Patent Application: US-11594302-A

Abstract:
disclosed are methods for treating and preventing neurological disorders which have a significant inflammatory component . the methods of the present invention involve extracting blood from a patient , subjecting the blood ex vivo to at least one stressor selected from the group consisting of an oxidative environment , thermal stress and uv light , and then re - administering the blood to the patient , thereby reducing inflammation .

Description:
“ therapeutic treatment ” refers to the treatment of a disease wherein the treatment reduces or eliminates the symptoms of that disease . “ prophylactic treatment ” or “ prophylaxis ” refers to the prevention or hindrance of development of disease . the terms “ aliquot ,” “ aliquot of blood ” or similar terms used herein include whole blood , separated cellular fractions of the blood including platelets , separated non - cellular fractions of the blood including plasma , and combinations thereof . the method of this invention provides for the prophylactic or therapeutic treatment of a neurological brain disease mediated by reactive oxygen species . in this method a patient is first identified as having such a disease condition or is at risk of having such a disease condition mediated by reactive oxygen species . the patient is then evaluated to determine whether that disease condition or risk of disease condition can be effectively treated by reducing the concentration of reactive oxygen species . such evaluation is made by the attending clinician based upon the disease to be treated and the progression of the disease . such factors are well within the skill of the art . if , in the opinion of the attending clinician , a reduction in the concentration of reactive oxygen species would be suitable for the prophylactic or therapeutic treatment of such a disease , then the patient is administered an aliquot of blood which has been treated ex vivo with at least two stressors selected from the group consisting of an oxidative environment , thermal stress and electromagnetic radiation . the ex vivo treatment of the aliquot of blood is described below . the method provides a reduced concentration of the reactive oxygen species in said patient . in this preferred method , the patient is evaluated to determine whether the neurological brain disease condition or risk of neurological brain disease condition could be effectively treated by reducing the concentration of reactive oxygen species , e . g . whether its inflammation component associated with the presence of reactive oxygen species can be effectively reduced by reducing the concentration of reactive oxygen species . in this regard , the reduction of the reactive oxygen species is reduced in the patient at the time when a reduction of reactive oxygen species effectively treats ( either prophylactically or therapeutically ) the disease . the concentration of reactive oxygen species may be measured by a variety of methods known in the art . for example , one can determine them from measurements of depletion of anti - oxidative enzymes ( glutathione , catalase ) in the patient &# 39 ; s blood ( see layton et . al .). an alternative is to test the serum of a patient for oxidized low density lipoproteins , using anti - oldl elisa immunoassay ( see wilburgur et . al .). one can also measure lipid peroxidation products such as tiabarbituric acid and its derivatives in plasma , or measure arachidonic acid oxidation products in a patient &# 39 ; s blood . the treated blood is administered to the mammal by a method suitable for vaccination selected from the group consisting of intra - arterial injection , intramuscular injection , intravenous injection , subcutaneous injection , intraperitoneal injection , and oral , nasal or rectal administration . intramuscular injection is preferred . according to a preferred process of the present invention , an aliquot of blood is extracted from a mammalian subject , preferably a human , and the aliquot of blood is treated ex vivo with certain stressors , described in more detail below . the effect of the stressors is to modify the blood , and / or the cellular or non - cellular fractions thereof , contained in the aliquot . the modified aliquot is then re - introduced into