Molecular Antioxidant Properties and In Vitro Cell Toxicity of the p-Aminobenzoic Acid (PABA) Functionalized Peptide Dendrimers.

BACKGROUND: Exposure to ozone level and ultraviolet (UV) radiation is one of the major concerns in the context of public health. Numerous studies confirmed that abundant free radicals initiate undesired processes, e.g. carcinogenesis, cells degeneration, etc. Therefore, the design of redox-active molecules with novel structures, containing radical quenchers molecules with novel structures, and understanding their chemistry and biology, might be one of the prospective solutions. Methods: We designed a group of peptide dendrimers carrying multiple copies of p-aminobenzoic acid (PABA) and evaluated their molecular antioxidant properties in 1,1'-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) tests. Cytotoxicity against human melanoma and fibroblast cells as well as against primary cerebral granule cells (CGC) alone and challenged by neurotoxic sodium glutamate and production of reactive oxygen species (ROS) in presence of dendrimers were measured. Results: PABA-terminated dendrimers express enhanced radical and radical cation scavenging properties in relation to PABA alone. In cellular tests, the dendrimers at 100 M fully suppress and between 20⁻100 M reduce proliferation of the human melanoma cell line. In concentration 20 M dendrimers generate small amount of the reactive oxygen species (<25%) but even in their presence human fibroblast and mouse cerebellar granule cells remain intact Moreover, dendrimers at 0.2⁻20 µM concentration (except one) increased the percentage of viable fibroblasts and CGC cells treated with 100 M glutamate. Conclusions: Designed PABA-functionalized peptide dendrimers might be a potential source of new antioxidants with cationic and neutral radicals scavenging potency and/or new compounds with marked selectivity against human melanoma cell or glutamate-stressed CGC neurons. The scavenging level of dendrimers depends strongly on the chemical structure of dendrimer and the presence of other groups that may be prompted into radical form. The present studies found different biological properties for dendrimers constructed from the same chemical fragments but the differing structure of the dendrimer tree provides once again evidence that the structure of dendrimer can have a significant impact on drug⁻target interactions.

Bioinspired Amphiphilic Peptide Dendrimers as Specific and Effective Compounds against Drug Resistant Clinical Isolates of E. coli.

Evolution-derived natural compounds have been inspirational for design of numerous pharmaceuticals, e.g., penicillins and tetracyclines. Herein, we present a bioinspired strategy to design peptide dendrimers for the effective therapy of E. coli infections where the selection of appropriate amino acids and the mode of their assembly are based on the information gained from research on membranolytic natural antimicrobial peptides (AMP's). On the molecular level two opposite effects were explored: the effect of multiple positive charges necessary for membrane disintegration was equilibrated by the anchoring role of tryptophanes. Indeed, a series of Trp-terminated dendrimers exhibited high potency against clinical isolates of antibiotic resistant ESBL E. coli strains, stability in human plasma along with very low hemo- and genotoxicity. Investigation of the underlying antimicrobial mechanism indicated that the dendrimers studied at minimal inhibitory concentration showed weak permeability toward membranes. Solid-state 2D NMR studies revealed their presence on and inside the model membranes. Therefore, their biological properties might be explained by targeting of extra- or intracellular receptors. Our results point to a new approach to design novel branched antimicrobials with high therapeutic index.

Cardiovascular and respiratory activity of PK20, opioid and neurotensin hybrid peptide in anesthetized and awake rats.

AIM: Chimeric compound - PK20 despite its therapeutic activity on nociceptive and inflammatory processes may affect respiration and blood pressure. Our objective was to evaluate influence of the hybrid composed of endomorphin-2 and neurotensin fragments on ventilation, heart rate and blood pressure in anesthetized and awake rats. METHODS: The effects of PK20 (1mg/kg) were studied either after its intravenous administration in anesthetized rats or intraperitoneal injection in awake state. Tidal volume and the timing components of the breathing pattern, arterial blood pressure, and heart rate were recorded. RESULTS: Intravenous administration of PK20 in the neurally intact rats evoked a dose-dependent apnoea followed by a transient insignificant increase in tidal volume and breathing rate. The blood pressure changes were biphasic: transient increase was replaced by prolonged hypotension. Midcervical vagotomy abrogated all post-PK20 respiratory effects. Hypotension was eliminated after blockade of neurotensin NTS1 receptor, while respiratory changes were reduced by blockade of both: NTS1 and µ opioid receptors. After PK20 intraperitoneal injection awake rats did not show any significant changes in ventilation and blood pressure. CONCLUSION: This chimeric peptide should be used with care via intravenous administration in anesthetized animals since PK20 may evoke respiratory apnoea and hypotension. Nevertheless, applied intraperitoneally in the same dose in conscious rats induced no adverse effects.

