Effects of nongenetic factors on immune cell dynamics in early childhood: The Generation R Study.

BACKGROUND: Numbers of blood leukocyte subsets are highly dynamic in childhood and differ greatly between subjects. Interindividual variation is only partly accounted for by genetic factors. OBJECTIVE: We sought to determine which nongenetic factors affect the dynamics of innate leukocytes and naive and memory lymphocyte subsets. METHODS: We performed 6-color flow cytometry and linear mixed-effects modeling to define the dynamics of 62 leukocyte subsets from birth to 6 years of age in 1182 children, with 1 to 5 measurements per subject. Subsequently, we defined the effect of prenatal maternal lifestyle-related or immune-mediated determinants, birth characteristics, and bacterial/viral exposure-related determinants on leukocyte subset dynamics. RESULTS: Functionally similar leukocyte populations were grouped by using unbiased hierarchical clustering of patterns of age-related leukocyte dynamics. Innate leukocyte numbers were high at birth and predominantly affected by maternal low education level. Naive lymphocyte counts peaked around 1 year, whereas most memory lymphocyte subsets more gradually increased during the first 4 years of life. Dynamics of CD4+ T cells were predominantly associated with sex, birth characteristics, and persistent infections with cytomegalovirus (CMV) or EBV. CD8+ T cells were predominantly associated with CMV and EBV infections, and T-cell receptor γδ+ T cells were predominantly associated with premature rupture of membranes and CMV infection. B-cell subsets were predominantly associated with sex, breast-feeding, and Helicobacter pylori carriership. CONCLUSIONS: Our study identifies specific dynamic patterns of leukocyte subset numbers, as well as nongenetic determinants that affect these patterns, thereby providing new insights into the shaping of the childhood immune system.

Determinants of Ethnic Differences in Cytomegalovirus, Epstein-Barr Virus, and Herpes Simplex Virus Type 1 Seroprevalence in Childhood.

OBJECTIVE: To identify whether there are ethnic differences in cytomegalovirus (CMV), Epstein-Barr virus (EBV), and herpes simplex virus type 1 (HSV-1) seroprevalence rates in children at 6 years of age, and when present, to evaluate how these differences can be explained by sociodemographic and environmental factors. STUDY DESIGN: This study was embedded within a multi-ethnic population-based prospective cohort study. Serum IgG levels against CMV, EBV, and HSV-1 were measured by enzyme-linked immunosorbent assay in 4464 children (median age 6.0 years). Information on demographics and characteristics were assessed by questionnaires. Herpesvirus seroprevalences between Surinamese-Creole, Surinamese-Hindustani, Turkish, Moroccan, Cape Verdean Antillean, and Native Dutch children were compared. RESULTS: Non-Western ethnicity was an independent risk factor for CMV (aOR, 2.16; 95% CI 1.81-2.57), EBV (1.76; 1.48-2.09), and HSV-1 seropositivity (1.52; 1.39-1.66). Among the ethnic groups, CMV seroprevalences ranged between 29% and 65%, EBV between 43% and 69%, and HSV-1 between 13% and 39%. Low family net household income, low maternal educational level, crowding, and lifestyle factors explained up to 48% of the ethnic differences in HSV-1 seroprevalences, and up to 39% of the ethnic differences in EBV seroprevalences. These factors did not explain ethnic differences in CMV seroprevalences. CONCLUSIONS: Socioeconomic position and factors related to lifestyle explain only a part of the large ethnic differences in EBV and HSV-1 seroprevalences, whereas they do not explain ethnic differences in CMV seroprevalences in childhood.

Herpesvirus Infections and Transglutaminase Type 2 Antibody Positivity in Childhood: The Generation R Study.

