Does a Little Difference Make a Big Difference? Bovine ß-Casein A1 and A2 Variants and Human Health-An Update.

For over 20 years, bovine beta-casein has been a subject of increasing scientific interest because its genetic A1 variant during gastrointestinal digestion releases opioid-like peptide ß-casomorphin-7 (ß-CM-7). Since ß-CM-7 is involved in the dysregulation of many physiological processes, there is a growing discussion of whether the consumption of the ß-casein A1 variant has an influence on human health. In the last decade, the number of papers dealing with this problem has substantially increased. The newest clinical studies on humans showed a negative effect of variant A1 on serum glutathione level, digestive well-being, cognitive performance score in children, and mood score in women. Scientific reports in this field can affect the policies of dairy cattle breeders and the milk industry, leading to the elimination of allele A1 in dairy cattle populations and promoting milk products based on milk from cows with the A2A2 genotype. More scientific proof, especially in well-designed clinical studies, is necessary to determine whether a little difference in the ß-casein amino acid sequence negatively affects the health of milk consumers.

Stability of interleukin-1ß, -4, -6, -8, -10, -13, interferon-γ and tumor necrosis factor-α in human sera after repetitive freeze-thaw cycles and long storage.

INTRODUCTION: Cytokines (CKs) are one of the key components of the molecular network modulating multiple immunological interactions. Within such biological systems, CKs functions are associated with several processes, thus quantification of these analytes in serum samples, as well as a faithful determination of its concentration, are crucial for the translational aspect of many studies. AIM: This study is focused on the evaluation of the effects of storage duration and multiple freeze-thaw cycles on CKs stability. MATERIALS AND METHODS: Serum samples were obtained from 24 healthy participants. Samples were prospectively stored at 4 °C for 1-7 and 30 days, and also underwent multiple freeze-thaw cycles. Afterwards, CK levels were determined by enzyme-linked immunosorbent assay. RESULTS: Among the 8 examined CKs all of them showed significant degradation (determined with the two-way ANOVA and post-hoc test) after 4 days of sample storage at 4 °C. Serum were affected by freezing at -20 °C and thawing, and 2 of CKs (IL-1ß and IL-8) showed significant concentration decrease after following 2 freeze-thaw cycles. It has been also determineded that CKs in serum samples after multiple freeze-thaw cycles had better stability, when samples were stored at -80 °C (compared to storage at -20 °C). CONCLUSIONS: This study demonstrates that long storage at 4 °C, as well as multiple freeze-thaw cycles of serum samples, must be avoided and CK concentrations should be measured immediately after sample collection.

Vitamin D Receptor (VDR) Gene Polymorphism in Patients Diagnosed with Colorectal Cancer.

Colorectal cancer (CRC) is one of the most commonly occurring neoplasias in humans. The prevalence of CRC rates is still rising. Although the exact background of the disease still remains unknown, it is believed that CRC may not only be a result of environmental factors, but also genetic ones. One of the mechanisms underlying CRC might be the vitamin D pathway, as CRC is the most closely linked neoplasia to vitamin D deficiency. This study shows a possible association of the vitamin D receptor (VDR) polymorphisms FokI, BsmI, ApaI, and TaqI with CRC susceptibility. A total of 103 patients diagnosed with CRC (61 men and 42 women, aged 57-82 years) and 109 healthy people (50 men and 59 women, aged 47-68 years) were genotyped using PCR-RFLP for FokI, BsmI, ApaI, and TaqI. None of the single nucleotide polymorphisms (SNPs) individually increased or decreased the risk of CRC. The evaluation of haplotypes revealed two that might enhance the likelihood of CRC development: taB (OR = 30.22; 95% CI 2.81-325.31; p = 0.01) and tAb (OR = 3.84; 95% CI 1.29-11.38; p = 0.01). In conclusion, genotyping is an easy and robust procedure that needs to be performed only once in a lifetime. A creation of a relevant SNP's panel might contribute to the identification of the groups that are at the greatest risk of CRC.

Vitamin D Binding Protein (VDBP) and Its Gene Polymorphisms-The Risk of Malignant Tumors and Other Diseases.

Vitamin D is an important component of the endocrine system that controls calcium homeostasis and bone mineralization. Because of the very short half-life of free serum vitamin D it is stabilized and transported to target tissues by being bound to the vitamin D binding protein (VDBP). The most common polymorphisms: rs4588 and rs7041 in the vitamin D binding protein gene may correlate with differences in vitamin D status in the serum. This review presents data that relate to the presence of genetic variants in the VDBP gene in correlation with certain diseases, mostly concerning cancers (breast, prostate, pancreatic, lung, colorectal, basal cell carcinoma cancer and cutaneous melanoma) or other related diseases (thyroid autoimmunity disorders, obesity, diabetes mellitus, bone metabolism, rheumatoid arthritis, ankylosing spondylitis, asthma, chronic obstructive pulmonary disease, tuberculosis and coronary artery diseases).

CYP27B1 Gene Polymorphism rs10877012 in Patients Diagnosed with Colorectal Cancer.

Colorectal cancer (CRC) is the third most commonly occurring cancer worldwide. Intestinal cells are CYP27B1 gene expression sites and, as a consequence, they are capable of converting pro-vitamin D into the active paracrine and autocrine forms. It was demonstrated that rs10877012 polymorphism in the CYP27B1 gene influenced the circulating vitamin D level. This provided a rationale for determining the role that this polymorphism plays in the risk of developing colon cancer. In this study, we investigated the association of rs10877012 (T/G) polymorphism in the CYP27B1 gene with CRC susceptibility. The study population (n = 325) included CRC patients (n = 106) and healthy controls (n = 219). DNA was extracted from peripheral leukocytes and analyzed for the CYP27B1 polymorphism using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. We found an association between the presence of the T allele at the polymorphic site (odds ratio (OR) = 2.94; 95% CI 1.77-4.86; p < 0.0001) and a decreased CRC incidence.

