Patent Application: US-93121892-A

Abstract:
exposing the skin to uv radiation interferes with the induction of the t - cell mediated immune response , including both delayed and contact hyper - sensitivity immune responses initiated at non - irradiated sites . the present inventors have discovered that dna is at least one of the targets for uv - induced hypersensitivity , and demonstrate that the application of dna repair enzymes can reverse the damaging effects of uv irradiation on both the dhs and chs response . the usefulness of the invention in this regard was tested using a model immunosuppression system in mice . in these studies , mice were first exposed to uv radiation and then liposomes were used to deliver a dimer - specific excision repair enzyme to their epidermis in situ . the application of liposomal t4 endonuclease v encapsulated to the uv - irradiated skin both decreased the number of cyclobutane pyrimidine dimers in the epidermis and prevented suppression of both delayed and contact hypersensitivity responses . moreover , the formation of suppressor lymphoid cells was inhibited . these studies illustrate that the delivery of lesion - specific dna repair enzymes to living skin after uv irradiation is an effective tool for restoring immune function and suggest that this approach may be broadly applicable to preventing other alterations caused by dna damage , including preventing or reversing viral activation , oncogene expression , or autoimmune episodes .

Description:
the following examples illustrate techniques discovered by the inventors for the treatment of immunosuppression , particularly immunosuppression of the chs type , through the use of liposome encapsulated dna repair enzymes such as t4 endonuclease v . it should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent laboratory techniques that are illustrative in their design . the presentation of these studies are believed to be readily applicable to the use of such compositions in the clinic for the treatment and / or prevention of uv immunosuppression . specific - pathogen - free c3h / hen ( mammary tumor virus negative ) female mice were obtained from the frederick cancer research facility animal production area ( frederick , md .). age - matched mice between 10 and 12 wk old were used for each experiment . they were housed in filter - protected cages , and ambient lighting was controlled to provide 12 - h light - 12 - h dark cycles . autoclaved national institutes of health open formula mouse chow and water were provided ad libitum . the animal facility is accredited by the aaalac , and all procedures were approved by the institutional animal care and use committee . the uv source was a bank of six fs40 sunlamps ( national biological corp ., twinsburg , ohio ), which emit approximately 60 % of their radiation within the uv - b ( 280 - 320 nm ) range and have a peak emission at 313 nm . the average irradiance of the source was approximately 9 w / m 2 , as measured by an il - 700 radiometer ( international light , inc ., newburyport , mass .) equipped with a pt171c uv - b 320 filter , and an a127 quartz diffuser , which detects radiation in the wavelength range between 280 and 320 nm . the dose - rate of the incident radiation received by animals was 4 . 5 w / m 2 because of screening by the cage lid . before irradiation , the dorsal fur of the mice was shaved with electric clippers , and the animals were placed in individual compartments in cages that were located 20 cm below the light source . mice used for measuring chs had their ears covered with electrical tape during irradiation . except for exposure to uv radiation , control mice were treated exactly the same as the irradiated mice . t4n5 liposomes were prepared by encapsulating purified , recombinant t4 endonuclease v in liposomes composed of phosphatidylcholine , phosphatidylethanolamine , oleic acid , and cholesterol hemisuccinate ( 2 : 2 : 1 : 5 molar ratio ) by the detergent dialysis method ( 14 ). the concentration of the entrapped enzyme was determined by elisa ( 16 ) and is expressed as mg t4 endonuclease v per ml of vehicle . the encapsulated activity was assayed by nicking of uv - supercoiled dna with and without dissolution of the liposomes ( 16 ). control preparations of liposomes contained boiled ( enzymatically inactive ) t4 endonuclease v ( 14 ). the liposomes were mixed into a 1 % hydrogel ( hypan ss201 , kingston hydrogels , dayton , n . j .) made with phosphate - buffered saline and applied to shaved mouse skin with a moist cotton swab . immediately after uv irradiation , 0 . 