Xylosyltransferase-Deficiency in Human Dermal Fibroblasts Induces Compensatory Myofibroblast Differentiation and Long-Term ECM Reduction.

Desbuquois dysplasia type 2 (DBQD2) and spondylo-ocular syndrome (SOS) are autosomal recessive disorders affecting the extracellular matrix (ECM) and categorized as glycosaminoglycan (GAG) linkeropathies. Linkeropathies result from mutations within glycosyltransferases involved in the synthesis of the tetrasaccharide linker, a linker between the core protein of proteoglycan (PG) and GAG. DBQD2 and SOS are caused by the isolated mutations of the xylosyltransferase (XT) isoforms. In this work, we successfully generated XYLT1- as well as XYLT2-deficient GAG linkeropathy model systems in human dermal fibroblasts using a ribonucleoprotein-based CRISPR/Cas9-system. Furthermore, it was possible to generate a complete XYLT-knockdown. Short- and long-term XT activity deficiency led to the mutual reduction in all linker transferase-encoding genes, suggesting a potential multienzyme complex with mutual regulation. Fibroblasts compensated for ECM misregulation initially by overexpressing ECM through the TGFß1 signaling pathway, akin to myofibroblast differentiation patterns. The long-term reduction in one XT isoform induced a stress response, reducing ECM components. The isolated XYLT1-knockout exhibited α-smooth muscle actin overexpression, possibly partially compensated by unaltered XT-II activity. XYLT2-knockout leads to the reduction in both XT isoforms and a strong stress response with indications of oxidative stress, induced senescence and apoptotic cells. In conclusion, introducing XYLT-deficiency revealed temporal and isoform-specific regulatory differences.

A novel SPE-UPLC-MS/MS-based assay for the selective, simultaneous quantification of xylosyltransferase-I and -II activity.

Xylosyltransferase-I and -II (XT-I, -II) possess a central role during the glycosylation of proteoglycans (PGs). They catalyze the formation of an O-glycosidic bond between the xylosyl residue of uridinediphosphate-xylose and the core protein of a PG. Thereafter, three following glycosyltransferases lead to the generation of a tetrasaccharide linker, which connects the PG core protein to the respective glycosaminoglycan. The selective quantification of XT-I and XT-II activity is of biological and clinical interest due to their association with fibrotic processes and skeletal dysplasia. There is no assay available to date that simultaneously determines the activity of the two XT isoforms. Although an XT-I selective UPLC MS/MS-based assay was published by Fischer et al., in 2021, the determination of XT-II activity can only be performed simultaneously by the improved assay presented here. To establish the assay, two synthetic peptides, selectively xylosylated by the respective isoform, were identified and the associated measurement parameters for the mass spectrometer were optimized. In addition, the quantitative range of the xylosylated peptides were validated, and the incubation time of the enzyme reaction was optimized for cell culture samples and human sera. The specific enzyme kinetics (KM and Vmax) of the respective XT isoform for the two peptides were also determined. Subsequently, a mathematical model was developed, allowing the simultaneous determination of XT-I and XT-II activity from the chromatographic results. Summarized, a mass spectrometric method suitable for the simultaneous analysis of XT-I and XT-II activity in cell culture lysates, supernatants and human sera was successfully developed.

Dyspareunia, signs of epithelial disruption, sexual abstinence, and HIV status in female sex workers in Nairobi: a cross-sectional study.

