University of Copenhagen

Impact of Staphylococcus aureus colonization and skin abscesses on formation of human anti-αGal antibodies.

IgG antibodies against terminal galactose-α-1,3-galactose (anti-αGal antibodies) are naturally occurring in humans, but their origins remain poorly understood. These antibodies target various microorganisms including Staphylococcus aureus, a common nasal commensal and the major cause of skin abscesses. This study investigates the impact of S. aureus colonization and abscess events on plasma anti-αGal antibody levels. We measured plasma anti-αGal antibody levels using a quantitative immunoassay in: (i) 101 pairs of healthy individuals with and without nasal S. aureus colonization, (ii) 106 healthy individuals before and after abscess formation, and (iii) 43 patients with recurrent skin abscesses compared with 75 patient controls and 60 healthy controls. We observed a 35% reduction (95%CI: 7-54%) in anti-αGal antibody levels in nasal S. aureus carriers. Conversely, we found a 30% increase (95%CI: 4-66%) in individuals within 187 days post-skin abscess, and patients with recurrent skin abscesses exhibited 81% higher (95%CI: 14-190%) levels than patient controls, and 110% higher (95%CI: 39-230%) than healthy controls. This study suggests that skin abscesses lead to elevated plasma anti-αGal antibody levels and that these antibodies might convey or correlate with mucosal immunity to S. aureus.

Increasing prevalence of galactose-α-1,3-galactose sensitization in the Danish general adult population.

Alpha-gal syndrome is a novel food allergy to the oligosaccharide galactose-α-1,3-galactose (alpha-gal) present in mammalian meat. Tick bites are considered an important route of sensitization to alpha-gal. Data on alpha-gal sensitization in the general population is scant. We utilized a unique data source of repeated population-based health examination studies to assess prevalence, time trends, risk factors, and characteristics of alpha-gal sensitization. Alpha-gal sensitization was assessed in >11.000 adults from four health examination studies of randomly invited residents in the Copenhagen region conducted in 1990-1991, 2011-2012, 2012-2015, and 2016-2017. Alpha-gal sensitization was defined as serum specific IgE (sIgE) to alpha-gal ≥0.1 kU/L; ≥0.35 kU/L; ≥0.7 kU/L; ≥3.5 kU/L. The population was characterized according to genetically determined ABO blood group, aeroallergen sensitization, and pets at home. The prevalence of sIgE to alpha-gal ≥0.1 kUA/L was 1.3% in 1990-1991, 3.7% in 2012-2015 and 3.2% in 2016-2017. Of those sensitized to alpha-gal >97% reported to consume red meat at least once a week, even for sIgE to alpha-gal ≥3.5 kU/L. Male sex, older age, aeroallergen sensitization, cat at home, and blood group A were associated with increased odds of alpha-gal sensitization. The known protective effect of blood group B was confirmed. In this general adult population, the prevalence of alpha-gal sensitization had doubled from 1990-1991 to 2016-2017. This could potentially be due to increased tick exposure and an increased atopic predisposition.

Living close to the forest is a risk factor for galactose-α-1,3-galactose sensitization.

The α-gal syndrome is a food allergy to galactose-α-1,3-galactose present in mammalian meat; it usually develops after tick bites. Despite being considered an emerging health problem, little is known about the geographic risk factors and patterns of α-gal sensitization. The aim of this study was to assess geographic clusters of α-gal sensitization and determine whether distance from home address to the forest and tick abundance are risk factors for α-gal sensitization in a Danish adult population. We utilized 3 population-based health examination studies that were conducted in 2011-2017 and covered a total of 8742 participants living in a suburban area. All participants were screened for a level of serum-specific IgE to α-gal of 0.1 kU/L or higher. Their home addresses were linked with CORINE Land Cover data and a tick prediction model. The associations of (1) distance to the forest and (2) tick abundance with α-gal sensitization were assessed by using multivariable logistic regression. There were 344 cases of galactose-α-1,3-galactose-specific IgE level being 0.1 kU/L; we identified 1 geographic cluster of cases. For every 1 km closer a participant lived to the forest, the odds ratio of α-gal sensitization was 1.09 (95% CI = 1.05-1.13). There were no associations between predicted tick nymph abundance and α-gal sensitization. In a general, individuals residing in a suburban area had 9% higher odds of being sensitized to α-gal the closer they lived to a forest, thus supporting exposure to ticks as a risk factor for sensitization to α-gal.