Hydrazone Linker as a Useful Tool for Preparing Chimeric Peptide/Nonpeptide Bifunctional Compounds.

The area of multitarget compounds, joining two pharmacophores within one molecule, is a vivid field of research in medicinal chemistry. Not only pharmacophoric elements are essential for the design and activity of such compounds, but the type and length of linkers used to connect them are also crucial. In the present contribution, we describe compound 1 in which a typical opioid peptide sequence is combined with a fragment characteristic for neurokinin-1 receptor (NK1R) antagonists through a hydrazone bridge. The compound has a high affinity for µ- and δ-opioid receptors (IC50= 12.7 and 74.0 nM, respectively) and a weak affinity for the NK1R. Molecular modeling and structural considerations explain the observed activities. In in vivo test, intrathecal and intravenous administrations of 1 exhibited a strong analgesic effect, which indicates potential BBB penetration. This letter brings an exemplary application of the hydrazone linker for fast, facile, and successful preparation of chimeric compounds.

The role of allogenic keratin-derived dressing in wound healing in a mouse model.

Keratin is an interesting protein needed for wound healing and tissue recovery. We have recently proposed a new, simple and inexpensive method to obtain fur and hair keratin-derived biomaterials suitable for medical application. The aim of the study was to evaluate the role of the fur keratin-derived protein (FKDP) dressing in the allogenic full-thickness surgical skin wound model. The data obtained using scanning electron microscopy showed that employed processed biomaterial had higher surface porosity compared with control raw material. From the MTS test, it was found keratin biomaterial is not only toxic to the NIH/3T3 cell line (p < 0.05), but also enhances cell proliferation compared with the control. In vivo studies have shown keratin dressings are tissue biocompatible, accelerate wound closure and epithelialization to the statistically significant differences on day 5 (p < 0.05) in comparison to control wounds. Histological examination revealed, that in FKDP-treated wounds the inflammatory response contained predominantly macrophages whilst their morphological untreated variants showed mixed cell infiltrates rich in neutrophils. Predominant macrophages based response creates more favorable environment for healing. In FKDP-dressed wounds the number of microhemorrhages was also significantly decreased (p < 0.05) as compared with undressed wounds. Applied keratin dressing favors reconstruction of a more regular skin structure and assures better cosmetic effect in terms of scar formation and appearance. In conclusion, fur keratin-derived protein dressings significantly accelerated wound healing in the mouse model. Further studies are needed to determine the molecular mechanisms involved in the multilayer wound healing process and to assess the possible use of these dressings for medical purposes.

Neurotensin-based hybrid peptide's anti-inflammatory activity in murine model of a contact sensitivity response.

The objective of the study was to investigate the possibility of modulation of skin inflammation by topical treatment with a novel compound: an opioid-neurotensin hybrid peptide PK20 encompassing endomorphin-2 analog and modified fragment of neurotensin (8-13). Contact sensitivity response was induced in mice by skin sensitization with dinitrofluorobenzene (DNFB) followed by topical hapten application on ears. Mice were treated locally with PK20 or pure cream 2h after the challenge with DNFB. 2 and 24h after hapten exposure, ear thickness was determined. Ears were collected for histology and homogenization. Supernatants were used for measurement of contents of cytokines and lipid peroxidation products. Treatment with PK20 reduced significantly the late phase of contact sensitivity response, which was revealed by ear thickness diminution and reduction of inflammatory cell infiltration. The average concentrations of IL-1α, MCP-1, TNF-α and thiobarbituric acid-reactive substances were significantly decreased in the ears treated with the chimera in comparison to the control cream treated ears in DNFB sensitized/DNFB challenged group. We found that PK20 topical treatment alleviates hypersensitivity responses triggered by DNFB challenge and usage of the hybrid peptide may be a novel therapeutic strategy in the treatment of chronic inflammatory diseases. However, the mechanism remains unclear and needs further investigation.

