Studies of the Impact of the Bifidobacterium Species on Inducible Nitric Oxide Synthase Expression and Nitric Oxide Production in Murine Macrophages of the BMDM Cell Line.

Bifidobacterium species are one of the most important probiotic microorganisms which are present in both, infants and adults. Nowadays, growing data describing their healthy properties arise, indicating they could act at the cellular and molecular level. However, still little is known about the specific mechanisms promoting their beneficial effects. Nitric oxide (NO), produced by inducible nitric oxide synthase (iNOS), is involved in the protective mechanisms in the gastrointestinal tract, where it can be provided by epithelial cells, macrophages, or bacteria. The present study explored whether induction of iNOS-dependent NO synthesis in macrophages stems from the cellular action of Bifidobacterium species. The ability of ten Bifidobacterium strains belonging to 3 different species (Bifidobacterium longum, Bifidobacterium adolescentis, and Bifidobacterium animalis) to activate MAP kinases, NF-κB factor, and iNOS expression in a murine bone-marrow-derived macrophages cell line was determined by Western blotting. Changes in NO production were determined by the Griess reaction. It was performed that the Bifidobacterium strains were able to induce NF-қB-dependent iNOS expression and NO production; however, the efficacy depends on the strain. The highest stimulatory activity was observed for Bifidobacterium animalis subsp. animals CCDM 366, whereas the lowest was noted for strains Bifidobacterium adolescentis CCDM 371 and Bifidobacterium longum subsp. longum CCDM 372. Both TLR2 and TLR4 receptors are involved in Bifidobacterium-induced macrophage activation and NO production. We showed that the impact of Bifidobacterium on the regulation of iNOS expression is determined by MAPK kinase activity. Using pharmaceutical inhibitors of ERK 1/2 and JNK, we confirmed that Bifidobacterium strains can activate these kinases to control iNOS mRNA expression. Concluding, the induction of iNOS and NO production may be involved in the protective mechanism of action observed for Bifidobacterium in the intestine, and the efficacy is strain-dependent.

Mechanism of Molecular Activity of Yolkin-a Polypeptide Complex Derived from Hen Egg Yolk-in PC12 Cells and Immortalized Hippocampal Precursor Cells H19-7.

Food-derived bioactive peptides able to regulate neuronal function have been intensively searched and studied for their potential therapeutic application. Our previous study showed that a polypeptide complex yolkin, isolated from hen egg yolk as a fraction accompanying immunoglobulin Y (IgY), improved memory and cognitive functions in rats. However, the mechanism activated by the yolkin is not explained. The goal of the present study was to examine what molecular mechanism regulating brain-derived neurotrophic factor (BDNF) expression is activated by the yolkin complex, using in vitro models of PC12 cell line and fetal rat hippocampal cell line H19-7. It was shown that yolkin increased the proliferative activity of rat hippocampal precursor cells H19-7 cells and upregulated the expression/production of BDNF in a cyclic adenosine monophosphate (cAMP)-response element-binding protein (CREB)-dependent manner. Additionally the upregulation of carboxypeptidase E/neurotrophic factor-α1 (CPE/(NF-α1) expression was shown. It was also determined that upregulation of CREB phosphorylation by yolkin is dependent on cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) and phosphoinositide 3-kinases/protein kinase B (PI3K/Akt) signaling pathway activation. Moreover, the impact of yolkin on the level of intracellular Ca2+, nitric oxide, and activation of extracellular signal-regulated kinases 1/2 (ERK 1/2 kinase) was excluded. These results emphasize that yolkin can act comprehensively and in many directions and may participate in the regulation of neurons' survival and activity. Therefore, it seems that the yolkin specimen can be used in the future as a safe, bioavailable, natural nutraceutical helping to improve the cognition of older people.

Ceramide is implicated in humoral peripheral and intrathecal autoimmune response in MS patients.

BACKGROUND: The disturbed metabolism of ceramide (Cer) is supposed to evoke the autoimmune response, contributing to MS pathology. OBJECTIVES: To determine levels of anti-Cer immunoglobulins G (IgGs) in the CSF and serum of subjects with various phenotypes of MS, and to investigate relationships between levels of anti-Cer antibodies and MS-related variables. METHODS: IgGs isolated from serum and the CSF of 68 MS patients and appropriate controls were examined for their reactivity to Cer subspecies. Their levels were compared between the studied groups and compartments, and analyzed with regard to clinical variables. RESULTS: Increased levels of anti-C16:0-, C18:0-, C18:1-, C24:0- and C24:1-Cer IgGs were detected in the CSF and serum of MS patients in comparison with controls. For IgGs against particular Cer subspecies, correlations were found between their CSF and serum level, as well as with the Link index. Serum and the CSF anti-Cer IgGs differed between patients with clinically isolated syndrome (CIS) and relapsing-remitting MS from those with progressive MS. No correlations were found between anti-Cer IgGs and other MS-related clinical variables. CONCLUSION: Patients with MS have shown altered panels of anti-Cer IgGs in the CSF and serum, which might suggest a relevant, though limited role of Cer as a target for autoimmune humoral response. Utility of antibodies against Cer subspecies as potential markers for MS activity and progression deserves further investigations.

