Ewa Fiedorowicz

Impact of Maillard Reaction on Immunoreactivity and Allergenicity of the Hazelnut Allergen Cor a 11

Few studies exist on the influence of processing methods on structural changes and allergenic potential of hazelnut proteins. This study focused on the effect of glycation (Maillard reaction) on the immunoreactivity and degranulation capacity of the purified hazelnut 7S globulin, Cor a 11. After heating, the extent of the Maillard reaction, sensitivity to proteolysis, binding of human IgE or rabbit IgG, and degranulation capacity were analyzed. Changes in electrophoretic mobility, amount of free amino groups, and contents of bound sugar and fructosamine indicated that glycation of Cor a 11 occurred at all conditions. Glycation at 37 °C did not influence the specific IgG or IgE binding and was decreased after heating at 60 and 145 °C. Heating, with or without glucose, at 145 °C increased basophil degranulation capacity. The results suggest that glycation of Cor a 11 at 60 and 145 °C may decrease the IgE/IgG binding properties but not the degranulation capacity of basophils. This is possibly related to aggregation of the proteins as a result of the Maillard reaction.

The influence of μ-opioid receptor agonist and antagonist peptides on peripheral blood mononuclear cells (PBMCs).

Milk is one of the main source of biologically-active peptides that may function as regulatory substances called food hormones. After passing the gut-blood barrier, the μ-opioid receptor agonist and antagonist peptides may become the new factors influencing various functions of the human organism. The aim of the conducted research was to determine the influence of μ-opioid receptor agonist peptides: human and bovine β-casomorphin-7 (h/bBCM-7) and antagonistic peptides: casoxin-6 and- D (CXN-6/D) on proliferation and cytokine secretion of human peripheral blood mononuclear cells (PBMCs). The PBMCs proliferation was measured by the use of the BrdU test, which assesses the DNA synthesis activity and the WST-1 test which assesses the activity of mitochondrial dehydrogenase enzymes. The influence of all the investigated peptides on secretion of IL-4, IL-8, IL-13 and IFN-γ was determined by the use of the ELISA tests. Incubating the cells with the peptides has not caused any changes to their enzymatic activity, which has been proved by a WST-1 test. When using a BrdU test, however, it has been observed that there appear changes to proliferation of PBMCs correlated to amounts of bromodeoxyuridine incorporated into the cellular DNA. Moreover, changes to secretion of IL-4 and IL-13 by the cells under the influence of agonists were detected, as well as changes to secretion of IFN-gamma under the influence of all the examined substances. The obtained results provide information on immunomodulatory effects of food-derived opioid peptides, which may be of clinical significance especially in the case of allergic diseases in newborns.

Milk from cows of different β-casein genotypes as a source of β-casomorphin-7.

The aim of this study was to quantify β-casomorphin-7 in raw, hydrolyzed and processed milk in different stages of the cow lactation. The obtained results lead to the following conclusion: the highest amount of β-casomorphin-7 released from the hydrolyzed and processed milk is related to the β-casein A1 allele, irrespective of a lactation period. Some traces of β-casomorphin-7 in milk from cows with the β-casein A2 variant are probably a result of the acid hydrolysis of β-casein during its digestion with pepsin. It has been shown that processing of raw milk at high temperatures affects, in a slight degree, the differences between β-casomorphins-7 originating from different β-casein genotypes. The obtained results suggest a possibility to provide a new nutritional factor for milk quality based on the content of β-casomorphin-7 liberated in vivo from milk digested by a mixture of the gastrointestinal enzymes.

Influence of candidate polymorphisms on the dipeptidyl peptidase IV and μ-opioid receptor genes expression in aspect of the β-casomorphin-7 modulation functions in autism

Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder with population prevalence of approximately 60-70 per 10,000. Data shows that both opioid system function enhancement and opiate administration can result in autistic-like symptoms. Cow milk opioid peptides, including β-casomorphin-7 (BCM7, Tyr-Pro-Phe-Pro-Gly-Pro-Ile), affect the μ-opioid receptor (MOR) and are subjected to degradation resulting from the proline dipeptidyl peptidase IV (DPPIV, EC 3.4.14.5) enzyme activity. The presence of MOR and DPPIV activity are crucial factors determining biological activity of BCM7 in the human body. Our study examined the effect of β-casomorphin-7 on the MOR and DPPIV genes expression according to specific point mutations in these genes. In addition, we investigated frequency of A118G SNP in the MOR gene and rs7608798 of the DPPIV (A/G) gene in healthy and autistic children. Our research indicated correlation in DPPIV gene expression under the influence of BCM7 and hydrolyzed milk between healthy and ASD-affected children with genotype GG (P<0.0001). We also observed increased MOR gene expression in healthy children with genotype AG at polymorphic site A118G under influence of BCM7 and hydrolyzed milk. The G allele frequency was 0.09 in MOR gene and 0.68 in the DPPIV gene. But our results suggest no association between presence of the alleles G and A at position rs7608798 in DPPIV gene nor alleles A and G at position A118G of the MOR and increased incidence of ASD. Our studies emphasize the compulsion for genetic analysis in correlation with genetic factors affecting development and enhancement of autism symptoms.

