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This review aims to make clinicians aware of the newly described syndrome, VEXAS. VEXAS should become an obvious differential diagnosis in cases of unexplained inflammation, anemia, and rheumatological and/or hematological manifestations. Patients with VEXAS are typically male agedgt; 60, with inflammation, and macrocytic anaemia. On suspicion of cancer or infections patients have frequently been exposed to extensive diagnostic procedures and hospital admissions. In this review, we summarise the current knowledge of VEXAS regarding pathogenesis, symptoms, diagnosis, and treatment.

Naturally occurring antibodies are abundant in human plasma, but their importance in the defense against bacterial pathogens is unclear. We studied the role of the most abundant of such antibodies, the antibody against terminal galactose-α-1,3-galactose (anti-αGal), in the protection against pneumococcal infections (Streptococcus pneumonia). All known pneumococcal capsular polysaccharides lack terminal galactose-α-1,3-galactose, yet highly purified human anti-αGal antibody of the IgG class reacted with 48 of 91 pneumococcal serotypes. Anti-αGal was found to contain multiple antibody subsets that possess distinct specificities beyond their general reactivity with terminal galactose-α-1,3-galactose. These subsets in concert targeted a wide range of microbial polysaccharides. We found that anti-αGal constituted up to 40% of the total antibody reactivity to pneumococci in normal human plasma, that anti-αGal drives phagocytosis of pneumococci by human neutrophils, and that the anti-αGal level was 2-fold lower in patients prone to pneumococcal infections compared to controls. Moreover, during a 48-year period in Denmark, the 48 anti-αGal-reactive serotypes caused fewer invasive pneumococcal infections (n = 10,927) than the 43 non-reactive serotypes (n = 18,107), supporting protection on the population level. Our findings explain the broad-spectrum pathogen reactivity of anti-αGal and support that these naturally occurring polyreactive antibodies contribute significantly to human protective immunity.

Patients with common variable immunodeficiency (CVID) display low antibody levels and associated symptoms, including an increased risk of infections. The causes of CVID are uncertain and likely heterogeneous. The complement system protects against pathogens and plays essential roles in homeostasis and development. The influence of the complement system in CVID is not established. We investigated CVID patients and healthy individuals for plasma levels of the complement proteins: MASP-1, MASP-2, MASP-3, MAp19, and MAp44. We also tested other patients with symptoms similar to the CVID patients. CVID patients had lower average MASP-2 and MAp44 levels than healthy individuals (p

Functional antibody deficiency is clinically assessed from antibody responses to vaccination. However, diagnostic vaccination is complex and may fail in practice. We hypothesized that the levels of naturally occurring antibodies against galactose-α-1,3-galactose (αGal) may represent alternative markers of functional antibody capacity. We included data from 229 patients with suspected primary immunodeficiency in a retrospective study. Antibody levels against αGal and twelve pneumococcal serotypes were determined with solid-phase immunoassays. Pneumococcal vaccinations and treatment with normal human immunoglobulin were assessed from medical records. Anti-αGal antibody levels correlated positively with anti-pneumococcal antibody levels measured before and after pneumococcal vaccination. Contrary to the anti-pneumococcal antibody levels, the anti-αGal antibody level showed potential for predicting subsequent immunoglobulin treatment - a marker of disease severity. Naturally occurring antibodies may reflect the functional capacity tested by diagnostic vaccination but add more useful clinical data. The clinical utility of this easy test should be evaluated in prospective studies.

Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is a rare immune deficiency with a broad clinical presentation. IPEX syndrome causes dysfunctional regulatory T cells, increasing the risk of autoimmune diseases. In this case report, we describe a 7-year-old boy with lymphocytic interstitial pneumonia and bullous pemphigoid who was recently diagnosed with IPEX syndrome.