BACKGROUND: Epithelial trauma is a risk factor of HIV infection in men who have sex with men (MSM) and female sex workers (FSWs). Painful intercourse may be indicative of epithelial tissue disruption. Previous studies on a cohort of Kenyan FSWs established an association between prolonged sexual abstinence and late HIV seroconversion. Our research objective was to establish whether there is a relationship between HIV serostatus and signs of epithelial disruption and between HIV serostatus and sexual abstinence behaviour. METHODS: Participants were selected from a Nairobi health facility. A structured questionnaire was administered to 322 FSWs, who provided data on HIV status, sexual behaviour, abstinence intervals and the level of sexual dysfunction. Sexual dysfunction scores were created using parts of the Female Sexual Function Index (FSFI-19). Additional questions addressed epithelial trauma signs. Descriptive data analysis, bivariate and multivariate logistic regression were used to describe the study population and determine factors associated with living with HIV. Potential factors influencing sexual dysfunction were assessed by FSWs via self-rating. RESULTS: 36% of FSWs reported discomfort or pain during vaginal penetration half the time. 44% noticed genital bleeding half the time. Vaginal tenderness was experienced by 70.6% half the time during or after intercourse. Variables predictive of living with HIV on multivariate analysis included a medium and high score of discomfort or pain during and following vaginal penetration (medium: AOR 2.288, p-value 0.032, 95% CI 1.075-4.871; high: AOR 3.044, p-value 0.031, 95% CI 1.110-8.348). No significant association of HIV status with past abstinence durations as reported by participants could be established in the multivariate analysis. A majority of FSWs agreed that steady partnerships (81% agreement), regularity of intercourse (74%), foreplay (72%) and lubricants (65%) alleviated dyspareunia. CONCLUSIONS: Recurrent exposure to blood during sex was highly prevalent in FSWs, as was sexual dysfunction. Complaint levels were associated with living with HIV, providing evidence that reducing sexual dysfunctions may prevent HIV transmission. Preventive initiatives may be created that address sexual dysfunction in key populations and general populations with a high HIV prevalence. Subjective assessments indicate that prevention may include the promotion of sexual intercourse regularity, foreplay, and lubricant use.

Understanding of arthrofibrosis: New explorative insights into extracellular matrix remodeling of synovial fibroblasts.

Arthrofibrosis following total knee arthroplasty is a fibroproliferative joint disorder marked by dysregulated biosynthesis of extracellular matrix proteins, such as collagens and proteoglycans. The underlying cellular events remain incompletely understood. Myofibroblasts are highly contractile matrix-producing cells characterized by increased alpha-smooth muscle actin expression and xylosyltransferase-I (XT-I) secretion. Human XT-I has been identified as a key mediator of arthrofibrotic remodeling. Primary fibroblasts from patients with arthrofibrosis provide a useful in vitro model to identify and characterize disease regulators and potential therapeutic targets. This study aims at characterizing primary synovial fibroblasts from arthrofibrotic tissues (AFib) regarding their molecular and cellular phenotype by utilizing myofibroblast cell culture models. Compared to synovial control fibroblasts (CF), AFib are marked by enhanced cell contractility and a higher XT secretion rate, demonstrating an increased fibroblast-to-myofibroblast transition rate during arthrofibrosis. Histochemical assays and quantitative gene expression analysis confirmed higher collagen and proteoglycan expression and accumulation in AFib compared to CF. Furthermore, fibrosis-based gene expression profiling identified novel modifier genes in the context of arthrofibrosis remodeling. In summary, this study revealed a unique profibrotic phenotype in AFib that resembles some traits of other fibroproliferative diseases and can be used for the future development of therapeutic interventions.

Human Xylosyltransferase I-An Important Linker between Acute Senescence and Fibrogenesis.

The human xylosyltransferase isoform XT-I catalyzes the initial step in proteoglycan biosynthesis and represents a biomarker of myofibroblast differentiation. Furthermore, XT-I overexpression is associated with fibrosis, whereby a fibrotic process initially develops from a dysregulated wound healing. In a physiologically wound healing process, extracellular matrix-producing myofibroblasts enter acute senescence to protect against fibrosis. The aim of this study was to determine the role of XT-I in acute senescent proto-myofibroblasts. Normal human dermal fibroblasts were seeded in a low cell density to promote myofibroblast differentiation and treated with H2O2 to induce acute senescence. Initiation of the acute senescence program in human proto-myofibroblasts resulted in a suppression of XYLT mRNA expression compared to the control, whereby the isoform XYLT1 was more affected than XYLT2. Moreover, the XT-I protein expression and enzyme activity were also reduced in H2O2-treated cells compared to the control. The examination of extracellular matrix remodeling revealed reduced expression of collagen I, fibronectin and decorin. In summary, acute senescent proto-myofibroblasts formed an anti-fibrotic phenotype, and suppression of XT-I during the induction process of acute senescence significantly contributed to subsequent ECM remodeling. XT-I therefore plays an important role in the switch between physiological and pathological wound healing.

Third dose of the BNT162b2 vaccine in cardiothoracic transplant recipients: predictive factors for humoral response.