25 ml of liposome suspension containing 0 . 5 mg / ml t4 endonuclease v was applied to the uv - irradiated skin of each mouse . the number of pyrimidine dimers in epidermal dna was measured by the endonuclease - sensitive site assay using alkaline agarose gels ( 17 ). in this assay , the frequency of dimers in dna is measured by the change in average single - stranded dna length in alkaline gels after treatment with t4 endonuclease v . the lengths of dna in the control and endonuclease - digested samples are calculated using dna restriction fragments of defined length , run in adjacent lanes of the gel , and the average molecular length is computed by the same ( weight - average molecular weight )/ 2 calculation used in the well - established alkaline sucrose gradient method ( 18 ). under these conditions , dimer induction is directly proportional to uv - b fluence up to 100 dimers per million bases ( 17 ), and all measurements reported here are within this range . the dimer measurements in the alkaline agarose gel assay agree closely with results from the alkaline sucrose gradient method ( 18 ), confirming the accuracy of this technique . the epidermis was isolated by overnight digestion of excised dorsal skin in 0 . 25 % trypsin on ice , and the dna was purified by two rounds of proteinase k digestion ( 100 mg / ml in 1 % sds , 37 ° c . for 30 min ); phenol , phenol / chloroform ( 1 : 1 v / v ), and chloroform extraction ; and ethanol precipitation . the dna was then treated with purified t4 endonuclease v ( 10 mg / ml ) to produce breaks at all dimer sites , and the single strands were separated by alkaline agarose gel electrophoresis . photographic negatives of the ethidium - bromide stained gels were scanned by a hoefer gs300 scanning densitometer , and the output was digitized by a data translations 2805 analog / digital converting board in a compaq deskpro computer . the stored files were used to calculate the average molecular length for each sample , and the change in average molecular length was used to calculate the dimer frequency . the approximation of linear film response was used to calculate average molecular length because the t4 endonuclease v - treated samples showed fluorescence that fell within the linear portion of the film response curve ; the untreated dna samples showed fluorescence of single high molecular weight bands , most affected by a non - linear film response , but nearly identical in all samples and subject to the same small error . at each uv dose , the average dimer number per unit length of dna from at least two samples were compared in skin treated with either active or inactive liposomes or not treated with liposomes . formalin - fixed candida albicans was prepared as described previously ( 19 ). mice were sensitized by injecting 0 . 1 ml of formalin - fixed c . albicans cells ( 1 × 10 7 ) s . c . into each flank . nine days later , the hind footpad thickness was measured with a spring - loaded micrometer ( swiss precision instruments , los angeles , calif . ), and the animals were challenged by intradermal injection of 50 ml of candida antigen ( antigen supply house , northridge , calif .) in both hind footpads . footpad thickness was measured again 24 hr later , and the swelling was determined by subtracting prechallenge from postchallenge measurements . the thickness of the footpads before challenge ranged from 1 . 7 to 2 . 0 mm . for both dhs and chs , % suppression was calculated from formula i as follows : ## equ1 ## where n = negative control ( response of unsensitized mice to challenge ); p = positive control ( response of sensitized mice ± liposomes to challenge ); t = test group ( response of mice given uv ± liposomes before sensitization and challenge ). animals were sensitized by the epicutaneous application of 2 , 4 - dinitrofluorobenzene ( dnfb ) ( 50 ml of a 0 . 3 % v / v solution in acetone ) on shaved abdominal skin . five days later , the mice were challenged by applying 5 ml of a 0 . 