Effect of oral administration of pig spinal cord hydrolysate on clinical and histopathological symptoms of experimental allergic encephalomyelitis in rats.

Oral tolerance is the natural occurring phenomenon of a decreased immune response to previously fed antigens, which prevents induction of a response to dietary antigens. One of the mechanisms is deletion of T lymphocytes reactive to the fed antigen. Knowing that phenomenon, it seems appropriate to engage this mechanism for treatment of autoimmune diseases. Multiple sclerosis (MS) is an autoimmunological disease which causes neurological impairment in humans. Autoreactive T lymphocytes migrate through the open blood-brain barrier into the central nervous system (CNS), where they recognize myelin antigens as foreign, and induce an inflammatory response against the myelin sheath, which causes demyelination and even axonal loss. Experimental allergic encephalomyelitis (EAE), an animal model of MS, resembles the autoimmunological aspect of the disease. We used a broad spectrum of myelin antigens to induce EAE, and also to induce oral tolerance by giving myelin epitopes intragastrically to rats. The aim of our study was to evaluate whether pig spinal cord hydrolysate given intragastrically is able to evoke oral tolerance in rats with an animal model of MS - EAE. In our experiments we fed female Lewis rats with pig spinal cord hydrolysate at doses of 5, 20 and 100 mg per kg of body weight. We observed diminished clinical symptoms of ongoing EAE in rats fed with all doses of pig spinal cord hydrolysate. In the histopathological study, intensity of the inflammatory process in spinal cord was similar in rats not fed with EAE and in rats fed with lower doses of pig spinal cord hydrolysate. In animals fed with the highest dose of pig spinal cord hydrolysate, intensification of the inflammatory response was observed. These results were confirmed by morphometric evaluations. We found that feeding animals with preparations containing myelin antigens can reduce EAE symptoms, which may indicate oral tolerance induction, but the obtained results also underline the importance of dose of the orally given antigens, because of the possibility of enhancement of the inflammatory process in the CNS.

Oral Administration of Lactococcus lactis Expressing Synthetic Genes of Myelin Antigens in Decreasing Experimental Autoimmune Encephalomyelitis in Rats.

BACKGROUND: Multiple sclerosis is a human autoimmunological disease that causes neurodegeneration. One of the potential ways to stop its development is induction of oral tolerance, whose effect lies in decreasing immune response to the fed antigen. It was shown in animal models that administration of specific epitopes of the three main myelin proteins - myelin oligodendrocyte glycoprotein (MOG), myelin basic protein (MBP), and proteolipid protein (PLP) - results in induction of oral tolerance and suppression of disease symptoms. Use of bacterial cells to produce and deliver antigens to gut mucosa seems to be an attractive method for oral tolerance induction in treatment of diseases with autoimmune background. MATERIAL AND METHODS: Synthetic genes of MOG35-55, MBP85-97, and PLP139-151 myelin epitopes were generated and cloned in Lactococcus lactis under a CcpA-regulated promoter. The tolerogenic effect of bacterial preparations was tested on experimental autoimmune encephalomyelitis, which is the animal model of MS. EAE was induced in rats by intradermal injection of guinea pig spinal cord homogenate into hind paws. RESULTS: Rats were administered preparations containing whole-cell lysates of L. lactis producing myelin antigens using different feeding schemes. Our study demonstrates that 20-fold, but not 4-fold, intragastric administration of autoantigen-expressing L. lactis cells under specific conditions reduces the clinical symptoms of EAE in rats. CONCLUSIONS: The present study evaluated the use of myelin antigens produced in L. lactis in inhibiting the onset of experimental autoimmune encephalomyelitis in rats. Obtained results indicate that application of such recombinant cells can be an attractive method of oral tolerance induction.

Spinal cord hydrolysate ameliorate immunological reaction in experimental allergic encephalomyelitis.

The aim of this study was to use the hydrolysate of pig spinal cord proteins to induce oral tolerance in the animal model of sclerosis multiplex - experimental allergic encephalomyelitis. The female Lewis rats were fed with hydrolysate of pig spinal cord proteins in two doses for one week before immunization, which was induced by injection of guinea pig spinal cord homogenate. At the peak of clinical symptoms (the 13th day post immunization) the rats were sacrificed and the spleen removed. Splenocytes were suspended in a culture medium and placed in microculture plates. The cells were stimulated with homogenate. The cells were cultured for seven days. Proliferation of splenocytes was estimated by means of methyl-3H thymidine incorporation. In supernatants of cultures of splenocytes the level of cytokines INF-gamma, IL-10, IL-4, and TGF-gamma was measured. It was demonstrated that homogenate-induced splenocytes of hydrolysate-fed rats gave rise to low proliferation as compared to the controls used. The IFN-gamma was inhibited in hydrolysate-fed animals. The hydrolysate of pig spinal cord proteins has a modulatory effect on the immune reaction, particularly on the orally-induced antigen-specific modulation of autoimmune response.