In clinical practice, the capacity for producing anti-carbohydrate antibodies is regarded as an entity, but supportive evidence is lacking. We hypothesized that the outcome of the gold standard test for clinical assessment of this capacity, antibody response to polysaccharide vaccination, correlated with the level of the abundant naturally occurring anti-carbohydrate antibody, anti-αGal. To perform an exploratory study, 47 HIV-infected adults were recruited from a vaccine trial. Participants received a 23-valent pneumococcal capsular polysaccharide vaccine. Plasma samples obtained just before and median 4 weeks after the vaccination were quantified for IgG anti-αGal antibody and IgG antibodies to polysaccharides present in the vaccine (serotypes 1, 7F, and 19A) by solid-phase type immunoassays. The vaccination responses were assessed as a categorical variable (based on criteria defined by The American Academy of Allergy, Asthma & Immunology and the American College of Allergy, Asthma & Immunology) and as three different continuous variables (antibody increment, geometrical average, and standard normal deviates of the achieved antibody concentrations). The baseline anti-αGal level predicted the vaccine response as a categorical variable (ROC-curve analysis, AUC = 0.71; 95%CI: 0.55-0.86) and as the three continuous variables (e.g., slope of linear regression of geometrical average = 0.37; 95%CI: 0.15-0.59). The correlation between the anti-αGal level and antibody responses to polysaccharide vaccination fits with a shared underlying capacity. Thus, the present study supports the notion of a measurable capacity for the production of anti-carbohydrate antibodies in each individual. Firm conclusions on the generalizability and clinical utility require further studies.

The complement system is an intricate cascade of the innate immune system and plays a key role in microbial defense, inflammation, organ development, and tissue regeneration. There is increasing interest in developing complement regulatory and inhibitory agents to treat complement dysfunction. In this study, we describe the nanobody hC3Nb3, which is specific for the C-terminal C345c domain of human and mouse complement component C3/C3b/C3c and potently inhibits C3 cleavage by the alternative pathway. A high-resolution structure of the hC3Nb3–C345c complex explains how the nanobody blocks proconvertase assembly. Surprisingly, although the nanobody does not affect classical pathway–mediated C3 cleavage, hC3Nb3 inhibits classical pathway–driven hemolysis, suggesting that the C-terminal domain of C3b has an important function in classical pathway C5 convertase activity. The hC3Nb3 nanobody binds C3 with low nanomolar affinity in an SDS-resistant complex, and the nanobody is demonstrated to be a powerful reagent for C3 detection in immunohistochemistry and flow cytometry. Overall, the hC3Nb3 nanobody represents a potent inhibitor of both the alternative pathway and the terminal pathway, with possible applications in complement research, diagnostics, and therapeutics.

Antibodies of the IgG class to terminal Galα3Gal (IgG anti-αGal) is abundant in human plasma and are reported to bind most sepsis-causing Gram-negative bacteria. However, these seminal findings, made more than two decades ago, have not been reexamined. Our aim was to assess IgG anti-αGal´s pathogen reactivity. We affinity purified IgG anti-αGal from a therapeutic grade normal human IgG pool applying two rounds of positive selection with Galα3Gal-coupled beads and included removal of column matrix reactive antibodies. The purified antibodies were rigorously characterized in terms of specificity and purity in various solid-phase immunoassays. We used flow cytometry to study reactivity against 100 consecutive clinical isolates diagnosed as cause of sepsis in humans. We found that the purified IgG anti-αGal displays high specificity for Galα3Gal. Also, IgG anti-αGal at 5 mg/L bound 56 out of 100 pathogens with predilection for Gram-positive bacteria binding 39 out of 52 strains. We confirm that although IgG anti-αGal comprise a small fraction of the human antibody pool (~0.1%), these antibodies targets an impressively large part of pathogens causing invasive disease.

