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Abstract Mitochondrial DNA copy number (mtDNA-CN) is a biomarker for mitochondrial dysfunction associated with several diseases. Previous genome-wide association studies (GWAS) have been performed to unravel underlying mechanisms of mtDNA-CN regulation. However, the identified gene regions explain only a small fraction of mtDNA-CN variability. Most of this data has been estimated from microarrays based on various pipelines. In the present study we aimed to (1) identify genetic loci for qPCR-measured mtDNA-CN from three studies (16,130 participants) using GWAS, (2) identify potential systematic differences between our qPCR derived mtDNA-CN measurements compared to the published microarray intensity-based estimates, and (3) disentangle the nuclear from mitochondrial regulation of the mtDNA-CN phenotype. We identified two genome-wide significant autosomal loci associated with qPCR-measured mtDNA-CN: at HBS1L (rs4895440, p = 3.39 × 10–13) and GSDMA (rs56030650, p = 4.85 × 10–08) genes. Moreover, 113/115 of the previously published SNPs identified by microarray-based analyses were significantly equivalent with our findings. In our study, the mitochondrial genome itself contributed only marginally to mtDNA-CN regulation as we only detected a single rare mitochondrial variant associated with mtDNA-CN. Furthermore, we incorporated mitochondrial haplogroups into our analyses to explore their potential impact on mtDNA-CN. However, our findings indicate that they do not exert any significant influence on our results.

IntroductionHematopoietic stem cell transplantation (HSCT) is associated with immune complications and endothelial dysfunction due to intricate donor-recipient interactions, conditioning regimens, and inflammatory responses.MethodsThis study investigated the role of the complement system during HSCT and its interaction with the cytokine network. Seventeen acute myeloid leukemia patients undergoing HSCT were monitored, including blood sampling from the start of the conditioning regimen until four weeks post-transplant. Clinical follow-up was 200 days.ResultsTotal complement functional activity was measured by WIELISA and the degree of complement activation by ELISA measurement of sC5b-9. Cytokine release was measured using a 27-multiplex immuno-assay. At all time-points during HSCT complement functional activity remained comparable to healthy controls. Complement activation was continuously stable except for two patients demonstrating increased activation, consistent with severe endotheliopathy and infections. In vitro experiments with post-HSCT whole blood challenged with Escherichia coli, revealed a hyperinflammatory cytokine response with increased TNF, IL-1β, IL-6 and IL-8 formation. Complement C3 inhibition markedly reduced the cytokine response induced by Staphylococcus aureus, Aspergillus fumigatus, and cholesterol crystals.DiscussionIn conclusion, HSCT patients generally retained a fully functional complement system, whereas activation occurred in patients with severe complications. The complement-cytokine interaction indicates the potential for new complement-targeting therapeutic strategies in HSCT.

IntroductionThe complement system is part of innate immunity and is comprised of an intricate network of proteins that are vital for host defense and host homeostasis. A distinct mechanism by which complement defends against invading pathogens is through the membrane attack complex (MAC), a lytic structure that forms on target surfaces. The MAC is made up of several complement components, and one indispensable component of the MAC is C7. The role of C7 in MAC assembly is well documented, however, inherent characteristics of C7 are yet to be investigated.MethodsTo shed light on the molecular characteristics of C7, we examined the properties of serum-purified C7 acquired using polyclonal and novel monoclonal antibodies. The properties of serum‑purified C7 were investigated through a series of proteolytic analyses, encompassing Western blot and mass spectrometry. The nature of C7 protein-protein interactions were further examined by a novel enzyme-linked immunosorbent assay (ELISA), as well as size‑exclusion chromatography. ResultsProtein analyses showcased an association between C7 and clusterin, an inhibitory complement regulator. The distinct association between C7 and clusterin was also demonstrated in serum-purified clusterin. Further assessment revealed that a complex between C7 and clusterin (C7-CLU) was detected. The C7-CLU complex was also identified in healthy serum and plasma donors, highlighting the presence of the complex in circulation. DiscussionClusterin is known to dissociate the MAC structure by binding to polymerized C9, nevertheless, here we show clusterin binding to the native form of a terminal complement protein in vivo. The presented data reveal that C7 exhibits characteristics beyond that of MAC assembly, instigating further investigation of the effector role that the C7-CLU complex plays in the complement cascade.

Der zweitägige Workshop „Data Stewardship goes Germany“ wurde 2022 von der FDM-AG der TU9 organisiert mit der primären Zielgruppe Data Stewards1. Bei dem Workshop stand nicht nur die Informationsvermittlung durch Vorträge, sondern vor allem die interaktive Arbeit und die Vernetzung der Teilnehmenden im Vordergrund. Neben den zwei Keynotes von der TU Delft und der Aalto University zu Data Stewardship-Konzepten gab es Beiträge zu den beiden Themenblöcken „Aufbau von FDM-Dienstleistungen” und „Technische Tools”. Die Interaktion und Vernetzung wurden durch Raum für Gespräche an Postern und in Pausen sowie durch die Barcamp-Methode gefördert. Auffällig war, dass Data Stewards sich oft als Einzelkämpfer:innen fühlen und viele Strukturen und Überlegungen parallel entwickelt und getätigt werden. Eine Nachnutzung bestehender Strukturen und Dienste ist aktuell noch wenig etabliert und vor allem auch wenig bekannt. Der Workshop stellt einen wichtigen Beitrag dar, um solche Lücken zu schließen und Awareness zur Nachnutzung von FDM-Strukturen und-Diensten zu schaffen. Außerdem wurde deutlich, dass es in Deutschland noch kein einheitliches Verständnis von Data Stewardship gibt und dass eine Unterstützung und Förderung von Data Stewards stark von der Universität bzw. den außeruniversitären Einrichtungen abhängt. Eine Vernetzung über verschiedene Einrichtungen fand bisher kaum statt, so dass der Workshop ein wichtiger Schritt in diese Richtung ist.