OBJECTIVES: Persistent viral infections have been implicated in the etiology of autoimmune diseases in adulthood, but it is not known whether herpesviruses are associated with the development of celiac disease autoimmunity in childhood. We assessed whether herpesvirus infections are associated with transglutaminase type 2 antibody (TG2A) concentrations in children at 6 years of age. METHODS: The present study was embedded within a population-based prospective cohort study. Serum immunoglobulin G levels against Epstein-Barr virus, cytomegalovirus (CMV), and herpes simplex virus type 1 were measured by enzyme-linked immunosorbent assay , and TG2A concentrations with fluorescence enzyme immunoassay in 4420 children at 6 years of age. Children were categorized based on TG2A concentrations into negative (<7 U/mL), positive (≥7-70 U/mL), and strongly positive (≥70 U/mL), that is, 10 times upper limit normal. RESULTS: Fifty-nine children (1.3%) were TG2A positive, and of these 31 (53%) had concentrations 70 U/mL or more. Children with TG2A concentrations 70 U/mL or more were less often infected with CMV (adjusted odds ratio (aOR) 0.38, 95% CI 0.14-0.98, P = 0.04) and with any of the 3 viruses (aOR 0.38, 95% CI 0.18-0.78, P < 0.01) than children with TG2A negative concentrations. In addition, children with TG2A concentrations 70 U/mL or more were less often infected with 2 or more viruses than children with TG2A negative concentrations (aOR 0.15, 95% CI 0.03-0.65, P = 0.01). CONCLUSIONS: Both CMV single infection and combined CMV, Epstein-Barr virus and/or herpes simplex virus type 1 infections are inversely associated with strongly TG2A positivity. This may indicate a protective effect of herpesvirus infections in the pathogenesis of celiac disease autoimmunity.

Growth trajectories and bone mineral density in anti-tissue transglutaminase antibody-positive children: the Generation R Study.

BACKGROUND & AIMS: Increased levels of anti-tissue transglutaminase (tTG) have been associated with reduced weight and bone mineral density (BMD) in symptomatic patients with celiac disease. Little is known about the effects of these antibodies in patients with subclinical or other forms of celiac disease. We examined associations between anti-tTG positivity and growth and BMD. METHODS: In a population-based prospective cohort study, serum samples were collected from children (median age, 6 years; n = 4442) and analyzed for anti-tTG. All children were born between April 2002 and January 2006 and were not previously diagnosed with celiac disease. Children were categorized as anti-tTG negative (<7 U/mL, n = 4249) or anti-tTG positive (≥7 U/mL, n = 57). Children's levels of anti-tTG were further categorized on the basis of ≥10 times upper limit of normal (70 U/mL). Height, weight, and body mass index (BMI) age- and sex-adjusted standard deviation scores (SDS) ([observed value - mean]/SD) were obtained by using Dutch reference growth charts. BMD was measured by dual-energy x-ray absorptiometry. Multivariable linear regression and linear mixed models were performed. RESULTS: Children who tested positive for anti-tTG had reduced growth in weight SDS/year (reduction of 0.05; 95% CI, reductions of 0.09-0.01) and BMI SDS/year (reduction of 0.10; 95% CI, reductions of 0.18-0.01) from 6 months until 6 years, compared with children without anti-tTG; they also tended to have reduced growth in height from 6 months until 6 years (reduction of 0.02 SDS/year; 95% CI, reductions of 0.06-0.02). Children who tested positive for anti-tTG were shorter (0.29 SDS shorter; 95% CI, reductions of 0.55-0.04 SDS), weighed less (0.38 SDS less; 95% CI, reductions of 0.64-0.12), and had lower BMIs (0.26 SDS less; 95% CI, reductions of 0.49-0.03) and BMDs (0.26 SDS less; 95% CI, reductions of 0.45-0.08) at 6 years of age than anti-tTG negative children. CONCLUSIONS: Anti-tTG positive children without gastrointestinal symptoms have lower BMDs and reduced growth trajectories until they are 6 years old. This suggests that subclinical or potential celiac disease can affect BMD and growth.

Neopterin: a potential biomarker for delirium in elderly patients.

