Natural moisturizing factors in children with and without eczema: Associations with lifestyle and genetic factors.

BACKGROUND: Filaggrin-derived natural moisturizing factors (NMF) play an important role in skin barrier function and in atopic dermatitis (AD). Its deficiency is associated with dry skin and increased surface pH. Studies on childhood environmental exposures and associations with NMF levels are scarce. OBJECTIVES: To investigate previous exposures and genetic factors and their associations with NMF levels in young children. METHODS: In a case-control study nested in a prospective birth cohort (Odense Child Cohort), 169 healthy controls (HC) and 99 children with AD were included consecutively at the age of 7 years based on previous responses from questionnaires administered at 18 months, 3 years and 5 years, pertaining to past medical history, including allergy-specific questions. NMF levels were measured via a stratum corneum tape-stripping technique, genotyping for filaggrin (FLG) gene variants was performed and data on external exposures, including usage of moisturizer and topical steroids, antibiotics and early pet exposures, were obtained from questionnaires. RESULTS: Natural moisturizing factors levels were significantly lower in AD participants compared to HC (P < 0.001). This significance persisted after stratifying for AD subgroups of present AD, current AD during the last year and previous AD (P < 0.001, P = 0.039, P = 0.009 respectively). There was a significant association between NMF and FLG genotype (P = 0.016, P = 0.002 for HC, AD respectively). NMF levels were negatively correlated with early age moisturizer use (<18 months, P = 0.001) in HC but not significant in AD. CONCLUSIONS: We found decreased levels of NMF with early moisturizer use and a genetic influence of the FLG variant on these levels. NMF was decreased in the AD subgroup with previous AD compared with HC, which could suggest the persistence of a Th2 cytokine milieu suppressing these levels.

No diagnostic utility of antibody patterns against Klebsiella pneumoniae capsular serotypes in patients with axial spondyloarthritis vs. patients with non-specific low back pain: a cross-sectional study.

OBJECTIVES: To investigate whether antibody response patterns against Klebsiella pneumoniae capsular serotypes can discriminate patients with axial spondyloarthritis (axSpA) from patients with non-specific low back pain (LBP). METHOD: Immunoglobulin (Ig)G and IgA antibodies against K. pneumoniae capsular serotypes K2, K26, K36, and K50 were measured, and antibody seropositivity compared between groups and analysed for patient correlation in five different groups: (a) 96 patients fulfilling the Assessment of SpondyloArthritis International Society (ASAS) classification criteria for axSpA; (b) 38 patients with either a positive magnetic resonance imaging (MRI) scan as defined by ASAS or a positive human leucocyte antigen (HLA)-B27 status plus one clinical SpA feature, characterized as 'non-axSpA'; (c) 82 non-specific LBP patients; (d) 40 healthy blood donors and (e) 43 patients with diagnosed ankylosing spondylitis (AS) served as the negative and positive control groups. RESULTS: There was no difference in IgG and IgA seropositivity against all serotypes between the axSpA, non-axSpA, and LBP groups. No significant correlations were found between anti-Klebsiella antibodies and age, gender, HLA-B27, or high-sensitivity C-reactive protein (hsCRP). IgG seropositivity against K50 was more frequent in AS (25.6%) than in axSpA (13.5%, p < 0.05). axSpA patients with radiographic sacroiliitis and AS controls concordantly had the highest frequency of seropositivity for ≥ 2 serotypes (21%). CONCLUSIONS: The antibody patterns against K. pneumoniae serotypes K2, K26, K36, and K50 did not discriminate between early axSpA and non-specific LBP.

Atopic dermatitis from adolescence to adulthood in the TOACS cohort: prevalence, persistence and comorbidities.

