HIV-1 mRNA electroporation of PBMC: a simple and efficient method to monitor T-cell responses against autologous HIV-1 in HIV-1-infected patients

Efficient monitoring of HIV-1-specific T-cells is crucial for the development of HIV-1 vaccines and immunotherapies. Currently, mainly peptides and vaccinia vectors are used for detection of HIV-1-specific cytotoxic T-lymphocytes (CTL), however, as HIV-1 is a variable virus, it is unknown to what extent the T-cell response against the autologous virus is under- or overestimated by using antigens from heterologous viral strains. Therefore, we established a new method for immunomonitoring of CTL using electroporation of peripheral blood mononuclear cells (PBMC) with mRNA derived from autologous viral strains. From six HIV-1-infected patients virus derived mRNA was produced after PCR-based cloning of autologous gag (n=5) and/or nef genes (n=3) from plasma and electroporated into PBMC from patients and healthy donors. Electroporation of PBMC with mRNA resulted in efficient protein expression with good induction of γ-interferon (γ-IFN) release by specific T-cells comparable to peptide pools and better than recombinant vaccinia viruses. Three mRNA encoded autologous Gag proteins and one autologous mRNA encoded Nef protein were better recognized by autologous PBMC in comparison to heterologous mRNA encoded Gag or Nef proteins (SF2 or HXB2). However, in one case each, mRNA encoded autologous Gag or Nef, respectively, was recognized less efficiently due to the presence of CTL escape mutations. In summary, electroporation of PBMC with mRNA is a very efficient, easy and rapid method for immunomonitoring of HIV-1-specific T-cell responses against autologous viral strains. Our data demonstrate that patients' CTL responses against autologous viral strains may be under- or overestimated by using antigens from heterologous viral strains.     

Sequetyping: serotyping Streptococcus pneumoniae by a single PCR sequencing strategy

The introduction of pneumococcal conjugate vaccines necessitates continued monitoring of circulating strains to assess vaccine efficacy and replacement serotypes. Conventional serological methods are costly, labor-intensive, and prone to misidentification, while current DNA-based methods have limited serotype coverage requiring multiple PCR primers. In this study, a computer algorithm was developed to interrogate the capsulation locus (cps) of vaccine serotypes to locate primer pairs in conserved regions that border variable regions and could differentiate between serotypes. In silico analysis of cps from 92 serotypes indicated that a primer pair spanning the regulatory gene cpsB could putatively amplify 84 serotypes and differentiate 46. This primer set was specific to Streptococcus pneumoniae, with no amplification observed for other species, including S. mitis, S. oralis, and S. pseudopneumoniae. One hundred thirty-eight pneumococcal strains covering 48 serotypes were tested. Of 23 vaccine serotypes included in the study, most (19/22, 86%) were identified correctly at least to the serogroup level, including all of the 13-valent conjugate vaccine and other replacement serotypes. Reproducibility was demonstrated by the correct sequetyping of different strains of a serotype. This novel sequence-based method employing a single PCR primer pair is cost-effective and simple. Furthermore, it has the potential to identify new serotypes that may evolve in the future.     

Pattern of time-dependent reduction of histologically determined infarct volume after focal ischaemia in mice

The mouse model of transcranial permanent occlusion of the middle cerebral artery (tpMCAO) is widely used in stroke research. Here we quantified infarct size using a conventional histological method at several post-ischaemic times, going beyond the commonly analysed period of up to 2 days, following artery occlusion. Two different mouse strains, which are widely used for pharmacological studies of neuroprotection and for genetic engineering, were used. A drill whole was made into the skull of anaesthetised mice and ischaemia was induced by electrocoagulation of the middle cerebral artery. In both mouse strains tested (C57Black/6 and NMRI), the measured infarct volumes decreased significantly during the first days after tpMCAO. Notably, 13 days after surgery, ischaemic and sham-operated animals had indistinguishably small lesions, which where in the range of only 5% of the infarct size on day 2 post-ischaemia. The standard method of calculating oedema and shrinkage correction provided no sufficient explanation for this significant decrease in infarct volume. There was, however, evidence that structural changes in the residual ipsilateral hemisphere may compromise the significance of results arising from the method of calculating oedema and shrinkage correction. In conclusion, our study indicates that the pronounced and fast, time-dependent decrease in histologically defined infarct volume can compromise results when studying the lasting neuroprotective effects of potential drugs.     