Summary: Complement is a fundamental innate immune response component. Its alterations are associated with severe systemic diseases. To illuminate the complement’s genetic underpinnings, we conduct genome-wide association studies of the functional activity of the classical (CP), lectin (LP), and alternative (AP) complement pathways in the Cooperative Health Research in South Tyrol study (n = 4,990). We identify seven loci, encompassing 13 independent, pathway-specific variants located in or near complement genes (CFHR4, C7, C2, MBL2) and non-complement genes (PDE3A, TNXB, ABO), explaining up to 74% of complement pathways’ genetic heritability and implicating long-range haplotypes associated with LP at MBL2. Two-sample Mendelian randomization analyses, supported by transcriptome- and proteome-wide colocalization, confirm known causal pathways, establish within-complement feedback loops, and implicate causality of ABO on LP and of CFHR2 and C7 on AP. LP causally influences collectin-11 and KAAG1 levels and the risk of mouth ulcers. These results build a comprehensive resource to investigate the role of complement in human health.

IntroductionThe rapid evolution of SARS-CoV-2 has posed a challenge to long-lasting immunity against the novel virus. Apart from neutralizing function, binding antibodies induced by vaccination or infection play an important role in containing the infection. MethodsTo determine the proportion of wild-type (WT)–generated antibodies recognizant of more recent variants, plasma samples from either SARS-CoV-2 WT-infected (n = 336) or double-mRNA (Comirnaty)–vaccinated individuals (n = 354, age and sex matched to the convalescent group) were analyzed for binding antibody capacity against the S1 protein of the BA.1 omicron variant. ResultsOverall, 38.59% (95% CI, 37.01– 40.20) of WT-generated antibodies recognized Omicron BA.1 S1 protein [28.83% (95% CI, 26.73–30.91) after infection and 43.46% (95% CI, 41.61–45.31) after vaccination; p < 0.001]. Although the proportion of WT-generated binding and neutralizing antibodies also binding to BA.1 is substantially reduced, the avidity of the remaining antibodies against the Omicron variant was non-inferior to that of the ancestral virus: Omicron: 39.7% (95% CI: 38.1–41.3) as compared to the avidity to WT: 27.0% (95% CI, 25.5–28.4), respectively (p < 0.001). Furthermore, we noticed a modestly yet statistically significant higher avidity toward the Omicron epitopes among the vaccinated group (42.2%; 95% CI, 40.51–43.94) as compared to the convalescent counterparts (36.4%; 95% CI, 33.42–38.76) (p = 0.003), even after adjusting for antibody concentration. DiscussionOur results suggest that an aspect of functional immunity against the novel strain was considerably retained after WT contact, speculatively counteracting the impact of immune evasion toward neutralization of the strain. Higher antibody levels and cross-binding capacity among vaccinated individuals suggest an advantage of repeated exposure in generating robust immunity.

Typical hemolytic uremic syndrome (HUS) is mainly caused by Shiga toxin-producing Escherichia coli (STEC) releasing Shiga toxin 2 (Stx2). Two different structures of this AB5 toxin have been described: uncleaved, with intact B and A chains, and cleaved, with intact B and a nicked A chain consisting of two fragments, A1 and A2, connected by a disulfide bond. Despite having the same toxic effect on sensitive cells, the two forms differ in their binding properties for circulating cells, serum components and complement factors, thus contributing to the pathogenesis of HUS differently. The outcome of STEC infections and the development of HUS could be influenced by the relative amounts of uncleaved or cleaved Stx2 circulating in patients’ blood. Cleaved Stx2 was identified and quantified for the first time in four out of eight STEC-infected patients’ sera by a method based on the inhibition of cell-free translation. Cleaved Stx2 was present in the sera of patients with toxins bound to neutrophils and in two out of three patients developing HUS, suggesting its involvement in HUS pathogenesis, although in association with other bacterial or host factors.

Shiga toxins (Stxs), especially the Stx2a subtype, are the major virulence factors involved in enterohemorrhagic Escherichia coli (EHEC)-associated hemolytic uremic syndrome (eHUS), a life-threatening disease causing acute kidney injury, especially in children. After oral transmission and colonization in the gut, EHEC release Stx. Intracellular cleavage of the Stx A subunit, when followed by reduction, boosts the enzymatic activity that causes damage to targeted cells. This cleavage was assumed to be mostly mediated by furin during Stx intracellular trafficking. To investigate whether this cleavage could occur in the intestine, even prior to entering target cells, Stx2a A subunit structure (intact or cleaved) was characterized after its exposure to specific host factors present in human stool. The molecular weight of Stx2a A subunit/fragments was determined by immunoblotting after electrophoretic separation under reducing conditions. In this study, it was demonstrated that Stx2a is cleaved by certain human stool components. Trypsin and chymotrypsin-like elastase 3B (CELA3B), two serine proteases, were identified as potential candidates that can trigger the extracellular cleavage of Stx2a A subunit directly after its secretion by EHEC in the gut. Whether the observed cleavage indeed translates to natural infections and plays a role in eHUS pathogenesis has yet to be determined. If so, it seems likely that a host’s protease profile could affect disease development by changing the toxin’s biological features.

IntroductionWhile complement is a contributor to disease severity in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections, all three complement pathways might be activated by the virus. Lectin pathway activation occurs through different pattern recognition molecules, including mannan binding lectin (MBL), a protein shown to interact with SARS-CoV-2 proteins. However, the exact role of lectin pathway activation and its key pattern recognition molecule MBL in COVID-19 is still not fully understood.MethodsWe therefore investigated activation of the lectin pathway in two independent cohorts of SARS-CoV-2 infected patients, while also analysing MBL protein levels and potential effects of the six major single nucleotide polymorphisms (SNPs) found in the MBL2 gene on COVID-19 severity and outcome.ResultsWe show that the lectin pathway is activated in acute COVID-19, indicated by the correlation between complement activation product levels of the MASP-1/C1-INH complex (p=0.0011) and C4d (p

