Pools of overlapping synthetic peptides are routinely used for ex vivo monitoring of antigen-specific T-cell responses. However, it is rather unlikely that these peptides match those resulting from ...naturally processed antigens. T-activated proteins have been described as immunogenic and more natural stimulants, since they have to pass through antigen processing and comprise activation of all clinically relevant effector cell populations.
We performed comparative analysis of numbers and cytokine expression pattern of CD4 and CD8 T cells after stimulation with recombinant, urea-formulated T-activated EBV-BZLF1, -EBNA3A, and HCMV-IE1, and -pp65 proteins or corresponding overlapping peptide pools. Freshly isolated and cryopreserved PBMC of 30 EBV- and 19 HCMV-seropositive and seven EBV- and HCMV-seronegative subjects were stimulated ex vivo and analysed for IFN-γ, TNF and IL-2 production by flow cytometry-based intracellular cytokine staining.
T-activated proteins showed a high specificity of 100% (EBV-BZLF1, HCMV-IE1, and -pp65) and 86% (EBV-EBNA3A), and a high T-cell stimulatory capacity of 73-95% and 67-95% using freshly isolated and cryopreserved PBMC, respectively. The overall CD4 T-cell response rates in both cohorts were comparable after stimulation with either T-activated protein or peptide pools with the exception of lower numbers of CD8 T cells detected after stimulation with T-activated EBV-EBNA3A- (p = 0.038) and HCMV-pp65- (p = 0.0006). Overall, the number of detectable antigen-specific T cells varied strongly between individuals. Cytokine expression patterns in response to T-activated protein and peptide pool-based stimulation were similar for CD4, but significantly different for CD8 T-cell responses.
EBV and HCMV-derived T-activated proteins represent innovative, highly specific recall antigens suitable for use in immunological endpoint assays to evaluate success or failure in immunotherapy clinical trials (e.g. to assess the risk of EBV and/or HCMV reactivation after allogenic hematopoietic stem cell transplantation). T-activated proteins could be of particular importance, if an impaired antigen processing (e.g. in a post-transplant setting) must be taken into account.
Beside positive effects on athlete's health, competitive sport can be linked with an increased risk of illness and injury. Because of high relative increases in training, additional physical and ...psychological strains, and an earlier specialization and professionalization, adolescent athletes needs an increased attention. Training can alter the immune system by inducing a temporary immunosuppression, finally developing infection symptoms. Previous studies identified Epstein Barr Virus (EBV) as potential indicator for the immune status. In addition to the identification of triggering risk factors for recurrent infections, the aim was to determine the interaction between training load, stress sense, immunological parameters, and clinical symptoms.
A controlled, prospective, longitudinal study on young athletes (
= 274, mean age: 13.8 ± 1.5 yrs) was conducted between 2010 and 2014. Also 285 controls (students, who did not perform competitive sports, mean age: 14.5 ± 1.9 yrs) were recruited. Athletes were examined 3 times each year to determine the effects of stress factors (training load: training hours per week Th/w) on selected outcome parameters (clinical susceptibility to infection, WURSS-21: 21-item
, immunological, psychological end points). As part of each visit, EBV serostatus and EBV-specific IgG tiers were studied longitudinally as potential immune markers.
Athletes (A) trained 14.9 ± 5.6 h weekly. Controls (C) showed no lower stress levels compared to athletes (
= 0.387). Twelve percent of athletes reported recurrent infections (C: 8.5%,
= 0.153), the presence of an upper respiratory tract infection (URTI) was achieved in 30.7%. EBV seroprevalence of athletes was 60.3% (C: 56.6%,
= 0.339). Mean EBV-specific IgG titer of athletes was 166 ± 115 U/ml (C: 137 ± 112 U/ml,
= 0.030). With increasing Th/w, higher stress levels were observed (
< 0.001). Analyzes of WURSS-21 data revealed no relationship to training load (
= 0.323). Also, training load had no relation to EBV serostatus (
= 0.057) or the level of EBV-specific IgG titers (
= 0.364).
Young elite athletes showed no increased sense of stress, no higher prevalence of recurrent infections, and no different EBV-specific serological parameters compared to controls. Also, no direct relationship between training loads, clinical complaints, and EBV-specific immune responses was found. With increasing training loads athletes felt more stressed, but significant associations to EBV-specific serological parameters were absent. In summary, EBV serostatus and EBV-specific IgG titers do not allow risk stratification for impaired health. Further investigations are needed to identify additional risk factors and immune markers, with the aim to avoid inappropriate strains by early detection and following intervention.