Different blood pressure responses to opioids in 3 rat hypertension models: role of the baseline status of sympathetic and renin-angiotensin systems.

Opioids interact with sympathetic and renin-angiotensin systems in control of mean arterial pressure (MAP). Our earlier finding that biphalin, a synthetic enkephalin analogue, decreased MAP in anaesthetized spontaneously hypertensive rats (SHR) prompted us to further explore this action, to get new insights into pathogenesis of various forms of hypertension. Biphalin effects were studied in SHR, uninephrectomized rats on a high-salt diet (HS/UNX), and rats with angiotensin-induced hypertension (Ang-iH). Besides MAP, renal and iliac blood flows (RBF, IBF) and vascular resistances were measured. In anaesthetized and conscious SHR, biphalin (300 µg·h-1·kg-1 i.v.) decreased MAP by ∼10 and ∼20 mm Hg, respectively (P < 0.001). In anaesthetized HS/UNX and normotensive rats, MAP increased by ∼6-7 mm Hg (P < 0.02); without anaesthesia, only transient decreases occurred. MAP never changed in Ang-iH rats. Morphine (1.5 mg·h-1·kg-1 i.v.) decreased MAP in HS/UNX but only transiently so without anaesthesia; such anaesthesia dependence of response was also seen in normotensive rats. Ang-iH rats never responded to morphine. Hypotensive effect in SHR only depends primarily on the reduction by biphalin of vascular responsiveness to increased sympathetic stimulation; such increase is well documented for SHR. No MAP response to biphalin or morphine in Ang-iH could depend on angiotensin-induced alterations of the vascular wall morphology and function.

Antinociceptive effect induced by a combination of opioid and neurotensin moieties vs. their hybrid peptide [Ile(9)]PK20 in an acute pain treatment in rodents.

Hybrid compounds are suggested to be a more effective remedy for treatment of various diseases than combination therapy, since the attenuation or total disappearance of side effects, typically induced by a single moiety, can be observed. This is of great importance, especially when we consider problems resulting from the use of opioid analgesics. However, although it seems that such compounds can be valuable therapeutic tools, the lack of conviction among the public as to the appropriateness of their use still remains; therefore patients are commonly treated with polypharmacy. Thus, in the presented paper we show a comparison of the antinociceptive effect between a novel opioid-neurotensin chimera called [Ile(9)]PK20 and a mixture of its structural elements, delivered intrathecally and systemically. Additionally, motor coordination was assessed in the rotarod test. The results clearly indicate that spinal administration of the examined compounds, resulted in a long-lasting, dose- and time-dependent antinociceptive effect. Although the mixture of both pharmacophores was found to be more active than [Ile(9)]PK20, motor impairments surfaced as a side effect. This in turn illustrates the advantageous use of hybrid structures over drug cocktails.

Biological evaluation and molecular docking studies of AA3052, a compound containing a µ-selective opioid peptide agonist DALDA and d-Phe-Phe-d-Phe-Leu-Leu-NH2, a substance P analogue.

The design of novel drugs for pain relief with improved analgesic properties and diminished side effect induction profile still remains a challenging pursuit. Tolerance is one of the most burdensome phenomena that may hamper ongoing opioid therapy, especially in chronic pain patients. Therefore, a promising strategy of hybridizing two pharmacophores that target distinct binding sites involved in pain modulation and transmission was established. Previous studies have led to the development of opioid agonist/NK1 agonist hybrids that produce sufficient analgesia and also suppress opioid-induced tolerance development. In our present investigation we assessed the antinociceptive potency of a new AA3052 chimera comprised of a potent MOR selective dermorphin derivative (DALDA) and an NK1 agonist, a stabilized substance P analogue. We have shown that AA3052 significantly prolonged responses to both mechanical and noxious thermal stimuli in rats after intracerebroventricular administration. Additionally, AA3052 did not trigger the development of tolerance in a 6-day daily injection paradigm nor did it produce any sedative effects, as assessed in the rotarod performance test. However, the antinociceptive effect of AA3052 was independent of opioid receptor stimulation by the DALDA pharmacophore as shown in the agonist-stimulated G-protein assay. Altogether the current results confirm the antinociceptive effectiveness of a novel opioid/SP hybrid agonist, AA3052, and more importantly its ability to inhibit the development of tolerance.

