Development and validation of a minimally invasive and image-guided tape stripping method to sample atopic skin in children.

BACKGROUND: Molecular skin profiling techniques, typically performed on skin samples taken by punch biopsy, have enhanced the understanding of the pathophysiology of atopic dermatitis (AD), thereby enabling the development of novel targeted therapeutics. However, punch biopsies are not always feasible or desirable, and novel minimally invasive methods such as skin tape stripping have been developed. AIM: To develop, optimize and validate a novel tape stripping method guided by noninvasive in vivo skin imaging to sample atopic skin in children. METHODS: Skin tape stripping-based procedures were compared and optimized using data from 30 healthy controls (HCs: 5 adults, 25 children) and 39 atopic children. Evaluations were guided by high-resolution photography, reflectance confocal microscopy, optical coherence tomography and transepidermal water loss measurements. We assessed and compared adverse events (AEs), the time needed to perform the sampling and the cDNA levels obtained from the tapes. RESULTS: Tape stripping methods based on previously described protocols resulted in erosions in all participants and required a median time of 65 min to perform (range 60-70 min), but provided good cDNA yield. Shorter durations appeared less invasive but provided lower cDNA yield. The final optimized tape stripping protocol, using 11 tapes of 22 mm in diameter, each applied twice for 5 s with 90° rotation, did not produce significant AEs, was completed within a median time of 7 min (range 5-15 min) and provided good cDNA yield both in HCs and atopic children. CONCLUSION: Our minimally invasive method is safe and reliable, and provides reproducible acquisition of cDNA in atopic children. In addition, it enables rapid sample collection, a crucial factor in clinical practice.

A biomarker of collagen type I degradation is associated with cardiovascular events and mortality in patients with atherosclerosis.

OBJECTIVE: Atherosclerosis is characterized by accumulation of lipids, cells and extracellular matrix (ECM) proteins in the arterial wall. Collagen type I (COL1), a component of the arterial ECM, is cleaved by matrix metalloproteinases (MMPs) and known to be remodelled in atherosclerosis. We explored whether the MMP-mediated COL1 biomarker, C1M, was associated with cardiovascular events, cardiovascular mortality and all-cause mortality in a large prospective cohort of patients with known atherosclerosis. METHODS: Serum from 787 patients who underwent a carotid endarterectomy was included. Circulating levels of C1M were measured in serum. A total of 473 patients were followed for 6 years after surgery. Associations between C1M and incidence of cardiovascular events, cardiovascular mortality and all-cause mortality were assessed by Kaplan-Meier curves and Cox regression analysis. RESULTS: A total of 101 (21.4%) patients suffered from nonfatal cardiovascular events during the follow-up period, and 64 (13.5%) patients died. Of these, 39 (60.9%) died from cardiovascular diseases. Patients with C1M levels above the median were significantly associated with cardiovascular events, cardiovascular mortality and all-cause mortality (P < 0.001, P = 0.004 and P < 0.001, respectively). C1M was included in the final model for prediction of cardiovascular events (HR 2.15, 95% CI 1.40-3.32, P = 0.001), cardiovascular mortality (HR 2.20, 95% CI 1.07-4.51, P = 0.031) and all-cause mortality (HR 2.98 95% CI 1.67-5.33, P = < 0.001). CONCLUSIONS: In patients with atherosclerotic carotid lesions, high levels of C1M predicted cardiovascular events, cardiovascular mortality and all-cause mortality. These findings emphasize the importance of remodelling mechanisms in atherosclerosis that are now becoming more and more explored.

A fragment of SPARC reflecting increased collagen affinity shows pathological relevance in lung cancer - implications of a new collagen chaperone function of SPARC.