BACKGROUND: While much is known about childhood atopic dermatitis, little is known about persistence of atopic dermatitis into adult life. We report, to our knowledge for the first time, the clinical course of atopic dermatitis in an unselected cohort of adolescents followed into adulthood. METHODS: The course of atopic dermatitis from adolescence to adulthood was studied prospectively in a cohort of unselected 8th-grade schoolchildren established in 1995 and followed up in 2010 with questionnaire and clinical examination. RESULTS: The lifetime prevalence of atopic dermatitis was high (34.1%), and a considerable number of adults still suffered from atopic dermatitis evaluated both by questionnaire (17.1%) and clinical examination (10.0%). Persistent atopic dermatitis was found in 50% of those diagnosed in school age, and persistent atopic dermatitis was significantly associated with early onset, childhood allergic rhinitis and hand eczema. A close association was also found with allergic contact dermatitis and increased specific IgE to Malassezia furfur, but not with filaggrin gene defect. CONCLUSION: Persistence of atopic dermatitis in adulthood is common and affects quality of life. Persistent atopic dermatitis is particularly prevalent in those with early onset, allergic rhinitis and hand eczema in childhood. It is important to recognizing atopic dermatitis as a common and disabling disease not only in children but also in adults.

Parity and tanned white skin as novel predictors of vitamin D status in early pregnancy: a population-based cohort study.

CONTEXT: In pregnancy, vitamin D insufficiency and deficiency, defined as serum 25-hydroxyvitamin D (25(OH)D) <50 nM, and <25 nM, respectively, may have adverse effects for both mother and child. Prevalence estimates, and identification of subgroups at special risk, may be useful for the planning of preventive strategies. OBJECTIVE: To study the prevalence and risk factors of hypovitaminosis D in early pregnancy. DESIGN AND METHODS: In a cross-sectional study of 1348 women in early pregnancy from the Odense Child Cohort, Denmark, 25(OH)D was determined and correlated to demographic and lifestyle variables (age, nationality, skin tone, parity, prepregnancy body mass index (BMI), smoking and sun exposure), using multiple linear and logistic regression analyses for all year, or stratified for summer and winter. The risk of vitamin D insufficiency was expressed as odds ratios (OR) with 95% confidence intervals in brackets. RESULTS: The prevalence of vitamin D insufficiency and deficiency was estimated to 27·8% and 3·5% respectively. In adjusted analyses, vitamin D insufficiency was directly associated with winter season, OR = 1·89 (1·35-2·63); increasing prepregnancy BMI, OR = 1·06 (1·03-1·10); and smoking, OR = 2·7 (1·34-5·41); but was less frequent in nulliparous, OR = 0·47 (0·33-0·68) and tanned Caucasians, OR = 0·63 (0·41-0·97). Season-specific associations having parental origin from outside Europe in summer, OR = 4·13 (1·41-12·13); in winter smoking, OR = 3·15 (1·19-8·36); and prepregnancy BMI, OR = 1·12 (1·06-1·18). CONCLUSIONS: Vitamin D insufficiency was widespread in early pregnancy. Associations to smoking, prepregnancy BMI and origin outside Europe varied with season. Multiparity and not being tanned in Caucasians represent new risk factors of vitamin D insufficiency.

Genetical analysis of all Danish patients diagnosed with chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a rare inherited disorder of the innate immune system caused by a defect in NADPH oxidase, leaving the granulocytes unable to kill invading microorganisms. CGD is caused by mutation in one of the five components gp91phox, p22phox, p47phox, p67phox and p40phox, encoded by the X-linked CYBB gene and the autosomal CYBA, NCF1, NCF2 and NCF4 genes respectively. We have collected samples from all Danish patients with known CGD followed in the clinic or newly diagnosed during a 5-year period, a cohort of 27 patients, and characterized them genetically. The cohort includes 10 male patients with X-linked CGD and one female with extremely lyonized expression of a defective CYBB allele. Six patients had mutation in CYBA. Seven of 10 patients with a defect in NCF1 were homozygous for the common GT deletion, one was compound heterozygous for the GT deletion and a splice-site mutation, and two patients were homozygous for a nonsense mutation in exon 7. Three novel mutations were detected, a deletion of exon 6 in CYBA, a duplication of exon 8-13 in CYBB and a splice site mutation in intron 7 of NCF1.

An improved haemolytic plaque assay for the detection of cells secreting antibody to bacterial antigens.