The most commonly used markers to assess complement activation are split products that are produced through activation of all three pathways and are located downstream of C3. In contrast, C4d derives from the cleavage of C4 and indicates either classical (CP) or lectin pathway (LP) activation. Although C4d is perfectly able to distinguish between CP/LP and alternative pathway (AP) activation, no well-established markers are available to differentiate between early CP and LP activation. Active enzymes of both pathways (C1s/C1r for the CP, MASP-1/MASP-2 for the LP) are regulated by C1 esterase inhibitor (C1-INH) through the formation of covalent complexes. Aim of this study was to develop validated immunoassays detecting C1s/C1-INH and MASP-1/C1-INH complex levels. Measurement of the complexes reveals information about the involvement of the respective pathways in complement-mediated diseases. Two sandwich ELISAs detecting C1s/C1-INH and MASP-1/C1-INH complex were developed and tested thoroughly, and it was investigated whether C1s/C1-INH and MASP-1/C1-INH complexes could serve as markers for either early CP or LP activation. In addition, a reference range for these complexes in healthy adults was defined, and the assays were clinically validated utilizing samples of 414 COVID-19 patients and 96 healthy controls. The immunoassays can reliably measure C1s/C1-INH and MASP-1/C1-INH complex concentrations in EDTA plasma from healthy and diseased individuals. Both complex levels are increased in serum when activated with zymosan, making them suitable markers for early classical and early lectin pathway activation. Furthermore, measurements of C1-INH complexes in 96 healthy adults showed normally distributed C1s/C1-INH complex levels with a physiological concentration of 1846 ± 1060 ng/mL (mean ± 2SD) and right-skewed distribution of MASP-1/C1-INH complex levels with a median concentration of 36.9 (13.18 - 87.89) ng/mL (2.5-97.5 percentile range), while levels of both complexes were increased in COVID-19 patients (p

Activation of the complement system is mediated by the interaction between pathogens and pattern recognition molecules (PRMs); mannose-binding lectin (MBL), ficolins, and collectin-10/-11 from the lectin pathway and C1q from the classical pathway. Lectin pathway activation specifically depends on proteases named MBL-associated serine proteases (MASPs) that are found in complexes with PRMs. In this study, we hypothesize that MASPs can recognize selected pathogens independently of PRMs. Using different clinical strains of opportunistic fungi, we have observed that MASPs directly recognize certain fungal pathogens in a way that can facilitate complement activation. Among these were Aspergillus fumigatus – a dangerous pathogen, especially for immunocompromised patients. In flow cytometry and fluorescence microscopy, we found that MASP-1 and -3 bound to all A. fumigatus growth stages (conidia, germ tubes, and hyphae), whereas rMASP-2 and the nonproteolytic rMAP-1 did not. Bound rMASPs could recruit rMBL and rficolin-3 to A. fumigatus conidia in a nonclassical manner and activate complement via rMASP-2. In experiments using recombinant and purified components, rMASP-1 increased the neutrophilic phagocytosis of conidia. In serum where known complement activation pathways were blocked, phagocytosis could be mediated by rMASP-3. We have encountered an unknown pathway for complement activation and found that MASP-1 and MASP-3 have dual functions as enzymes and as PRMs.

Summary: Background: Several COVID-19 vaccines have been approved. The mRNA vaccine from Pfizer/BioNTech (Comirnaty, BNT162b2; BNT) and the vector vaccine from AstraZeneca (Vaxzevria, ChAdOx1; AZ) have been widely used. mRNA vaccines induce high antibody and T cell responses, also to SARS-CoV-2 variants, but are costlier and less stable than the slightly less effective vector vaccines. For vector vaccines, heterologous vaccination schedules have generally proven more effective than homologous schedules. Methods: In the HEVACC three-arm, single-blinded, adaptive design study (ClinicalTrials.gov Identifier: NCT04907331), participants between 18 and 65 years with no prior history of SARS-CoV-2 infection and a first dose of AZ or BNT were included. The AZ/AZ and the AZ/BNT arms were randomized (in a 1:1 ratio stratified by sex and trial site) and single-blinded, the third arm (BNT/BNT) was observational. We compared the reactogenicity between the study arms and hypothesized that immunogenicity was higher for the heterologous AZ/BNT compared to the homologous AZ/AZ regimen using neutralizing antibody titers as primary endpoint. Findings: This interim analysis was conducted after 234 participants had been randomized and 254 immunized (N=109 AZ/AZ, N=115 AZ/BNZ, N=30 BNT/BNT). Heterologous AZ/BNT vaccination was well tolerated without study-related severe adverse events. Neutralizing antibody titers on day 30 were statistically significant higher in the AZ/BNT and the BNT/BNT groups than in the AZ/AZ group, for B.1.617.2 (Delta) AZ/AZ median reciprocal titer 75.9 (99.9% CI 58.0 - 132.5), AZ/BNT 571.5 (99.9% CI 396.6 - 733.1), and BNT/BNT 404.5 (99.9% CI 68.3 - 1024). Similarly, the frequency and multifunctionality of spike-specific T cell responses was comparable between the AZ/BNT and the BNT/BNT groups, but lower in the AZ/AZ vaccinees. Interpretation: This study clearly shows the immunogenicity and safety of heterologous AZ/BNT vaccination and encourages further studies on heterologous vaccination schedules. Funding: This work was supported by the Medical University of Innsbruck, and partially funded by NIAID contracts No. 75N9301900065, 75N93021C00016, and 75N93019C00051.

The transition metals iron and copper are required by virtually all organisms but are toxic in excess. Acquisition of both metals and resistance to copper excess have previously been shown to be important for virulence of the most common airborne human mold pathogen, Aspergillus fumigatus. Here we demonstrate that the ambient availability of amino acids and proteins increases the copper resistance of A. fumigatus wild type and particularly of the ΔcrpA mutant that lacks export-mediated copper detoxification. The highest-protecting activity was found for L-histidine followed by L-asparagine, L-aspartate, L-serine, L-threonine, and L-tyrosine. Other amino acids and proteins also displayed significant but lower protection. The protecting activity of non-proteinogenic D-histidine, L-histidine-mediated growth inhibition in the absence of high-affinity copper uptake, determination of cellular metal contents, and expression analysis of copper-regulated genes suggested that histidine inhibits low-affinity but not high-affinity copper acquisition by extracellular copper complexation. An increase in the cellular copper content was found to be accompanied by an increase in the iron content, and, in agreement, iron starvation increased copper susceptibility, which underlines the importance of cellular metal balancing. Due to the role of iron and copper in nutritional immunity, these findings are likely to play an important role in the host niche.