Infection with the Epstein-Barr virus (EBV) elicits a complex T-cell response against a broad range of viral proteins. Hence, identifying potential differences in the cellular immune response of ...patients with different EBV-associated diseases or different courses of the same disorder requires interrogation of a maximum number of EBV antigens. Here, we tested three novel EBV-derived antigen formulations for their ability to reactivate virus-specific T cells ex vivo in patients with EBV-associated infectious mononucleosis (IM). We comparatively analyzed EBV-specific CD4+ and CD8+ T-cell responses to three EBV-derived antigen formulations in 20 pediatric patients during the early phase of IM: T-activated EBV proteins (BZLF1, EBNA3A) and EBV-like particles (EB-VLP), both able to induce CD4+ and CD8+ T-cell responses ex vivo, as well as an EBV-derived peptide pool (PP) covering 94 well-characterized CD8+ T-cell epitopes. We assessed the specificity, magnitude, kinetics, and functional characteristics of EBV-specific immune responses at two sequential time points (v1 and v2) within the first six weeks after IM symptom onset (T.sub.onset). All three tested EBV-derived antigen formulations enabled the detection of EBV-reactive T cells during the early phase of IM without prior T-cell expansion in vitro. EBV-reactive CD4+ and CD8+ T cells were mainly mono-functional (CD4+: mean 64.92%, range 56.15-71.71%; CD8+: mean 58.55%, range 11.79-85.22%) within the first two weeks after symptom onset (v1) with IFN-gamma and TNF-secreting cells representing the majority of mono-functional EBV-reactive T cells. By contrast, PP-reactive CD8+ T cells were primarily bi-functional (>60% at v1 and v2), produced IFN-gamma and TNF and had more tri-functional than mono-functional components. We observed a moderate correlation between viral load and EBNA3A, EB-VLP, and PP-reactive CD8+ T cells (r.sub.s = 0.345, 0.418, and 0.356, respectively) within the first two weeks after T.sub.onset, but no correlation with the number of detectable EBV-reactive CD4+ T cells. All three EBV-derived antigen formulations represent innovative and generic recall antigens suitable for monitoring EBV-specific T-cell responses ex vivo. Their combined use facilitates a thorough analysis of EBV-specific T-cell immunity and allows the identification of functional T-cell signatures linked to disease development and severity.
Background
The cellular mechanisms involved in the lack of protective antibody response after hepatitis B vaccination are still rather unclear. Regulatory B cells (Breg) known as modulators of B-and ...T-cell responses may contribute to poor vaccine responsiveness. The current study aimed to investigate the role of regulatory B cells (Breg) in hepatitis B vaccine non-responsiveness after immunization with second- or third-generation hepatitis B vaccines.
Method
We performed comparative phenotypic and frequency analysis of Breg subsets (CD24
+
CD27
+
and CD24
high
CD38
high
Breg) in second-generation hepatitis B vaccine non-responders (2
nd
HBvac NR, n = 11) and responders (2
nd
HBvac R, n = 8) before (d0), on day 7 (d7), and 28 (d28) after booster vaccination. Cryopreserved peripheral blood mononuclear cells were stimulated
ex vivo
with a combination of CpG, PMA, and Ionomycin (CpG+P/I) and analyzed for numbers and IL-10 expression levels of Breg by flow cytometry-based analyses.
Results
Flow cytometry-based analyses revealed elevated frequencies of CD24
+
CD27
+
Breg at all time points and significantly higher frequencies of CD24
high
CD38
high
Breg on d0 (
p
= 0.004) and 28 (
p
= 0.012) in 2
nd
HBvac NR compared to 2
nd
HBvac R. In parallel, we observed significantly lower levels of CpG+P/I-induced IL-10 expression levels of CD24
+
CD27
+
and CD24
high
CD38
high
Breg (d0:
p
< 0.0001; d7:
p
= 0.0004; d28:
p
= 0.0003 and d0:
p
= 0.016; d7:
p
= 0.016, respectively) in 2
nd
HBvac NR compared to 2
nd
HBvac R before and after booster immunization.
Frequencies of CD24
+
CD27
+
and CD24
high
CD38
high
Breg significantly decreased after third-generation hepatitis B booster vaccination (d7:
p
= 0.014; d28:
p
= 0.032 and d7:
p
= 0.045, respectively), whereas IL-10 expression levels of both Breg subsets remained stable.