BACKGROUND/AIMS: The diagnosis of delirium is not supported by specific biomarkers. In a previous study, high neopterin levels were found in patients with a postoperative delirium. In the present study, we investigated levels of neopterin, interleukin-6 (IL-6) and insulin-like growth factor-1 (IGF-1) in acutely ill admitted elderly patients with and without a delirium. METHODS: Plasma/serum levels of neopterin, IL-6 and IGF-1 were determined in patients aged ≥65 years admitted to the wards of Internal Medicine and Geriatrics. Differences in biomarker levels between patients with and without a delirium were investigated by the analysis of variance in models adjusted for age, gender, comorbidities and eGFR (when appropriate). RESULTS: Eighty-six patients were included; 23 of them with a delirium. In adjusted models, higher mean levels of neopterin (70.5 vs. 45.9 nmol/l, p = 0.009) and IL-6 (43.1 vs. 18.5 pg/ml, p = 0.034) and lower mean levels of IGF-1 (6.3 vs. 9.3 nmol/l, p = 0.007) were found in patients with a delirium compared to those without. CONCLUSIONS: The findings of this study suggest that neopterin might be a potential biomarker for delirium which, through oxidative stress and activation of the immune system, may play a role in the pathophysiology of delirium.

Levels of antibodies against tissue transglutaminase during pregnancy are associated with reduced fetal weight and birth weight.

BACKGROUND & AIMS: Celiac disease in pregnant women has been associated with poor growth of the fetus, but little is known about how the level of celiac disease affects fetal growth or birth outcomes. We assessed the associations between levels of antibodies against tissue transglutaminase (anti-tTG, a marker of celiac disease) and fetal growth and birth outcomes for pregnant women. METHODS: We performed a population-based prospective birth cohort study of 7046 pregnant women. Serum samples were collected during the second trimester of pregnancy and analyzed for levels of anti-tTG. Based on these levels, the women were categorized into 3 groups: negative anti-tTG (≤0.79 U/mL; n = 6702), intermediate anti-tTG (0.8 to ≤6 U/mL; n = 308), or positive anti-tTG (>6 U/mL; n = 36). Data on fetal growth and birth outcomes were collected from ultrasound measurements and medical records. RESULTS: Fetuses of women in the positive anti-tTG group weighed 16 g less than those of women in the negative anti-tTG group (95% confidence interval [CI], -32 to -1 g) during the second trimester and weighed 74 g less (95% CI, -140 to -8 g) during the third trimester. Newborns of women in the intermediate and positive anti-tTG groups weighed 53 g (95% CI, -106 to -1 g) and 159 g (95% CI, -316 to -1 g) less at birth, respectively, than those of women in the negative anti-tTG group. The reduction in birth weight in offspring of mothers in the intermediate anti-tTG group was 2-fold greater among mothers who carried HLA-DQ2 or -DQ8 than among those without HLA-DQ2 or -DQ8. CONCLUSIONS: Levels of anti-tTG in pregnant women are inversely associated with fetal growth. Growth was reduced to the greatest extent in fetuses of women with the highest levels of anti-tTG (>6 U/mL). Birth weight was also reduced in women with intermediate levels of anti-tTG (0.8 to ≤6 U/mL) and further reduced in those carrying HLA-DQ2 and -DQ8.

Women with high early pregnancy urinary iodine levels have an increased risk of hyperthyroid newborns: the population-based Generation R Study.

OBJECTIVE: Iodine deficiency during pregnancy results in thyroid dysfunction and has been associated with adverse obstetric and foetal effects, leading to worldwide salt iodization programmes. As nowadays 69% of the world's population lives in iodine-sufficient regions, we investigated the effects of variation in iodine status on maternal and foetal thyroid (dys)function in an iodine-sufficient population. DESIGN, PARTICIPANTS AND MEASUREMENTS: Urinary iodine, serum TSH, free T4 (FT4) and TPO-antibody levels were determined in early pregnancy (13·3 (1·9) week; mean (SD)) in 1098 women from the population-based Generation R Study. Newborn cord serum TSH and FT4 levels were determined at birth. RESULTS: The median urinary iodine level was 222·5 µg/l, indicating an iodine-sufficient population. 30·8% and 11·5% had urinary iodine levels <150 and >500 µg/l, respectively. When comparing mothers with urinary iodine levels <150 vs ≥150 µg/l, and >500 vs ≤500 µg/l, there were no differences in the risk of maternal increased or decreased TSH, hypothyroxinaemia or hyperthyroidism. Mothers with urinary iodine levels >500 µg/l had a higher risk of a newborn with decreased cord TSH levels (5·6 ± 1·4 (mean ± SE) vs 2·1 ± 0·5%, P = 0·04), as well as a higher risk of a hyperthyroid newborn (3·1 ± 0·9 vs 0·6 ± 0·3%, P = 0·02). These mothers had newborns with higher cord FT4 levels (21·7 ± 0·3 vs 21·0 ± 0·1 pm, P = 0·04). Maternal urinary iodine levels <150 µg/l were not associated with newborn thyroid dysfunction. CONCLUSIONS: In an iodine-sufficient population, higher maternal urinary iodine levels are associated with an increased risk of a hyperthyroid newborn.