The matricellular protein SPARC (secreted proteome acidic and rich in cysteine) is known to bind collagens and regulate fibrillogenesis. Cleavage of SPARC at a single peptide bond, increases the affinity for collagens up to 20-fold. To investigate if this specific cleavage has pathological relevance in fibrotic disorders, we developed a competitive ELISA targeting the generated neo-epitope on the released fragment and quantified it in serum from patients with lung cancer, idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD) and healthy subjects. Furthermore, the ability of SPARC to protect fibrillar collagens from proteolytic degradation was investigated in vitro, potentially adding a new collagen chaperone function to SPARC. The fragment was significantly elevated in lung cancer patients when compared to healthy subjects measured in a discovery cohort (p = 0.0005) and a validation cohort (p < 0.0001). No significant difference was observed for IPF and COPD patients compared to healthy subjects. When recombinant SPARC was incubated with type I or type III collagen and matrix metalloproteinase-9, collagen degradation was completely inhibited. Together, these data suggest that cleavage of SPARC at a specific site, which modulates collagen binding, is a physiological mechanism increased during pathogenesis of lung cancer. Furthermore, inhibition of fibrillar collagen degradation by SPARC adds a new chaperone function to SPARC which may play additional roles in the contribution to increased collagen deposition leading to a pro-fibrotic and tumorigenic environment.

Neonates colonized with pathogenic bacteria in the airways have a low-grade systemic inflammation.

BACKGROUND AND OBJECTIVES: The development of childhood asthma is associated with neonatal colonization with pathogenic bacteria in hypopharynx. Furthermore, established asthma is associated with systemic low-grade inflammation. We here report on the association between neonatal colonization with pathogenic bacteria in hypopharynx and the development of systemic low-grade inflammation. METHODS: Bacterial colonization of the hypopharynx with Moraxella catharralis, Haemophilus influenzae, and/or Streptococcus pneumoniae was assessed in asymptomatic children from the Copenhagen Prospective Studies on Asthma in Childhood2000 (COPSAC2000 ) cohort at age 1 month by culturing technique (N = 238) and by quantitative polymerase chain reaction (qPCR) technique (N = 249) and in the COPSAC2010 cohort by culturing at age 1 month (N = 622) and again at age 3 months (N = 613). Systemic low-grade inflammation was determined in both cohorts at age 6 months by measuring plasma levels of high-sensitivity C-reactive protein (hs-CRP), tumor necrosis factor-α (TNF-α), and interleukin-6 (lL-6). RESULTS: In both cohorts, bacterial colonization was associated with increased levels of hs-CRP: COPSAC2000 , 1 month culturing (geometric mean ratio of colonized/noncolonized [95% CI]), 1.39 [0.97-2.01], P = .08; 1 month qPCR, 1.55 [1.14-2.10], P < .01; COPSAC2010 , 1 month, 1.52 [1.23-1.87], P < .01; and 3 month, 1.57 [1.30-1.90], P < .01. A multiparametric principal component analysis incorporating hs-CRP, TNF-α, and IL-6 confirmed a systemic inflammatory profile in children colonized with M. catharralis, H. influenzae. and/or S. pneumoniae in the hypopharynx compared to noncolonized children (P-values < .05). CONCLUSION: The composition of the upper airway microbiome in early life may cause systemic low-grade inflammation.

Objective Cognitive Impairment and Progression to Dementia in Women: The Prospective Epidemiological Risk Factor Study.

BACKGROUND: Identification of subjects with a progressive disease phenotype is an urgent need in the pharmaceutical industry where most of the recent clinical trials in Alzheimer's disease have failed. OBJECTIVES: The objective of this study was to identify subgroups of individuals with objective cognitive impairment (OCI), who were most likely to progress to dementia and to identify the risk factors associated with progression. DESIGN: Prospective cohort study. SETTING: Population-based. PARTICIPANTS: 5,380 elderly women from Denmark. MEASUREMENTS: The Short Blessed Test and a category fluency test with animal naming, was used to assess cognitive function, and to classify them into different groups of OCI. RESULTS: OCI was identified in 852 subjects at baseline. The risk of dementia was elevated for OCI subjects as compared to subjects with normal cognition (HR 1.46[1.19-1.79]). The courses of OCI were studied in a sub-cohort who completed the cognitive assessment at both the baseline and the follow-up visit (n = 1,933). Of these subjects 203 had OCI at baseline. The multi-domain subtypes of OCI were associated with progressive OCI. Subjects most likely to progress were older, physically inactive, had a higher level of total cholesterol (>6.5 mmol/L) and had a history of depression as compared to subjects with a non-progressive course of OCI. CONCLUSIONS: In this cohort we identified a risk profile associated with progression from OCI in older women. The degree of impairment at baseline was an important predictor of conversion to dementia, additionally several modifiable risk factors were associated with progression.