Bifunctional Peptide-Based Opioid Agonist-Nociceptin Antagonist Ligands for Dual Treatment of Acute and Neuropathic Pain.

Herein, the opioid pharmacophore H-Dmt-d-Arg-Aba-ß-Ala-NH2 (7) was linked to peptide ligands for the nociceptin receptor. Combination of 7 and NOP ligands (e.g., H-Arg-Tyr-Tyr-Arg-Ile-Lys-NH2) led to binding affinities in the low nanomolar domain. In vitro, the hybrids behaved as agonists at the opioid receptors and antagonists at the nociceptin receptor. Intravenous administration of hybrid 13a (H-Dmt-d-Arg-Aba-ß-Ala-Arg-Tyr-Tyr-Arg-Ile-Lys-NH2) to mice resulted in potent and long lasting antinociception in the tail-flick test, indicating that 13a was able to permeate the BBB. This was further supported by a cell-based BBB model. All hybrids alleviated allodynia and hyperalgesia in neuropathic pain models. Especially with respect to hyperalgesia, they showed to be more effective than the parent compounds. Hybrid 13a did not result in significant respiratory depression, in contrast to an equipotent analgesic dose of morphine. These hybrids hence represent a promising avenue toward analgesics for the dual treatment of acute and neuropathic pain.

Biphalin preferentially recruits peripheral opioid receptors to facilitate analgesia in a mouse model of cancer pain - A comparison with morphine.

The search for new drugs for cancer pain management has been a long-standing goal in basic and clinical research. Classical opioid drugs exert their primary antinociceptive effect upon activating opioid receptors located in the central nervous system. A substantial body of evidence points to the relevance of peripheral opioid receptors as potential targets for cancer pain treatment. Peptides showing limited blood-brain-barrier permeability promote peripheral analgesia in many pain models. In the present study we examined the peripheral and central analgesic effect of intravenously administered biphalin - a dimeric opioid peptide in a mouse skin cancer pain model, developed by an intraplantar inoculation of B16F0 melanoma cells. The effect of biphalin was compared with morphine - a golden standard in cancer pain management. Biphalin produced profound, dose-dependent and naloxone sensitive spinal analgesia. Additionally, the effect in the tumor-bearing paw was largely mediated by peripheral opioid receptors, as it was readily attenuated by the blood-brain-barrier-restricted opioid receptor antagonist - naloxone methiodide. On the contrary, morphine facilitated its analgesic effect primarily by activating spinal opioid receptors. Both drugs induced tolerance in B16F0 - implanted paws after chronic treatment, however biphalin as opposed to morphine, showed little decrease in its activity at the spinal level. Our results indicate that biphalin may be considered a future alternative drug in cancer pain treatment due to an enhanced local analgesic activity as well as lower tolerance liability compared with morphine.

An endomorphine analog ([d-Ala(2)]-Endomorphin 2, TAPP) lowers blood pressure and enhances tissue nitric oxide in anesthetized rats.

BACKGROUND: Activation of opioid receptors can alter cardiovascular function, an action possibly mediated by nitric oxide (NO). In this study we examined the effects of ([d-Ala(2)]-Endomorphin 2, TAPP), a synthetic opioid µ-receptor agonist, on blood pressure (MABP), tissue NO bioavailability and renal hemodynamics and excretion. METHODS: In acute experiments with anesthetized normotensive male Sprague-Dawley rats TAPP was given as a short iv infusion at a dose of 1.2 or 12mg/kg and then MABP, renal medullary NO signal (polarographic electrode), total renal blood flow (RBF, renal artery Transonic probe), renal regional perfusion (laser-Doppler fluxes) and renal excretion were simultaneously measured over 2h. RESULTS: After 1.2mg/kg dose MABP decreased progressively from 121±7 to 114±9mmHg (-6%, p<0.05) while kidney tissue NO signal increased from 29.1±2.7 to 31.7±3.1nA (6%, p<0.04). Both effects were prevented by Naloxone methiodide, a peripheral opioid receptor inhibitor. RBF and renal regional perfusion were not altered by either dose of TAPP; renal sodium excretion changes were highly variable and were not affected by Naloxone pretreatment. CONCLUSIONS: Briefly, we found that in anesthetized normotensive rats stimulation of peripheral opioid receptors with TAPP caused a prolonged decrease in arterial pressure, a change that was associated and probably causally related to an increase in tissue NO. The data suggest that synthetic opioids that do not penetrate the blood-brain barrier and are potentially non-addictive could be considered for antihypertensive therapy.