2 % solution of dnfb onto each ear surface . alternatively , mice were sensitized with fluorescein isothiocyanate ( fitc ), according to the method of thomas et al . ( 20 ). a 0 . 5 % fitc solution ( aldrich chemicals , milwaukee , wis .) was prepared in a solvent composed of equal volumes of acetone and dibutyl phthalate ( aldrich ). for sensitization , 0 . 4 ml of the solution was applied to shaved abdominal skin . five days later , the chs reaction was elicited by applying 5 ml of the 0 . 5 % fitc solution to each surface of both ears . the thickness of each ear was measured with a micrometer immediately before challenge and 24 hr later . the ear thickness before challenge ranged from 0 . 24 to 0 . 30 mm . on day 10 after sensitization ( day 6 for chs ), spleens were removed from mice that exhibited a decreased dhs or chs response and from appropriate control animals . single - cell suspensions were prepared from pooled spleens by teasing the spleens apart in hanks &# 39 ; balanced salt solution . clumps were removed by filtration through nylon mesh . the cells were washed once and refiltered , and 10 8 viable , nucleated cells were injected i . v . into syngeneic recipients . immediately thereafter , the recipients were sensitized with c . albicans or fitc ; the dhs or chs response was measured 10 or 6 days later , respectively , as described above , and % suppression was calculated according to formula 1 . the significance of differences in the dhs or chs response between control and treatment groups was determined using an anova for repeated measures . each group contained at least five mice , and each experiment was performed at least twice and usually three times . a . effect of t4n5 liposomes on uv - induced suppression of the induction of dhs to candida exposure of mice to uv - b radiation before immunization inhibits the induction of the dhs response to c . albicans injected s . c . at an unirradiated site ; this treatment also causes the formation of suppressor t lymphocytes that suppress the induction of dhs to candida ( 19 ). to determine whether liposomes containing t4 endonuclease v would abrogate these immunosuppressive effects of uv irradiation , groups of mice were exposed to a single dose of 5 kj / m 2 uv - b radiation , and liposomes containing active ( t4n5 ) or heat - inactivated ( hi ) enzyme were immediately applied to the uv - irradiated dorsal skin . the mice were immunized 3 days later by s . c . injection of formalin - fixed candida . as shown in fig1 a , application of t4n5 liposomes completely abrogated the suppressive effect of uv irradiation ; in contrast , liposomes containing heat - inactivated endonuclease had no effect . this experiment was performed six times with very similar results . transfer of spleen cells from the immunized , uv - irradiated mice to normal , syngeneic recipients rendered the recipients unresponsive to immunization with candida , indicating that suppressor cells were present in the spleen cell suspension ( fig1 b ). treatment of the spleen cell donors with t4n5 liposomes after uv irradiation prevented the development of suppressor cells ; transfer of spleen cells from these animals to normal mice did not affect the magnitude of their dhs response to candida . it is conceivable that the t4n5 liposomes could be blocking the release or action of the immunosuppressive mediators produced by uv - irradiated cells ( 3 - 5 ) or activating an immunostimulatory mechanism , rather than acting at the level of dna repair . to test these possibilities , control experiments were performed in which the t4n5 liposomes were applied to ventral , unirradiated skin or were applied to dorsal , uv - irradiated skin 2 days after uv treatment . application of t4n5 liposomes on ventral ( unirradiated ) skin had no effect on uv - induced suppression of the dhs response : 51 % suppression in mice treated on ventral skin with t4n5 liposomes versus 57 % in uv - irradiated mice and 50 % in mice treated on ventral skin with liposomes containing heat - inactivated enzyme . in a second experiment , mice were exposed to uv radiation and t4n5 liposomes were applied immediately or 2 days after uv ; all groups were immunized with candida 5 days after uv . as before , applying t4n5 liposomes