Immunothrombosis, an excessive inflammatory response with simultaneous overactivation of the coagulation system, is a central pathomechanism in sepsis and COVID-19. It is associated with cellular activation, vascular damage, and microvascular thrombosis, which can lead to multiple organ failure and death. Here, we characterized factors related to immunothrombosis in plasma samples from 78 sepsis patients. In the course of routine clinical testing, SARS-CoV-2 was detected in 14 of these patients. Viral infection was associated with a higher mortality. Both, COVID-19 negative and COVID-19 positive sepsis patients showed increased levels of effectors of immunothrombosis, including platelet factor 4, D-dimer, nucleosomes, citrullinated histone H3, high mobility group box-1 protein, as well as phosphatidylserine-expressing platelet-derived extracellular vesicles, compared to healthy controls (n = 25). Using a 27-plex cytokine bead array, we found that Interleukin (IL)-1ra, IL-6, IL-8, IL-13, tumor necrosis factor (TNF)-α, interferon inducible protein (IP)-10, monocyte chemotactic protein (MCP)-1, macrophage inflammatory protein (MIP)-1α, and granulocyte-colony stimulating factor (G-CSF) were elevated in both, COVID-19 negative and COVID-19 positive sepsis patients, as compared to healthy controls. SARS-CoV-2 infection was associated with elevated levels of IP-10, MCP-1, and IL-13, while all other mediators widely overlapped between COVID-19 negative and COVID-19 positive patients.

Background: Mucormycetes, a heterogeneous group of fungi, induce a life-threatening disease called mucormycosis. Immune deficiencies represent a major risk factor; hence, we wanted to illuminate the role of complement and platelets in the defense against mucormycetes. Methods: Rhizopus arrhizus (Ra), Rhizopus microsporus (Rm), Lichtheimia ramosa (Lr), Lichtheimia corymbifera (Lc), Rhizomucor pusillus (Rmp), and Mucor circinelloides (Mc) spores were opsonized with human and mouse serum, and C1q, C3c, and terminal complement complex (C5b-9) deposition was measured. Additionally, thrombocytopenic, C3-deficient, or C6-deficient mice were intravenously infected with selected isolates. Survival and immunological parameters were monitored, and fungal burden was determined and compared to that of immunocompetent and neutropenic mice. Results: In vitro experiments showed significant differences in complement deposition between mucormycetes. Mc isolates bound up to threefold more human C5b-9 than other mucormycetes. Lr, Lc, and Mc bound high levels of murine C3c, whereas human C3c deposition was reduced on Mc compared to Lr and Lc. Murine C3c deposition negatively correlated with virulence. Complement deficiencies and neutropenia, but not thrombocytopenia, were shown to be a risk factor for a lethal outcome. Conclusion: Complement deposition varies between mucormycetes. Additionally, we demonstrated that complement and neutrophilic granulocytes, but not platelets, play an important role in a murine model of disseminated mucormycosis.

Background In early March 2020, a SARS-CoV-2 outbreak in the ski resort Ischgl in Austria triggered the spread of SARS-CoV-2 throughout Austria and Northern Europe. In a previous study, we found that the seroprevalence in the adult population of Ischgl had reached 45% by the end of April, representing an exceptionally high level of local seropositivity in Europe. We performed a follow-up study in Ischgl, which is the first to show persistence of immunity and protection against SARS-CoV-2 and some of its variants at a community level.Methods Of the 1259 adults that participated in the baseline study, 801 have been included in the follow-up in November 2020. The study involved the analysis of binding and neutralizing antibodies and T cell responses. In addition, the incidence of SARS-CoV-2 and its variants in Ischgl was compared to the incidence in similar municipalities in Tyrol until April 2021.Findings For the 801 individuals that participated in both studies, the seroprevalence declined from 51.4% (95% confidence interval (CI) 47.9–54.9) to 45.4% (95% CI 42.0–49.0). Median antibody concentrations dropped considerably (5.345, 95% CI 4.833 – 6.123 to 2.298, 95% CI 2.141 – 2.527) but antibody avidity increased (17.02, 95% CI 16.49 – 17.94 to 42.46, 95% CI 41.06 – 46.26). Only one person had lost detectable antibodies and T cell responses. In parallel to this persistent immunity, we observed that Ischgl was relatively spared, compared to similar municipalities, from the prominent second COVID-19 wave that hit Austria in November 2020. In addition, we used sequencing data to show that the local immunity acquired from wild-type infections also helped to curb infections from variants of SARS-CoV-2 which spread in Austria since January 2021.Interpretation The relatively high level of seroprevalence (40–45%) in Ischgl persisted and might have been associated with the observed protection of Ischgl residents against virus infection during the second COVID-19 wave as well as against variant spread in 2021.Funding Funding was provided by the government of Tyrol and the FWF Austrian Science Fund.

Abstract Background Increasing bacterial resistance to antibiotics is a serious problem worldwide. We sought to record the acquisition of antibiotic-resistant Escherichia coli (E. coli) in healthy infants in Northern Thailand and investigated potential determinants. Methods Stool samples from 142 infants after birth, at ages 2wk, 2mo, 4 to 6mo, and 1y, and parent stool samples were screened for E. coli resistance to tetracycline, ampicillin, co-trimoxazole, and cefazoline by culture, and isolates were further investigated for multiresistance by disc diffusion method. Pulsed-field gel electrophoresis was performed to identify persistent and transmitted strains. Genetic comparison of resistant and transmitted strains was done by multilocus sequence typing (MLST) and strains were further investigated for extra- and intra-intestinal virulence factors by multiplex PCR. Results Forty-seven (33%) neonatal meconium samples contained resistant E. coli. Prevalence increased continuously: After 1y, resistance proportion (tetracycline 80%, ampicillin 72%, co-trimoxazole 66%, cefazoline 35%) almost matched those in parents. In 8 infants (6%), identical E. coli strains were found in at least 3 sampling time points (suggesting persistence). Transmission of resistant E. coli from parents to child was observed in only 8 families. MLST showed high diversity. We could not identify any virulence genes or factors associated with persistence, or transmission of resistant E. coli. Full-term, vaginal birth and birth in rural hospital were identified as risk factors for early childhood colonization with resistant E. coli. Conclusion One third of healthy Thai neonates harboured antibiotic-resistant E. coli in meconium. The proportion of resistant E. coli increased during the first year of life almost reaching the value in adults. We hypothesize that enhancement of infection control measures and cautious use of antibiotics may help to control further increase of resistance.