Single Nucleotide Polymorphisms in 25-Hydroxyvitamin D3 1-Alpha-Hydroxylase (CYP27B1) Gene: The Risk of Malignant Tumors and Other Chronic Diseases.

: Vitamin D is widely known for its roles in the promotion of apoptosis and differentiation, with simultaneous inhibition of proliferation, inflammation, angiogenesis, invasion, and metastasis. Modern literature lacks complete information on polymorphisms in CYP27B1, the only enzyme capable of vitamin D activation. This review presents gathered data that relate to genetic variants in CYP27B1 gene in correlation to multiple diseases, mostly concerning colorectal, prostate, breast, lung, and pancreatic cancers, as well as on other pathologies, such as non-Hodgkin's lymphoma, oral lichen planus, or multiple sclerosis.

Modulatory Effects of Osthole on Lipopolysaccharides-Induced Inflammation in Caco-2 Cell Monolayer and Co-Cultures with THP-1 and THP-1-Derived Macrophages.

Lipopolysaccharydes (LPS) are responsible for the intestinal inflammatory reaction, as they may disrupt tight junctions and induce cytokines (CKs) secretion. Osthole has a wide spectrum of pharmacological effects, thus its anti-inflammatory potential in the LPS-treated Caco-2 cell line as well as in Caco-2/THP-1 and Caco-2/macrophages co-cultures was investigated. In brief, Caco-2 cells and co-cultures were incubated with LPS to induce an inflammatory reaction, after which osthole (150-450 ng/mL) was applied to reduce this effect. After 24 h, the level of secreted CKs and changes in gene expression were examined. LPS significantly increased the levels of IL-1ß, -6, -8, and TNF-α, while osthole reduced this effect in a concentration-dependent manner, with the most significant decrease when a 450 ng/mL dose was applied (p < 0.0001). A similar trend was observed in changes in gene expression, with the significant osthole efficiency at a concentration of 450 ng/µL for IL1R1 and COX-2 (p < 0.01) and 300 ng/µL for NF-κB (p < 0.001). Osthole increased Caco-2 monolayer permeability, thus if it would ever be considered as a potential drug for minimizing intestinal inflammatory symptoms, its safety should be confirmed in extended in vitro and in vivo studies.

Role of Milk-Derived Opioid Peptides and Proline Dipeptidyl Peptidase-4 in Autism Spectrum Disorders.

Opioid peptides released during digestion of dietary proteins such as casein, were suggested to contribute to autism development, leading to the announcement of opioid excess hypothesis of autism. This paper examines role of enzyme proline dipeptidyl peptidase-4 (DPPIV; EC 3.4.14.5) and it is exogenous substrate, ß-casomorphin-7 (BCM7) in autism etiology. Our study included measurements of DPPIV and BCM7 concentrations in serum and urine, which were analyzed with ELISA assays and activity of DPPIV was measured by colorimetric test. The effect of opioid peptides from hydrolysed bovine milk on DPPIV gene expression in peripheral blood mononuclear cells (PBMC) in autistic and healthy children was determined using the Real-Time PCR (Polymerase Chain Reaction) method. Our research included 51 healthy children and 86 children diagnosed with autism spectrum disorder (ASD, ICDF84). We determined that the concentration of BCM7 in serum was significantly, 1.6-fold, higher in the ASD group than in controls (p < 0.0001). Concentration of DPPIV was found to also be significantly higher in serum from ASD children compared to the control group (p < 0.01), while we did not notice significant difference in enzymatic activity of serum DPPIV between the two study groups. We confirmed correlation according to the gender between analyzed parameters. The inspiration for this study emanated from clinical experience of the daily diet role in relieving the symptoms of autism. Despite this, we have concluded that milk-derived opioid peptides and DPPIV are potentially factors in determining the pathogenesis of autism; conducted studies are still limited and require further research.

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.

µ-Opioid receptor gene (OPRM1) polymorphism in patients with breast cancer.

Structure-dependent µ-opioid receptor (MOR) activity is an important element in cancer opioid analgesic effectiveness. It is widely accepted that guanine (G) substitution for adenine (A) at OPRM1 gene sequence position 118 changes receptor glycosylation pattern. This is associated with decreased binding ability in both exogenous and endogenous opioids, resulting in increased human pain resistance. The endogenous opioid system's function in body homeostasis maintenance is considered mainly regulatory, so its participation in breast tumor formation and progression is identified herein. We examine the association of the most frequent MOR (A118G) gene polymorphism on breast cancer risk in a Northeastern Polish population by PCR-RFLP comparison of A and G allele frequency at OPRM1 gene A118G polymorphic site in breast cancer-diagnosed patients with healthy control group frequencies. Our results highlight a strong association between G allele presence at µ-opioid receptor A118G and increased breast cancer incidence (OR = 3.3, 95 % CI 2.2-5.0, p < 0.0001) and female gender (OR = 2.0, 95 % CI 1.4-2.9, p = 0.0004). Consequently, OPRM1 G allele presence at that site is a highly significant risk factor in breast cancer development.

Influence of candidate polymorphisms on the dipeptidyl peptidase IV and µ-opioid receptor genes expression in aspect of the ß-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 ß-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 ß-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.

ß-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 ß-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 ß-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.