Cathepsin-S degraded decorin are elevated in fibrotic lung disorders - development and biological validation of a new serum biomarker.

BACKGROUND: Decorin is one of the most abundant proteoglycans of the extracellular matrix and is mainly secreted and deposited in the interstitial matrix by fibroblasts where it plays an important role in collagen turnover and tissue homeostasis. Degradation of decorin might disturb normal tissue homeostasis contributing to extracellular matrix remodeling diseases. Here, we present the development and validation of a competitive enzyme-linked immunosorbent assay (ELISA) quantifying a specific fragment of degraded decorin, which has potential as a novel non-invasive serum biomarker for fibrotic lung disorders. METHODS: A fragment of decorin cleaved in vitro using human articular cartilage was identified by mass-spectrometry (MS/MS). Monoclonal antibodies were raised against the neo-epitope of the cleaved decorin fragment and a competitive ELISA assay (DCN-CS) was developed. The assay was evaluated by determining the inter- and intra-assay precision, dilution recovery, accuracy, analyte stability and interference. Serum levels were assessed in lung cancer patients, patients with idiopathic pulmonary fibrosis (IPF), patients with chronic obstructive pulmonary disease (COPD) and healthy controls. RESULTS: The DCN-CS ELISA was technically robust and was specific for decorin cleaved by cathepsin-S. DCN-CS was elevated in lung cancer patients (p < 0.0001) and IPF patients (p < 0.001) when compared to healthy controls. The diagnostic power for differentiating lung cancer patients and IPF patients from healthy controls was 0.96 and 0.77, respectively. CONCLUSION: Cathepsin-S degraded decorin could be quantified in serum using the DCN-CS competitive ELISA. The clinical data indicated that degradation of decorin by cathepsin-S is an important part of the pathology of lung cancer and IPF.

Allergic sensitization at school age is a systemic low-grade inflammatory disorder.

BACKGROUND: Systemic low-grade inflammation has been demonstrated in a range of the frequent noncommunicable diseases (NCDs) proposing a shared mechanism, but is largely unexplored in relation to allergic sensitization. We therefore aimed to investigate the possible association with childhood allergic sensitization. METHODS: High-sensitivity C-reactive protein (hs-CRP), interleukin-1ß (IL-1ß), IL-6, tumor necrosis factor-α (TNF-α), and chemokine (C-X-C motif) ligand 8 (CXCL8) were measured in plasma at age 6 months (N = 214) and 7 years (N = 277) in children from the Copenhagen Prospective Studies on Asthma in Childhood2000 (COPSAC2000 ) birth cohort. Allergic sensitization against common inhalant and food allergens was determined longitudinally at ages ½, 1½, 4 and 6 years by specific IgE assessments and skin prick tests. Associations between inflammatory biomarkers and sensitization phenotypes were tested with logistic regression and principal component analyses (PCAs). RESULTS: Adjusted for gender, recent infections, and a CRP genetic risk score, hs-CRP at 7 years was associated with concurrent elevated specific IgE against any allergen [adjusted OR (aOR) = 1.40; 95% CI, 1.14-1.72; P = 0.001], aeroallergens (aOR, 1.43; 1.15-1.77; P = 0.001), food allergens (aOR, 1.31; 95% CI, 1.02-1.67; P = 0.04), sensitization without any clinical allergy symptoms (aOR = 1.40; 1.06-1.85; P = 0.02), and with similar findings for skin prick tests. The other inflammatory markers were not univariately associated with sensitization, but multiparametric PCA suggested a specific inflammatory response among sensitized children. Inflammatory markers at age 6 months were not associated with subsequent development of sensitization phenotypes. CONCLUSIONS: Elevated hs-CRP is associated with allergic sensitization in school-aged children suggesting systemic low-grade inflammation as a phenotypic characteristic of this early-onset NCD.