Common variable immunodeficiency (CVID) is a primary immunodeficiency characterized by recurrent bacterial infections and defined by reduced levels of IgG, IgA, and/or IgM, insufficient response to polysaccharide vaccination, and an abnormal B-cell immunophenotype with a significantly reduced fraction of isotype-switched memory B cells. In addition to this infectious phenotype, at least one third of the patients experience autoimmune, autoinflammatory, granulomatous, and/or malignant complications. The very heterogeneous presentation strongly suggests a collection of different disease entities with somewhat different pathogeneses and most likely diverse genetic etiologies. Major progress has been made during recent years with the advent and introduction of next-generation sequencing, initially for research purposes, but more recently in clinical practice. In the present study, we performed whole exome sequencing on 20 CVID patients with autoimmunity, autoinflammation, and/or malignancy from the Danish CVID cohort with the aim to identify gene variants with a certain, possible, or potential disease-causing role in CVID. Through bioinformatics analyses, we identified variants with possible/probable disease-causing potential in nine of the patients. Of these, three patients had four variants in three different genes classified as likely pathogenic (NFKB1, TNFAIP3, and TTC37), whereas in six patients, we identified seven variants of possible pathogenic potential classified as variants of unknown significance (STAT3, IL17F, IRAK4, DDX41, NLRC3, TNFRSF1A, and PLCG2). In the remaining 11 patients, we did not identify possible genetic causes. Genetic findings were correlated to clinical disease presentation, clinical immunological phenotype, and disease complications. We suggest that the variants identified in the present work should lay the ground for future studies to functionally validate their disease-causing potential and to investigate at the mechanistic and molecular level their precise role in CVID pathogenesis. Overall, we believe that the present work contributes important new insights into the genetic basis of CVID and particular in the subset of CVID patients with a complex phenotype involving not only infection, but also autoimmunity, autoinflammation, and malignancy.

As much as one percent of antibodies in human plasma are reported to be specific for the non-human disaccharide structure αGal. Various microbes express αGal. However, the implications of anti-αGal antibodies for the anti-microbial defenses are poorly established. With the perspective of studying the biological importance of the antibodies, we have established a sensitive Time-Resolved ImmunoFluorometric Assay (TRIFMA) for quantification of such antibodies. Two versions were developed, one for IgM antibodies and one for IgG antibodies. Samples were collected from plasma donations of healthy adults (n = 120) of known gender (60 + 60), AB0-type (0: 15 + 15, A: 15 + 15, B: 15 + 15, and AB: 15 + 15) and age (19–64 yrs). We subsequently examined the potential association between antibody concentration and AB0-type, gender, age, and titers of antibodies to blood type antigens. We found that IgG and IgM anti-αGal concentrations are, 1) stable over time within the individual, 2) vary more than 400-fold between individuals, 3) negatively correlated with age for IgM but not for IgG antibodies, 4) IgM antibodies are 2-fold higher in females whereas no gender difference was observed for the IgG antibodies, 5) inter-mutual correlated, 6) lowest in individuals expressing B-antigen, and 7) AB0-type A individuals may constitute an intermediate group. Our established method and findings pave the way for further studies of the involvement of anti-αGal antibodies in immunity and may be a method to examine the potential of an individual to mount an anti-carbohydrate response.

Preeclampsia is a major cause of maternal and perinatal deaths. The aetiology of preeclampsia is largely unknown but a polygenetic component is assumed. To explore this hypothesis, we performed an in-depth whole-exome sequencing study in women with (cases, N = 50) and without (controls, N = 50) preeclampsia. The women were identified in an unselected cohort of 2,545 pregnant women based on data from the Danish National Patient Registry and the Medical Birth Registry. Matching DNA was obtained from a biobank containing excess blood from routine antenatal care visits. Novogene performed the whole-exome sequencing blinded to preeclampsia status. Variants for comparison between cases and controls were filtered in the Ingenuity Variant Analysis software. We applied two different strategies; a disease association panel approach, which included variants in single genes associated with established clinical risk factors for preeclampsia, and a gene panel approach, which included biological pathways harbouring genes previously reported to be associated with preeclampsia. Variant variability was compared in cases and controls at the level of biological processes, signalling pathways, and in single genes. Regardless of the applied strategy and the level of variability examined, we consistently found positive correlations between variant numbers in cases and controls (all R2s>0.88). Contrary to what was expected, cases carried fewer variants in biological processes and signalling pathways than controls (all p-values ≤0.02). In conclusion, our findings challenge the hypothesis of a polygenetic aetiology for preeclampsia with a common network of susceptibility genes. The greater genetic diversity among controls may suggest a protective role of genetic diversity against the development of preeclampsia.