Conclusion
Here we report significantly higher frequencies of CD24
high
CD38
high
Breg in parallel with significantly lower IL-10 expression levels of CD24
+
CD27
+
and CD24
high
CD38
high
Breg in 2
nd
HBvac NR compared to 2
nd
HBvac R. Anti-HBs seroconversion accompanied by a decrease of Breg numbers after booster immunization with a third-generation hepatitis B vaccine could indicate a positive effect of third-generation hepatitis B vaccines on Breg-mediated immunomodulation in hepatitis B vaccine non-responders.
Polyfunctional CD4 or CD8 T cells are proposed to represent a correlate of immune control for persistent viruses as well as for vaccine mediated protection against infection. A well-suited ...methodology to study complex functional phenotypes of antiviral T cells is the combined staining of intracellular cytokines and phenotypic marker expression using polychromatic flow cytometry. In this study we analyzed the effect of an overnight resting period at 37 °C on the quantity and functionality of HIV-1, EBV, CMV, HBV and HCV specific CD4 and CD8 T-cell responses in a cohort of 21 individuals. We quantified total antigen specific T cells by multimer staining and used 10-color intracellular cytokine staining (ICS) to determine IFNγ, TNFα, IL2 and MIP1β production. After an overnight resting significantly higher numbers of functionally active T cells were detectable by ICS for all tested antigen specificities, whereas the total number of antigen specific T cells determined by multimer staining remained unchanged. Overnight resting shifted the quality of T-cell responses towards polyfunctionality and increased antigen sensitivity of T cells. Our data suggest that the observed effect is mediated by T cells rather than by antigen presenting cells. We conclude that overnight resting of PBMC prior to ex vivo analysis of antiviral T-cell responses represents an efficient method to increase sensitivity of ICS-based methods and has a prominent impact on the functional phenotype of T cells.
Kidney transplant recipients (KTRs) are at high risk for a severe course of coronavirus disease 2019 (COVID-19); thus, effective vaccination is critical. However, the achievement of protective ...immunogenicity is hampered by immunosuppressive therapies. We assessed cellular and humoral immunity and breakthrough infection rates in KTRs vaccinated with homologous and heterologous COVID-19 vaccination regimens.
We performed a comparative in-depth analysis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T-cell responses using multiplex Fluorospot assays and SARS-CoV-2-specific neutralizing antibodies (NAbs) between three-times homologously (n = 18) and heterologously (n = 8) vaccinated KTRs.
We detected SARS-CoV-2-reactive T cells in 100% of KTRs upon third vaccination, with comparable frequencies, T-cell expression profiles, and relative interferon γ and interleukin 2 production per single cell between homologously and heterologously vaccinated KTRs. SARS-CoV-2-specific NAb positivity rates were significantly higher in heterologously (87.5%) compared to homologously vaccinated (50.0%) KTRs (
< 0.0001), whereas the magnitudes of NAb titers were comparable between both subcohorts after third vaccination. SARS-CoV-2 breakthrough infections occurred in equal numbers in homologously (38.9%) and heterologously (37.5%) vaccinated KTRs with mild-to-moderate courses of COVID-19.
Our data support a more comprehensive assessment of not only humoral but also cellular SARS-CoV-2-specific immunity in KTRs to provide an in-depth understanding about the COVID-19 vaccine-induced immune response in a transplant setting.
Anomaly Detection in Scratch Assignments Korber, Nina
2021 IEEE/ACM 43rd International Conference on Software Engineering: Companion Proceedings (ICSE-Companion)
Conference Proceeding
For teachers, automated tool support for debugging and assessing their students' programming assignments is a great help in their everyday business. For block-based programming languages which are ...commonly used to introduce younger learners to programming, testing frameworks and other software analysis tools exist, but require manual work such as writing test suites or formal specifications. However, most of the teachers using languages like Scratch are not trained for or experienced in this kind of task. Linters do not require manual work but are limited to generic bugs and therefore miss potential task-specific bugs in student solutions. In prior work, we proposed the use of anomaly detection to find project-specific bugs in sets of student programming assignments automatically, without any additional manual labour required from the teachers' side. Evaluation on student solutions for typical programming assignments showed that anomaly detection is a reliable way to locate bugs in a data set of student programs. In this paper, we enhance our initial approach by lowering the abstraction level. The results suggest that the lower abstraction level can focus anomaly detection on the relevant parts of the programs.