The immune system is a tightly regulated network which allows the development of defense mechanisms against foreign antigens and tolerance toward self-antigens. Regulatory T cells (Treg) contribute to immune homeostasis by maintaining unresponsiveness to self-antigens and suppressing exaggerated immune responses. Dysregulation of any of these processes can lead to serious consequences. Classically, Treg cell functions have been described in CD4+ T cells, but other immune cells also harbour the capacity to modulate immune responses. Regulatory functions have been described for different CD8+ T cell subsets, as well as other T cells such as γδT cells or NKT cells. In this review we describe the diverse populations of Treg cells and their role in different scenarios. Special attention is paid to the aging process, which is characterized by an altered composition of immune cells. Treg cells can contribute to the development of various age-related diseases but they are poorly characterized in aged individuals. The huge diversity of cells that display immune modulatory functions and the lack of universal markers to identify Treg make the expanding field of Treg research complex and challenging. There are still many open questions that need to be answered to solve the enigma of regulatory T cells.

The complement system has developed different strategies to clear infections by several effector mechanisms, such as opsonization, which supports phagocytosis, attracting immune cells by C3 and C5 cleavage products, or direct killing of pathogens by the formation of the membrane attack complex (MAC). As the Zika virus (ZIKV) activates the classical complement pathway and thus has to avoid clearance by the complement system, we analyzed putative viral escape mechanisms, which limit virolysis. We identified binding of the recombinant viral envelope E protein to components of the terminal pathway complement (C5b6, C7, C8, and C9) by ELISA. Western blot analyses revealed that ZIKV E protein interfered with the polymerization of C9, induced on cellular surfaces, either by purified terminal complement proteins or by normal human serum (NHS) as a source of the complement. Further, the hemolytic activity of NHS was significantly reduced in the presence of the recombinant E protein or entire viral particles. This data indicates that ZIKV reduces MAC formation and complement-mediated lysis by binding terminal complement proteins to the viral E protein.

Invasive fungal infections caused by Aspergillus (A.) and Mucorales species still represent life-threatening diseases in immunocompromised individuals, and deeper knowledge about fungal interactions with elements of innate immunity, such as complement and platelets, appears essential for optimized therapy. Previous studies showed that galactosaminogalactan secreted by A. fumigatus and A. flavus is deposited on platelets, thereby inducing their activation. Since the altered platelet surface is a putative trigger for complement activation, we aimed to study the interplay of platelets with complement in the presence of fungal GAG. Culture supernatants (SN) of A. fumigatus and A. flavus both induced not only GAG deposition but also subsequent deposition of complement C3 fragments on the platelet surface. The SN of a Δuge3 mutant of A. fumigatus, which is unable to synthesize GAG, did not induce complement deposition on platelets, nor did the SN of other Aspergillus species and all tested Mucorales. Detailed analysis revealed that GAG deposition itself triggered the complement cascade rather than the GAG-induced phosphatidylserine exposure. The lectin pathway of complement could be shown to be crucially involved in this process. GAG-induced complement activation on the platelet surface was revealed to trigger processes that might contribute to the pathogenesis of invasive aspergillosis by A. fumigatus or A. flavus. Both pro-inflammatory anaphylatoxins C3a and C5a arose when platelets were incubated with SN of these fungal species; these processes might favor excessive inflammation after fungal infection. Furthermore, platelets were stimulated to shed microparticles, which are also known to harbor pro-inflammatory and pro-coagulant properties. Not only did early processes of the complement cascade proceed on platelets, but also the formation of the terminal complement C5b-9 complex was detected on platelets after incubation with fungal SN. Subsequently, reduced viability of the platelets could be shown, which might contribute to the lowered platelet numbers found in infected patients. In summary, fungal GAG initiates an interplay between complement and platelets that can be supposed to contribute to excessive inflammation, thrombocytopenia, and thrombosis, which are important hallmarks of fatal invasive mycoses.

Candida is a dominant fungal pathogen in immunocompromised hosts, leading to opportunistic infections. Complement with its multifaceted functions is involved in the immune defense against this yeast, and recently several novel aspects have emerged in this old battle. It is clear that Candida can adopt both roles as a colonizer or as a pathogen. In our article, we focus on the molecular mechanisms of the Candida-complement interplay, which occur in disseminated disease as well as locally on skin or on mucous membranes in mouth and vagina; the mechanisms can be supposed to be the same. Activation of the complement system by Candida is facilitated by directly triggering the three dominant pathways, but also indirectly via the coagulation and fibrinolysis systems. The complement-mediated anti-Candida effects induced thereby clearly extend chemotaxis, opsonization, and phagocytosis, and even the membrane attack complex formed on the fungal surface plays a modulatory role, although lysis of the yeast per se cannot be induced due to the thick fungal cell wall. In order to avoid the hostile action of complement, several evasion mechanisms have evolved during co-evolution, comprising the avoidance of recognition, and destruction. The latter comes in many flavors, in particular the cleavage of complement proteins by yeast enzymes and the exploitation of regulatory proteins by recruiting them on the cell wall, such as factor H. The rationale behind that is that the fluid phase regulators on the fungal cell surface down-regulate complement locally. Interestingly, however, evasion protein knockout strains do not necessarily lead to an attenuated disease, so it is likely more complex in vivo than initially thought. The interactions between complement and non-albicans species also deserve attention, especially Candida auris, a recently identified drug-resistant species of medical importance. This is in particular worth investigating, as deciphering of these interactions may lead to alternative anti-fungal therapies directly targeting the molecular mechanisms.