A case of alpha-gal syndrome: Recall urticaria and 10 years of measurements of IgE to galactose-α-1,3-galactose.

Alpha-gal IgE level can change rapidly. Reassessment of a patient's alpha-gal IgE level may be helpful in the patient's clinical follow-up. Pruritus related to the site of a previous tick bite strengthens the diagnosis of alpha-gal syndrome.

Prevalence, predictors, and clinical relevance of α-gal sensitization in patients with chronic urticaria.

Little is known about α-gal (galactose-α-1,3-galactose) sensitization in patients with chronic urticaria (CU). The aim of this study was to examine the prevalence, predictors and clinical relevance of α-gal sensitization in patients with CU. Two consecutive cohorts of newly referred patients with CU from a primary care allergology practice and a tertiary hospital dermatology department, plus a control group with allergic disease, but not CU, from the allergology practice, were interviewed and screened for α-gal sensitization (serum specific-IgE ≥0.35 KU/L). Of 733 patients included, 21 (5.6%) and 11 (3.9%) of CU patients from private practice and hospital, respectively, were α-gal sensitized. In total, 8 patients (38.1% of sensitized patients, and 2.1% of all CU patients) from private practice, and 2 patients (18.2% of sensitized patients, and 0.7% of all CU patients) from hospital, had clinically relevant α-gal allergy. In private practice, male sex (47.6 vs. 24.7%), p = 0.020, obesity (33.3 vs. 23.6%), p = 0.302, and frequency of angioedema (61.9 vs. 51.4%), p = 0.350; and in hospital, male sex (72.7 vs. 27.9%), p = 0.003, and high total immunoglobulin E (median 168 vs. 70.5 KU/L), p = 0.022 were associated with α-gal sensitization. α-gal sensitization is observed in a small fraction of CU patients with only few patients experiencing clinically relevant sensitization. Certain patients, particularly from primary care, may constitute a relevant population for aimed testing.

Delayed reaction in alpha-gal allergy is reflected in serum levels after ingestion of pork kidney, and absorption is dependent on food processing.

Cetuximab plus irinotecan administered biweekly with reduced infusion time to heavily pretreated patients with metastatic colorectal cancer and related RAS and BRAF mutation status.

Metastatic colorectal cancer (mCRC) is treated with cetuximab 250 mg/m administered weekly over 1 hour or biweekly (q2w) over 3.5 hours when combined with irinotecan. This prospective study investigated cetuximab 500 mg/m plus irinotecan 180 mg/m administered q2w over 1.5 hours independent of RAS or BRAF mutation status in mCRC patients in a third-line setting. The intention-to-treat population included 181 patients. No patients had complete response, 18% had partial responses (PR) and 48% stable disease (SD). For cetuximab, a relative dose intensity of ≥90% was reached in 78% and for irinotecan in 67% of the patients. Grade 3 to 4 toxicities were pain (17%), fatigue (9%), neutropenia (8%), diarrhea (8%), rash (8%), infection (7%) and hypersensitivity (3%). No deaths occurred. Next-generation sequencing in 96.7% of the patients revealed that 50.3% had RAS and BRAF wild type (WT), with a mutation type (MT) in 45.1% of the RAS and 4.4% of the BRAF genes. In patients with RAS-WT and RAS-MT tumors, a PR was obtained in 32% and 4% (P = .000003) and an SD in 43% and 53%, respectively, with a superior PFS (6.2 vs 3.7 months; hazard ratio [HR] 2.12, P = .00001) and OS (12.9 vs 8.8 months; HR 1.71, P = .0008). Treatment efficacy was poor in 7.4% of patients with an RAS mutation outside KRAS exon 2 and in 38% of patients with KRAS exon 2 mutations. Administration of cetuximab and irinotecan q2w, shortening treatment time from 3.5 to 1.5 hours, is recommended as standard therapy.