A Novel Mechanism of Macrophage Activation by the Natural Yolkin Polypeptide Complex from Egg Yolk.

Ageing is accompanied by the inevitable changes in the function of the immune system. It provides increased susceptibility to chronic infections that have a negative impact on the quality of life of older people. Therefore, rejuvenating the aged immunity has become an important research and therapeutic goal. Yolkin, a polypeptide complex isolated from hen egg yolks, possesses immunoregulatory and neuroprotective activity. Considering that macrophages play a key role in pathogen recognition and antigen presentation, we evaluated the impact of yolkin on the phenotype and function of mouse bone marrow-derived macrophages of the BMDM cell line. We determined yolkin bioavailability and the surface co-expression of CD80/CD86 using flow cytometry and IL-6, IL-10, TGF-ß and iNOS mRNA expression via real-time PCR. Additionally, the impact of yolkin on the regulation of cytokine expression by MAPK and PI3K/Akt kinases was determined. The stimulation of cells with yolkin induced significant changes in cell morphology and an increase in CD80/CD86 expression. Using pharmaceutical inhibitors of ERK, JNK and PI3K/Akt, we have shown that yolkin is able to activate these kinases to control cytokine mRNA expression. Our results suggest that yolkin is a good regulator of macrophage activity, priming mainly the M1 phenotype. Therefore, it is believed that yolkin possesses significant therapeutic potential and represents a promising possibility for the development of novel immunomodulatory medicine.

C-Terminal Fragment of Vitellogenin II, a Potential Yolkin Polypeptide Complex Precursor Protein-Heterologous Expression, Purification, and Immunoregulatory Activity.

The aim of this research was to analyze the heterologous expression, purification, and immunoregulatory activity of recombinant YGP40 (rYGP40), the potential precursor of the yolkin peptide complex. The ygp40 coding sequence was codon optimized, successfully expressed in the E. coli system, and purified from inclusion bodies with a yield of about 1.1 mg/L of culture. This study showed that the protein exhibits immunomodulatory activity, expressed by the stimulation of TNF-α and IL-10 production and nitric oxide induction at a level comparable to that of the natural yolkin peptide complex obtained by other authors from hen egg yolk. At the highest dose of 100 µg/mL, rYGP40 also caused the up-regulation of iNOS expression in murine bone marrow-derived macrophages (BMDM). Moreover, no cytotoxic effects of rYGP40 on the BMDM cell line were observed.

Comparative Studies of Yolkin Preparations Isolated from Egg Yolks of Selected Bird Species.

The results of our research have proven that yolkin preparations isolated from eggs of different bird species show a high similarity in polypeptide composition. Despite the small differences in protein patterns, all of yolkin preparations showed also strong immunomodulatory activity, comparable with yolkin obtained previously from hen egg yolk. It can therefore be deducted that the presence of this polypeptide complex in the egg is not accidental and performs an important biological function for developing embryo.

Isolation and Characterization of NP-POL Nonapeptide for Possible Therapeutic Use in Parkinson's Disease.

Colostrum and milk are the initial mammalian nourishment and rich reservoir of essential nutrients for newborn development. Bioactive peptides isolated from natural sources, such as colostrum, serve as endogenous regulators and can be used as alternative therapeutic agents in the treatment of neurodegenerative diseases. One example is the previously unknown NP-POL nonapeptide isolated from Colostrinin. In the present study, we investigated a method of NP-POL nonapeptide isolation using Bio-Gel P2 molecular sieve chromatography. We showed the protective effect of NP-POL on 6-hydroxydopamine- (6-OHDA-) induced neurotoxicity using rat adrenal pheochromocytoma (PC12 Tet On) cells. Treatment of PC12 cells with NP-POL nonapeptide reduced 6-OHDA-induced apoptosis and caused transient phosphorylation of extracellular signal-regulated kinases (ERK 1/2), which were shown to promote cell survival. NP-POL nonapeptide also protected neuronal cells against oxidative injury induced by 6-OHDA. These results showed a potential use of NP-POL in the therapy of Parkinson's disease.

Neurotrophic Activity of Cultured Cell Line U87 is Up-Regulated by Proline-Rich Polypeptide Complex and Its Constituent Nonapeptide.

Neurotrophins such as nerve growth factor (NGF) and brain-derived neurotrophic factor, as well as cytokines, for example, interleukin-6 (IL-6) play an important role in neuroprotection and in the control of the central nervous system (CNS) function. Reduced expression of neurotrophic factors can lead to dysregulation of neuron function and neuronal death. There is also evidence for mutual interactions between neurotrophins and IL-6. Therefore, the up-regulating the level of neuroprotective substances is one of the key manners to control the nervous system development and function. It can be a promising aim in the therapy of neurodegenerative disease in which the decreased level of neurotrophins is observed. In our recent studies, the role of proline-rich polypeptide complex (PRP) and its nonapeptide fragment (NP) in the regulation of neurotrophic activity in cultured astrocytes was shown. PRP and NP stimulate human astrocytoma cell line U87 to release the significant amounts of NGF to the extracellular space both in its precursor and mature form. We also provide the evidence that in NP-treated cells, the level of ßNGF mRNA was increased. NP-treated cells used in this study produced also increasing amounts of IL-6. This finding indicates that PRP and its nonapeptide fragment NP up-regulate neurotrophic activity of U87 cell line by increase of NGF synthesis and its release into the extracellular space. It was also shown that NP-dependent increased production of IL-6 can enhance the NGF activity.