Udgivelsesdato: 2010-null BACKGROUND: Real-Time quantitative PCR is an important tool in research and clinical settings. Here, we describe two new approaches that broaden the scope of real-time quantitative PCR; namely, run-internal mini standard curves (RIMS) and direct real-time relative quantitative PCR (drqPCR). RIMS are an efficient alternative to traditional standard curves and provide both run-specific and target-specific estimates of PCR parameters. The drqPCR enables direct estimation of target ratios without reference to conventional control samples. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we compared RIMS-based drqPCR with classical quantifications based on external standard curves and the "comparative Ct method". Specifically, we used a raw real-time PCR dataset as the basis for more than two-and-a-half million simulated quantifications with various user-defined conditions. Compared with classical approaches, we found that RIMS-based drqPCR provided superior precision and comparable accuracy. CONCLUSIONS/SIGNIFICANCE: The obviation of referencing to control samples is attractive whenever unpaired samples are quantified. This may be in clinical and research settings; for instance, studies on chimerism, TREC quantifications, copy number variations etc. Also, lab-to-lab comparability can be greatly simplified.

Polysaccharide responsiveness is tested by measuring antibody responses to polysaccharide vaccines to diagnose for humoral immunodeficiency. A common assumption is that this responsiveness is biased by any previous exposure to the polysaccharides in the form of protein-coupled polysaccharide vaccines, such as those used in many childhood vaccination programmes. To examine this assumption, we investigated the effect of protein-coupled polysaccharide vaccination on subsequent polysaccharide responsiveness. HIV-infected adults (n = 47) were vaccinated twice with protein-coupled polysaccharides and six months later with pure polysaccharides. We measured immunoglobulin G responses against three polysaccharides present in only the polysaccharide vaccine (non-memory polysaccharides) and seven recurring polysaccharides (memory polysaccharides). Responsiveness was evaluated according to the consensus guidelines published by the American immunology societies. Impaired responsiveness to non-memory polysaccharides was more frequent than to memory polysaccharides (51% versus 28%, P = 0.015), but the individual polysaccharides did not differ in triggering sufficient responses (74% versus 77%, P = 0.53). Closer analysis revealed important shortcomings of the current evaluation guidelines. The interpreted responseś number and their specificities influenced the likelihood of impaired responsiveness in a complex manor. This influence was propelled by the dichotomous approaches inherent to the American guidelines. We therefore define a novel more robust polysaccharide responsiveness measure, the Z-score, which condenses multiple, uniformly weighted responses into one continuous variable. Using the Z-score, responsiveness to non-memory polysaccharides and memory-polysaccharides were found to correlate (R2 = 0.59, P

In recent years an increasing number of novel genetic defects have been ascribed an important role in susceptibility to infection. Two examples are: I) Defects in the genes encoding the subunits of the NADPH oxidase which allow production of reactive oxygen species in phagocytes combating microorganisms. These defects may result in life-threatening bacterial and fungal infections and excessive inflammatory reactions leading to granulomatous lesions. II) Mutations in DNA double strand break repair genes which impacts V(D)J recombination, class switching and lymphocyte maturation leading to hypogammaglobulinaemia, and increased risk of both infections as well as cancer. We employed whole exome sequencing (WES) to identify mutations associated with primary immunodeficiency in severely affected children. We present WES data on 2 patients with severe immunodeficiency. WES was performed using TruSeq exome kit and Illumina HiSeq, SNP and Indels were called and subsequently filtered using Cartagenia software. First, we identified a heterozygous missense, gain of function mutation in the NLRP3 gene along with compound heterozygosity for MPO defect in a 10 months old girl with juvenile psoriasiform dermatitis and severe infections including sepsis. Second, we identified compound heterozygote stopgain mutations in RAD52 and a heterozygote mutation in LRRC8A in a 7 year-old girl with T-cell deficiency, reduced T-cell mediated B-cell activity, hypogammaglobulinaemia, prolonged splenomegali and benign adenopathy. RAD52 has not previously been linked to immunodeficiency and we are currently investigating the functional consequences. Knowledge of the mechanisms underlying immunodeficiencies is a prerequisite for understanding disease pathogenesis. WES allows the demonstration of immune defects that may result from combined genetic variations contributing to an overall functional defect. This explains the fact that monogenic defects are rare among clinically immune compromised patients, and that patients present with a high degree of clinical variability due to private compound immune defects.