim of this study is to assess the change of incidence and characteristics of EoE in children in the same population between 2005 and 2015 and compare the findings to those reported in the previous study. We retrospectively reviewed the electronic medical records from Olmsted Medical Center and Mayo Clinic between 2005 and 2015, using Rochester Epidemiology Project (REP) resources. All children with EoE diagnosis based on the North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition (NASPGHAN) guidelines were included. The incidence and characteristics of children with EoE during the study period were compared to those diagnosed between 1995 and 2005. The incidence of EoE in children adjusted for age and sex was 5.31 per 100,000 population person-years in 1995, 15.2 in 2005, and 19.2 in 2015. Change in annual incidence and seasonal variation were not significant, (P = .48) and (P = .32), respectively. Between 2005 and 2015, 73 children received an EoE diagnosis (boys 49; 67%) compared to 16 children (boys 10; 62.5) between 1995 and 2005. Mean (SD) age at diagnosis was 7.5 (5.2) and 12.8 (4.3) years, respectively. Symptoms differed by age of presentation, with vomiting the most common in children younger than 5 years (41.1% and 43.5%) and dysphagia in those older than 5 years (35.6% and 60.9%). The incidence of EoE was not increased for any specific age-group during the study period (P = .49). This study showed increased incidence of EoE in children in Olmsted County between 2005 and 2015 compared to the incidence between 1995 and 2005 (5.31 per 100,000 population person-years in 1995, 15.2 in 2005, and 19.2 in 2015). However, between 2005 and 2015, the change of incidence was not statically significant, (P = .48) despite the steady increase of EGD performed during the same time frame (64 in 2005 to 144 in 2015). By comparing children diagnosed between 2005 and 2015 to those diagnosed between 1995 and 2005, the mean age at diagnosis was younger in the former group, 7.5 versus 12.8 years. Vomiting replaced dysphagia as the most common clinical presentation. Otherwise, the presenting symptom of EoE in children remained consistent across specific age groups.

Effect of Low-Dose Rapamycin on Senescence Markers and Physical Functioning in Older Adults with Coronary Artery Disease: Results of a Pilot Study.

Rapamycin, an mTOR inhibitor affects senescence through suppression of senescence-associated secretory phenotype (SASP). We studied the safety and feasibility of low-dose rapamycin and its effect on SASP and frailty in elderly undergoing cardiac rehabilitation (CR). 13 patients; 6 (0.5mg), 6 (1.0mg), and 1 patient received 2mg oral rapamycin (serum rapamycin <6ng/ml) daily for 12 weeks. Median age was 73.9±7.5 years and 12 were men. Serum interleukin-6 decreased (2.6 vs 4.4 pg/ml) and MMP-3 (26 vs 23.5 ng/ml) increased. Adipose tissue expression of mRNAs (arbitrary units) for MCP-1 (3585 vs 2020, p=0.06), PPAR-γ (1257 vs 1166), PAI-1 (823 vs 338, p=0.08) increased, whereas interleukin-8 (163 vs 312), TNF-α (75 vs 94) and p16 (129 vs 169) decreased. Cellular senescence-associated beta galactosidase activity (2.2% vs 3.6%, p=0.18) tended to decrease. We observed some correlation between some senescence markers and physical performance but no improvement in frailty with rapamycin was noted. (NCT01649960).

Beneficial effects of polyphenol-rich olive oil in patients with early atherosclerosis.

PURPOSE: Diets rich in plant-derived polyphenols such as olive oil (OO) and/or catechins such as epigallocatechin 3-gallate (EGCG) have been shown to reduce the incidence of cardiovascular diseases, potentially by improving endothelial function, an important surrogate for atherosclerosis. The possible augmentation of endothelial function with the combined efforts of OO and EGCG is intriguing, yet unknown. METHODS: Eighty-two patients with early atherosclerosis (presence of endothelial dysfunction) were enrolled in this double-blind, randomized trial with 52 completing the study. The aim of the study was to compare the effect of a daily intake of 30 ml simple OO, with 30 ml of EGCG-supplemented OO, on endothelial function as well as on inflammation and oxidative stress after a period of 4 months. Endothelial function was assessed noninvasively via peripheral arterial tonometry (Endo-PAT®). RESULTS: After 4 months, when OO and EGCG-supplemented OO groups were combined, OO significantly improved endothelial function (RHI, 1.59 ± 0.25-1.75 ± 0.45; p < 0.05). However, there were no significant differences in results between the two olive oil groups. Interestingly, with OO supplementation there was a significant reduction in inflammatory parameters: sICAM (196 to 183 ng/mL, p = < 0.001); white blood cells (WBCs) (6.0 × 109/L-5.8 × 109/L, p < 0.05); monocytes (0.48 × 109/L to 0.44 × 109/L, p = 0.05); lymphocytes (1.85 × 109/L to 1.6 × 109/L, p = 0.01); and platelets (242-229 × 109/L, p = 0.047). CONCLUSIONS: Improvement in endothelial dysfunction in patients with early atherosclerosis in association with significant reduction in leukocytes may suggest an important role of early cellular inflammatory mediators on endothelial function. The current study supports one potential mechanism for the role of olive oil, independent of EGCG, modestly supplemented to a healthy cardiovascular diet.

