Anna Cieślińska

Polymorphism of bovine beta-casein and its potential effect on human health.

Proteins in bovine milk are a common source of bioactive peptides. The peptides are released by the digestion of caseins and whey proteins. In vitro the bioactive peptide beta-casomorphin 7 (BCM-7) is yielded by the successive gastrointestinal proteolytic digestion of bovine beta-casein variants A1 and B, but this was not seen in variant A2. In hydrolysed milk with variant A1 of beta-casein, BCM-7 level is 4-fold higher than in A2 milk. Variants A1 and A2 of beta-casein are common among many dairy cattle breeds. A1 is the most frequent in Holstein-Friesian (0.310-0.660), Ayrshire (0.432-0.720) and Red (0.710) cattle. In contrast, a high frequency of A2 is observed in Guernsey (0.880-0.970) and Jersey (0.490-0.721) cattle. BCM-7 may play a role in the aetiology of human diseases. Epidemiological evidence from New Zealand claims that consumption of beta-casein A1 is associated with higher national mortality rates from ischaemic heart disease. It seems that the populations that consume milk containing high levels of beta-casein A2 have a lower incidence of cardiovascular disease and type 1 diabetes. BCM-7 has also been suggested as a possible cause of sudden infant death syndrome. In addition, neurological disorders, such as autism and schizophrenia, seem to be associated with milk consumption and a higher level of BCM-7. Therefore, careful attention should be paid to that protein polymorphism, and deeper research is needed to verify the range and natureof its interactions with the human gastrointestinal tract and whole organism.

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.

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.

Impact of fexofenadine, osthole and histamine on peripheral blood mononuclear cell proliferation and cytokine secretion

This paper compares results of peripheral blood mononuclear cell (PBMC) incubation with fexofenadine (FXF) and osthole. FXF is a third-generation antihistamine drug and osthole is assumed a natural antihistamine alternative. To our best knowledge, this is the first comparative study on FXF, osthole and histamine cytokine secretion and cytotoxicity in PBMC in vitro cultures using cell proliferation ELISA BrdU. The cultures were treated 12, 42, 48 and 72h with FXF and osthole at 150, 300 and 450ng/ml concentrations and histamine at 50, 100 and 200ng/ml. Our study results confirm that FXF, osthole and histamine exert no cytotoxic effect on PBMCs and that IL-6, IL-10 and TNF-α cytokine secretion following osthole cell stimulation was similar to that by FXF stimulation.This confirms our hypothesis that osthole is a natural histamine antagonist, and can therefore be beneficially applied in antihistamine treatment.

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.

Cytokine production by PBMC and serum from allergic and non-allergic subjects following in vitro histamine stimulation to test fexofenadine and osthole anti-allergic properties

FXF is a third-generation antihistamine drug and osthole is assumed a natural antihistamine alternative. This paper compares peripheral blood mononuclear cell (PBMC) incubation with FXF and osthole, by studying FXF, osthole and histamine cytokine secretion in PBMC in vitro cultures. Mabtech kits determined the interleukins IL-1β, IL-4, IL-10, IL-13 and TNF-α. The influence of the above active substances on cytokine secretion in PBMCs and serum was assessed: cytokines were IL-1β, IL-4, IL-10, IL-13 and TNF-α; and cytokine levels secreted by untreated PBMCs in pure culture medium formed the absolute control (ctrl). We determined that osthole affects PBMC cytokine secretion to almost precisely the same extent as FXF (IL-1β, IL-4, IL-10 and TNF). In addition osthole had greater IL-13 blocking ability than FXF. Moreover, we observed significantly decreased IL-4 level in histamine/osthole theatment compared to histamine alone. Meanwhile, FXF not significantly decrease the level of IL-4 increased by histamine. This data indicates ostholes strong role in allergic inflamation. All results confirm our hypothesis that osthole is a natural histamine antagonist and therefore can be beneficially used in antihistamine treatment of conditions such as allergies.

Changes in gene expression induced by histamine, fexofenadine and osthole: Expression of histamine H1 receptor, COX-2, NF-κB, CCR1, chemokine CCL5/RANTES and interleukin-1β in PBMC allergic and non-allergic patients

INTRODUCTION Fexofenadine (FXF) is a third-generation antihistamine drug and osthole is assumed as a natural antihistamine alternative. This paper compares results of histamine, FXF and osthole impact on HRH-1, COX-2, NF-κB-p50, CCR1 mRNA expression. We also measured mRNA expression of IL-1β and CCL5/RANTES in incubated peripheral blood mononuclear cells (PBMC) to compared how histamine, FXF and osthole had influence on expression level and interacts on product secretion. OBJECTIVE The purpose was to investigate expression pattern in asthma PBMC. METHODS The cultures were treated 72h with FXF and osthole. We measured mRNA expression of histamine HRH-1, COX-2, NF-κB-p50, CCR1, IL-1β and CCL5/RANTES with Real-Time PCR (RT-PCR). RESULTS The present study suggest that osthole may be a potential inhibitor of histamine H1 receptor activity. We also demonstrated that cells cultured with histamine increase COX-2 mRNA expression and osthole reduce it. CONCLUSION Allergy remains one of the most common chronic diseases in Europe and it is rapidly approaching epidemic proportions; with current predictions estimating that the number of allergy-afflicted will equal the healthy population by 2020. It is therefore paramount to find new pharmaceuticals which successfully combat allergic disease.

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.