Candidiasis is common in diabetic patients. Complement evasion is facilitated by binding complement factor H (FH). Since the expression of high-affinity glucose transporter 1 (Hgt1), a FH-binding molecule, is glucose-dependent, we aimed to study its relevance to the pathogenesis of Candida albicans. Euglycemic and diabetic mice were intravenously challenged with either Candida albicans lacking Hgt1 (hgt1-/-) or its parental strain (SN152). Survival and clinical status were monitored over 14 days. In vitro, Candida albicans strains were grown at different glucose concentrations, opsonized with human serum, and checked for C3b/iC3b and FH deposition. Phagocytosis was studied by fluorescein isothiocyanate-labeled opsonized yeast cells incubated with granulocytes. The murine model demonstrated a significantly higher virulence of SN152 in diabetic mice and an overall increased lethality of mice challenged with hgt1-/-. In vitro lower phagocytosis and C3b/iC3b deposition and higher FH deposition were demonstrated for SN152 incubated at higher glucose concentrations, while there was no difference on hgt1-/- at physiological glucose concentrations. Despite C3b/iC3b and FH deposition being glucose-dependent, this effect has a minor influence on phagocytosis. The absence of Hgt1 is diminishing this dependency on complement deposition, but it cannot be attributed to being beneficial in a murine model.

Overactivation of the complement system has been characterized in severe COVID-19 cases. Complement components are known to trigger NETosis via the coagulation cascade and have also been reported in human tracheobronchial epithelial cells. In this longitudinal study, we investigated systemic and local complement activation and NETosis in COVID-19 patients that underwent mechanical ventilation. Results confirmed significantly higher baseline levels of serum C5a (24.5 ± 39.0 ng/mL) and TCC (11.03 ± 8.52 µg/mL) in patients compared to healthy controls (p < 0.01 and p < 0.0001, respectively). Furthermore, systemic NETosis was significantly augmented in patients (5.87 (±3.71) × 106 neutrophils/mL) compared to healthy controls (0.82 (±0.74) × 106 neutrophils/mL) (p < 0.0001). In tracheal fluid, baseline TCC levels but not C5a and NETosis, were significantly higher in patients. Kinetic studies of systemic complement activation revealed markedly higher levels of TCC and CRP in nonsurvivors compared to survivors. In contrast, kinetic studies showed decreased local NETosis in tracheal fluid but comparable local complement activation in nonsurvivors compared to survivors. Systemic TCC and NETosis were significantly correlated with inflammation and coagulation markers. We propose that a ratio comprising systemic inflammation, complement activation, and chest X-ray score could be rendered as a predictive parameter of patient outcome in severe SARS-CoV-2 infections.

Complement is usually seen to largely originate from the liver to accomplish its tasks systemically – its return to the production site has long been underestimated. Recent progress in genomics, therapeutic effects on complement, standardised possibilities in medical laboratory tests and involvement of complosome brings the complement system with its three major functions of opsonization, cytolysis and phagocytosis back to liver biology and pathology. The LOINC™ system features 20 entries for the C3 component of complement to anticipate the application of artificial intelligence data banks algorythms of which are fed with patient-specific data connected to standard lab assays for liver function. These advancements now lead to increased vigilance by clinicians. This reassessment article will further elucidate the distribution of synthesis sites to the three germ layer-derived cell systems and the role complement now known to play in embryogenesis, senescence, allotransplantation and autoimmune disease. This establishes the liver as part of the gastro-intestinal system in connection with nosological entities never thought of, such as the microbiota-liver-brain axis. In neurological disease etiology infectious and autoimmune hepatitis play an important role in the context of causative viz reactive complement activation. The mosaic of autoimmunity, i.e. multiple combinations of the many factors producing varying clinical pictures, leads to the manifold facets of liver autoimmunity. Keywords: Hepatocyte, Complement system, Complosome, Autoantibody, Diagnostic tests

ABSTRACT We have recently shown that the catecholamine dopamine regulates cellular iron homeostasis in macrophages. As iron is an essential nutrient for microbes, and intracellular iron availability affects the growth of intracellular bacteria, we studied whether dopamine administration impacts the course of Salmonella infections. Dopamine was found to promote the growth of Salmonella both in culture and within bone marrow-derived macrophages, which was dependent on increased bacterial iron acquisition. Dopamine administration to mice infected with Salmonella enterica serovar Typhimurium resulted in significantly increased bacterial burdens in liver and spleen, as well as reduced survival. The promotion of bacterial growth by dopamine was independent of the siderophore-binding host peptide lipocalin-2. Rather, dopamine enhancement of iron uptake requires both the histidine sensor kinase QseC and bacterial iron transporters, in particular SitABCD, and may also involve the increased expression of bacterial iron uptake genes. Deletion or pharmacological blockade of QseC reduced but did not abolish the growth-promoting effects of dopamine. Dopamine also modulated systemic iron homeostasis by increasing hepcidin expression and depleting macrophages of the iron exporter ferroportin, which enhanced intracellular bacterial growth. Salmonella lacking all central iron uptake pathways failed to benefit from dopamine treatment. These observations are potentially relevant to critically ill patients, in whom the pharmacological administration of catecholamines to improve circulatory performance may exacerbate the course of infection with siderophilic bacteria. IMPORTANCE Here we show that dopamine increases bacterial iron incorporation and promotes Salmonella Typhimurium growth both in vitro and in vivo. These observations suggest the potential hazards of pharmacological catecholamine administration in patients with bacterial sepsis but also suggest that the inhibition of bacterial iron acquisition might provide a useful approach to antimicrobial therapy.