Crystal structure of α-galactosidase from Lactobacillus acidophilus NCFM: insight into tetramer formation and substrate binding.

Lactobacillus acidophilus NCFM is a probiotic bacterium known for its beneficial effects on human health. The importance of α-galactosidases (α-Gals) for growth of probiotic organisms on oligosaccharides of the raffinose family present in many foods is increasingly recognized. Here, the crystal structure of α-Gal from L. acidophilus NCFM (LaMel36A) of glycoside hydrolase (GH) family 36 (GH36) is determined by single-wavelength anomalous dispersion. In addition, a 1.58-Å-resolution crystallographic complex with α-d-galactose at substrate binding subsite -1 was determined. LaMel36A has a large N-terminal twisted β-sandwich domain, connected by a long α-helix to the catalytic (β/α)(8)-barrel domain, and a C-terminal β-sheet domain. Four identical monomers form a tightly packed tetramer where three monomers contribute to the structural integrity of the active site in each monomer. Structural comparison of LaMel36A with the monomeric Thermotoga maritima α-Gal (TmGal36A) reveals that O2 of α-d-galactose in LaMel36A interacts with a backbone nitrogen in a glycine-rich loop of the catalytic domain, whereas the corresponding atom in TmGal36A is from a tryptophan side chain belonging to the N-terminal domain. Thus, two distinctly different structural motifs participate in substrate recognition. The tetrameric LaMel36A furthermore has a much deeper active site than the monomeric TmGal36A, which possibly modulates substrate specificity. Sequence analysis of GH36, inspired by the observed structural differences, results in four distinct subgroups having clearly different active-site sequence motifs. This novel subdivision incorporates functional and architectural features and may aid further biochemical and structural analyses within GH36.

Six cases of treatment with panitumumab in patients with severe hypersensitivity reactions to cetuximab.

Biweekly cetuximab and irinotecan as third-line therapy in patients with advanced colorectal cancer after failure to irinotecan, oxaliplatin and 5-fluorouracil.

Standard weekly cetuximab and irinotecan (CetIri) is an effective regimen in heavily pretreated patients with advanced colorectal cancer (ACRC). Inspired by a pharmacokinetic study demonstrating no differences between weekly and biweekly cetuximab, we present the results of 74 consecutive patients treated with biweekly CetIri. Biweekly CetIri schedule: cetuximab 500 mg/m(2), first course was given as a 120-min infusion followed 1 h later by irinotecan 180 mg/m(2) as a 30-min infusion. Subsequent courses of cetuximab were given in 60 min, immediately followed by irinotecan-resulting in an overall treatment time of 90 min. All patients had ACRC resistant to 5-fluorouracil and irinotecan and 95% to oxaliplatin. Median age was 63 years, median performance status was 0. Median duration of therapy was 4.3 months. Response rate was 25%. Median progression-free survival and overall survival were 5.4 months and 8.9 months, respectively, comparable to own historical controls receiving weekly CetIri. Grade 3-4 toxicity was rare (skin 7%, nail 3%, diarrhoea 10%, fatigue 3%, neutropenia 9%). One patient experienced severe allergic reaction. Salvage therapy with simplified biweekly CetIri is a convenient, effective and well-tolerated regimen in heavily pretreated patients with ACRC. A confirmatory phase II study is ongoing.

The galactophilic lectin (PA-IL, gene LecA) from Pseudomonas aeruginosa. Its binding requirements and the localization of lectin receptors in various mouse tissues.