the subject &# 39 ; s body by any route suitable for vaccination . the stressors to which the aliquot of blood is subjected ex vivo according to the method of the present invention are selected from temperature stress ( blood temperature above or below body temperature ), an oxidative environment and an electromagnetic emission , individually or in any combination , simultaneously or sequentially . suitably , in human subjects , the aliquot has a volume sufficient that , when re - introduced into the subject &# 39 ; s body , at least partial alleviation of the reactive oxygen species mediated disorder is achieved in the subject . preferably , the volume of the aliquot is up to about 400 ml , preferably from about 0 . 1 to about 100 ml , more preferably from about 5 to about 15 ml , even more preferably from about 8 to about 12 ml , and most preferably about 10 ml , along with an anticoagulant , e . g ., 2 ml sodium citrate . it is preferred , according to the invention , to apply all three of the aforementioned stressors simultaneously to the aliquot under treatment , in order to ensure the appropriate modification to the blood . it may also be preferred in some embodiments of the invention to apply any two of the above stressors , for example to apply temperature stress and oxidative stress , temperature stress and an electromagnetic emission , or an electromagnetic emission and oxidative stress . care must be taken to utilize an appropriate level of the stressors to thereby effectively modify the blood to alleviate the reactive oxygen species mediated disorder in the subject . the temperature stressor warms the aliquot being treated to a temperature above normal body temperature or cools the aliquot below normal body temperature . the temperature is selected so that the temperature stressor does not cause excessive hemolysis in the blood contained in the aliquot and so that , when the treated aliquot is injected into a subject , alleviation of the reactive oxygen species mediated disorder will be achieved . preferably , the temperature stressor is applied so that the temperature of all or a part of the aliquot is up to about 55 ° c ., and more preferably in the range of from about − 5 ° c . to about 55 ° c . in some preferred embodiments of the invention , the temperature of the aliquot is raised above normal body temperature , such that the mean temperature of the aliquot does not exceed a temperature of about 55 ° c ., more preferably from about 40 ° c . to about 50 ° c ., even more preferably from about 40 ° c . to about 44 ° c ., and most preferably about 42 . 5 ± 1 ° c . in other preferred embodiments , the aliquot is cooled below normal body temperature such that the mean temperature of the aliquot is within the range of from about − 5 ° c . to about 36 . 5 ° c ., more preferably from about 10 ° c . to about 30 ° c ., and even more preferably from about 15 ° c . to about 25 ° c . the oxidative environment stressor can be the application to the aliquot of solid , liquid or gaseous oxidizing agents . preferably , it involves exposing the aliquot to a mixture of medical grade oxygen and ozone gas , most preferably by bubbling through the aliquot , at the aforementioned temperature range , a stream of medical grade oxygen gas having ozone as a minor component therein . the ozone content of the gas stream and the flow rate of the gas stream are preferably selected such that the amount of ozone introduced to the blood aliquot , either on its own or in combination with other stressors , does not give rise to excessive levels of cell damage such that the therapy is rendered ineffective . suitably , the gas stream has an ozone content of up to about 300 μg / ml , preferably up to about 100 μg / ml , more preferably about 30 μg / ml , even more preferably up to about 20 μg / ml particularly preferably from about 10 μg / ml to about 20 μg / ml , and most preferably about 14 . 5 ± 1 . 0 μg / ml . the gas stream is suitably supplied to the aliquot at a rate of up to about 2 . 0 liters / min , preferably up to about 0 . 5 liters / min , more preferably up to about 0 . 4 liters / min , even more preferably up to about 0 . 33 liters / min , and most preferably about 0 . 24 ± 0 . 024 liters / min , at stp . the lower limit of the flow rate of the gas stream is preferably not lower than 0 . 