BACKGROUND: We report the results of a prospective study on the immunogenicity of a 3rd dose of BNT162b2 in thoracic organ recipients with no or minimal response following a two-dose BNT162b2 vaccination scheme. METHODS: A total of 243 transplant recipients received a homologue 3rd dose. Anti-SARS-CoV2-immunoglobulins (IgGs) were monitored immediately before (T1), 4 weeks (T2) as well as 2 and 4 months after the 3rd dose. Neutralizing antibody capacity (NAC) was determined at T2. To reveal predictors for detectable humoral response, patients were divided into a positive response group (n = 129) based on the combined criteria of IgGs and NAC above the defined cut-offs at T2-and a group with negative response (n = 114), with both, IgGs and NAC beyond the cut-offs. RESULTS: The 3rd dose induced a positive humoral response in 53% of patients at T2, 47% were still non-responsive. Sero-positivity was significantly stronger in patients who presented with weak, but detectable IgGs already prior to the booster (T1), when compared to those with no detectable response at T1. Multivariable analysis identified age > 55 years, a period since transplantation < 2 years, a reduced glomerular filtration rate, a triple immunosuppressive regimen, and the use of tacrolimus and of mycophenolate as independent risk factors for lack of humoral response. CONCLUSIONS: Our data indicate that a lack of immunogenicity is linked to the type and extent of maintenance immunosuppression. The necessity of the cumulative immunosuppressive regimen might individually be questioned and possibly be reduced to enhance the chance of an immune response following an additional booster dose.

Analysis of a German blood donor cohort reveals a high number of undetected SARS-CoV-2 infections and sex-specific differences in humoral immune response.

Seroprevalence studies can contribute to a better assessment of the actual incidence of infection. Since long-term data for Germany are lacking, we determined the seroprevalence of immunoglobulin G (IgG) antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in residual plasma samples of 3,759 German regular blood donors between July 2020 and June 2021. Over almost the entire study period, the incidences determined based on our data were higher than those officially reported by the Robert Koch Institute, the public health institute in Germany. Using our serological testing strategy, we retrospectively detected natural infection in 206/3,759 (5.48%; 95% confidence interval (CI): 4.77-6.25) individuals. The IgG seroprevalence ranked from 5.15% (95% CI: 3.73-6.89) in Lower Saxony to 5.62% (95% CI: 4.57-6.84) in North Rhine Westphalia. The analyses of follow-up samples of 88 seropositive blood donors revealed a comparable fast decay of binding and neutralizing anti-SARS-CoV-2 IgG antibodies. The antibody avidity remained at a low level throughout the whole follow-up period of up to 181 days. Interestingly, female donors seem to express a stronger and longer lasting humoral immunity against the new coronavirus when compared to males. Conclusion: Overall, our data emphasizes that seroprevalence measurements can and should be used to understand the true incidence of infection better. Further characterization of follow-up samples from seropositive donors indicated rapid antibody waning with sex-specific differences concerning the strength and persistence of humoral immune response.

Humoral and cellular immune response levels at a 1-year follow-up after mild COVID-19.

The primary objective of this study was to establish a 1-year follow-up of patients after mild COVID-19 with no or only short-term detection of antibodies shortly after disease. At 1 year after disease, cellular memory against SARS-CoV-2, as measured by IFN-γ release by T cells, was detected in 76% (38/50) of participants. The data suggest that even if antibody levels decline after the primary infection has resolved, a cellular immune response may be detectable for longer.

Cellular and Humoral Immune Response to a Third Dose of BNT162b2 COVID-19 Vaccine - A Prospective Observational Study.