The selective mineralocorticoid receptor antagonist eplerenone is protective in mild anti-GBM glomeru-lonephritis

Background: Growing evidence suggests that blockade of the aldosterone-receptor may preserve kidney function by anti-inflammatory effects independent of the blood pressure. We hypothesized that the selective aldosterone-receptor antagonist eplerenone has a profound anti-inflammatory effect in the autologous phase of anti-glomerular basement membrane (GBM) glomerulonephritis (GN). Methods: Mice received ≈200mg/kg body wt/day eplerenone via supplemented chow diet or standard chow starting at the day of immunization with rabbit IgG. Three days later the anti-GBM antibody was injected and the experiments were stopped at day 7 and 14. Results: Mice receiving eplerenone showed significantly decreased albuminuria and glomerular sclerosis at day 7 and 14 after induction of anti-GBM GN. Eplerenone treatment significantly inhibited the infiltration of CD4+, CD8+ T cells and macrophages into the kidneys. Circulating levels and glomerular deposition of autologous IgG were comparable in both groups. At day 7 the pro-inflammatory cytokines MCP-1 and IL-6 were found to be significantly decreased in regional draining lymph nodes of eplerenone-treated mice, whereas the anti-inflammatory cytokine IL-10 was significantly upregulated. In line, splenocytes from eplerenone-treated nephritic mice produced significantly increased IL-10. Conclusion: Aldosterone-receptor blockade by eplerenone effectively attenuated proteinuria, kidney damage and the inflammatory response in anti-GBM GN by significantly decreasing pro-inflammatory cytokines in the regional draining lymph nodes of the kidney. Our results suggest that this selective aldosterone receptor antagonist is a possible additional tool in the treatment of GN.     

Peanut-induced anaphylaxis in children and adolescents: Data from the European Anaphylaxis Registry

Background

Peanut allergy has a rising prevalence in high-income countries, affecting 0.5%–1.4% of children. This study aimed to better understand peanut anaphylaxis in comparison to anaphylaxis to other food triggers in European children and adolescents.

Methods

Data was sourced from the European Anaphylaxis Registry via an online questionnaire, after in-depth review of food-induced anaphylaxis cases in a tertiary paediatric allergy centre.

Results

3514 cases of food anaphylaxis were reported between July 2007 - March 2018, 56% in patients younger than 18 years. Peanut anaphylaxis was recorded in 459 children and adolescents (85% of all peanut anaphylaxis cases). Previous reactions (42% vs. 38%; p = .001), asthma comorbidity (47% vs. 35%; p < .001), relevant cofactors (29% vs. 22%; p = .004) and biphasic reactions (10% vs. 4%; p = .001) were more commonly reported in peanut anaphylaxis. Most cases were labelled as severe anaphylaxis (Ring&Messmer grade III 65% vs. 56% and grade IV 1.1% vs. 0.9%; p = .001). Self-administration of intramuscular adrenaline was low (17% vs. 15%), professional adrenaline administration was higher in non-peanut food anaphylaxis (34% vs. 26%; p = .003). Hospitalization was higher for peanut anaphylaxis (67% vs. 54%; p = .004).

Conclusions

The European Anaphylaxis Registry data confirmed peanut as one of the major causes of severe, potentially life-threatening allergic reactions in European children, with some characteristic features e.g., presence of asthma comorbidity and increased rate of biphasic reactions. Usage of intramuscular adrenaline as first-line treatment is low and needs to be improved. The Registry, designed as the largest database on anaphylaxis, allows continuous assessment of this condition.

     

Why are young Canadians afraid of birth? A survey study of childbirth fear and birth preferences among Canadian University students

Objective

to examine attitudes towards birth that may be common among young adults who have been socialised into a medicalised birth culture. Specifically, we were interested in examining factors that might be associated with fear of birth and preferences for elective obstetric interventions among the next generation of maternity care consumers.

Design

secondary analysis of an online survey of university students.

Setting

British Columbia, Canada.

Participants

students from the University of British Columbia (n=3680). A quarter of the sample comprised Asian students, which allowed for analysis of cultural differences in attitudes towards birth. Both male and female students participated in the study; results are reported for the full sample, and by gender.