Complement is a tightly controlled arm of the innate immune system, facilitating phagocytosis and killing of invading pathogens. Factor H (FH) is the main fluid-phase inhibitor of the alternative pathway. Many pathogens can hijack FH from the host and protect themselves from complement-dependent killing. Candida albicans is a clinically important opportunistic yeast, expressing different FH binding molecules on its cell surface, which allow complement evasion. One such FH binding molecule is the transmembrane protein “High affinity glucose transporter 1” (Hgt1p), involved in glucose metabolism. This study demonstrated that Hgt1p transcription and expression is induced and highest at the low, but physiological glucose concentration of 0.1%. Thus, this concentration was used throughout the study. We also demonstrated the transport of Hgt1p to the fungal cell wall surface by vesicle trafficking and its release by exosomes containing Hgt1p integrated in the vesicular membrane. We corroborated Hgt1p as FH binding molecule. A polyclonal anti-Hgt1p antibody was created which interfered with the binding of FH, present in normal human serum to the fungal cell wall. A chimeric molecule consisting of FH domains 6 and 7 fused to human IgG1 Fc (FH6.7/Fc) even more comprehensively blocked FH binding, likely because FH6.7/Fc diverted FH away from fungal FH ligands other than Hgt1p. Reduced FH binding to the yeast was associated with a concomitant increase in C3b/iC3b deposition and resulted in significantly increased in vitro phagocytosis and killing by human neutrophils. In conclusion, Hgt1p also exhibits non-canonical functions such as binding FH after its export to the cell wall. Blocking Hgt1p-FH interactions may represent a tool to enhance complement activation on the fungal surface to promote phagocytosis and killing of C. albicans.

Shortly before his 70th birthday, Robert B [...]

Whole genome sequencing is a useful tool to monitor the spread of resistance mechanisms in bacteria. In this retrospective study, we investigated genetic resistance mechanisms, sequence types (ST) and respective phenotypes of linezolid-resistant Staphylococcus epidermidis (LRSE, n = 129) recovered from a cohort of patients receiving or not receiving linezolid within a tertiary hospital in Innsbruck, Austria. Hereby, the point mutation G2603U in the 23S rRNA (n = 91) was the major resistance mechanism followed by the presence of plasmid-derived cfr (n = 30). The majority of LRSE isolates were ST2 strains, followed by ST5. LRSE isolates expressed a high resistance level to linezolid with a minimal inhibitory concentration of ≥256 mg/L (n = 83) in most isolates, particularly in strains carrying the cfr gene (p < 0.001). Linezolid usage was the most prominent (but not the only) trigger for the development of linezolid resistance. However, administration of linezolid was not associated with a specific resistance mechanism. Restriction of linezolid usage and the monitoring of plasmid-derived cfr in LRSE are potential key steps to reduce linezolid resistance and its transmission to more pathogenic Gram-positive bacteria.

With deep sadness, we have to accept that two cornerstones, not just of British immunology, but also world-famous scientists in the field of complement research, passed away within the margin of a few weeks: Peter Julius Lachmann on 26 December 2020 (Figure 1), and Robert Braidwood Sim (known to everyone as Bob) on 6 February 2021 (Figure 2) [...]

The rapid spread of the virus in Latin America and the association of the infection with microcephaly in newborns or Guillain–Barré Syndrome in adults prompted the WHO to declare the Zika virus (ZIKV) epidemic to be an international public health emergency in 2016. As the virus was first discovered in monkeys and is spread not only by mosquitos but also from human to human, we investigated the stability to the human complement of ZIKV derived from mosquito (ZIKVInsect), monkey (ZIKVVero), or human cells (ZIKVA549 and ZIKVFibro), respectively. At a low serum concentration (10%), which refers to complement concentrations found on mucosal surfaces, the virus was relatively stable at 37 °C. At higher complement levels (up to 50% serum concentration), ZIKV titers differed significantly depending on the cell line used for the propagation of the virus. While the viral titer of ZIKVInsect decreased about two orders in magnitude, when incubated with human serum, the virus derived from human cells was more resistant to complement-mediated lysis (CML). By virus-capture assay and Western blots, the complement regulator protein CD55 was identified to be incorporated into the viral envelope. Blocking of CD55 by neutralizing Abs significantly increased the sensitivity to human complement. Taken together, these data indicate that the incorporation of CD55 from human cells contributes to the stability of ZIKV against complement-mediated virolysis.

With great sadness and sympathy for his family, especially for his wife and companion for many decades, Sylvia, I convey the news of the peaceful passing of Professor Sir Peter Lachmann on 26 December 2020, three days after his 89th birthday and after a Christmas celebration—as far as this was possible these days—in the heart of his family [...]

The pathogenesis of Escherichia coli-induced hemolytic uremic syndrome (eHUS) caused by infections with pathogenic Shiga toxin (Stx) producing E. coli (STEC) is centered on bacterial (e.g., Stx) and host factors (circulating cells, complement system, serum proteins) whose interaction is crucial for the immediate outcome and for the development of this life-threatening sequela. Stx2a, associated to circulating cells (early toxemia) or extracellular vesicles (late toxemia) in blood, is considered the main pathogenic factor in the development of eHUS. Recently, it was found that the functional properties of Stx2a (binding to circulating cells and complement components) change according to modifications of the structure of the toxin, i.e., after a single cleavage of the A subunit resulting in two fragments, A1 and A2, linked by a disulfide bridge. Herein, we describe a method to be used for the detection of the cleaved form of Stx2a in the serum of STEC-infected or eHUS patients. The method is based on the detection of the boosted inhibitory activity of the cleaved toxin, upon treatment with reducing agents, on a rabbit cell-free translation system reconstituted with human ribosomes. The method overcomes the technical problem caused by the presence of inhibitors of translation in human serum that have been stalled by the addition of RNAase blockers and by treatment with immobilized protein G. This method, allowing the detection of Stx2a at concentrations similar to those found by ELISA in the blood of STEC-infected patients, could be a useful tool to study the contribution of the cleaved form of Stx2a in the pathogenesis of eHUS.