Segmental coronary endothelial dysfunction in patients with minimal atherosclerosis is associated with necrotic core plaques.

BACKGROUND/OBJECTIVE: Endothelial dysfunction and atherosclerosis are systemic disorders, but are often characterised by segmental involvement and complications. A potential mechanism for local involvement early in the disease process may be related to plaque composition. This study was designed to test the hypothesis that in patients with minimal coronary atherosclerosis, coronary artery segments with abnormal endothelial function have specific plaque characteristics. METHODS: Intravascular ultrasound (IVUS) images were obtained from 30 patients who underwent coronary endothelial function assessment. Spectral analysis of the IVUS radiofrequency data was used for assessment of plaque composition. IVUS findings of the coronary sections were compared according to the corresponding endothelial response to acetylcholine. RESULTS: Sections with a decrease epicardial coronary arterial diameter in response to acetylcholine had smaller baseline lumen (7.5 (2.4) mm(2) vs 8.8 (3.3) mm(2), p = 0.006) but larger plaque burden (37.1% (9.4%) vs 31% (7%), p = 0.003) than sections with normal endothelial response. Sections with endothelial dysfunction had larger necrotic core plaques: 0.13 (0.03-0.33) mm(2) vs 0.0 (0.0-0.07), p<0.001 and more dense calcium: 0.03 (IQR 0.0-0.13) mm(2) vs 0.0 (0.0-0.10) mm(2), p<0.01), than those with normal endothelial response. Only necrotic core area was associated with endothelial dysfunction (p<0.001) after adjusting for other measures. CONCLUSIONS: This study suggests that local coronary endothelial dysfunction in patients with minimal coronary atherosclerosis is associated with plaque characteristics that are typical of vulnerable plaques.

Deliberate Self-Harm (DSH): a follow-up study of Irish children.

This study aimed to look at rates of repetition in children presenting with Deliberate Self-Harm (DSH) to a paediatric A&E department. Children presenting with DSH to a paediatric A&E between 2000 and 2005 were invited to participate in the study. Telephone interviews collected information on demographic details and mental health functioning, including repetition of DSH. Data was obtained from 39 parents and 10 children (31 girls and 8 boys, mean age 15) 1 in 5 females (20%) had made a repeat attempt of DSH and 1 in 10 (10%) had repeated more than once. No males repeated self-harm. On going parental concern rather than clinician risk assessment at index episode predicted subsequent DSH. Given the poor predictive value of clinician risk assessment, all attempts of DSH must be taken seriously, especially those associated with ongoing parental concern.

Rosiglitazone enhances glucose uptake in glomerular podocytes using the glucose transporter GLUT1.