Background: Since the introduction of various vaccines against SARS-CoV-2 at the end of 2020, infection rates have continued to climb worldwide. This led to the establishment of a third dose vaccination in several countries, known as a booster. To date, there has been little real-world data about the immunological effect of this strategy. Methods: We compared the humoral- and cellular immune response before and after the third dose of BioNTech/Pfizer vaccine BNT162b2, following different prime-boost regimen in a prospective observational study. Humoral immunity was assessed by determining anti-SARS-CoV-2 binding antibodies using a standardized quantitative assay. In addition, neutralizing antibodies were measured using a commercial surrogate ELISA-assay. Interferon-gamma release was measured after stimulating blood-cells with SARS-CoV-2 specific peptides using a commercial assay to evaluate the cellular immune response. Results: We included 243 health-care workers who provided blood samples and questionnaires pre- and post- third vaccination. The median antibody level increased significantly after the third vaccination dose to 2663.1 BAU/ml vs. 101.4 BAU/ml (p < 0.001) before administration of the booster dose. This was also detected for neutralizing antibodies with a binding inhibition of 99.68% ± 0.36% vs. 69.06% ± 19.88% after the second dose (p < 0.001). 96.3% of the participants showed a detectable T-cell-response after the booster dose with a mean interferon-gamma level of 2207.07 mIU/ml ± 1905 mIU/ml. Conclusion: This study detected a BMI-dependent antibody increase after the third dose of BNT162b2 following different vaccination protocols. All participants showed a significant increase in their immune response. This, in combination with the low rate of post-vaccination-symptoms underlines the potential beneficial effect of a BNT162b2-booster dose.

The Impact of Inflammatory Stimuli on Xylosyltransferase-I Regulation in Primary Human Dermal Fibroblasts.

Inflammation plays a vital role in regulating fibrotic processes. Beside their classical role in extracellular matrix synthesis and remodeling, fibroblasts act as immune sentinel cells participating in regulating immune responses. The human xylosyltransferase-I (XT-I) catalyzes the initial step in proteoglycan biosynthesis and was shown to be upregulated in normal human dermal fibroblasts (NHDF) under fibrotic conditions. Regarding inflammation, the regulation of XT-I remains elusive. This study aims to investigate the effect of lipopolysaccharide (LPS), a prototypical pathogen-associated molecular pattern, and the damage-associated molecular pattern adenosine triphosphate (ATP) on the expression of XYLT1 and XT-I activity of NHDF. We used an in vitro cell culture model and mimicked the inflammatory tissue environment by exogenous LPS and ATP supplementation. Combining gene expression analyses, enzyme activity assays, and targeted gene silencing, we found a hitherto unknown mechanism involving the inflammasome pathway components cathepsin B (CTSB) and caspase-1 in XT-I regulation. The suppressive role of CTSB on the expression of XYLT1 was further validated by the quantification of CTSB expression in fibroblasts from patients with the inflammation-associated disease Pseudoxanthoma elasticum. Altogether, this study further improves the mechanistic understanding of inflammatory XT-I regulation and provides evidence for fibroblast-targeted therapies in inflammatory diseases.

First Characterization of Human Dermal Fibroblasts Showing a Decreased Xylosyltransferase-I Expression Induced by the CRISPR/Cas9 System.

BACKGROUND: Xylosyltransferases-I and II (XT-I and XT-II) catalyze the initial and rate limiting step of the proteoglycan (PG) biosynthesis and therefore have an import impact on the homeostasis of the extracellular matrix (ECM). The reason for the occurrence of two XT-isoforms in all higher organisms remains unknown and targeted genome-editing strategies could shed light on this issue. METHODS: XT-I deficient neonatal normal human dermal fibroblasts were generated by using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated proteins (Cas) 9 system. We analyzed if a reduced XT-I activity leads to abnormalities regarding ECM-composition, myofibroblast differentiation, cellular senescence and skeletal and cartilage tissue homeostasis. RESULTS: We successfully introduced compound heterozygous deletions within exon 9 of the XYLT1 gene. Beside XYLT1, we detected altered gene-expression levels of further, inter alia ECM-related, genes. Our data further reveal a dramatically reduced XT-I protein activity. Abnormal myofibroblast-differentiation was demonstrated by elevated alpha-smooth muscle actin expression on both, mRNA- and protein level. In addition, wound-healing capability was slightly delayed. Furthermore, we observed an increased cellular-senescence of knockout cells and an altered expression of target genes knowing to be involved in skeletonization. CONCLUSION: Our data show the tremendous relevance of the XT-I isoform concerning myofibroblast-differentiation and ECM-homeostasis as well as the pathophysiology of skeletal disorders.

Short-Term Drop in Antibody Titer after the Third Dose of SARS-CoV-2 BNT162b2 Vaccine in Adults.