01 liters / min , more preferably not lower than 0 . 1 liters / min , and even more preferably not lower than 0 . 2 liters / min . the electromagnetic emission stressor is suitably applied by irradiating the aliquot under treatment from a source of an electromagnetic emission while the aliquot is maintained at the aforementioned temperature and while the oxygen / ozone gaseous mixture is being bubbled through the aliquot . preferred electromagnetic emissions are selected from photonic radiation , more preferably uv , visible and infrared light , and even more preferably uv light . the most preferred uv sources are uv lamps emitting primarily uv - c band wavelengths , i . e ., at wavelengths shorter than about 280 nm . such lamps may also emit amounts of visible and infrared light . ultraviolet light corresponding to standard uv - a ( wavelengths from about 315 to about 400 nm ) and uv - b ( wavelengths from about 280 to about 315 ) sources can also be used . for example , an appropriate dosage of such uv light , applied simultaneously with the aforementioned temperature and oxidative environment stressors , can be obtained from lamps with a combined power output of from about 15 to about 25 watts , arranged to surround the sample container holding the aliquot , each lamp providing an intensity at a distance of one meter , of from about 45 - 65 mw / cm 2 . up to eight such lamps surrounding the sample bottle , with a combined output at 253 . 7 nm of 15 - 25 watts , operated at an intensity to deliver a total uv light energy at the surface of the blood of from about 0 . 025 to about 10 joules / cm 2 , preferably from about 0 . 1 to about 3 . 0 joules / cm 2 , may advantageously be used . preferably , four such lamps are used . the time for which the aliquot is subjected to the stressors is normally within the time range of up to about 60 minutes . the time depends to some extent upon the chosen intensity of the electromagnetic emission , the temperature , the concentration of the oxidizing agent and the rate at which it is supplied to the aliquot . some experimentation to establish optimum times may be necessary on the part of the operator , once the other stressor levels have been set . under most stressor conditions , preferred times will be in the approximate range of from about 2 to about 5 minutes , more preferably about 3 or about 3½ minutes . the starting blood temperature , and the rate at which it can be warmed or cooled to a predetermined temperature , tends to vary from subject to subject . such a treatment provides a modified blood aliquot which is ready for injection into the subject . in the practice of the preferred process of the present invention , the blood aliquot may be treated with the stressors using an apparatus of the type described in u . s . pat . no . 4 , 968 , 483 to mueller . the aliquot is placed in a suitable , sterile , uv light - transmissive container , which is fitted into the machine . the uv lamps re switched on for a fixed period before the gas flow is applied to the aliquot providing the oxidative stress , to allow the output of the uv lamps to stabilize . the uv lamps are typically switched on while the temperature of the aliquot is adjusted to the predetermined value , e . g ., 42 . 5 ± 1 ° c . then the oxygen / ozone gas mixture , of known composition and controlled flow rate , is applied to the aliquot , for the predetermined duration of up to about 60 minutes , preferably 2 to 5 minutes and most preferably about 3 minutes as discussed above , so that the aliquot experiences all three stressors simultaneously . in this way , blood is appropriately modified according to the present invention to achieve the desired effects . a subject preferably undergoes a course of treatments , such individual treatment comprising removal of a blood aliquot , treatment thereof as described above and re - administration of the treated aliquot to the subject . a course of such treatments may comprise daily administration of treated blood aliquots for a number of consecutive days , or may comprise a first course of daily treatments for a designated period of time , followed by an interval and then one or more additional courses of daily treatments . in one preferred