Excessive collagen turnover products are released during colorectal cancer progression and elevated in serum from metastatic colorectal cancer patients.

During cancer progression, the homeostasis of the extracellular matrix becomes imbalanced with an excessive collagen remodeling by matrix metalloproteinases. As a consequence, small protein fragments of degraded collagens are released into the circulation. We have investigated the potential of protein fragments of collagen type I, III and IV as novel biomarkers for colorectal cancer. Specific fragments of degraded type I, III and IV collagen (C1M, C3M, C4M) and type III collagen formation (Pro-C3) were assessed in serum from colorectal cancer patients, subjects with adenomas and matched healthy controls using well-characterized and validated ELISAs. Serum levels of the biomarkers were significantly elevated in colorectal cancer patients compared to subjects with adenomas (C1M, Pro-C3, C3M) and controls (C1M, Pro-C3). When patients were stratified according to their tumour stage, all four biomarkers were able to differentiate stage IV metastatic patients from all other stages. Combination of all markers with age and gender in a logistic regression model discriminated between metastatic and non-metastatic patients with an AUROC of 0.80. The data suggest that the levels of these collagen remodeling biomarkers may be a measure of tumour activity and invasiveness and may provide new clinical tools for monitoring of patients with advanced stage colorectal cancer.

High breast milk IL-1ß level is associated with reduced risk of childhood eczema.

BACKGROUND: We recently demonstrated a dual effect of breastfeeding with increased risk of eczema and decreased risk of wheezing in early childhood by increasing breastfeeding length. We hypothesize that immune mediators in breast milk could explain such association either through a direct effect or as a surrogate marker of maternal immune constitution. OBJECTIVE: To investigate the possible association between cytokine and chemokine levels in breast milk and development of eczema and recurrent wheeze during early childhood. METHODS: Levels of 19 pro-inflammatory and immunoregulatory cytokines and chemokines were measured in 223 breast milk samples from mothers in the Copenhagen Prospective Study on Asthma in Childhood2000 (COPSAC) high-risk birth cohort. Eczema and recurrent wheeze at the age of 0-3 years were prospectively diagnosed by COPSAC physicians adherent to predefined validated algorithms. Association analyses were performed by Cox regression adjusting for potential confounding factors and by multivariable principal component analysis. RESULTS: Increased IL-1ß in breast milk (≥ 0.7 pg/mL) was associated with more than a halved risk of eczema before age three (aHR = 0.41; 95% CI = 0.24-0.68; P < 0.001), which remained significant after false discovery rate adjustment (P = 0.008). The principal component analysis confirmed that a mediator pattern dominated by high levels of IL-1ß, IL-17A, and CCL17 and low levels of CXCL1 and TSLP in breast milk protected against eczema (aHR = 0.82; 95% CI = 0.68-0.98; P = 0.03). No associations were observed for recurrent wheeze. CONCLUSIONS AND CLINICAL RELEVANCE: Elevated breast milk IL-1ß level was associated with decreased risk of early childhood eczema suggesting either a direct protective effect of IL-1ß or IL-1b acting as a proxy for a healthy maternal immune system protecting high-risk offspring from eczema.

Siblings Promote a Type 1/Type 17-oriented immune response in the airways of asymptomatic neonates.

BACKGROUND: Siblings have been shown to reduce the risk of childhood asthma and allergy, but the mechanism driving this association is unknown. The objective was to study whether siblings affect the airway immune response in healthy neonates, which could represent an underlying immune modulatory pathway. METHODS: We measured 20 immune mediators related to the Type 1, Type 2, Type 17, or regulatory immune pathways in the airway mucosa of 571 one-month-old asymptomatic neonates from the Copenhagen Prospective Studies on Asthma in Childhood2010 birth cohort (COPSAC2010 ). The association between airway mediator levels and presence of siblings was investigated using conventional statistics and principle component analysis (PCA). RESULTS: Neonates with siblings had an upregulated level of airway immune mediators, with predominance of Type 1- and Type 17-related mediators. This was supported by the PCA showing a highly significant difference between children with vs without siblings: P < 10(-10) , which persisted after adjustment for potential confounders including pathogenic airway bacteria and viruses: P < 0.0001. The immune priming effect was inversely associated with time since last childbirth: P = 0.0015. CONCLUSIONS: Siblings mediate a Type 1/Type 17-related immune-stimulatory effect in the airways of asymptomatic neonates, also after adjustment for pathogenic bacteria and viruses, indicating that siblings exert a transferable early immune modulatory effect. These findings may represent an in utero immune priming effect of the fetal immune system caused by previous pregnancies as the effect was attenuated with time since last childbirth, or it could relate to the presence of unidentified microbes, but further studies are needed to confirm our findings.