Little is known about the longevity of antibodies after a third dose of the mRNA-based SARS-CoV-2 vaccine BNT162b2 (BioNTech/Pfizer, Mainz, Germany). Therefore, serum antibody levels were evaluated after a third dose of BNT162b2 in healthy adult healthcare workers in Germany. These antibody levels dropped significantly within a short period of 11 weeks from 4155.59 ± 2373.65 BAU/mL to 2389.10 ± 1433.90 BAU/mL, p-value < 0.001 but remained higher than after the second dose (611.92 ± 450.31 BAU/mL). To evaluate the quality of the humoral immune response, we additionally measured neutralizing antibodies, which also showed a small but significant decrease within this short period. These data underline the positive effect of a third dose of BNT162b2 concerning antibody re-induction but also shows a drop of Anti-SARS-CoV-2-IgG within a short span of time.

Persistence of Immune Response in Health Care Workers After Two Doses BNT162b2 in a Longitudinal Observational Study.

Background: The mRNA-based vaccine BNT162b2 of BioNTech/Pfizer has shown high efficacy against SARS-CoV-2 infection and a severe course of the COVID-19 disease. However, little is known about the long-term durability of the induced immune response resulting from the vaccination. Methods: In a longitudinal observational study in employees at a German hospital we compared the humoral and cellular immune response in 184 participants after two doses of the BioNTech/Pfizer vaccine (BNT162b2) with a mid-term follow-up after 9 months. Anti-SARS-CoV-2 binding antibodies were determined using both a quantitative and a semi-quantitative assay. For a qualitative assessment of the humoral immune response, we additionally measured neutralizing antibodies. Cellular immune response was evaluated by measuring Interferon-gamma release after stimulating blood-cells with SARS-CoV-2 specific peptides using a commercial assay. Results: In the first analysis, a 100% humoral response rate was described after two doses of BNT162b2 vaccine with a mean antibody ratio of 8.01 ± 1.00. 9 months after the second dose of BNT162b2, serological testing showed a significant decreased mean antibody ratio of 3.84 ± 1.69 (p < 0.001). Neutralizing antibodies were still detectable in 96% of all participants, showing an average binding inhibition value of 68.20% ± 18.87%. Older age (p < 0.001) and obesity (p = 0.01) had a negative effect on the antibody persistence. SARS-CoV-2 specific cellular immune response was proven in 75% of individuals (mean Interferon-gamma release: 579.68 mlU/ml ± 705.56 mlU/ml). Conclusion: Our data shows a declining immune response 9 months after the second dose of BNT162b2, supporting the potentially beneficial effect of booster vaccinations, the negative effect of obesity and age stresses the need of booster doses especially in these groups.

SARS-CoV-2-antibody response in health care workers after vaccination or natural infection in a longitudinal observational study.

BACKGROUND: Following a year of development, several vaccines have been approved to contain the global COVID-19 pandemic. Real world comparative data on immune response following vaccination or natural infection are rare. METHODS: We conducted a longitudinal observational study in employees at a secondary care hospital affected by the COVID-19 pandemic. Comparisons were made about the presence of anti-SARS-CoV-2 immunglobulin G (IgG) antibody ratio after natural infection, or vaccination with one or two doses of BioNTech/Pfizer (BNT162b2), or one dose of AstraZenca (Vaxzevria) vaccine. RESULTS: We found a 100% humoral response rate in participants after 2 doses of BNT162b2 vaccine. The antibody ratio in participants with one dose BNT162b2 and Vaxzevria did not differ significantly to those with previous PCR-confirmed infection, whereas this was significantly lower in comparison to two doses of BioNTech/Pfizer. We could not identify a correlation with previous comorbidities, obesity or age within this study. Smoking showed a negative effect on the antibody response (p = 0.006) CONCLUSION: Our data provide an overview about humoral immune response after natural SARS-CoV-2 infection or following vaccination, and supports the usage of booster vaccinations, especially in patients after a natural SARS-CoV-2 infection.

After-Action Reviews and Long-Term Performance: An Experimental Examination in the Context of an Emergency Simulation.