Recent advances in the development of conjugate polysaccharide vaccines for human use have stimulated interest in the use of assays detecting antibody-secreting cells (AbSC) with specificity for bacterial antigens. Here we present improved haemolytic plaque-forming cell (PFC) assays detecting AbSC with specificity for tetanus and diphtheria toxoid as well as for Haemophilus influenzae type b and pneumococcal capsular polysaccharides. These assays were found to be less time consuming, more economical and yielded 1.9-3.4-fold higher plaque numbers than traditional Jerne-type PFC assays. In the case of anti-polysaccharide AbSC of the IgG isotype, the increase was as high as 7.4-11.8 times. Evidence is presented that the pronounced improvement in the detection of the latter is due to the presence of aggregating anti-IgG antibody from the beginning of the assay. It is proposed that in the case of low affinity of anti-polysaccharide antibodies aggregation of secreted monomeric antibody (IgG) is critical for plaque formation and increases the avidity of binding to target cells.

A simplification of the enzyme-linked immunospot technique. Increased sensitivity for cells secreting IgG antibodies to Haemophilus influenzae type b capsular polysaccharide.

A simplified enzyme-linked immunospot (ELISPOT) technique is described for the detection of cells secreting antibodies to tetanus toxoid (TT), diphtheria toxoid (DT) or Haemophilus influenzae type b capsular polysaccharide (PRP). By combining the cell suspension with the enzyme-linked secondary antibodies in one incubation, the second incubation and washing procedure could be omitted from the original technique. The simplified assay had the same sensitivity for anti-TT and anti-DT spot-forming cells as the ordinary ELISPOT assay. The IgG anti-PRP spots were, however, improved both in quality and in quantity (median: 40% more spots), while the detection of IgM and IgA anti-PRP spot-forming cells was the same in the two techniques. This simplified technique can probably also be used to save time in other antigen systems and should be considered when designing ELISPOT assays for the detection of polysaccharide-specific antibody-secreting cells.

Quantitation of human IgG subclass antibodies to Haemophilus influenzae type b capsular polysaccharide. Results of an international collaborative study using enzyme immunoassay methodology.

An international collaborative study was conducted at ten sites to examine the performance of enzyme immunoassays (EIAs) for the quantitation of IgG1, IgG2, IgG3, IgG4 and total IgG anti-Haemophilus influenzae type b (Hib) capsular polysaccharide in human serum. All groups used the same reagents: microtiter plates coated with polyribosylribitol phosphate (PRP) conjugated to poly-L-lysine (PLL), reference, control and test human sera, biotin-conjugated International Union of Immunological Societies (IUIS)-documented monoclonal anti-human IgG1-4 and IgG Pan detection antibodies, avidin-peroxidase and TMB substrate. Initial mixing of soluble PRP antigen or an equal volume of buffer with the 20 test sera prior to analysis confirmed PRP antigen specificity in all five EIAs with greater than 80% competitive inhibition at most sites. Positive correlation between the total IgG anti-Hib and sum of IgG1-4 anti-Hib was demonstrated (r2 = 0.99, Y = 1.13X -0.15). Good agreement was shown between the total IgG anti-Hib as measured by EIA and the total Hib-specific antibodies measured by the current radiolabeled antigen binding assay (r2 = 0.97, Y = 4.6X -5.8). Assay parallelism was demonstrated with an average interdilutional %CV of 22% and parallel dose-response curve slopes. The interdilutional %CVs were calculated as an average per sample of the variation of microgram/ml (corrected for dilution) at different dilutions per laboratory for all participating sites. The interlaboratory variation was the only performance parameter studied that exceeded the target level of 35% CV in all IgG1-4 and total IgG anti-Hib assays. IgG subclass distributions in the test sera demonstrated a predominance of IgG1 anti-Hib in the pediatric serum pools and IgG2 anti-Hib in the adult sera, with low but detectable levels of IgG3 and IgG4 anti-Hib in each group.

Antibody response to Haemophilus influenzae type b capsular polysaccharide conjugated to tetanus toxoid in preterm infants.