The opportunistic pathogen Pseudomonas aeruginosa contains lectins of which one of them, PA-IL (gene lecA), shows preference for alpha-galactosylated glycans. The bacterial lectin is probably important in the carbohydrate-mediated adhesion of the microorganism to endothelia and epithelia and thereby the lectin facilitates entering and damaging of the cells. The requirements for the interaction between PA-IL and the carbohydrate epitopes to which the bacterial lectin may bind were here studied using alpha-galactosylated neoglycoproteins that were immobilized on Microtiter plates. It is concluded that the carbohydrate recognizing site of the lectin can have a binding requirement of only one saccharide. Lectin histochemistry was performed on sections from wild type mice and from knock-out mice, which lack function of the alpha1,3-galactosyltransferase gene. All assays with the P. aeruginosa lectin were compared with the results obtained using an isolectin from the legume shrub Griffonia simplicifolia: the GSI-B4 isolectin, which is highly specific for glycans terminating in Galalpha1-R. In the wild-type mice, lectin histochemistry showed a strong capillary reaction in heart, kidney and adrenal gland while none of the two lectins were able to detect capillaries in the pancreas. This could indicate a differential glycosylation with respect to endothelial cell Galalpha epitopes among different organs. Further, since no PA-IL binding to the endothelial cells in the KO mouse was observed, it seems that, in the mouse, the Pseudomonas lectin adheres to the Galalpha1-3Galbeta1-4GlcNAc carbohydrate on endothelial cells in most organs and tissues. Finally, lectin staining of the basement membrane of the acini in the exocrine pancreas suggests the presence of Galalpha1-3Gal epitopes in WT mice basement membranes that are not detected by the P. aeruginosa lectin.

Re-treatment with cetuximab in patients with severe hypersensitivity reactions to cetuximab. Two case reports.

The bacterial flora of alpha-Gal knockout mice express the alpha-Gal epitope comparable to wild type mice.

The human genome possesses pseudogenes for the enzyme alpha1,3 galactosyltransferase and hence, human cells and tissues do not express the Galalpha terminated trisaccharide structure Galalpha1-3Galbeta1-4GlcNAc, the so-called alpha-Gal epitope. Circulating antibodies specific for this carbohydrate epitope are, however, present in high amounts in humans. It has previously been hypothesized that the antibody production is induced by the presence of the alpha-Gal epitope in the cell walls of the enteric flora, especially Enterobacteriaceae spp. However, in mice, in which the epitope has been deleted by targeted mutation of the gal-transferase gene, alpha-Gal antibodies do not appear without prior immunization, although the mice through their growth probably have been exposed to a normal bacterial flora of e.g. Enterobacteriaceae spp. It is unknown whether there are different types of immune reactions to antigenic carbohydrate expressing bacteria and whether there are discrepancies in the enteric flora between these knockout mice and their wild type litter mates. In this study the enteric flora of alpha-Gal knockout and wild type mice was compared both in relation to the prevalence of different types of bacteria in the two groups of mice, as well as in relation to the expression of the epitope on the surface of Enterobacteriaceae spp. Our results showed that the enteric flora did not differ significantly between knockout and wild type mice and that it was comparable to the flora known to be present in the intestines of other mice. All Enterobacteriaceae spp. examined expressed the alpha-Gal epitope no matter whether they were isolated from knockout or wild type mice. It is, therefore, discussed whether it is more reasonable to assume that alpha-Gal antibodies in mammals that do not produce alpha1,3 galactosyltransferase such as in the knock mice and in humans are the result of another antigen stimulant than these common representatives of the enteric flora, that we isolated from the two types of mice. Possible candidates for a carrier in humans could be bacteria or viruses not isolated from barrier-bred mice.

Analyses of Pseudomonas aeruginosa lectin binding to alpha-galactosylated glycans.

The specificity and binding capacity of the galactophilic lectin from the Gram negative bacterium Pseudomonas aeruginosa (PA-IL) was determined by solid phase measurements using galactosylated neoglycoproteins immobilized on microtiter plates. The bacterial lectin reacted with both short chain (monosaccharide) and long chain (pentasaccharide) glycoconjugates. Among the Galalpha1-XGal disaccharides, the highest affinity was observed towards the Galalpha1-3Gal structure. Raising the incubation temperature enhanced the lectin-polysaccharide agglutination, and it is suggested that binding to certain conformations of polysaccharides could vary between lectins with the same monocarbohydrate specificity and that this activity may, in part, be temperature dependent. Histochemical examination of lectin binding to different porcine tissues suggests a differential glycosylation of the carbohydrate antigens on endothelial cells in various parts of the vascular system. In the pancreas, PA-IL also adhered to the excretory ducts. These observations on PA-IL binding could be of importance both to determine infection foci in P. aeruginosa-mediated vacuities and to determine its role for pancreatic involvement in cystic fibrosis.