Heterogeneous nuclear RNPs as targets of autoantibodies in systemic rheumatic diseases.

OBJECTIVE: To investigate the abundance of autoantibodies to heterogeneous nuclear RNPs (hnRNPs) in systemic rheumatic diseases. METHODS: Recombinant human hnRNPs A1, B1, C1, E1, F, Gi, H1, I, K, and P2 were prepared. Antibodies to these antigens were determined by Western blotting and by enzyme-linked immunosorbent assay (ELISA) (for hnRNPs B1, E1, F, and H1) in serum samples obtained from patients with chronic fatigue syndrome (control subjects) and from patients with various connective tissue diseases. RESULTS: Western blotting analysis in 106 control subjects and 298 patients with a connective tissue disease revealed that antibodies to all tested hnRNP antigens, except hnRNP Gi, were significantly more prevalent in patients with Sjögren's syndrome (SS) than in control subjects. The highest reactivity was observed for hnRNPs B1, E1, F, and H1 (reactivity in >45% of patients with SS and in 2.8% of control subjects). Reactivity with hnRNPs B1, E1, F, and H1 was also evaluated by ELISA in 89 control subjects and 228 patients with a connective tissue disease. Reactivity with at least 2 of the 4 tested antigens was observed in 1.1% of control subjects, 16% of patients with systemic lupus erythematosus (SLE), and 18% of patients with SS. Reactivity with at least 3 of the 4 antigens was observed in 0% of the control subjects, 3.2% of patients with SLE, and 15% of patients with SS. CONCLUSION: Several hnRNPs are target antigens in SS. The combined presence of antibodies to several hnRNPs was strongly associated with connective tissue disease in general and with SS in particular.

Factor Xa and thrombin stimulate proinflammatory and profibrotic mediator production by retinal pigment epithelial cells: a role in vitreoretinal disorders?

BACKGROUND: Vitreoretinal disorders, including proliferative vitreoretinopathy (PVR), proliferative diabetic retinopathy (PDR) and exudative age-related macular degeneration (AMD), are a major cause of visual impairment worldwide and can lead to blindness when untreated. Loss of blood-retinal barrier (BRB) integrity associated with vitreoretinal fibrin deposition, inflammation, fibrosis and neovascularization contribute to the pathophysiological processes in these disorders. Retinal pigment epithelial (RPE) cells are well recognized to contribute to vitreoretinal inflammation/fibrosis and are likely to encounter contact with coagulation factor upon loss of BRB integrity. METHODS: An extensive study was performed in which we examined the effect of factor Xa and thrombin on the production of a broad panel of cytokines/chemokines and growth factors by RPE cells. For this purpose we used the ARPE-19 cell line as well as primary RPE cells, a glass slide based array that allows simultaneous detection of 120 cytokines/chemokines and growth factors, ELISA and real-time-quantitative PCR. The involved signaling cascade was examined using specific inhibitors for protease activated receptor (PAR)1, PAR2 and nuclear factor kappa-B (NF-κB). RESULTS: Factor Xa and thrombin regulated the production of cytokines and growth factors (including GM-CSF, IL-6, IL-8, MCP-3, PDGF-AA, PDGF-BB, TIMP-1 and TGF-α) that fit well in the pathobiology of vitreoretinal disease. Blocking studies revealed that the effects were mediated via PAR1 induced NF-κB activation. CONCLUSIONS: Our findings suggest that factor Xa and thrombin can drive vitreoretinal inflammation and fibrosis and should be considered as treatment targets in vitreoretinal disorders such as PVR, PDR and AMD.

Psychiatric phenomena as initial manifestation of encephalitis by anti-NMDAR antibodies.