Divergent response profile in activated cord blood T cells from first-born child implies birth-order-associated in utero immune programming.

BACKGROUND: First-born children are at higher risk of developing a range of immune-mediated diseases. The underlying mechanism of 'birth-order effects' on disease risk is largely unknown, but in utero programming of the child's immune system may play a role. OBJECTIVE: We studied the association between birth order and the functional response of stimulated cord blood T cells. METHOD: Purified cord blood T cells were polyclonally activated with anti-CD3-/anti-CD28-coated beads in a subgroup of 28 children enrolled in the COPSAC2010 birth cohort. Expression levels of seven activation markers on helper and cytotoxic T cells as well as the percentage of CD4(+) CD25(+) T cells were assessed by flow cytometry. Production of IFN-γ, TNF-α, IL-17, IL-4, IL-5, IL-13, and IL-10 was measured in the supernatants. RESULTS: IL-10 secretion (P = 0.007) and CD25 expression on CD4(+) helper T cells (P = 0.0003) in the activated cord blood T cells were selectively reduced in first-born children, while the percentage of circulating CD4(+) CD25(+) cord blood T cells was independent of birth order. CONCLUSION: First-born infants display a reduced anti-inflammatory profile in T cells at birth. This possible in utero 'birth-order' T-cell programming may contribute to later development of immune-mediated diseases by increasing overall immune reactivity in first-born children as compared to younger siblings.

The effect of maternal Inflammation on foetal programming of metabolic disease.

Maternal obesity during pregnancy increases the child's risk of developing obesity and obesity-related diseases later in life. Key components in foetal programming of metabolic risk remain to be identified; however, chronic low-grade inflammation associated with obesity might be responsible for metabolic imprinting in the offspring. We have therefore surveyed the literature to evaluate the role of maternal obesity-induced inflammation in foetal programming of obesity and related diseases. The literature on this topic is limited, so this review also includes animal models where maternal inflammation is mimicked by single injections with lipopolysaccharide (LPS). An LPS challenge results in an immunological response that resembles the obesity-induced immune profile, although LPS injections provoke a stronger response than the subclinical obesity-associated response. Maternal LPS or cytokine exposures result in increased adiposity and impaired metabolic homeostasis in the offspring, similar to the phenotype observed after exposure to maternal obesity. The cytokine levels might be specifically important for the metabolic imprinting, as cytokines are both transferable from maternal to foetal circulation and have the capability to modulate placental nutrient transfer. However, the immune response associated with obesity is moderate and therefore potentially weakened by the pregnancy-driven immune modulation, dominated by anti-inflammatory Treg and Th2 cells. We know from other low-grade inflammatory diseases, such as rheumatoid arthritis, that pregnancy can improve disease state. If pregnancy is also capable of suppressing the obesity-associated inflammation, the immunological markers might be less likely to affect metabolic programming in the developing foetus than otherwise implied.

Effects of pregnancy on obesity-induced inflammation in a mouse model of fetal programming.