Mixed MOP/DOP agonist biphalin elicits anti-transit effect in mouse models mimicking diarrhea-predominant irritable bowel syndrome symptoms.

BACKGROUND: Opioid receptors play a crucial role in the maintenance of homeostasis in the gastrointestinal (GI) tract. The aim of this study was to characterize the effect of biphalin, a mixed MOP/DOP agonist, on mouse intestinal contractility in vitro and GI motility in vivo and in animal models mimicking symptoms of diarrhea-predominant irritable bowel syndrome (IBS-D). METHODS: The effect of biphalin on muscle contractility in vitro was characterized in the ileum and colon. The anti-transit activity of biphalin in vivo was assessed in the following tests: whole gastrointestinal transit, colonic bead expulsion, fecal pellet output and castor oil-induced diarrhea, alone and in the presence of naloxone, and MOP and DOP antagonists. RESULTS: In vitro, biphalin (10(-10)-10(-6)M) inhibited colonic and ileal smooth muscle contractions in a concentration-dependent, opioid antagonist-reversible manner. In vivo, biphalin at the dose of 5mg/kg ip prolonged the whole GI transit and inhibited colonic bead expulsion. Biphalin reversed hypermotility and exerted anti-diarrheal effect in mouse models mimicking IBS-D symptoms. CONCLUSION: Biphalin is an interesting template for novel opioid-based agents to be used in therapy of functional GI diseases.

An antihypertensive opioid: Biphalin, a synthetic non-addictive enkephalin analog decreases blood pressure in spontaneously hypertensive rats.

BACKGROUND: Endogenous opioid systems may be engaged in the control of arterial pressure (MAP), however, given the risk of addiction, opioid receptor agonists are not used in antihypertensive therapy. We examined cardiovascular effects of biphalin, a potentially non-addictive dimeric enkephalin analog, an agonist of opioid µ and δ receptors. METHODS: Biphalin was infused iv at 150µg/kg/h to anesthetized spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). Along with MAP and heart rate (HR), renal blood flow (RBF) and iliac blood flow (IBF, a measure of hind limb perfusion) were measured using Transonic probes on renal and iliac artery, respectively. The effects of biphalin were compared with those of intravenous morphine (1.5mg/kg/h). RESULTS: In two SHR groups biphalin decreased MAP from 143±2 to 130±2 and from 177±4 to 167±3mmHg (p<0.001) while HR did not change or modestly decreased. The renal blood flow (RBF) increased modestly and both renal and hind limb vascular resistances decreased significantly (p<0.001). The responses were blocked by inhibition of peripheral opioid receptors with naloxone methiodide. Unlike in SHR, in WKY rats biphalin did not change MAP or vascular resistances. Morphine infusion decreased MAP in SHR from 169±6 to 150±6mmHg (less decrease in WKY) and significantly decreased RBF and IBF. CONCLUSION: Since biphalin, a non-addictive synthetic opioid, lowers MAP in SHR, a model of hypertension with pronounced neurogenic component, such analogs might find therapeutic application in human stress-induced hypertensive states. Biphalin's advantage is no associated reduction of renal perfusion.

Synthesis and binding characteristics of [(3)H]neuromedin N, a NTS2 receptor ligand.

Neurotensin (NT) and its analog neuromedin N (NN) are formed by the processing of a common precursor in mammalian brain tissue and intestines. The biological effects mediated by NT and NN (e.g. analgesia, hypothermia) result from the interaction with G protein-coupled receptors. The goal of this study consisted of the synthesis and radiolabeling of NN, as well as the determination of the binding characteristics of [(3)H]NN and G protein activation by the cold ligand. In homologous displacement studies a weak affinity was determined for NN, with IC50 values of 454nM in rat brain and 425nM in rat spinal cord membranes. In saturation binding experiments the Kd value proved to be 264.8±30.18nM, while the Bmax value corresponded to 3.8±0.2pmol/mg protein in rat brain membranes. The specific binding of [(3)H]NN was saturable, interacting with a single set of homogenous binding sites. In sodium sensitivity experiments, a very weak inhibitory effect of Na(+) ions was observed on the binding of [(3)H]NN, resulting in an IC50 of 150.6mM. In [(35)S]GTPγS binding experiments the Emax value was 112.3±1.4% in rat brain and 112.9±2.4% in rat spinal cord membranes and EC50 values of 0.7nM and 0.79nM were determined, respectively. NN showed moderate agonist activities in stimulating G proteins. The stimulatory effect of NN could be maximally inhibited via use of the NTS2 receptor antagonist levocabastine, but not by the opioid receptor specific antagonist naloxone, nor by the NTS1 antagonist SR48692. These observations allow us to conclude that [(3)H]NN labels NTS2 receptors in rat brain membranes.