OBJECTIVE: Autoimmune encephalitis associated with autoantibodies against the N-methyl-d-aspartate receptor (NMDAR) often presents with behavioural change. Our objective was to describe in detail the psychiatric presentation and pathways to care in order to aid the early diagnosis of NMDAR encephalitis. METHODS: Sera and cerebrospinal fluid (CSF) from patients with suspected NMDAR encephalitis were tested on HEK 293 cells transfected with the NR1 subunit of the NMDAR. Clinical information was obtained from the referring psychiatrists and neurologists and by review of the clinical records. RESULTS: Samples from 15 patients (13 female, 2 male, mean age 24 years, range 5-56 years) tested anti-NMDAR positive. Twelve of the 15 patients (80%) presented with prominent psychiatric symptoms and 8 were initially referred to a psychiatric service. The most prominent initial psychiatric symptoms were anxiety in seven (47%), behavioural change (often bizarre) in six (40%) and agitation in five (33%). All patients developed psychiatric symptoms in the first 6 weeks of illness. Thirteen patients received psychotropic medications: antipsychotics in 12 and benzodiazepines in 11. Treating physicians considered the psychotropic medication not effective in 11 patients resulting in many drug switches. At nadir, all patients were in a very poor condition. However, eight patients (53%) recovered (almost) completely. Outcome tended to be better in patients who had received early immunotherapy or tumour removal. CONCLUSIONS: Autoimmune encephalitis and anti-NMDAR testing in serum and CSF should be considered in patients, especially young females, presenting with atypical psychiatric phenomena. Early diagnosis and treatment will likely improve the prognosis of NMDAR encephalitis.

Low urinary iodine excretion during early pregnancy is associated with alterations in executive functioning in children.

The rare but deleterious effects of severe iodine deficiency during pregnancy on cognitive functioning of children are well known. Reports on possible associations between mild-to-moderate maternal iodine deficiency and child development, however, are scarce. In a population-based cohort we examined the association between maternal urinary iodine during early pregnancy and executive functioning in children at 4 y of age. In addition, we investigated the modification of this association by maternal diet and thyroid function. During pregnancy, we measured urinary iodine and thyroid hormone concentrations in 1156 women. In 692 of their children impairment of executive functioning was assessed by the Behavior Rating Inventory of Executive Function. Five hundred mothers of Dutch national origin completed an FFQ. Analyses were performed by using regression models. The children of mothers with low urinary iodine showed higher scores on the problem scales of inhibition [ß = 0.05 (95% CI: 0.01, 0.10), P = 0.03] and working memory [ß = 0.07 (95% CI: 0.02, 0.12), P = 0.003]. Although maternal dietary intake and thyroid hormone concentration did not significantly modify these associations, the associations between urinary iodine and problems of inhibition were attenuated after adjustment for maternal psychological symptoms. In addition, the consumption of bread [ß = 0.61 (95% CI: 0.27, 0.95), P < 0.001] and eggs (ß = 1.87 (95% CI: 0.13, 3.62), P = 0.04] was associated with higher urinary iodine. Thus, low maternal urinary iodine during pregnancy is associated with impaired executive functioning in children. Because these symptoms were subclinical and occurred at an early age, future studies are needed to show whether these children are more vulnerable to develop later clinical disorders.

Natural antibodies against several pneumococcal virulence proteins in children during the pre-pneumococcal-vaccine era: the generation R study.

The currently available pneumococcal vaccines do not protect against all serotypes of Streptococcus pneumoniae. A shift toward nonvaccine serotypes causing colonization and invasive disease has occurred, and studies on protein-based vaccines have been undertaken. We assessed the association between specific antibodies against pneumococcal virulence proteins and colonization and respiratory tract infections (RTIs). Additionally, we assessed the extent to which colonization induces a humoral immune response. Nasopharyngeal swabs collected from children at 1.5, 6, 14, and 24 months of age were cultured for pneumococcus. Serum samples were obtained at birth and at 6, 14, and 24 months (n = 57 children providing 177 serum samples). Data were collected prior to the pneumococcal vaccine era. IgG, IgA, and IgM levels against 17 pneumococcal protein vaccine candidates were measured using a bead-based flow cytometry technique (xMAP; Luminex Corporation). Information regarding RTIs was questionnaire derived. Levels of IgG against all proteins were high in cord blood, decreased in the first 6 months and increased again thereafter, in contrast to the course of IgA and IgM levels. Specific antibodies were induced upon colonization. Increased levels of IgG against BVH-3, NanA, and SP1003 at 6 months, NanA, PpmA, PsaA, SlrA, SP0189, and SP1003 at 14 months, and SlrA at 24 months were associated with a decreased number of RTIs in the third year of life but not with colonization. Maternal antipneumococcal antibodies did not protect against pneumococcal colonization and infection. Certain antibodies against pneumococcal virulence proteins, some of which are induced by colonization, are associated with a decreased number of RTIs in children. This should be taken into account in future pneumococcal vaccine studies.