The effect of endurance exercise on intestinal integrity in well‐trained healthy men

Exercise is one of the external factors associated with impairment of intestinal integrity, possibly leading to increased permeability and altered absorption. Here, we aimed to examine to what extent endurance exercise in the glycogen‐depleted state can affect intestinal permeability toward small molecules and protein‐derived peptides in relation to markers of intestinal function. Eleven well‐trained male volunteers (27 ± 4 years) ingested 40 g of casein protein and a lactulose/rhamnose (L/R) solution after an overnight fast in resting conditions (control) and after completing a dual – glycogen depletion and endurance – exercise protocol (first protocol execution). The entire procedure was repeated 1 week later (second protocol execution). Intestinal permeability was measured as L/R ratio in 5 h urine and 1 h plasma. Five‐hour urine excretion of betacasomorphin‐7 (BCM7), postprandial plasma amino acid levels, plasma fatty acid binding protein 2 (FABP‐2), serum pre‐haptoglobin 2 (preHP2), plasma glucagon‐like peptide 2 (GLP2), serum calprotectin, and dipeptidylpeptidase‐4 (DPP4) activity were studied as markers for excretion, intestinal functioning and recovery, inflammation, and BCM7 breakdown activity, respectively. BCM7 levels in urine were increased following the dual exercise protocol, in the first as well as the second protocol execution, whereas 1 h‐plasma L/R ratio was increased only following the first exercise protocol execution. FABP2, preHP2, and GLP2 were not changed after exercise, whereas calprotectin increased. Plasma citrulline levels following casein ingestion (iAUC) did not increase after exercise, as opposed to resting conditions. Endurance exercise in the glycogen depleted state resulted in a clear increase of BCM7 accumulation in urine, independent of DPP4 activity and intestinal permeability. Therefore, strenuous exercise could have an effect on the amount of food‐derived bioactive peptides crossing the epithelial barrier. The health consequence of increased passage needs more in depth studies.

Glucose and calcium ions may modulate the efficiency of bovine β-casomorphin-7 permeability through a monolayer of Caco-2 cells.

Milk and dairy products provide a lot of valuable nutritive elements. They are also sources of biologically active peptides, including β-casomorphins that manifest the properties of morphine. An activity of DPPIV seems to be most crucial factor decreasing the efficiency of the β-casomorphin-7 (BCM7) transport. The increase of BCM7 concentration in blood may intensify symptoms of apparent life threatening events (ALTE), autism, schizophrenia, and allergy. This study aimed at identifying the influence of several selected substances on a transport efficiency of bovine BCM7 through an intestinal monolayer in a Caco-2 cell model system. Applying the ELISA method, the permeability coefficient of BCM7 through the Caco-2 monolayer was calculated. TEER values were used to evaluate the integrity of Caco-2 cell monolayers. An increase of glucose and Ca(2+) concentrations in the culture medium was accompanied by an increase of the BCM7 transport efficiency. The lowest permeability coefficients of BCM7 were observed for the membranes with high electrical resistances. The transport was enhanced in the presence of milk infant formulas, whereas no changes were observed when using μ-opioid receptor antagonist (casoxin-6). The results may be useful in understanding the pathogenesis of inflammation and food allergy in infants.

Vitamin D Receptor Gene Polymorphisms Associated with Childhood Autism

Background: Autism spectrum disorder (ASD) is a group of heterogeneous, behaviorally defined disorders whereby currently no biological markers are common to all affected individuals. A deregulated immune response may be contributing to the etiology of ASD. The active metabolite of vitamin D3 has an immunoregulatory role mediated by binding to the vitamin D receptor (VDR) in monocyte, macrophages, and lymphocytes. The effects of vitamin D and interaction with the VDR may be influenced by polymorphism in the VDR gene. Methods: Genetic association of four different VDR polymorphisms (Apa-I, Bsm-I, Taq-I, Fok-I) associated with susceptibility to the development of autism in children was investigated. Results: We uniquely found an association between the presence of the T allele at position Taq-I and presence of the a allele at position Apa-I of the VDR gene with decreased ASD incidence. There was also an association between female gender and the presence of the T allele. We found no statistical significant correlation between VDR single nucleotide polymorphisms (SNPs) and vitamin D3 concentration in serum of ASD children. Conclusion: Genetic polymorphism in two SNP in VDR may be correlated with development of ASD symptoms by influencing functionality of vitamin D3 metabolism, while vitamin D3 levels were not significantly different between ASD and non-ASD children.