Biphalin protects against cognitive deficits in a mouse model of mild traumatic brain injury (mTBI).

Traumatic brain injury (TBI) is often a result of traffic accidents, contact sports or battlefield explosions. A mild form of traumatic brain injury (mTBI) is frequently underestimated, as the immediate physical symptoms decrease rapidly and conventional neuroimaging studies often do not show visible evidence of brain lesions. However, cognitive impairments persist for weeks, months or even years after the incident. Endogenous opioids were documented to play a role in thmodulation of mTBI pathology, whereas exogenous opioids were shown to possess neuroprotective properties. In the present study, biphalin, a dimeric enkephalin analog, improved cognitive performance in the Morris Water Maze and Novel Object Recognition tests in a mouse weight-drop model of mTBI. The effect of a single systemic injection of 10 mg/kg biphalin immediately after trauma was reversed by naltrexone, suggesting an opioid receptor-mediated mechanism. Biphalin also reduced cortical and hippocampal neurodegeneration, as shown by silver staining. Our data indicates that opioid receptor activation by biphalin may provide neuroprotection of post-traumatic neurodegeneration processes and may protect against memory impairments.

Dual Alleviation of Acute and Neuropathic Pain by Fused Opioid Agonist-Neurokinin 1 Antagonist Peptidomimetics.

Herein, the synthesis and biological evaluation of dual opioid agonists-neurokinin 1 receptor (NK1R) antagonists is described. In these multitarget ligands, the two pharmacophores do not overlap, and this allowed maintaining high NK1R affinity and antagonist potency in compounds 12 and 13. Although the fusion of the two ligands resulted in slightly diminished opioid agonism at the µ- and δ-opioid receptors (MOR and DOR, respectively), as compared to the opioid parent peptide, balanced MOR/DOR activities were obtained. Compared to morphine, compounds 12 and 13 produced more potent antinociceptive effects in both acute (tail-flick) and neuropathic pain models (von Frey and cold plate). Similarly to morphine, analgesic tolerance developed after repetitive administration of these compounds. To our delight, compound 12 did not produce cross-tolerance with morphine and high antihyperalgesic and antiallodynic effects could be reinstated after chronic administration of each of the two compounds.

Effects of the Hybridization of Opioid and Neurotensin Pharmacophores on Cell Survival in Rat Organotypic Hippocampal Slice Cultures.

Several neurotransmitter and neuromodulatory systems can control physiological glutamatergic activity. For example, opioid receptor ligands were shown to partially inhibit N-methyl-D-aspartic acid (NMDA) receptor-dependent glutamatergic excitotoxicity. Also, the endogenous tridecapeptide neurotensin (NT) was found to modulate excessive glutamate release and glutamate receptor activity in neurons. Alternatively to the one target-one drug approach, it has been well documented that hybrid compounds encompassing two pharmacophores in one molecular scaffold can represent more potent drugs. Moreover, such structures with dual activity can potentially enable a reduction of undesirable side effects and/or improved bioavailability. Herein, we describe the neuroprotective potential of an opioid-NT hybrid peptide (PK20), which was recently designed and synthesized within our group. The protective properties of PK20, assessed in an in vitro model of excitotoxic injury in organotypic hippocampal slice cultures subjected to NMDA, were compared to the effects caused by NT. Our results indicate that PK20 is a potent anti-neurodegenerative agent. Moreover, co-administered with NMDA, PK20 (25-100 ng/ml) dose-dependently reduced hippocampal cell death, determined by a decrease in the propidium iodide signal. We also report for the first time the significant NT-induced neuroprotective effect, as its application (50-100 ng/ml) to hippocampal slice cultures protected CA1 damage against neurotoxicity caused by NMDA.

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.