embodiment , the subject is given an initial course of treatments comprising the administration of 4 to 6 aliquots of treated blood . in another preferred embodiment , the subject is given an initial course of therapy comprising administration of from 2 to 4 aliquots of treated blood , with the administration of any pair of consecutive aliquots being either on consecutive days , or being separated by a rest period of from 1 to 21 days on which no aliquots are administered to the patient , the rest period separating one selected pair of consecutive aliquots being from about 3 to 15 days . in a more specific , preferred embodiment , the dosage regimen of the initial course of treatments comprises a total of three aliquots , with the first and second aliquots being administered on consecutive days and a rest period of 11 days being provided between the administration of the second and third aliquots . it may be preferred to subsequently administer additional courses of treatments following the initial course of treatments . preferably , subsequent courses of treatments are administered at least about three weeks after the end of the initial course of treatments . in one particularly preferred embodiment , the subject receives a second course of treatment comprising the administration of one aliquot of treated blood every 30 days following the end of the initial course of treatments , for a period of 6 months . it will be appreciated that the spacing between successive courses of treatments should be such that the positive effects of the treatment of the invention are maintained , and may be determined on the basis of the observed response of individual subjects . the invention is demonstrated and illustrated by the following animal experiments , conducted on wistar rats , according to ethically - approved procedures and protocols . the experiments investigated the effect of pre - treatment of peripheral blood exposed to immune modulation therapy on lps - induced impairment of ltp in rat hippocampal tissue . preliminary studies were also carried out in cortical tissue to explore the consequence of immune modulation therapy on the accumulation of ros , the concentration of the cytokines tnfα and il - 10 , as well as il - 1 receptor type i , and on the activity of the stress - activated protein kinase , jnk . four groups of eight male wistar rats ( 300 - 350 g ; bioresources unit , trinity college dublin , republic of ireland ) were used in these experiments . animals were housed in groups of four under a 12 - hour light schedule , ambient temperature was controlled between 22 and 23 ° c . and rats were maintained under veterinary supervision . whole blood was obtained by cardiac puncture from donor rats and anticoagulated with sodium citrate ( 10 ml of blood + 2 ml of 3 . 13 % sodium citrate solution ). the anticoagulated blood was divided into two aliquots ; 2 ml to be used for sham treatment and 10 ml to undergo immune modulation treatment . for immune modulation treatment , 10 ml of anticoagulated blood was transferred to a custom - built sterile , low - density polyethylene disposable blood container ( vasogen inc , toronto , on , canada ) and exposed to a combination of controlled physiochemical stress factors in a medical device ( vasogen inc ). during processing , the temperature of the blood was raised to 42 . 5 ° c ., during which time blood was exposed to uv light ( maximum emission spectrum at 254 nm ). when this temperature was reached , a gas mixture of 14 . 5 ± 1 . 0 μg / ml of ozone in medical oxygen was bubbled through the blood at a flow rate of 240 ± 24 ml per min for 3 minutes , after which time the heat and uvc light sources were turned off . the foaming action caused by bubbling the gas through the blood increased the surface area exposed to the uvc light . the blood was then allowed to settle to the bottom of the blood container and was then ready to be used . two groups of 16 rats were treated by intramuscular injection with 150 μl of processed blood or untreated blood ( sham treatment ). injections were administered 14 days , 13 days and 1 day before lps challenge / induction of ltp . rats were anesthetized by intraperitoneal injection of urethane ( 1 . 