Maternal thyroid function during pregnancy and behavioral problems in the offspring: the generation R study.

Maternal thyroid function during pregnancy is implicated in the neurodevelopment of the offspring, yet little is known about the effect of maternal thyroid parameters on the behavior of children. We investigated the association of maternal thyroid function during the first half of pregnancy with parent-reported problem behavior of the offspring up to age of 3 y. In the Generation R study, a population-based cohort of 3736 children and their mothers, data on maternal thyroid function and child's behavior were examined. The degree of internalizing and externalizing problems in the children were assessed with the Child Behavior Checklist at ages 1½ and 3 y. Higher levels of maternal TSH during pregnancy predicted a higher externalizing scores in children at 1½ and 3 y (B = 0.22 per SD of TSH; 95% CI: 0.04, 0.40; B = 0.10 per SD for internalizing scores; 95% CI: -0.01, 0.21). Maternal free thyroxine (T4) and total T4 were not associated with internalizing or externalizing scores of children. The linear relationship with more externalizing scores was across the range of TSH; this implies that subtle impairments of maternal thyroid function may affect the child. The results suggest that thyroid function is crucial for fetal brain development, which determines problem behavior later in life.

Dietary restriction modifies certain aspects of the postoperative acute phase response.

BACKGROUND: Lifespan extension is achieved through long-term application of dietary restriction (DR), and benefits of short-term dietary restriction on acute stress and inflammation have been observed. So far, the effects of short-term DR in humans are relatively unknown. We hypothesized that short-term DR in humans reduces the acute phase response following a well defined surgical trauma. METHODS: Thirty live kidney donors were randomized between 30% preoperative dietary restriction followed by 1 d of fasting (n=17) or a 4 d ad libitum regimen (n=13) prior to surgery. Leukocyte subsets and numbers and serum cytokine levels were determined. Whole blood was stimulated with lipopolysaccharide (LPS) and cytokine production was determined. RESULTS: A clear trend towards lower numbers of postoperative circulating leukocytes was observed in the DR group. IL-8 serum levels were significantly higher in the DR group over the first 6 postoperative d (P=0.018). After LPS stimulation, significantly less TNF-α (P=0.001) was produced by blood obtained postoperatively compared with preoperative blood from the DR group. This was not observed in the control group. CONCLUSIONS: A relatively short preoperative dietary restriction regimen was able to modify certain aspects of the postoperative acute phase response. These data warrant further studies into the dietary conditions that improve stress resistance in humans. (Dutch Trial Registry number: NTR1875).

Immunogenicity of toxins during Staphylococcus aureus infection.