OBJECTIVE: The present study examined the effectiveness of after-action reviews (AARs; also known as debriefing) in mitigating skill decay. BACKGROUND: Research on the long-term effectiveness of AARs is meager. To address this gap in the literature, we conducted an experimental study that also overcomes some research design issues that characterize the limited extant research. METHOD: Eighty-four participants were randomly assigned to an AAR or non-AAR condition and trained to operate a PC-based fire emergency simulator. During the initial acquisition phase, individuals in the AAR condition were allowed to review their performance after each practice session, whereas individuals in the non-AAR condition completed a filler task. About 12 weeks later, participants returned to the lab to complete four additional practice sessions using a similar scenario (i.e., the retention and reacquisition phase). RESULTS: The performance of participants in the AAR condition degraded more after nonuse but also recovered faster than the performance of participants in the non-AAR condition, although these effects were fairly small and not statistically significant. CONCLUSION: Consistent with the limited research on the long-term effectiveness of AARs, our findings failed to support their effectiveness as a decay-prevention intervention. Because the present study was conducted in a laboratory setting using a relatively small sample of undergraduate students, additional research is warranted. APPLICATION: Based on the results of the present study, we suggest some additional strategies that trainers might consider to support long-term skill retention when using AARs.

Half-Year Longitudinal Seroprevalence of SARS-CoV-2-Antibodies and Rule Compliance in German Hospital Employees.

COVID-19, which is caused by SARS-CoV-2, is an occupational health risk, especially for healthcare employees due to their higher exposure and consequently higher risk of symptomatic and asymptomatic infections. This study was designed to determine the longitudinal seroprevalence of specific immunoglobulin-G (IgG) antibodies in employees in a hospital setting. All employees in a secondary care hospital, including healthcare and non-healthcare workers, were invited to participate in this single-center study. After an initial screening, a 6-month follow-up was carried out, which included serological examination for SARS-CoV-2 IgG antibodies and a questionnaire for self-reported symptoms, self-perception, and thoughts about local and national hygiene and pandemic plans. The seroprevalence of SARS-CoV-2 IgG antibodies was 0.74% among 406 hospital employees (0.75% in healthcare workers, 0.72% in non-healthcare workers), initially recruited in April 2020, in their follow-up blood specimens in October 2020. In this study, 30.54% of the participants reported using the official German coronavirus mobile application and the majority were content with the local and national rules in relation to coronavirus-related restrictions. At the 6-month follow-up, the 0.74% seroprevalence was below the reported seroprevalence of 1.35% in the general German population. The prevalence in healthcare workers in direct patient care compared with that in workers without direct patient contact did not differ significantly. Further follow-up to monitor the seroprevalence in the high-risk healthcare sector during the ongoing global pandemic is essential.

A combined strategy to detect plasma samples reliably with high anti-SARS-CoV-2 neutralizing antibody titers in routine laboratories.

The determination of anti-SARS-CoV-2 neutralizing antibodies (NAbs) is of interest in many respects. High NAb titers, for example, are the most important criterion regarding the effectiveness of convalescent plasma therapy. However, common cell culture-based NAb assays are time-consuming and feasible only in special laboratories. Our data reveal the suitability of a novel ELISA-based surrogate virus neutralization test (sVNT) to easily measure the inhibition-capability of NAbs in the plasma of COVID-19 convalescents. We propose a combined strategy to detect plasma samples with high NAb titers (≥ 1:160) reliably and to, simultaneously, reduce the risk of erroneously identifying low-titer specimens. For this approach, results of the sVNT assay are compared to and combined with those acquired from the Euroimmun anti-SARS-CoV-2 IgG assay. Both assays are appropriate for high-throughput screening in standard BSL-2 laboratories. Our measurements further show a long-lasting humoral immunity of at least 11 months after symptom onset.

Evidence of Long-Lasting Humoral and Cellular Immunity against SARS-CoV-2 Even in Elderly COVID-19 Convalescents Showing a Mild to Moderate Disease Progression.

We here evaluate the humoral and cellular immune response against SARS-CoV-2 in 41 COVID-19 convalescents. As previous studies mostly included younger individuals, one advantage of our study is the comparatively high mean age of the convalescents included in the cohort considered (54 ± 8.4 years). While anti-SARS-CoV-2 antibodies were still detectable in 95% of convalescents up to 8 months post infection, an antibody-decay over time was generally observed in most donors. Using a multiplex assay, our data additionally reveal that most convalescents exhibit a broad humoral immunity against different viral epitopes. We demonstrate by flow cytometry that convalescent donors show a significantly elevated number of natural killer cells when compared to healthy controls, while no differences were found concerning other leucocyte subpopulations. We detected a specific long-lasting cellular immune response in convalescents by stimulating immune cells with SARS-CoV-2-specific peptides, covering domains of the viral spike, membrane and nucleocapsid protein, and measuring interferon-γ (IFN-γ) release thereafter. We modified a commercially available ELISA assay for IFN-γ determination in whole-blood specimens of COVID-19 convalescents. One advantage of this assay is that it does not require special equipment and can, thus, be performed in any standard laboratory. In conclusion, our study adds knowledge regarding the persistence of immunity of convalescents suffering from mild to moderate COVID-19. Moreover, our study provides a set of simple methods to characterize and confirm experienced COVID-19.