5 g / kg ). all rats received lps ( 100 μg / kg ) or saline , intraperitoneally and monitored for 3 h . a bipolar stimulating electrode and a unipolar recording electrode were placed in the perforant path ( 4 . 4 mm lateral to lambda ) and in the dorsal cell body region of the dentate gyrus ( 2 . 5 mm lateral and 3 . 9 mm posterior to bregma ), respectively , and 0 . 033 - hz test shocks were given for 10 min before , and 40 min after , titanic stimulation ( three trains of stimuli delivered at 30 - s intervals , 250 hz for 200 ms ( mcgahon & amp ; lynch , 1996 )). rats were killed by decapitation ; cross - chopped slices ( 350 × 350 μm ) were prepared from dentate gyri , entorhinal cortex , hippocampus and cortex and used to prepare dissociated cells ( see below ) or frozen separately in krebs solution containing 10 % dimethyl sulfoxide ( haan & amp ; bowen , 1981 ) and stored at − 80 ° c . for analysis , slices were thawed rapidly and rinsed in fresh oxygenated krebs solution before preparation of homogenate or the crude synaptosomal pellet p 2 ( mcgahon & amp ; lynch , 1996 ). the formation of reactive oxygen species was assessed by analyzing formation of the highly - fluorescent 2 ′, 7 - dichlorofluorescein ( dcf ) from the non - fluorescent probe , 2 ′, 7 - dichlorofluorescein diacetate ( dcfh - da ; molecular probes , usa ; lebel et al ., 1992 ). the synaptosomal pellet , p 2 , prepared from cortex , was resuspended in 1 ml ice - cold 40 mm tris buffer ( ph 7 . 4 ), incubated at 37 ° c . for 15 min with dcfh - da ( 10 μl ; final concentration 5 μm ; from a stock solution of 500 μm in methanol ) and the reaction was terminated by centrifugation at 13 , 000 × g for 8 min at 4 ° c . pellets were resuspended in 1 . 5 ml of ice - cold 40 mm tris buffer , ph 7 . 4 , and monitored for fluorescence at 37 ° c . ( excitation , 488 nm ; emission , 525 nm ). reactive oxygen species formation was quantified from a standard curve of dcf in methanol ( range 0 to 5 μm ). protein concentration was determined ( bradford , 1976 ) and the results were expressed as nmol / mg protein / min . a commercially available enzyme - linked immunosorbent assay was used to analyze cortical tnfα ( biosource international inc .) and cortical il - 10 was measured using an il - 10 cytoset antibody pair ( biosource international inc .). each tissue was added to 1 ml of iscove &# 39 ; s culture medium containing 5 % fetal bovine serum and a cocktail enzyme inhibitor ( 100 mm amino - n - caproic acid , 10 mm na 2 edta , 5 mm benzamidine hcl , 0 . 2 mm phenylmethylsulfonyl fluoride ). tissue was homogenized and centrifuged at 10 , 000 rpm at 4 ° c . for 10 min . supernatants were removed and analyzed for tnfα using elisa . protein concentration was determined ( bradford , 1976 ) and the results were expressed as pg / mg protein . jnk activity and il - 1 receptor type i concentration was analyzed using western blotting technique in samples prepared from cortical tissue . tissue homogenates were diluted to equalize for protein concentration ( bradford , 1976 ) and 10 μl aliquots ( 1 mg / ml ) were added to 5 μl of sample buffer ( 0 . 5 mm tris - hcl , ph 6 . 8 , 10 % glycerol , 10 % sds , 5 % b - mercaptoethanol , 0 . 05 % bromophenol blue , w / v ) and boiled for 5 min . samples were frozen until western blotting was performed . 10 μl of each sample was loaded onto gels ( 10 % sds ) for each analysis . proteins were separated by application of a 32 - ma constant current for 25 - 30 min , transferred onto nitrocellulose strips ( 225 ma for 75 min ), and immunoblotted with the appropriate antibody . to assess jnk activity , proteins were immunoblotted with an antibody that specifically targets phosphorylated jnk ( santa cruz biotechnology , inc ; 1 : 100 in tbs and 0 . 1 % tween 20 containing 1 % bsa ) for 2 h at room temperature . il - 1 receptor type i concentration was assessed by immunoblotting proteins with a rabbit polyclonal antibody raised against an epitope mapping at the carboxy terminus of il - 1ri of mouse origin ( santa cruz biotechnology , inc . ; 1 : 2000 in pbs and 0 . 