OBJECTIVE: To evaluate the antibody response to a Haemophilus influenzae type b capsular polysaccharide (HibCP) tetanus toxoid (TT) conjugate vaccine (HibCP-TT) in preterm infants. SUBJECTS: Thirty-five healthy preterm infants with gestational ages (GA) from 27 to 36 weeks and birth weights from 920 to 2550 g. Controls were 37 term infants. METHODS: All infants were immunized with HibCP-TT at 2, 4 and 12 months of age. Antibodies to HibCP and TT were determined at each immunization and 1 month after the second and third. RESULTS: After two doses of HibCP-TT the preterm infants with GAs < or = 30 weeks (n = 8; mean GA, 29.5 weeks) had a significantly lower HibCP antibody response than the preterm infants with GAs > 30 weeks (n = 23; mean GA, 34.2 weeks) (P = 0.004), who for their part had a response not significantly different from that of the term infants. After the third dose there were no significant differences among the groups. The response to the TT part of the vaccine showed the same pattern. CONCLUSION: Although the most immature infants may show an inadequate antibody response to the initial immunizations, many preterm infants can benefit from vaccination with HibCP-TT when starting immunization at the same chronologic age as term infants.

[Chronic fatigue syndrome--a controlled cross-sectional study]. / Kronisk traethedssyndrom--et kontrolleret tvoersnitsstudie.

Twenty-one patients fulfilling the Center for Disease Control criteria for chronic fatigue syndrome (CFS) were examined in a controlled study. Viral antibodies and tests evaluating the immune system were investigated in the patients and in a control group of 21 sex- and age-matched individuals. Production in vitro of the predominantly T-cell-derived cytokines interleukin-2 and interferon-gamma was significantly higher in patients with CFS compared the control group. Furthermore, the serum concentrations of IgA and IgE were significantly lower in patients with CFS; however, the values were within the normal reference range. All other variables were similar in the two groups. This study does not suggest a clearly disordered immune system or a chronic viral infection as a major pathogenetic factor in CFS. Longitudinal studies of immunological and virological parameters in CFS are warranted as are studies on patients that are severely handicapped.

Expression of human PTPN22 alleles.

Considering the female predominance in most of the autoimmune disorders that associate with the PTPN22 Trp620 variant and the complexity by which this variant influences immunologic tolerance, the objective of this study was to ascertain if the allele-specific expression of the disease-associated Arg620Trp polymorphism is affected by cis-acting or sex-specific trans-acting factor/s (e.g. sex-hormones). The use of the allele-specific transcript quantification of the Arg620Trp encoding 1858T polymorphism revealed no difference in the expression of the 1858C- and T-alleles in non-stimulated peripheral blood mononuclear cells (PBMCs) from non-pregnant female subjects, male subjects or pregnant female subjects in first or third trimester (P=0.70), respectively. While the transcription of PTPN22 in anti-CD3/anti-CD28 stimulated PBMCs increased fourfold (P<0.0001) and 13-fold (P<0.0001) after 48 and 72 h of activation, respectively, the expression of PTPN22 1858C- and T-alleles increased to the same extent (P=0.64). The present result essentially excludes such phenomena as a partial explanation for the female predominance in most of the autoimmune disorders that associate with the PTPN22 Trp620 variant.

Normal ICOS, ICOSL and AID alleles in Danish patients with common variable immunodeficiency.

Common variable immunodeficiency, CVID, is a primary antibody deficiency characterized by decreased levels of serum immunoglobulin G (IgG), decreased IgA and/or IgM and recurrent infections. It is assumed to be heterogeneous group of disorders caused by different genetic defects. Some patients have decreased levels of class switched memory B cells and/or decreased levels of somatic hypermutation which points to defects in the germinal centre (GC) reactions as cause of the disease in these patients. The inducible costimulator, ICOS, and its' ligand, ICOSL, are both involved in and necessary for the GC reaction and so is activation-induced cytidine deaminase, AID. Therefore, we sequenced the ICOS, ICOSL and AID genes in a cohort of 34 Danish CVID patients. We found 13 new single nucleotide polymorphisms (SNP) in the intron regions of the ICOSL gene as well as one SNP in exon 3. However, none of these polymorphisms were associated with CVID. We did not find a previously reported CVID-causing ICOS gene deletion or any other unique mutations in the ICOS or AID genes.

Distribution of kappa and lambda light chain isotypes among human blood immunoglobulin-secreting cells after vaccination with pneumococcal polysaccharides.