Measurements

a six item fear of childbirth scale was developed, as well as a 4 item index that measures students' concerns over physical changes following pregnancy and birth and a 2 item scale that assesses students' attitudes towards obstetric technology.

Findings

as we hypothesised, students who were more fearful of birth preferred epidural anaesthesia and birth by CS. Worries over physical changes following pregnancy and birth, favourable attitudes towards obstetric technology, and exposure to pregnancy and birth information via the media were also significantly associated with a preference for CS. Fear of birth scores were highest among students who reported that the media had shaped their attitudes towards pregnancy and birth. Asian students had significantly higher fear of birth scores and were more likely to prefer CS, compared to Caucasian students.

Implications for practice

young adults are contemplating pregnancy and birth in an increasingly technology-dependent society. Educational programmes aimed at reducing fear of childbirth and concerns over physical changes following pregnancy and childbirth might contribute to vaginal birth intentions among young adults. Midwives may use the findings to identify and counsel nulliparas who exhibit fear of birth and other childbirth attitudes that may predispose them to choose elective obstetric interventions.     

A design strategy to generate a SARS-CoV-2 RBD vaccine that abrogates ACE2 binding and improves neutralizing antibody responses

The structure-based design of antigens holds promise for developing vaccines with higher efficacy and improved safety profiles. We postulate that abrogation of host receptor interaction bears potential for the improvement of vaccines by preventing antigen-induced modification of receptor function as well as the displacement or masking of the immunogen. Antigen modifications may yet destroy epitopes crucial for antibody neutralization. Here, we present a methodology that integrates deep mutational scans to identify and score SARS-CoV-2 receptor binding domain variants that maintain immunogenicity, but lack interaction with the widely expressed host receptor. Single point mutations were scored in silico, validated in vitro, and applied in vivo. Our top-scoring variant receptor binding domain-G502E prevented spike-induced cell-to-cell fusion, receptor internalization, and improved neutralizing antibody responses by 3.3-fold in rabbit immunizations. We name our strategy BIBAX for body-inert, B-cell-activating vaccines, which in the future may be applied beyond SARS-CoV-2 for the improvement of vaccines by design.     

Corrective Experiences of Psychotherapists in Training

Although the concept of corrective experiences (CEs) is usually linked to the process of change in psychotherapy patients, we investigated them in the professional development of therapists‐in‐training. Inasmuch as psychotherapy is a relational process, it is important to look closely at how therapists reach the position of a competent partner in corrective experiencing. In this study, we interviewed 10 therapists‐in‐training undergoing their own training therapy. Responses to these semistructured interviews were analyzed using a computer‐assisted grounded theory method. The 499 first‐level categories were grouped into 5 main themes: therapist characteristics, therapist technical interventions, therapist relational interventions, relationship experience, and outcome experience. Two core categories representing corrective experiencing were (a) unexpected unconditional support from and trust in their own therapist and (b) unexpected confrontation and limitation with their therapist as well as awareness of self–other boundaries. Results are discussed in the broader context of the CE literature, relational theory, and relational practice.     

PulmoStent: <Emphasis Type="Italic">In Vitro</Emphasis> to <Emphasis Type="Italic">In Vivo</Emphasis> Evaluation of a Tissue Engineered Endobronchial Stent

Currently, there is no optimal treatment available for end stage tumour patients with airway stenosis. The PulmoStent concept aims on overcoming current hurdles in airway stenting by combining a nitinol stent with a nutrient-permeable membrane, which prevents tumour ingrowth. Respiratory epithelial cells can be seeded onto the cover to restore mucociliary clearance. In this study, a novel hand-braided dog bone stent was developed, covered with a polycarbonate urethane nonwoven and mechanically tested. Design and manufacturing of stent and cover were improved in an iterative process according to predefined requirements for permeability and mechanical properties and finally tested in a proof of concept animal study in sheep for up to 24 weeks. In each animal two stents were implanted, one of which was cell-seeded by endoscopic spraying in situ. We demonstrated the suitability of this membrane for our concept by glucose transport testing and in vitro culture of respiratory epithelial cells. In the animal study, no migration occurred in any of the twelve stents. There was only mild granulation tissue formation and tissue reaction; no severe mucus plugging was observed. Thus, the PulmoStent concept might be a step forward for palliative treatment of airway stenosis with a biohybrid stent device.