1 % tween 20 containing 2 % non - fat dried milk ) for 45 min at room temperature and 45 min at 37 ° c . nitrocellulose strips were washed and incubated with secondary antibody [ peroxide - linked anti - mouse igg ; 1 : 300 dilution ( sigma ) for 2 h at room temperature in the case of jnk and with hrp - linked anti - rabbit antibody ; 1 : 2000 dilution ( amersham , uk ) for 60 min at room temperature and 30 min at 37 ° c . in the case of il - 1 receptor type i . visualization of phosphorylated jnk was achieved using supersignal west dura extended duration substrate ( pierce , usa ). immunoblots were immersed in substrate for 5 min and subsequently exposed to film for 1 s . protein complexes of il - 1 receptor type i were visualized by ecl detection ( amersham , uk ) and immunoblots were exposed to film overnight at 4 ° c . in both cases films were processed using a fuji x - ray processor . quantification of protein bands was achieved by densitometric analysis using two software packages , grab it ( grab it annotating grabber , version 2 . 04 . 7 , synotics ; uvp ltd ) and gelworks ( gelworks id , version 2 . 51 ; uvp ltd ) for photography and densitometry , respectively . gelworks provides a single value ( in arbitrary units ) representing the density of each blot . glutamate release was assessed in the impure synaptosomal preparation , p 2 , obtained from dentate gyrus ; either freshly - prepared tissue was used or alternatively , p 2 was prepared from frozen slices of dentate gyri which were obtained from rats in which electrophysiological recordings were made ( mcgahon and lynch , 1996 ). in both cases , p 2 preparations were resuspended in oxygenenated krebs solution containing 2 mm cacl 2 and glutamate release was assessed as described previously ( mcgahon et al ., 1996 ). briefly , synaptosomal tissue was aliquotted onto millipore filters ( o45 μm ), rinsed under vacuum and the filtrate was discarded . synaptosomes were then incubated in 250 μl oxygenated krebs solution at 37 ° c . for 3 min , in the presence or absence of 40 mm kcl , and filtrate was collected and stored for analysis as described ( ordronneau et al ., 1991 ). in some experiments , synatosomes were incubated at 37 ° c . for 20 min in krebs solution containing il - 1β ( 1 ng / ml ) in the presence or absence of vasoactive intestinal peptide ( vip ; 1 μm ). triplicate samples ( 50 μl ) or glutamate standards ( 50 μl ; 25 nm to 1 μm prepared in 100 mm nah 2 po 4 buffer , ph 8 . 0 ) were added to glutaraldehyde - coated 96 - well plates , incubated for 60 min at 37 ° c ., and washed with 100 mm nah 2 po 4 buffer . ethanolamine ( 250 μl ; 0 . 1 m in 100 mm nah 2 po 4 4 buffer ) was used to bind unreacted aldehydes and donkey serum ( 200 μl ; 3 % in pbs - t ) was added to block non - specific binding . samples were incubated overnight at 4 ° c . in the presence of antiglutamate antibody ( raised in rabbit ; 100 μl ; 1 : 5 , 000 in pbs - t ; sigma , uk ), washed and reacted with secondary antibody ( anti - rabbit horseradish peroxidase ( hrp )- linked secondary antibody ; 100 μl ; 1 : 10 , 000 in pbs - t ; amersham , uk ) for 60 min at room temperature . 3 . 3 ′, 4 . 4 ′- tetramethylbenzidine liquid substrate was added as chromogen and incubation continued for exactly 60 min at room temperature , at which time the reaction was stopped by h 2 so 4 ( 4 m ; 30 μl ). optical densities were determined at 450 mm using a multiwell plate reader and values were calculated with reference to the standard curve , corrected for protein ( bradford , 1976 ) and expressed as μmol glutamate / mg protein . il - β concentration in hippocampal was analysed by elisa ( r & amp ; d systems , uk ). antibody - coated ( 100 μl ; 1 . 0 μg / ml final concentration , diluted in phosphate buffered saline ( pbs ), ph 7 . 3 ; goat anti - rat il - 1βantibody ) 96 = well plates were incubated overnight at room temperature , washed several times with pbs containing 0 . 05 % tween 20 , blocked for 1 hour at room temperature with 300 μl blocking buffer , ( pbs , ph 7 . 3 containing 5 % sucrose , 1 % bovine serum albumin ( bsa ), and 0 . 