Summary
Background: Sentinel lymph node biopsy (SLNB) for cutaneous malignancies usually carried out with radioactive nanocolloids (Tc‐99m). The SLNE is controversially discussed internationally. ...This is especially given to the high false‐negative rate up to 44 %. An alternative could be the fluorescent dye indocyanine green (ICG).
Material and Methods: We investigated the advantage of intraoperative fluorescence detection of lymphatic vessels and SLN with a Near‐Infrared (NIR) camera in comparison to conventional methods using preoperative lymphoscintigraphy and SPECT/CT in 22 patients with malignant melanoma.
Results: A total of 61 SLNs were removed in 22 operative procedures. In 7 SLN (10.3 %; 7/68) the histopathological assessment could demonstrate a metasta‐tic involvement. 11 additional SLN (19.1 %) in 8 patients were only identified using the fluorescent labeling. Two of these additional SLN (9.1 %; 2/22) showed metastatic involvement.
Conclusion: The ICG fluorescence‐guided SLNB is an innovative imaging technique for dermato‐oncology, reliable and providing additional information in the detection of SLN. Therefore SLNB with fluorescence‐dye is an attractive option with intraoperative real‐time lymphoscintigraphy to improve the detection of SLN in cutaneous malignancies and potential reduction of the false negative rate in SLN.
LitterBox Fraser, Gordon; Heuer, Ute; Körber, Nina ...
2021 IEEE/ACM 43rd International Conference on Software Engineering: Software Engineering Education and Training (ICSE-SEET),
05/2021
Conference Proceeding
Creating programs with block-based programming languages like Scratch is easy and fun. Block-based programs can nevertheless contain bugs, in particular when learners have misconceptions about ...programming. Even when they do not, Scratch code is often of low quality and contains code smells, further inhibiting understanding, reuse, and fun. To address this problem, in this paper we introduce LitterBox, a linter for Scratch programs. Given a program or its public project ID, LitterBox checks the program against patterns of known bugs and code smells. For each issue identified, LitterBox provides not only the location in the code, but also a helpful explanation of the underlying reason and possible misconceptions. Learners can access LitterBox through an easy to use web interface with visual information about the errors in the block-code, while for researchers LitterBox provides a general, open source, and extensible framework for static analysis of Scratch programs.
Vaccines are an important means to overcome the SARS-CoV-2 pandemic. They induce specific antibody and T-cell responses but it remains open how well vaccine-induced immunity is preserved over time ...following homologous and heterologous immunization regimens. Here, we compared the dynamics of humoral and cellular immune responses up to 180 days after homologous or heterologous vaccination with either ChAdOx1-nCoV-19 (ChAd) or BNT162b2 (BNT) or both.
Various tests were used to determine the humoral and cellular immune response. To quantify the antibody levels, we used the surrogate neutralization (sVNT) assay from YHLO, which we augmented with pseudo- and real virus neutralization tests (pVNT and rVNT). Antibody avidity was measured by a modified ELISA. To determine cellular reactivity, we used an IFN-γ Elispot, IFN-γ/IL Flurospot, and intracellular cytokine staining.
Antibody responses significantly waned after vaccination, irrespective of the regimen. The capacity to neutralize SARS-CoV-2 – including variants of concern such as Delta or Omicron – was superior after heterologous compared to homologous BNT vaccination, both of which resulted in longer-lasting humoral immunity than homologous ChAd immunization. All vaccination regimens induced stable, polyfunctional T-cell responses.
These findings demonstrate that heterologous vaccination with ChAd and BNT is a potent alternative to induce humoral and cellular immune protection in comparison to the homologous vaccination regimens.
The study was funded by the German Centre for Infection Research (DZIF), the European Union's “Horizon 2020 Research and Innovation Programme" under grant agreement No. 101037867 (VACCELERATE), the “Bayerisches Staatsministerium für Wissenschaft und Kunst” for the CoVaKo-2021 and the For-COVID projects and the Helmholtz Association via the collaborative research program “CoViPa”. Further support was obtained from the Federal Ministry of Education and Science (BMBF) through the “Netzwerk Universitätsmedizin”, project “B-Fast” and “Cov-Immune”. KS is supported by the German Federal Ministry of Education and Research (BMBF, 01KI2013) and the Else Kröner-Stiftung (2020_EKEA.127).
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