immediately after uv irradiation completely restored the dhs response ( 0 % suppression ). in contrast , applying the active liposomes 2 days after uv had no effect on uv - induced suppression ( 68 %) compared with mice given uv alone ( 68 % suppression ) or given inactive liposomes immediately ( 70 % suppression ) or 2 days after uv irradiation ( 72 % suppression ). it is also possible that at different doses of uv radiation , different mechanisms might be involved in immunosuppression . we therefore performed a dose - response study to determine whether liposome treatment would completely restore the dhs response across a wide range of uv doses . as illustrated in fig2 the t4n5 liposomes abrogated the uv - induced suppression of dhs at all doses of uv radiation tested . in contrast , the response of mice treated with liposomes containing heat - inactivated endo - nuclease did not differ significantly from that of mice given uv irradiation without liposomes as determined by linear regression analysis . c . effect of t4n5 liposomes on uv - induced inhibition of the elicitation of dhs to candida previous studies demonstrated that exposing mice to uv radiation after immunization also reduces the dhs response ; however , higher doses of uv radiation are required , and suppressor lymphocytes do not seem to be involved . to determine whether t4n5 liposomes would also prevent this form of uv - induced immunosuppression , experiments were performed in which mice were treated with uv radiation and liposomes 5 days after immunization . as shown in fig3 treatment with t4n5 liposomes , but not liposomes containing heat - inactivated enzyme , completely abrogated the inhibitory effect of uv radiation on elicitation of the dhs response . d . effect of t4n5 liposomes on uv - induced suppression of the induction of chs it has been postulated ( 6 ) that uv - induced systemic suppression of chs in the mouse is mediated by urocanic acid , which , when it absorbs uv radiation , undergoes a trans - to - cis isomerization . if this hypothesis were correct , an increase in the repair of uv - induced pyrimidine dimers in dna should not abrogate uv - induced suppression of chs . to test this possibility , we examined the effect of t4n5 liposomes on uv - induced suppression of the induction of chs to dnfb . mice were exposed to a single dose of 10 kj / m 2 uv - b radiation on shaved dorsal skin . three days later , they were sensitized by application of dnfb onto shaved ventral skin . some groups of mice were treated with t4n5 liposomes or liposomes containing heat - inactivated endonuclease on uv - irradiated skin immediately after irradiation ; unirradiated control groups were also treated with both liposome preparations . as shown in fig4 a , treatment of uv - irradiated mice with active liposomes significantly increased the chs response to nearly the level observed in unirradiated , t4n5 liposome - treated animals ( p = 0 . 027 for uv , t4n5 group vs . uv , hi group ; p = 0 . 036 for uv , t4n5 group vs . uv group ), but the response was not completely restored ( p = 0 . 04 for uv , t4n5 group vs . non - irradiated , t4n5 group ). in a similar study using fitc as a contact sensitizer , the chs response was also increased in uv - irradiated , t4n5 - treated mice , but not completely restored ; however , the difference between the uv , t4n5 - treated group and the nonirradiated , t4n5 - treated group was not statistically significant ( fig4 b ). transfer of spleen cells from the uv - irradiated , fitc - sensitized mice to normal , syngeneic recipients confirmed the presence of suppressor cells that inhibited the induction of chs to fitc ; such suppressor cells could not be detected in mice given t4n5 liposomes immediately after uv irradiation ( fig4 c ). t4n5 liposomes were shown previously to reduce the number of pyrimidine dimers in dna isolated from the epidermis of uv - irradiated hairless mice ( 14 ). these animals have an endogenous dna repair mechanism that removes up to 40 % of the cyclobutane pyrimidine dimers from epidermal dna during the first 6 hr after uv irradiation ( 17 ), and t4n5 liposomes reduce the number remaining by an additional 40 % during that time period ( 14 ). to ensure that the liposomes were also increasing the repair of such lesions in the epidermis of the inbred c3h mice used in these experiments , pyrimidine dimers were measured at 6 hr after uv irradiation in mice taken at random from treatment groups of the experiments depicted in fig2 . the pooled results of these measurements are presented in fig5 . the number of pyrimidine dimers per 10 6 bases was directly proportional to the uv dose ( 11 ± 1 dimers / 10 6 bases / kj / m 2 uv - b ; r = 0 . 