05 % nan 3 ). after several washes , plates were incubated with il - 1β standards ( 100 μl ; 0 - 1000 pg / ml in pbs containing 1 % bsa ) or samples ( homogenized in krebs solution containing 2 mm cacl 2 ) for 2 hours at room temperature . samples were incubated with secondary antibody ( 100 μl ; final concentration 350 ng / ml in pbs containing 1 % bsa and 2 % normal goat serum ; biotinylated goat anti - rat il - 1β antibody ) for 2 hours at room temperature , washed and incubated in detection agent ( 100 μl ; horseradish peroxidase conjugated streptavidin ; 1 : 200 dilution in pbs containing 1 % bsa ) for 20 min at room temperature . substrate solution ( 100 μl ; 1 : 1 mixture of h 2 o 2 and tetramethylbenzidine ) was added , samples were incubated at room temperature in the dark for 1 hour after which time the reaction was stopped using 50 μl 1 m h 2 so 4 . absorbance was read at 450 nm , values were corrected for protein ( bradford , 1976 ) and expressed as pg il - 1β / mg protein . dissociated cells were prepared by enzymatic and mechanical digestion of fresh hippocampal slices . slices were incubated with collagenase ( 0 . 125 %) in pbs for 30 min at room temperature , washed with pbs to terminate collagenase digestion , and then gently triturated with a glass pasteur pipette , before passing through a nylon mesh filter to remove tissue clumps . cells were than cytospun onto glass microscope slides , fixed with methanol and stored until use . tunel ( terminal deoxynucleotidyl transferase ( tdt )- mediated dutp nick - end labeling ) staining , which identifies nuclei with fragmented dna ( a characteristic of apoptotic cells ), was performed according to the manufacturer &# 39 ; s instructions . briefly , fixed cytospun cells were washed and permeabilized . cells were equilibrated in buffer ( 200 mm potassium cacodylate ( ph 6 . 6 at 25 ° c . ), 25 mm tris - hcl ( ph 5 . 5 at 25 ° c . ), 0 . 2 mm dtt , 0 . 25 mg / ml bsa , 2 . 5 mm cocl 2 ) for 5 min at room temperature and incubated in tdt reaction mixture ( 30 μl ; 98 μl equilibration buffer , 1 μl biotinylated nucleotide mix , 1 μl tdt enzyme ) at 37 ° c . for 1 hour . the reaction was terminated by adding 100 μl 2 × scc ( 1 : 10 ; 2 × scc : deionized water ), endogenous peroxidases were blocked by incubating with h 2 o 2 ( 100 μl ; 0 . 3 % in pbs ) for 5 min at room temperature , and washed cells were incubated for 30 min at room temperature in streptavidin hrp solution ( 100 μl ; 1 : 500 in pbs ) to allow binding to biotinylated nucleotides . diaminobenzidine solution was added to washed cells , and the incubation proceeded fo 10 min at room temperature . cells were washed with deionised water , dehydrated through graded ethanol , and then cleared with xylene after which slides were mounted in dpx mounting medium and coverslipped . tunel positive cells were expressed as a percentage of the total . data were analyzed , as appropriate , using either the student &# 39 ; s t - test for independent means , or by using a one - way analysis of variance ( anova ) followed by post hoc analysis using the student newman keuls test . mean body weight , dose of urethane administered to induce anaesthesia , and stimulus strength required to induce an epsp spike were calculated . there was no significant difference between groups in body weight ( fig1 a ) or urethane concentration ( fig1 b ) administered due to immune modulation therapy . [ 0103 ] fig1 demonstrates that immune modulation therapy does not significantly alter body weight ( a ) or dose of urethane administered to induce anaesthesia ( b ). there is however an increase in the amplitude required to induce an action potential ( c ). the data are expressed as means with standard errors . tetanic stimulation delivered to the perforant path 3 h after intraperitoneal injection of lps resulted in an increase in the mean slope of the population excitatory post - synaptic potential ( epsp ). the mean percentage change in the 2 min immediately following tetanic stimulation (± sem ; compared with the 5 min immediately before tetanic stimulation ) was 114 . 49 (± 2 . 79 ), but this was not maintained so that the mean percentage change in population epsp slope in the last 5 min of the experiment was 90 . 32 (± 2 . 42 ). the corresponding values in the saline - treated control rats were 170 . 15 (± 10 . 16 ) and 121 . 28 (± 1 . 