Enterohemorrhagic Escherichia coli (EHEC) infections can cause EHEC-associated hemolytic uremic syndrome (eHUS) via its main virulent factor, Shiga toxins (Stxs). Complement has been reported to be involved in the progression of eHUS. The aim of this study was to investigate the interactions of the most effective subtype of the toxin, Stx2a, with pivotal complement proteins C3b and C5. The study further examined the effect of Stx2a stimulation on the transcription and synthesis of these complement proteins in human target cell lines. Binding of Stx2a to C3b and C5 was evaluated by ELISA. Kidney and gut cell lines (HK-2 and HCT-8) were stimulated with varied concentrations of Stx2a. Subsequent evaluation of complement gene transcription was studied by real-time PCR (qPCR), and ELISAs and Western blots were performed to examine protein synthesis of C3 and C5 in supernatants and lysates of stimulated HK-2 cells. Stx2a showed a specific binding to C3b and C5. Gene transcription of C3 and C5 was upregulated with increasing concentrations of Stx2a in both cell lines, but protein synthesis was not. This study demonstrates the binding of Stx2a to complement proteins C3b and C5, which could potentially be involved in regulating complement during eHUS infection, supporting further investigations into elucidating the role of complement in eHUS pathogenesis.

In Austria, consent to receiving vaccines is regulated at the federal state level and in Tyrol, children aged 14 years are allowed to consent to receiving vaccination. In August 2017, we investigated determinants associated with vaccine hesitancy, having been vaccinated against measles and human papillomavirus (HPV) and the intention to vaccinate among schoolchildren born in 2002 and 2003. Those who consider measles and HPV a severe disease had a significantly higher intention to be vaccinated (prevalence ratio (PR) of 3.5 (95% CI 1.97–6.32) for measles and a PR of 3.2 (95% CI 1.62–6.35) for HPV). One-third of the participants (32.4%; 95% CI 27.8–37.4) were not aware that they are allowed to consent to receiving vaccines. The most common trusted source reported by respondents (n = 311) was the medical doctor (80.7%; 95% CI 75.7–84.7). The main finding related to the aim of the study was that the proportion of objectors is below 4% and therefore it should still be possible to reach measles elimination for which a 95% uptake is necessary. Although the proportion of objectors is not higher compared to adults, we recommend to intensify health education to increase health literacy.

Introduction: The complement system is essential for an adequate immune response. Much attention has been given to the role of complement in disease. However, to better understand complement in pathology, it is crucial to first analyze this system under different physiological conditions. The aim of the present study was therefore to investigate the inter-individual variation in complement activity and the influences of age and sex.Methods: Complement levels and functional activity were determined in 120 healthy volunteers, 60 women, 60 men, age range 20–69 year. Serum functional activity of the classical pathway (CP), lectin pathway activated by mannan (MBL-LP) and alternative pathway (AP) was measured in sera, using deposition of C5b-9 as readout. In addition, levels of C1q, MBL, MASP-1, MASP-2, ficolin-2, ficolin-3, C2, C4, C3, C5, C6, C7, C8, C9, factor B, factor D, properdin, C1-inhibitor and C4b-binding protein, were determined. Age- and sex-related differences were evaluated.Results: Significantly lower AP activity was found in females compared to males. Further analysis of the AP revealed lower C3 and properdin levels in females, while factor D concentrations were higher. MBL-LP activity was not influenced by sex, but MBL and ficolin-3 levels were significantly lower in females compared to males. There were no significant differences in CP activity or CP components between females and males, nevertheless females had significantly lower levels of the terminal components. The CP and AP activity was significantly higher in the elderly, in contrast to MBL-LP activity. Moreover, C1-inhibitor, C5, C8, and C9 increased with age in contrast to a decrease of factor D and C3 levels. In-depth analysis of the functional activity assays revealed that MBL-LP activity was predominantly dependent on MBL and MASP-2 concentration, whereas CP activity relied on C2, C1-inhibitor and C5 levels. AP activity was strongly and directly associated with levels of C3, factor B and C5.Conclusion: This study demonstrated significant sex and age-related differences in complement levels and functionality in the healthy population. Therefore, age and sex analysis should be taken into consideration when discussing complement-related pathologies and subsequent complement-targeted therapies.

Although neglected in the past, the interest on Zika virus (ZIKV) raised dramatically in the last several years. The rapid spread of the virus in Latin America and the association of the infection with microcephaly in newborns or Guillain-Barré Syndrome in adults prompted the WHO to declare the ZIKV epidemic to be an international public health emergency in 2016. As the virus gained only limited attention in the past, investigations on interactions of ZIKV with human complement are limited. This prompted us to investigate the stability of the virus to human complement. At low serum concentrations (10%) which refers to complement concentrations found on mucosal surfaces, the virus was relatively stable at 37°C, while at high complement levels (50% serum concentration) ZIKV titers were dramatically reduced, although the virus remained infectious for about 4–5 min under these conditions. The classical pathway was identified as the main actor of complement activation driven by IgM antibodies. In addition, direct binding of C1q to both envelope and NS1 proteins was observed. Formation of the MAC on the viral surface and thus complement-mediated lysis and not opsonization seems to be essential for the reduction of viral titers.

Measles elimination has been identified as a public health priority in Europe for a long time but has not yet been achieved. The World Health Organization (WHO) recommends identification of susceptible sub-populations to target supplementary immunization activities. We used three different sources of information: retrospective samples investigated for measles IgG between 1997 and 2016, vaccine coverage data from the existing electronic registry for birth cohorts 2015 to 1999, and surveillance data from 2009 until 20 July 2019. We calculated susceptibility by birth cohort using seroprevalence data, adjusting vaccine coverage data with reported effectiveness (93% for the first and 97% for the second dose, respectively), and compared it with measles incidence data, aggregated by birth cohorts and districts. Susceptibility levels for persons 10−41 years (birth cohorts 2007−1976) were 10.4% and thus far above the recommended values of WHO (5%). Older birth cohorts were sufficiently protected. Districts with the highest susceptibility estimates corresponded with districts with the highest incidence rates. Birth cohorts with susceptibility levels > 10% showed a 4.7 increased relative risk of having had more than one measles case. We conclude that retrospective serosurveys are a cheap and useful approach in identifying susceptible sub-populations, especially for older birth cohorts whose coverage data remain scarce.