The light chain isotype of immunoglobulin-secreting blood cells was investigated by means of monolayer plaque-forming cell assays allowing direct immunofluorescence staining for cytoplasmic kappa and lambda light chains in centre cells. The study revealed that cultured, polyclonally activated pokeweed mitogen (PWM) and Epstein-Barr virus (EBV), IgM-, IgG- and IgA-secreting cells expressed the kappa light chain isotype in approximately 65% of the cells. IgM- and IgG-secreting cells induced by vaccination with pneumococcal polysaccharides had a similar percentage of kappa light chain-containing cells. In contrast, IgA-secreting cells induced by vaccination with pneumococcal polysaccharides showed a different (bimodal) distribution as regards expression of kappa light chain. The majority (56%) of the investigated individuals expressed kappa light chain in approximately 50% of the cells and the rest expressed kappa light chains in approximately 80% of the cells. The percentage of cells containing kappa light chains among spontaneous IgA-secreting cells in unvaccinated individuals was approximately 50% and thus also differed from the general pattern for mitogen-activated B cells. The light chain pattern of IgA-secreting cells from individuals vaccinated with pneumococcal polysaccharides and from unvaccinated individuals probably indicates that these cells are being derived from B-cell clones with a limited idiotypic heterogeneity, which have been selected and clonally expanded by naturally occurring antigens at the mucosal membranes.

The first constant-domain (CH1) exon of human IGHG2 is polymorphic and in strong linkage disequilibrium with the CH2 exon polymorphism encoding the G2m(n+) allotype in Caucasians.

Here we describe a hitherto unknown proline/threonine polymorphism at residue 72 of the human IgG2 CH1 domain (EU numbering 189) and show that it is linked to the known valine/methionine polymorphism at residue 52 of CH2 (EU numbering 282) defining the G2m(n+)/G2m(n-) allotypes. We sequenced the entire constant region of the heavy-chain gene for secreted IgG2 in five IGHG2*02 homozygous individuals covering CH1, hinge, CH2, and CH3 regions (approximately 2 kb). Proline 72 in CH1 of G2m(n-) is changed to threonine in the G2m(n+) [G2m(23)] allotype. Based on the crystal structure of human IgG1, this amino acid position is expected to be surface exposed in IgG2. Besides this structural difference, we identified two silent nucleotide polymorphisms in the CH1 region and seven in the introns. Finally, we developed a sequence-specific PCR typing system detecting the polymorphisms in the CH1 and CH2 regions. We typed 64 Danish Caucasians and found that the CH1 and CH2 region polymorphisms are in complete linkage disequilibrium in this population.

A new apparently functional IGVK gene (VkLa) present in some individuals only.

We describe a hitherto unknown functional IGKV gene, VkLa, belonging to the IGKV1 subgroup with exon 2 having only 94% similarity to the closest known IGKV gene, 1-13/1D-13 (L4/L18a). Genomic DNA sequences spanning from 5' of the decanucleotide box to 3' of the heptamer (649 bp) were cloned and sequenced from four individuals. The new gene encodes the conserved amino acids in the exons and contains no apparent defects in known regulatory intron sequences such as pd-box, dc-box, TATA-box, CCCT-elements, splice-sequences, initiation codon, and heptamer sequence. VkLa is therefore potentially functional and, correspondingly, we found transcripts of properly rearranged VkLa with somatical hypermutations. VkLa was found in 12 of 57 (21%) healthy Caucasians by a nested polymerase chain reaction and subsequent sequencing of exon 2. This finding shows that there is more inter-individual variation in the available IGKV gene repertoire than was hitherto assumed. Finally, we describe a minor correction in the IGKV1D-43 (L23) gene sequence.

Subclass of individual IgA-secreting human lymphocytes. Investigation of in vivo pneumococcal polysaccharide-induced and in vitro mitogen-induced blood B cells by monolayer plaque-forming cell assays.

The subclass of individual human IgA B cells was investigated by means of monolayer plaque-forming cell assays permitting analysis of all IgA-secreting cells as well as of cells secreting IgA anti-pneumococcal polysaccharide antibody. Center cells were examined by indirect immunofluorescence staining with mouse mAb against either of the two IgA subclasses as primary antibodies and FITC-conjugated rabbit anti-mouse Ig as the second antibody. Blood lymphocytes spontaneously secreting IgA (mean 399/10(6) mononuclear cells) produced mainly IgA1 (73%). A similar distribution of subclasses was recorded among IgA-secreting blood cells in PWM- and EBV-stimulated cultures. In contrast, a predominance of IgA2 (54%) was found among IgA-secreting cells (2531/10(6)) isolated from the blood 7 days after in vivo stimulation with pneumococcal polysaccharides, and a similar proportion (51%) of IgA2 producing cells was found among IgA anti-pneumococcal polysaccharide-secreting cells. It was thus confirmed that IgA1 is the predominant subclass of blood IgA-secreting cells in general. However, the high percentage of IgA2-secreting cells found after vaccination with pneumococcal polysaccharides suggests that these Ag have an unusually high ability to activate IgA2 B cells, or that the B cells stimulated originate from lymphatic tissues with a high frequency of IgA2 committed cells.