AIMS/HYPOTHESIS: Peroxisome proliferator-activated receptor (PPAR) gamma agonists are used increasingly in the treatment of type 2 diabetes. In the context of renal disease, PPARgamma agonists reduce microalbuminuria in diabetic nephropathy; however, the mechanisms underlying this effect are unknown. Glomerular podocytes are newly characterised insulin-sensitive cells and there is good evidence that they are targeted in diabetic nephropathy. In this study we investigated the functional and molecular effects of the PPARgamma agonist rosiglitazone on human podocytes. METHODS: Conditionally immortalised human podocytes were cultured with rosiglitazone and functional effects were measured with glucose-uptake assays. The effect of rosiglitazone on glucose uptake was also measured in 3T3-L1 adipocytes, nephrin-deficient podocytes, human glomerular endothelial cells, proximal tubular cells and podocytes treated with the NEFA palmitate. The role of the glucose transporter GLUT1 was investigated with immunofluorescence and small interfering RNA knockdown and the plasma membrane expression of GLUT1 was determined with bis-mannose photolabelling. RESULTS: Rosiglitazone significantly increased glucose uptake in wild-type podocytes and this was associated with translocation of GLUT1 to the plasma membrane. This effect was blocked with GLUT1 small interfering RNA. Nephrin-deficient podocytes, glomerular endothelial cells and proximal tubular cells did not increase glucose uptake in response to either insulin or rosiglitazone. Furthermore, rosiglitazone significantly increased basal and insulin-stimulated glucose uptake when podocytes were treated with the NEFA palmitate. CONCLUSIONS/INTERPRETATION: In conclusion, rosiglitazone has a direct and protective effect on glucose uptake in wild-type human podocytes. This represents a novel mechanism by which PPARgamma agonists may improve podocyte function in diabetic nephropathy.

Are we following the European charter? Children, parents and staff perceptions.

BACKGROUND: In 1988, the European Association for Children in Hospital (EACH) established a charter of rights setting out the guiding principles for the treatment of children in hospital. Our aim was to ascertain whether children, parents and staff in a children's hospital believe the European Charter is conformed to. METHODS: A total of 111 parents (response rate = 90%), 50 children (response rate = 100%), 61 nurses (response rate = 55%) and 41 doctors (response rate = 25%) agreed to participate. Questionnaires based on the 10 rights in the EACH Charter were administered to children, parents and staff. RESULTS: The majority of children and parents were happy with the relationship they had with staff. However, the findings suggest that children, parents and staff are concerned with the lack of facilities in hospital, including parental accommodation, play, education, age-appropriate wards and lack of privacy. Staff felt that many children undergo unnecessary admission and treatment in hospital. Many staff are reluctant to discuss issues such as side effects of medications with patients and do not encourage children to ask questions. Contrary to expectations, clinicians were reluctant to consider children under 16 as capable of giving consent, and most parents and children felt that children should be over 17 and 18 respectively. CONCLUSION: This paper highlights patients' and staff's perceptions regarding children's rights in hospital and discusses the changes needed to fully conform to the EACH Charter.

Comorbid conditions and outcomes after percutaneous coronary intervention.

OBJECTIVE: To evaluate whether adding comorbid conditions to a risk model can help predict in-hospital outcome and long-term mortality after percutaneous coronary intervention (PCI). DESIGN: Retrospective chart review SETTING: Academic medical centre. PATIENTS: 7659 patients who had 9032 PCIs. INTERVENTIONS: PCI performed at Mayo Clinic between 1 January 1999 and 30 June 2004. MAIN OUTCOME MEASURES: The Mayo Clinic Risk Score (MCRS) and the coronary artery disease (CAD)-specific index for determination of comorbid conditions in all patients. RESULTS: The mean (SD) MCRS score was 6.5 (2.9). The CAD-specific index was 0 or 1 in 46%, 2 or 3 in 30% and 4 or higher in 24%. The rate of in-hospital major adverse cardiovascular events (MACE) increased with higher MCRS and CAD-specific index (Cochran-Armitage test, p<0.001 for both models). The c-statistic for the MCRS for in-hospital MACE was 0.78; adding the CAD-specific index did not improve its discriminatory ability for in-hospital MACE (c-statistic = 0.78; likelihood ratio test, p = 0.29). A total of 707 deaths after dismissal occurred after 7253 successful procedures. The c-statistic for all-cause mortality was 0.69 for the MCRS model alone and 0.75 for the MCRS and CAD-specific indices together (likelihood ratio test, p<0.001), indicating significant improvement in the discriminatory ability. CONCLUSIONS: Addition of comorbid conditions to the MCRS adds significant prognostic information for post-dismissal mortality but adds little prognostic information about in-hospital complications after PCI. Such health-status measures should be included in future risk stratification models that predict long-term mortality after PCI.

Effect of the introduction of a lumbar-puncture sticker and teaching manikin on junior staff documentation and performance of paediatric lumbar punctures.