20 ), respectively ( fig2 ). the lps induced inhibition of ltp was blocked by pre - treatment with immune modulation therapy . [ 0105 ] fig2 demonstrates that the lps - induced impairment of ltp , was inhibited by pre - treatment with immune modulation therapy . the mean population epsp slope immediately after tetanic stimulation was attenuated in lps - treated rats compared with saline - treated rats and was close to baseline at the end of the 40 min post - tetanus recording period . the inhibitory effect of lps on ltp was blocked by pre - treatment with immune modulation therapy , which exerted no significant effect in saline - challenged rats . the data presented are means of seven to eight observations in each treatment group . the mean percentage change in population epsp slope (± sem ) in the 2 min immediately after tetanic stimulation was 166 . 85 (± 4 . 54 ) in the sham pretreated , saline challenged group compared with 147 . 44 (± 5 . 84 ) in the group pretreated with immune modulation therapy and challenged with lps . in the last 5 min of the experiment the values were 121 . 96 (± 0 . 85 ) and 128 . 07 (± 1 . 46 ), respectively ( n = 7 - 8 ). dissocated cells prepared form fresh hippocampal tissue displayed an increased number of apoptotic cells after lps injection as evidenced by increased number of cells displaying dark brown stained nuclei i . e . tunel positive cells . this contrasts with cells prepared from hippocampus of saline - treated rats and rats treated with immune modulation therapy . treatment with immune modulation therapy reversed the effects of lps as shown by a reduction in the number of cells displaying tunel positive staining . the precentage of tunel positive cells was significantly increased in the lps - treated group compared with the control treated group ( p & lt ; 0 . 01 ; anova ) and demonstrates that the immune modulation therapy reversed the degenerative effect of lps ( p & lt ; 0 . 01 ; anova ). animals were administered either immune modulation therapy or sham treatments , as previously described , and the following measurements were made in the cortex : ros accumulation , tnf - α and il - 10 levels . these experiments were performed without lps stimulation of the animals . [ 0110 ] fig3 indicates that immune modulation therapy significantly reduces reactive oxygen species accumulation in the cortex ( p & lt ; 0 . 05 ; student &# 39 ; s t - test for independent means , n = 7 - 8 ). the data are expressed as means with standard errors . the concentration of pro - inflammatory cytokine , tnfα is significantly reduced in the cortex as a result of immune modulation therapy ( p & lt ; 0 . 01 ; student &# 39 ; s t - test for independent means , fig4 a ). in contrast , il - 10 concentration is significantly increased ( p & lt ; 0 . 01 ; student &# 39 ; s t - test for independent means ) ( fig4 b ). fig4 a and 4 b show that immune modulation therapy significantly reduces tnfα concentration ( a ) and significantly increases il - 10 concentration ( b ) in the cortex ( p & lt ; 0 . 01 ; student &# 39 ; s t - test for independent means ; n = 7 - 8 ). the data are expressed as means with standard errors . [ 0112 ] fig5 illustrates that immune modulation therapy decreased jnk activity as indicated by a decrease in the phosphorylated form of jnk . analysis of the mean data obtained from densitometric analysis indicated that vasocare ™ therapy significantly reduced kinase activity ( p & lt ; 0 . 05 ; student &# 39 ; s t - test for independent means ). fig5 shows that immune modulation therapy significantly reduces jnk activity in the cortex ( p & lt ; 0 . 05 ; student &# 39 ; s t - test for independent means ; n = 7 - 8 ). the data are expressed as means with standard errors . with respect to the concentration of il - 1 receptor type i in cortical tissue , pilot work indicates that immune modulation therapy reduces il - 1 receptor type i expression ( fig6 ). the concentration of ligand pro - inflammatory il - 1β itself is expected to be lower , and is under examination . fig6 shows that immune modulation therapy reduces the concentration of il - 1 receptor type i ( preliminary data ; n = 3 ). the data are expressed as means with standard errors . analysis of endogenous glutamate release in synatosomes prepared from tetanized and untetanized tissue obtained from these rats revealed a significant effect of lps injection . addition of 40 mm kcl to synaptosomes prepared from untetanized dentate gyrus obtained from saline - treated control rats , significantly increased glutamate release ( p & lt ; 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