Poor humoral and T-cell response to two-dose SARS-CoV-2 messenger RNA vaccine BNT162b2 in cardiothoracic transplant recipients.

AIMS: Immunocompromised patients have been excluded from studies of SARS-CoV-2 messenger RNA vaccines. The immune response to vaccines against other infectious agents has been shown to be blunted in such patients. We aimed to analyse the humoral and cellular response to prime-boost vaccination with the BNT162b2 vaccine (Pfizer-BioNTech) in cardiothoracic transplant recipients. METHODS AND RESULTS: A total of 50 transplant patients [1-3 years post heart (42), lung (7), or heart-lung (1) transplant, mean age 55 ± 10 years] and a control group of 50 healthy staff members were included. Blood samples were analysed 21 days after the prime and the boosting dose, respectively, to quantify anti-SARS-CoV-2 spike protein (S) immunoglobulin titres (tested by Abbott, Euroimmun and RocheElecsys Immunoassays, each) and the functional inhibitory capacity of neutralizing antibodies (Genscript). To test for a specific T-cell response, heparinized whole blood was stimulated with SARS-CoV-2 specific peptides, covering domains of the viral spike, nucleocapsid and membrane protein, and the interferon-γ release was measured (QuantiFERON Monitor ELISA, Qiagen). The vast majority of transplant patients (90%) showed neither a detectable humoral nor a T-cell response three weeks after the completed two-dose BNT162b2 vaccination; these results are in sharp contrast to the robust immunogenicity seen in the control group: 98% exhibited seroconversion after the prime dose already, with a further significant increase of IgG titres after the booster dose (average > tenfold increase), a more than 90% inhibition capability of neutralizing antibodies as well as evidence of a T-cell responsiveness. CONCLUSIONS: The findings of poor immune responses to a two-dose BNT162b2 vaccination in cardiothoracic transplant patients have a significant impact for organ transplant recipients specifically and possibly for immunocompromised patients in general. It urges for a review of future vaccine strategies in these patients.

Cytokine-mediated induction of human xylosyltransferase-I in systemic sclerosis skin fibroblasts.

Systemic sclerosis (SSc) is an inflammatory fibrotic disease characterized by an excessive extracellular matrix deposition in the skin and internal organs. One fibrotic key event remains the fibroblast-to-myofibroblast differentiation that is controlled by a combination of mechanical and soluble factors, such as transforming growth factor-ß1 (TGF-ß1) and interleukin-1ß (IL-1ß). One important myofibroblast biomarker is human xylosyltransferase-I (XT-I), the initial enzyme in proteoglycan biosynthesis. Increased serum XT activity was quantified in SSc, but the underlying cellular mechanisms remain elusive. This study aims to determine the cellular basis of XT-I induction in SSc by using a myofibroblast cell culture model with SSc fibroblasts (SScF) and healthy control fibroblasts. We found that SScF exhibit a higher extracellular XT-I activity compared to control fibroblasts. This increased XT-I activity in SScF was demonstrated to be mediated by an enhanced autocrine TGF-ß signaling. Upon IL-1ß treatment, SScF showed an increased mRNA expression level of XT-I and TGF-ß receptor II (TGFBR2), while healthy control fibroblasts did not, pointing towards an involvement of IL-1ß in the cytokine-mediated XT-I induction. Performing microRNA (miRNA) inhibition experiments in the presence of TGF-ß1, we showed that the pro-fibrotic effect of IL-1ß may be mediated by a miRNA-21/TGF-ß receptor II axis, enhancing the autocrine TGF-ß signaling in SScF. Taken together, this study improves the mechanistic understanding of fibrotic XT-I induction in SSc by identifying a hitherto unknown IL-1ß-mediated miRNA-21/TGFBR2 regulation contributing to the enhanced XYLT1 expression and XT-I activity in SScF.