BACKGROUND: Toxins are important Staphylococcus aureus virulence factors, but little is known about their immunogenicity during infection. Here, additional insight is generated. METHODS: Serum samples from 206 S. aureus-infected patients and 201 hospital-admitted control subjects were analyzed for immunoglobulin (Ig) G binding to 20 toxins, using flow-cytometry based technology. Antibody levels were associated with polymerase chain reaction-defined presence of toxin genes in homologous S. aureus isolates. RESULTS: IgG levels directed to exfoliative toxin (ET) A, ETB, gamma hemolysin B (HlgB), leukocidin (Luk) D, LukE, LukS, staphylococcal enterotoxin (SE) A, SEE, SEH, SEI, and SElM were higher in S. aureus-infected patients than in control subjects (P < .05). Furthermore, in the S. aureus-infected patient group, IgG levels were higher if genes encoding ETA, ETB, SEA, SEC, SEH, SElQ, toxic shock syndrome toxin-1 (TSST-1), or Panton-Valentine leukocidin (PVL) were present in the infectious isolate (P< .05). Levels of anti-SEA IgG increased during infections with sea-positive (median fluorescence intensity from 11,555 to 12,388; P<.05) but not sea-negative strains. In addition, anti-LukS IgG levels increased during skin and soft-tissue infections with luk-PV-positive (median fluorescence intensity from 15,231 to 15,911; P<.05) but not luk-PV-negative strains. Bacteremia was associated with sea (odds ratio, 3.4; 95% confidence interval, 1.2-10.0) and tst (odds ratio, 5.7; 95% confidence interval, 1.6-20.8). Skin and soft-tissue infections and bone and joint infections were associated with luk-PV (odds ratio, 2.5; 95% confidence interval, 1.2-5.2). CONCLUSIONS: Many toxins are expressed in vivo and recognized by the immune system during staphylococcal infections, suggesting their involvement in S. aureus pathogenesis.

Heterogeneous nuclear ribonucleoprotein h1, a novel nuclear autoantigen.

BACKGROUND: Serum samples from patients with autoimmune connective tissue diseases that show a finely speckled antinuclear antibody (ANA) on indirect immune-fluorescence often have antibodies against unknown nuclear target antigens. To search for such autoantigens we applied a proteomic approach using sera from patients with a high ANA titer (>or=640) and finely speckled fluorescence but in whom no antibodies to extractable nuclear antigens (ENA) could be identified. METHODS: Using an immunoproteomics approach we identified heterogeneous nuclear ribonucleoprotein H1 (hnRNP H1) as a novel nuclear target of autoantibody response. RESULTS: Recombinant rat hnRNP H1 reacted in Western blot analyses with 48% of 93 sera from patients with primary Sjögren syndrome and with 5.2% of 153 sera from patients with other connective tissue diseases (diseased controls). For comparison, the diagnostic sensitivity and specificity of anti-Sjögren syndrome A (SSA) antibodies for primary Sjögren syndrome in the same patient cohort were 88.2% and 76.3%, respectively. Interestingly, 5 of 11 primary Sjögren syndrome patients with no anti-SSA or anti-SSB antibodies had anti-hnRNP H1 antibodies. Anti-hnRNP H1 antibodies were preabsorbed by hnRNP H1, as demonstrated by indirect immunofluorescence. In an evaluation of the presence of anti-hnRNP H1 antibodies in 188 consecutive samples submitted to the clinical laboratory with positive ANA (titer >or=160), anti-hnRNP H1 antibodies were found in 3 of 7 (2 primary and 5 secondary) Sjögren syndrome patients and in 8.3% of the diseased controls. CONCLUSIONS: HnRNP H1 is a newly discovered autoantigen that could become an additional diagnostic marker.

Variation in the IGF-1 gene is associated with lymphocyte subset counts in neonates: the Generation R Study.

OBJECTIVE: IGF-1 stimulates growth, development and function of lymphocytes. The aim of this study was to examine whether functional variants of the IGF-1 gene are associated with absolute lymphocyte subset counts in neonates. STUDY DESIGN AND MEASUREMENTS: This study was embedded in the Generation R Study, a prospective cohort study from foetal life onwards. A polymorphism in the IGF-1 promoter region was genotyped in cord blood DNA. Lymphocytes (T, B and NK) and T lymphocyte subsets (helper, cytotoxic, naive and memory) in cord blood were immunophenotyped in 380 neonates by six-colour flow cytometry. RESULTS: In total, 39% of the neonates were homozygous for the 192-bp allele (wild-type), 48% were heterozygous and 13% were noncarrier. No differences in absolute lymphocyte and T lymphocyte subset counts were observed between the 192-bp allele heterozygous and homozygous groups. In noncarriers, we found 15% lower T lymphocyte (P = 0.03), 22% lower B lymphocyte (P = 0.04) and 10% lower NK lymphocyte counts (P = 0.36) than in the 192-bp allele homozygous group. Analyses of T lymphocyte subsets showed 16% lower helper T lymphocyte counts (P = 0.01) in noncarriers. No significant differences were found for cytotoxic, naive and memory T lymphocyte counts. All associations were adjusted for gravidity, mode of delivery, gestational age, birth weight, gender and 1- and 5- min Apgar scores. CONCLUSIONS: Our study showed associations between this IGF-1 promoter region polymorphism and absolute lymphocyte subset counts in neonates. These results should be regarded as hypothesis generating until they have been replicated in other studies.