Alterations in glutamate transport and group I metabotropic glutamate receptors in the rat brain during acute phase of experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is an animal model that mimics many aspects of multiple sclerosis (MS). In MS the immune system attacks the white matter of the brain and spinal cord, leading to disability and paralysis. Neurons, oligodendrocytes and myelin are lost due to the release of cytotoxic cytokines, autoantibodies and toxic amounts of the excitatory neurotransmitter glutamate. This study was designed to determine the changes in: a) glutamate transport in nerve endings and astroglial fraction, b) level of excitatory amino acid transporters (EAATs) and c) level of group I metabotropic glutamate receptors (mGluR G I) protein in the acute phase of EAE (12 d.p.i. - day post immunization), in the peak of neurological deficits. We have found that glutamate uptake in synaptosomes and GPV fraction increases by about 30% and 15%, respectively, compared to controls. We also observed an increase in KCl-dependent glutamate release from synaptosomes and GPV fraction obtained from EAE rats by about 20%. Western blots analysis of protein expression shows elevation of group I metabotropic glutamate receptors (mGluR G I) and excitatory amino acid transporters (EAATs) in EAE rats during the acute phase of the disease (12 d.p.i), when the level of proinflammatory cytokines (IL-1beta, IL-6, TNF-alpha) rises. The results suggest that during the inflammatory conditions in the acute phase of EAE, disturbances in glutamate transport take place that may lead to the excitotoxicity.

Matrix metalloproteases activity and ultrastructural changes in the early phase of experimental allergic encephalomyelitis. The effect of oral treatment with spinal cord hydrolysate [correction of hydrolisate] proteins in Lewis rat. The pilot study.

Matrix metalloproteinases (MMPs) are a family of genes of the neutral proteinases that are important to normal development and to a variety of pathological processes including neuroinflammation. In the central nervous system (CNS), MMPs degrade components of the basal lamina, leading to disruption of the blood-brain barrier (BBB), and contribute to the neuroinflammatory responses. Their concentration in experimental allergic encephalomyelitis (EAE) increases a few folds and is accompanied by a thinner basal membrane in the early phase of EAE. After induction of oral tolerance by pretreatment with spinal cord hydrolisate proteins, the concentration of MMPs decreased by 30%. Other ultrastructural changes were observed in the early phase of EAE, i.e., karioskeletal damage with vesicular structures in karioplasm, compartmentalisation of the endoplasmic reticulum in perikarium, large cisterns of the Golgi apparatus, increased activity of microglial cells with numbers of phagolisosomes, disorganisation of sheets of myelin, neoangiogenesis in parenchyma of the cerebral cortex. After oral pretreatment with spinal cord hydrolisate proteins, no changes in karioskeletal proteins were found. Still Golgi apparatus spheres were large. Many pores in the nuclear membranes were observed, which is probably a sign of increased genetic information transport. We also observed some collagen fibrils as a sign of reparative processes. These results show the diminishing of inflammation in the early phase of EAE after oral induction of immunological tolerance and some possibilities of clinical implication in multiple sclerosis treatment.

Influence of spinal cord protein hydrolysate upon the blood brain barrier changes due to experimental allergic encephalomyelitis in Lewis rats. Ultrastructural study.

A specific protein (antigen) given orally is a known method of introducing tolerance of immunological response to this antigen. This method has recently been reviewed by some authors as a possible tool in the treatment of autoaggressive diseases, such as multiple sclerosis. The experimental allergic encephalomyelitis (EAE) respected animal model for MS was used for the study. The aim of the study was the evaluation of the effect of pig spinal cord protein hydrolysate given orally upon the ultrastructural changes in the blood-brain barrier image in EAE. Changes of EAE are as follows: opened channels from basal membrane (tight junction) on the border with astrocytes, fragments of organelles of the cells, oedema of astrocytes, presence of vesicles with fluid, presence of macrophages with phagolysosomes. After pre-treatment with spinal cord hydrolysate up to 6 weeks all the above changes were normalised. These findings are promising as a possible tool in the clinical treatment of sclerosis multiplex.