OBJECTIVE: Maternal obesity is associated with increased risk of metabolic dysfunction in the offspring. It is not clear whether it is the metabolic changes or chronic low-grade inflammation in the obese state that causes this metabolic programming. We therefore investigated whether low-grade inflammation was present in obese dams compared with controls dams at gestation day 18 (GD18). METHODS: Female mice were fed either a standard chow diet or a highly palatable obesogenic diet for 6 weeks before conception. Mice were either kileed before mating (n=12 in each group) or on GD18 (n=8 in each group). Blood and tissues were collected for analysis. RESULTS: The obesogenic diet increased body weight and decreased insulin sensitivity before conception, while there was no difference between the groups at GD18. Local inflammation was assayed by macrophage count in adipose tissue (AT) and liver. Macrophage count in the AT was increased significantly by the obesogenic diet, and the hepatic count also showed a tendency to increased macrophage infiltration before gestation. This was further supported by a decreased population of monocytes in the blood of the obese animals, which suggested that monocytes are being recruited from the blood to the liver and AT in the obese animals. Gestation reversed macrophage infiltration, such that obese dams showed a lower AT macrophage count at the end of gestation compared with pre-pregnancy obese mice, and there were no longer a tendency toward increased hepatic macrophage count. Placental macrophage count was also similar in the two groups. CONCLUSION: At GD18, obese dams were found to have similar macrophage infiltration in placenta, AT and liver as lean dams, despite an incipient infiltration before gestation. Thus, the obesity-induced inflammation was reversed during gestation.

An elevated pro-inflammatory cytokine profile in multiple chemical sensitivity.

BACKGROUND: Multiple chemical sensitivity (MCS) is a medically unexplained condition characterized by reports of recurrent unspecific symptoms attributed to exposure to low levels of common volatile chemicals. The etiology of MCS is poorly understood, but dysregulation of the immune system has been proposed as part of the pathophysiology. OBJECTIVE: To compare plasma levels of cytokines in Danish MCS individuals with a healthy, sex- and age-matched control group. METHOD: Blood samples were obtained from 150 un-exposed MCS individuals and from 148 age- and sex-matched healthy controls. Plasma concentrations of 14 cytokines, chemokines and growth and allergen-specific IgE were measured. All participants completed a questionnaire including questions on MCS, psychological distress, morbidities and medication use at the time of the study. RESULTS: Plasma levels of interleukin-1ß, -2, -4, and -6 were significantly (P<0.001) increased in the MCS group compared with controls, tumor necrosis factor-α was borderline significantly (P=0.05) increased and interleukin-13 was significantly decreased (P<0.001). CONCLUSION: MCS individuals displayed a distinct systemic immune mediator profile with increased levels of pro-inflammatory cytokines and interleukin-2 and inverse regulation of Th2 associated cytokines interleukin-4 and interleukin-13 suggestive of low-grade systemic inflammation, along with a deviating Th2-associated cytokine response not involving IgE-mediated mechanisms.

Deep phenotyping of the unselected COPSAC2010 birth cohort study.

BACKGROUND: We hypothesize that perinatal exposures, in particular the human microbiome and maternal nutrition during pregnancy, interact with the genetic predisposition to cause an abnormal immune modulation in early life towards a trajectory to chronic inflammatory diseases such as asthma and others. OBJECTIVE: The aim of this study is to explore these interactions by conducting a longitudinal study in an unselected cohort of pregnant women and their offspring with emphasis on deep clinical phenotyping, exposure assessment, and biobanking. Exposure assessments focus on the human microbiome. Nutritional intervention during pregnancy in randomized controlled trials are included in the study to prevent disease and to be able to establish causal relationships. METHODS: Pregnant women from eastern Denmark were invited during 2008-2010 to a novel unselected 'COPSAC2010 ' cohort. The women visited the clinic during pregnancy weeks 24 and 36. Their children were followed at the clinic with deep phenotyping and collection of biological samples at nine regular visits until the age of 3 and at acute symptoms. Randomized controlled trials of high-dose vitamin D and fish oil supplements were conducted during pregnancy, and a trial of azithromycin for acute lung symptoms was conducted in the children with recurrent wheeze. RESULTS: Seven hundred and thirty-eight mothers were recruited from week 24 of gestation, and 700 of their children were included in the birth cohort. The cohort has an over-representation of atopic parents. The participant satisfaction was high and the adherence equally high with 685 children (98%) attending the 1 year clinic visit and 667 children (95%) attending the 2 year clinic visit. CONCLUSIONS: The COPSAC2010 birth cohort study provides longitudinal clinical follow-up with highly specific end-points, exposure assessments, and biobanking. The cohort has a high adherence rate promising strong data to elucidate the interaction between genomics and the exposome in perinatal life leading to lifestyle-related chronic inflammatory disorders such as asthma.