Quantitation of antibody-secreting cells in the blood after vaccination with Haemophilus influenzae type b conjugate vaccine.

The human B-lymphocyte response to protein-conjugated polysaccharide antigens has not previously been studied at the cellular level. In order to do so, we developed and evaluated haemolytic plaque-forming cell assays detecting Haemophilus influenzae type b (Hib) capsular polysaccharide-specific antibody-secreting cells (AbSC) of the isotypes IgM, IgG, and IgA. The appearance of AbSC in the blood after vaccination of adults with diphtheria toxoid-conjugated Hib polysaccharide was investigated. AbSC were detected from post-vaccination day 5 to day 14. IgA was the predominant isotype among these cells. IgM AbSC peaked slightly earlier (median day 7) than IgG and IgA AbSC (both day 8). On post-vaccination day 8 the numbers of AbSC were: IgA, 1217/10(6) mononuclear cells (median); IgG, 211; and IgM, 30 (n = 11). Similar isotype distribution has earlier been found after vaccination with pure capsular polysaccharides from Hib and pneumococci. The predominance of IgA AbSC in response to both conjugate and pure polysaccharide vaccines is probably due to reactivation of the same clones of IgA-committed memory B cells originally primed at the mucosa by natural exposure to the polysaccharide or cross-reacting antigens.

Influence of elevated body temperature on circulating immunoglobulin-secreting cells.

This work was designed to investigate the effect of in vivo hyperthermia in man on circulating immunoglobulin-secreting cells. Eight healthy male volunteers were immersed into a hot waterbath (WI) (water temperature 39.5 degrees C) for 2 h, whereby their body temperature rose to 39.5 degrees C. On another occasion they served as their own controls, being immersed into thermoneutral water (water temperature 34.5 degrees C) for 2 h. Blood samples were drawn before immersion, at body temperatures of 38, 39 and 39.5 degrees C, as well as 2 h after WI when their body temperatures were normalized. In the control experiments, blood samples were drawn at identical time points. A significant increase in the number of IgM-secreting cells per fixed number of blood mononuclear cells (BMNC) occurred 2 h after WI, whereas the number of IgA-secreting cells per fixed number of BMNC did not change. When the possible redistribution of BMNC was taken into account, the concentrations of IgM- and IgA-secreting cells (per ml blood) increased non-significantly during WI.

Detection of B-lymphocytes secreting antibodies to Dermatophagoides antigens.

An enzyme-linked immunospot assay (ELI-spot assay) has been established to count individual cells secreting antibodies to Dermatophagoides spp. allergens. Initial optimization of the assay was performed using Der p I-specific murine hybridoma cell lines. Inhibition with soluble purified allergen demonstrated that spot formation was specific. Addition of cycloheximide showed that spot formation was due to antibodies actively produced and secreted and not caused by preformed surface-associated Ig. An analogous method was used to count immunoglobulin-secreting cells (IgSC) of any specificity, and the number of circulating IgSC was measured in young healthy subjects. The results (means) were 331 IgM-SC, 532 IgG-SC, 850 IgA-SC and less than 1 IgE-SC per 10(6) mononuclear cells (MNC). Initial experiments performed with blood from patients undergoing immunotherapy with Dermatophagoides pteronyssinus Alutard, showed that 1 week after injection, circulating allergen-specific antibody-secreting cells (AbSC) were detected with this assay. The ranges were between 1 and 13 IgM-, 2 and 20 IgG- and 20 and 55 IgA allergen-specific AbSC per 10(6) MNC. It is expected that this assay will help to understand more thoroughly the basic immunological mechanisms behind the efficacy of immunotherapy.