Comparison of carboxylated and Penta-His microspheres for semi-quantitative measurement of antibody responses to His-tagged proteins.

The Luminex system is a flow cytometry based tool that permits the simultaneous measurement of many analytes from just a single serum sample. The technology uses microspheres, which are available in different colors and can be coated with different kinds of biomolecules. For the immobilisation of His-tagged proteins, two types of beads can be used: chemically activated carboxylated beads or Penta-His beads, which have antibodies against His-tags on their surface. In this study, we compared carboxylated and Penta-His beads. For carboxylated as compared to Penta-His beads, the non-specific background is lower (Median Fluorescence Intensity; MFI>250, 0% versus 15%), the specific signal intensity is higher (mean MFI 2860 versus 722) and not dependent on the configuration of the protein. Above all, the protein coupled carboxylated beads are useful over longer periods of time. Therefore, we conclude that for developing a multiplex assay for semi-quantitative measurement of antibody responses against His-tagged proteins the best microspheres to use are the carboxylated ones.

Heterophilic antibody interference in a tryptase immunoassay.

OBJECTIVE: Investigation of the susceptibility of a tryptase immunoassay to interference by heterophilic antibodies. METHODS: The effect of preincubation with a blocking agent was investigated on the levels of tryptase, human anti-mouse antibodies and IgM rheumatoid factor in sera with elevated IgM rheumatoid factor levels. RESULTS: In 5 of 30 sera with IgM rheumatoid factor, tryptase levels were reduced at least twofold after pre-incubation with blocking reagent. A significant association was observed between the presence of IgM rheumatoid factor in the sera and the interference of tryptase immunoassay. There was no quantitative correlation found between the reduction in serum tryptase level by treatment with a blocking agent, and the amount of IgM rheumatoid factor was present. However, this reduction in serum tryptase was significantly correlated with the amount of human anti-mouse antibodies in the sera. After incubation with blocking agent, there was no change in IgM Rheumatoid factor level, but a significant decrease in human anti-mouse antibodies. CONCLUSION: The Phadia tryptase assay method, in its present form, is sensitive to interference by heterophilic antibodies.

The identification of celiac disease in asymptomatic children: the Generation R Study.

BACKGROUND: The objective of our study was to assess whether TG2A levels in the healthy childhood population can be predictive of subclinical CD. METHODS: A total of 4442 children (median age, 6.0 years) participating in a population-based prospective cohort study were screened on serum TG2A. Those with positive TG2A (≥7 U/ml; n = 60, 1.4%) were invited for clinical evaluation (median age, 9.0 years). Medical history, physical examination, serum TG2A, and IgA-endomysium (EMA) were assessed, as well as HLA DQ 2.2/2.5/8 typing. Patients with positive serologies and genetic risk types underwent duodenal biopsies. TG2A levels at the time of biopsy were compared with the degree of enteropathy. RESULTS: Fifty-one TG2A-positive children were included in the follow-up: 31 (60.8%) children had CD, ten (19.6%) did not have CD, and ten (19.6%) were considered potential CD cases because of inconclusive serologies. Duodenal biopsies were performed in 26/31 children. CD with Marsh 3a/b enteropathy was observed in 75% (15/20) of children having TG2A levels ≥10ULN at 6 years of age, as well as in 75% (6/8) of children having a positive TG2A <10 ULN (OR 1.00; 95% CI 0.15-6.64). CD cases had a lower BMI SDS (mean -0.49, SD 0.92) than children without CD (mean 0.47, SD 1.37; p = 0.02). No differences were observed in gastrointestinal symptoms. CONCLUSIONS: Serum TG2A screening at 6 years of age in the healthy childhood population has a positive predictive value of 61% to detect subclinical CD. We did not find a positive correlation between serum TG2A levels and the degree of enteropathy.