β-casomorphin-7 alters μ-opioid receptor and dipeptidyl peptidase IV genes expression in children with atopic dermatitis.

Atopic dermatitis (AD) is a chronic inflammatory skin disease with heterogeneous clinical phenotypes reflecting genetic predisposition and exposure to environmental factors. Reactions to food may play a significant role especially in young children. Milk proteins are particularly strong allergens and are additional source of bioactive peptides including β-casomorphin-7 (BCM7, Tyr-Pro-Phe-Pro-Gly-Pro-Ile). BCM7 exerts its influence on nervous, digestive, and immune functions via the μ-opioid receptor (MOR). Proline dipeptidyl peptidase IV (DPPIV; EC 3.4.14.5) appears to be the primary degrading enzyme of BCM7. Moreover, DPPIV is known to restrict activity of proinflammatory peptides. BCM7 is considered to modulate an immune response by affecting MOR and DPPIV genes expression. In this study, we determined the MOR and DPPIV genes expression in children diagnosed with a severe form of AD. 40 healthy children and 62 children diagnosed with severe AD (AD score ≥60) were included in the study. Peripheral blood mononuclear cells (PBMCs) from the studied subjects were incubated with the peptide extracts of raw and hydrolysed cow milk with defined β-casein genotypes (A1A1, A2A2 and A1A2) and MOR and DPPIV genes expression was determined with real-time PCR. Incubation PBMCs with peptide extracts from cow milk caused an increase of the MOR gene expression (p<0.05; p<0.001) in AD children with a simultaneous decrease in the DPPIV gene expression (p<0.001). The obtained results supplement the knowledge on the BCM7 participation in AD etiology and provide an important diagnostic tool.

Single Nucleotide Polymorphisms in the Vitamin D Receptor Gene (VDR) May Have an Impact on Acute Pancreatitis (AP) Development: A Prospective Study in Populations of AP Patients and Alcohol-Abuse Controls

Vitamin D imbalance is suggested to be associated with the development of pancreatitis. Single nucleotide polymorphisms (SNPs), Apa-1, Bsm-1, Fok-1, and Taq-1, in the vitamin D receptor gene (VDR) are known in various diseases, but not yet in pancreatitis. The aim of this study was to explore possible associations of the four SNPs in the VDR receptor gene in a population of acute pancreatitis patients and alcohol-abuse controls, and to investigate the association with acute pancreatitis (AP) susceptibility. The study population (n = 239) included acute pancreatitis patients (n = 129) and an alcohol-abuse control group (n = 110). All patients met the Diagnostic and Statistical Manual of Mental Disorders (DSM IV) criteria for alcohol dependence. DNA was extracted from peripheral leukocytes and analyzed for VDR polymorphisms using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Odd ratios (ORs) and 95% confidence intervals (CIs) were calculated using logistic regression analysis. To date, we have found allele T in Taq-1 (OR = 2.61; 95% CI: 1.68–4.03; p < 0.0001) to be almost three times more frequent in the AP group compared to the alcohol-abuse control patients. Polymorphism Taq-1 occurring in the vitamin D receptor may have an impact on the development of acute pancreatitis due to the lack of the protective role of vitamin D.

Cytokines concentrations in serum samples from allergic children—Multiple analysis to define biomarkers for better diagnosis of allergic inflammatory process

BACKGROUND Allergic diseases can expand at any age as a result of complicated interaction of environmental and genetic factors. Through the years, studies have found that allergic diseases are primarily described by elevated Th2 pathway activation, leading to increased serum IgE levels, allergen reactivity, blood eosinophil counts and secreted interleukins. METHODS A total of 20 patients with allergy and 20 matched controls participants were recruited for the study. A study was designed with the framework of an ongoing project at the Regional Childrens Hospital in Olsztyn on the analysis of the immune profile of children with allergy and asthma. Diagnosis was conducted by medical specialists. Whole blood samples were collected and serum ILs and chemokin levels were made using ELISA kits. RESULTS Results demonstrated that in comparison to the controls, the individuals with allergy showed significantly higher concentration of IL-1β, IL-4, IL-6, IL-8, IL-10, IL-13 and TNF-α. We also demonstrated significant correlations between the levels of cytokines which implies the presence of an interactive network between them. The results of ROC analysis indicated the 3-factors (IL-1β, IL-4, IL-8) could be additional, helpful biomarkers in better diagnosis of allergy. CONCLUSIONS In this study, serum levels of cytokine differed among children with allergy. However, the findings of this support the possibility of using an appropriate selection of serum cytokine for the diagnosis allergy and emphasize the need to standardize quantitative methods for serum analysis.