BACKGROUND: Performing a lumbar puncture in an unwell child can cause anxiety in both the parent and the junior doctor. There is increasing evidence of post-lumbar-puncture complications in this age group. AIMS: To improve the documentation, consent for and technical performance of paediatric lumbar punctures to 100% of the required standard within 3 months. SETTING: The paediatric emergency department of a the Royal North Shore Hospital (University of Sydney, Sydney, Australia). PARTICIPANTS: Paediatric emergency staff, including residents, registrars and consultants. METHODS: Medical records of 40 consecutive children who had undergone a lumbar puncture in the 6 months before the introduction of the lumbar-puncture proforma were reviewed. After introduction of the proforma, the records of 25 consecutive patients were reviewed to assess changes in the outcome measures. Before introduction of the proforma, junior medical staff were instructed in the procedure using specialised lumbar puncture manikins (Baby Stap; Laerdel, USA). RESULTS: Before introduction of the proforma, the median number of documented indicators was 4, out of a maximum of 12. There was almost no documentation of parental consent, patient complications and analgesia. Introduction of the proforma resulted in a highly marked increase to a median of 12 documented indicators per patient (p<0.01, 95% confidence interval 6 to 8). CONCLUSIONS: The introduction of a lumbar-puncture proforma and formal teaching sessions using a paediatric manikin led to a marked improvement in the documentation of paediatric lumbar-punctures. Lumbar-punctures can be performed only by accredited medical officers who have achieved competency on the lumbar-puncture teaching manikin.

Mild renal insufficiency is associated with reduced coronary flow in patients with non-obstructive coronary artery disease.

Patients with chronic kidney disease (CKD) have increased risk for cardiovascular events. However, the association between these pathophysiological processes is unclear. Therefore, this study was designed to determine the association between early CKD and coronary microvascular disease in patients with normal or mildly diseased coronary arteries. A total of 605 patients with normal or mildly diseased coronary arteries based on angiography underwent coronary flow reserve (CFR) evaluation using intracoronary adenosine. Patients were divided based on glomerular filtration rate (GFR). CKD was defined as calculated GFR<60 ml/min/1.73 m(2). Patients with normal GFR (> or =60 ml/min/1.73 m(2), n=481) had higher CFR compared to those with CKD (n=124, CFR=3.0+/-0.8 vs 2.6+/-0.6, P<0.001, respectively). Patients with abnormal GFR were more likely to be older and of female gender, with greater prevalence of hypertension. Multiple logistic regression analysis adjusted for the aforementioned risk factors further supported the observed relationship. The current study shows that reduced renal function is associated with attenuated coronary vasodilator capacity in patients without obstructive coronary artery disease. The correlation between low GFR and reduced CFR may suggest parallel alterations in the renal and coronary microcirculation at the early stage of disease. Impairment in both microcirculatory beds may reflect an unmeasured risk factor induced by blunted renal function and add a burden to the increased propensity for cardiovascular events in CKD.

Comparative outcomes of percutaneous coronary interventions in diabetics vs non-diabetics with prior coronary artery bypass grafting.

AIMS: To determine the influence of diabetes on outcome after percutaneous coronary intervention in patients with prior coronary artery bypass grafting. METHODS AND RESULTS: Patients with prior coronary artery bypass grafting undergoing percutaneous coronary intervention from 1 January 1996, to 31 August 2000, were divided into two groups based on whether or not they had diabetes, excluding patients with acute infarction or shock. Cox proportional hazards models were utilized to estimate the association between diabetes and adverse events. One thousand one hundred and fifty-three post-coronary artery bypass grafting percutaneous coronary intervention patients were identified (326 diabetics and 827 non-diabetics). Diabetics were younger, more likely to have hypertension, heart failure, and lower ejection fraction. Procedural characteristics and angiographic and procedural success rates were similar. Diabetes was associated with increased mortality (hazard ratio 1.58, 95% confidence intervals 1.10-2.27). Diabetes did not have a significant effect on mortality in patients treated for single-territory coronary disease (hazard ratio 1.44, 95% confidence intervals 0.69-3.02), but did in patients with multi-territory disease (hazard ratio 1.79, 95% confidence intervals 1.16-2.76). However, in diabetics with multi-territory disease who were completely revascularized with percutaneous coronary intervention, mortality was comparable to non-diabetics (hazard ratio 1.32, 95% confidence intervals 0.57-3.03). CONCLUSION: Among percutaneous coronary intervention patients with prior coronary artery bypass grafting, diabetes portends an adverse prognosis.