A proline-rich polypeptide complex (PRP) influences inducible nitric oxide synthase in mice at the protein level.

A proline-rich polypeptide complex (PRP) with immunoregulatory and procognitive properties showed a beneficial effect in Alzheimer's disease (AD) when administered orally in the form of Colostrinin(R) tablets. The mechanism of action of PRP/Colostrinin in AD has not been yet clarified. It is known that oxidative stress enhances neurodegenerative processes. It was previously shown that the PRP regulates the secretion of cytokines and inhibits NO and O(2)(-) release in cell cultures. Since the results on isolated cells or cell lines frequently do not reflect events in vivo, the effect of PRP on NO release and iNOS protein synthesis in mice treated with LPS was studied. The PRP did not induce the production of NO. However, in the presence of PRP applied 6h after LPS, about 40% inhibition of NO release was observed. This effect was accompanied by lower iNOS protein expression in peritoneal cells. In the liver sections of mice treated with PRP 6h after LPS application, the number of iNOS-positive cells was significantly reduced. These results indicate that PRP can act as a regulator of inflammatory processes. The inhibition of iNOS activity/expression could be one of the mechanisms of the therapeutic effect of PRP/Colostrinin in AD.

Ovine colostrum nanopeptide affects amyloid beta aggregation.

A colostral proline-rich polypeptide complex (PRP) consisting of over 30 peptides shows beneficial effects in Alzheimer's disease (AD) patients when administered in the form of sublinqual tablets called Colostrinin. The aim of the present studies was to investigate whether nanopeptide fragment of PRP (NP) - one of the PRP complex components can affect aggregation of amyloid beta (Abeta1-42). The effect of NP on Abeta aggregation was studied using Thioflavin T (ThT) binding, atomic force microscopy, and analyzing circular dichroism spectra. Results presented suggest that NP can directly interact with amyloid beta, inhibit its aggregation and disrupt existing aggregates acting as a beta sheet breaker and reduce toxicity induced by aggregated forms of Abeta.

A proline-rich polypeptide complex (PRP) isolated from ovine colostrum. Modulation of H2O2 and cytokine induction in human leukocytes.

A proline-rich polypeptide complex (PRP) has immunoregulatory properties and also shows beneficial effects in Alzheimer's disease (AD). It is known that the unregulated activation of microglial cells in AD may result in chronic inflammatory response. There is a link between the activation of immune cells on the periphery and in the central nervous system (CNS). Therefore, we studied the effect of the PRP on human peripheral blood mononuclear cells (PBMCs) stimulated by LPS with PHA (LP) or PMA as proinflammatory activators. PRP and its nonapeptide fragment (NP) inhibited by 40-60% production of H(2)O(2) induced by PMA. The peptides also inhibited activity of superoxide dismutase. Both peptide preparations showed differential effects on the secretion of cytokines. NP induced TNF-alpha only while PRP induced IL-6, IL-10 and TNF-alpha. On the other hand, the release of TNF-alpha and IL-10 induced by LP in PBMCs was inhibited by PRP while NP inhibited the release of IFN-gamma and IL-10. The results obtained showed that PRP may affect not only adaptive immunity but also innate immunity and thus may regulate secretions of mediators of inflammation. The regulatory effect of the PRP on the innate immunity may shed some light on understanding the beneficial effects of this polypeptide complex in AD patients.

A proline-rich polypeptide complex and its nonapeptide fragment inhibit nitric oxide production induced in mice.

A proline-rich polypeptide complex (PRP) isolated from ovine colostrum shows immunoregulatory and procognitive activities. It shows beneficial effects in Alzheimer's disease (AD) patients when orally administered in the form of tablets called Colostrinin. The mechanism of action of PRP/Colostrinin in AD has not been yet clarified. It is known that oxidative stress and overproduction of NO may enhance neurodegenerative processes. PRP regulates the secretion of cytokines, inhibits NO and O2- release in cell cultures. Since the results on isolated cells or cell lines frequently do not reflect the events in vivo, the effect of PRP and its nonapeptide fragment (NP) on the level of NO2- in sera of mice untreated or intraperitoneally treated with LPS was studied. PRP and NP did not induce production of NO. However, when applicated 6 h after LPS, they inhibited the release of NO induced by LPS in about 30-50%. The results in vivo presented in this paper confirm the results obtained in cell cultures and indicate that the beneficial effects of PRP/Colostrinin observed in AD patients may be, among others, due to an inhibition of overproduction of NO.