Allergen specific responses in cord and adult blood are differentially modulated in the presence of endotoxins.

BACKGROUND: Endotoxins are common contaminants in allergen preparations and affect antigen-specific cellular responses. Distinct effects of endotoxin on cells in human umbilical cord and adult blood are poorly defined. OBJECTIVES: To examine the effect of endotoxins in allergen preparations on cellular responses in human cord and peripheral blood (PB). METHODS: The endotoxin content in beta lactoglobulin (BLG), the peanut allergen Ara h 1 and the major birch pollen allergen Bet v 1 was assessed. Proliferation and cytokine response of mononuclear cells towards contaminated and lipopolysaccharide (LPS)-free allergens were evaluated at different time-points. Fractions of contaminated BLG were generated and assayed on their immuno-stimulatory capacity. The involvement of toll-like receptor (TLR) 2 and 4 was investigated by blocking antibodies and TLR-transfected human embryonic kidney cells. RESULTS: The proliferative response of cord blood (CB)-derived mononuclear cells towards allergen-preparations at day 3 was related to the level of LPS contamination. At day 7, proliferation was also detected in the absence of endotoxin. Cytokine production in CB was strongly affected by the content of endotoxin, TLR-4 dependent and not related to the allergen content. Allergen- and endotoxin-induced proliferative responses were generally significantly higher in CB than in adult blood. CONCLUSION: Endotoxins in allergen preparations confound allergen-specific cellular responses. The impact of these contaminations varies with the blood source (CB vs. PB), the type of allergen and is time- and dose-dependent.

Milk-derived GM(3) and GD(3) differentially inhibit dendritic cell maturation and effector functionalities.

Gangliosides are complex glycosphingolipids, which exert immune-modulating effects on various cell types. Ganglioside GD(3) and GM(3) are the predominant gangliosides of human breast milk but during the early phase of lactation, the content of GD(3) decreases while GM(3) increases. The biological value of gangliosides in breast milk has yet to be elucidated but when milk is ingested, dietary gangliosides might conceptually affect immune cells, such as dendritic cells (DCs). In this study, we address the in vitro effect of GD(3) and GM(3) on DC effector functionalities. Treatment of bone marrow-derived DCs with GD(3) before lipopolysaccharide-induced maturation decreased the production of interleukin-6 (IL-6), IL-10, IL-12 and tumor necrosis factor-alpha as well as reduced the alloreactivity in mixed leucocyte reaction (MLR). In contrast, only IL-10 and IL-12 productions were significantly inhibited by GM(3,) and the potency of DCs to activate CD4(+) cells in MLR was unaffected by GM(3). However, both gangliosides suppressed expression of CD40, CD80, CD86 and major histocompatibility complex class II on DCs. Because GD(3) overall inhibits DC functionalities more than GM(3), the immune modulating effect of the ganglioside fraction of breast milk might be more prominent in the commencement of lactation during which the milk contains the most GD(3).

[Influence of different local anesthetics on the in vitro collagen synthesis of periodontal fibroblasts]. / Einfluss von Lokalanästhetika auf die In-vitro-Kollagensynthese von Desmodontalfibroblasten.

The influence of different local anesthetic fluids in an in-vitro-experiment of periodontal ligament cells was researched. Mepivacain, articain, lidocain and prilocain were used in the study. The common commercial local anesthetic fluids were in a proportion 1:1, 1:10 and 1:30 diluted, before they were placed in the incubator with cultures of periodontal ligament cells. At the 1st and 14th day after the experiment the synthesis of collagen type I, III, V and VI were determined with an ELISA. The mepivacain dilution of 1:30 had, compared with the controls the slightest influence on the collagen synthesis of periodontal ligament cells. Further clinical investigations must show if through dilution of the local anesthetic solution an adequate anesthesia can be reached.