986 ), and is expressed as a percentage of the dimers in epidermal dna from mice treated with uv radiation only . dimers in the epidermis of mice treated with inactive liposomes ranged from 97 to 135 % of that in the control animals ; in contrast , dimers in the epidermis of mice treated with t4n5 liposomes ranged from 44 to 74 % of the control values across the range of uv doses tested . these results demonstrate that at 6 hr after uv irradiation , fewer pyrimidine dimers were present in the epidermis of mice treated with t4n5 liposomes than in untreated mice or mice treated with inactive liposomes . based on these studies , the inventors conclude that uv - induced suppression of the dhs response to candida in c3h mice is triggered entirely by the dna - damaging effects of uv radiation . other possible explanations for the effect of the t4n5 liposomes were ruled out by using control liposome preparations containing heat - inactivated endonuclease and by performing control experiments in which liposomes were applied to unirradiated skin or were applied 2 days after uv irradiation . because the endonuclease used to repair dna damage is highly specific for pyrimidine dimers , these lesions are the most likely initiators of this immunosuppressive effect of uv irradiation . regardless of the specific dna lesion involved , however , the initiation of suppression of the dhs response by uv can be accounted for entirely by dna damage , and there is no evidence that other molecules altered by uv radiation , such as urocanic acid , can by themselves trigger photoimmunosuppression . systemic suppression of chs in the mouse following exposure to uv - b radiation is thought to involve different mechanisms than suppression of dhs ( 21 ). our studies indicated , however , that systemic suppression of the chs response in uv - irradiated c3h mice was also mediated mainly by dna damage in the form of pyrimidine dimers . unlike the studies with dhs , suppression of the chs response could not be restored completely by treatment with liposomes . a small component of the suppression , comprising 10 to 25 %, was not reversed by treatment with t4n5 liposomes , suggesting that an additional mechanism contributes to uv - induced systemic suppression of the chs response . one question raised by the foregoing studies is why the t4n5 liposomes cause complete , or nearly complete restoration of immunological function , while they appear in fig4 to repair only around 40 to 50 % of the pyrimidine dimers remaining in epidermal dna . most likely , this is due to the fact that in the studies depicted in fig4 dimers were measured 6 hr after uv irradiation , whereas immunization occurred 3 days later . in these studies , dna repair measurements were made at this early time point to avoid the complication introduced by subsequent uv - induced epidermal hyperplasia . however , an experiment in hairless mice was performed under the same conditions of uv irradiation , in which dimers were measured in dna pre - labeled with 3 h - thymidine in vivo . this study demonstrated that repair of 80 % of the dimers occurred over a 30 - hr period after exposure to 5 kj / m 2 uv - b radiation ( d . yarosh , unpublished data ), confirming that complete excision repair of dimers occurs slowly , over a period of days . alternatively , target dna involved in uv - induced immunosuppression may be repaired more rapidly than other dna regions , since it is known that actively transcribed dna is repaired preferentially after uv irradiation ( 22 ). the foregoing studies demonstrated that skin treated with active t4n5 liposomes exhibited repair of about 50 % of the remaining dimers , over a wide range of uv - b doses . this result is puzzling because one would expect a larger proportion of the dimers to be repaired at the lower uv - b doses where fewer dimers are induced . perhaps not all regions of the genome are equally accessible to t4 endonuclease v ; alternatively , some other factor released or activated in proportion to the uv dose ( e . g ., a dna ligase or helicase ) may become rate limiting after t4 endonuclease v has incised all the remaining dimers . these studies are consistent with those carried out in the opossum , which demonstrated by means of photoreactivation that dna damage in the skin , and particularly pyrimidine dimers , are primarily responsible for generating systemic suppression of chs ( 10 ). the target cell of the uv irradiation has not been identified ; however , the keratinocyte seems to be the most likely candidate . these cells produce a variety of cytokines involved in immune and inflammatory reactions ( 23 ), and they release immunosuppressive mediators after exposure to uv radiation in vitro ( 3 - 5 ). therefore , the inventors propose that uv - induced damage to the dna of keratinocytes triggers the synthesis and release of the immunosuppressive cytokines that inhibit the dhs and chs responses and cause the induction of suppressor t lymphocytes . the studies also demonstrate that the use of liposomes to deliver lesion - specific dna repair enzymes to the epidermis in situ provides an effective means of preventing uv - induced immunosuppression . in principle , this approach could be applied to the study of other biological endpoints induced by dna damage and may have practical applications in preventing pathological effects resulting from damage to dna . the following references are hereby incorporated herein by reference to the extent that they explain , teach or provide a basis for materials and methodology disclosed herein . 1 . parrish , j . a ., kripke , m . l . & amp ; 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( 1983 ) photoimmunology , eds . parrish , kripke , morison , plenum publishing corporation , new york . 3 . schwarz , t ., urbanska , a ., gschnait , f . & amp ; luger , t . a . ( 1986 ) j . invest . dermatol . 87 , 289 - 291 . 4 . kim , t - y ., kripke , m . l . & amp ; ullrich , s . e . ( 1990 ) j . invest . dermatol . 94 , 26 - 32 . 5 . ullrich , s . e ., mcintyre , b . w . & amp ; rivas , j . m . ( 1990 ) j . immunol . 145 , 489 - 498 . 6 . defabo , e . c . & amp ; noonan , f . p . ( 1983 ) j . exp . med . 157 , 84 - 98 . 7 . ross , j . a ., howie , s . e . m ., norval , m . & amp ; maingay , j . p . ( 1987 ) j . invest . dermatol . 89 , 230 - 233 . 8 . harriott - smith , t . g . & amp ; halliday , w . j . ( 1988 ) clin . exp . immunol . 72 , 174 - 177 . 9 . norval , m ., gilmour , j . w . & amp ; simpson , t . j . ( 1990 ) photodermatol , photoimmunol . photomed . 7 , 243 - 248 . 10 . applegate , l . a ., ley , r . d ., alcalay , j . & amp ; kripke , m . l . ( 1989 ) j . exp . med . 170 , 1117 - 1131 . 11 . ananthaswamy , h . & amp ; fisher , m . s . ( 1981 ) cancer res . 41 , 1829 - 1833 . 12 . gordon , l . & amp ; haseltine , w . ( 1980 ) j . biol . chem . 255 , 12047 - 12050 . 13 . yarosh , d . ( 1992 ) in liposome dermatics ( eds . braun - falco , o ., korting , h . and maibach , h .) springer - verlag ( in press ). 14 . yarosh , d . b ., tsimis , j . & amp ; yee , v . ( 1990 ) j . soc . cosmet . chem . 41 , 85 - 92 . 15 . yarosh , d . b ., kibitel , j ., green , l . & amp ; spinowitz , a . ( 1991 ) j . invest . dermatol . 97 , 147 - 150 . 16 . ceccoli , j ., rosales , n ., tsimis , j . & amp ; yarosh , d . b . ( 1989 ) j . invest . dermatol . 93 , 190 - 194 . 17 . yarosh , d . & amp ; yee , v . ( 1990 ) j . photochem . photodiol . 7 , 173 - 179 . 18 . freeman , s ., blackett , b ., moneteleone , d ., setlow , r ., sutherland , b . & amp ; sutherland , j . ( 1986 ) anal . biochem . 158 , 119 - 129 . 19 . denkins , y ., fidler , i . j . & amp ; kripke , m . l . ( 1989 ) photochem . photobiol . 49 , 615 - 619 . 20 . thomas , w . r ., edwards , a . j ., watkins , m . c . & amp ; asherson , g . l . ( 1980 ). immunol . 39 , 21 - 27 . 21 . kripke , m . l . & amp ; morison , w . l . ( 1986 ) j . invest . dermatol . 86 , 543 - 549 22 . bohr , v ., okumoto , d . & amp ; hanawalt , p . ( 1986 ) proc . natl . acad . sci . usa 83 , 3830 - 3833 . 23 . luger , t . a . & amp ; schwarz t . ( 1990 ) j . invest . dermatol . 95 , 100s - 104s .