Patent Application: US-51920006-A

Abstract:
the present invention relates to an isolated bioactive molecule caerulomycin a , derivatives and analogs thereof as effective immunosuppressive agents . the immunosuppressive property of the compound is targeted in particular against the lymphocytes , cd4 + t cells , cd8 + t cells and b cells and in the production of il - 4 and ifn - γ and antibodies . the compound operates through a mechanism by downregulating the expression of activation marker cd28 and upregulating the immunosuppressive marker ctla - 4 . caerulomycin a has previously been isolated from streptomyces caeruleus and found to have useful antifungal activity . prior to the present invention however , this compound had not been determined to have immunomodulatory activity .

Description:
the systematic study of the products from actinomycetes and fungi has led to the development of immunosuppressive drugs such as cyclosporin a ( csa ), fk506 ( tacrolimus ) and rapamycin ( sirolimus ). these drugs not only exert potent antifungal effects but are also used as potential immunosuppressants . by taking into consideration this point we started our study for screening of bioactive compounds with antifungal activities by isolating various microbes from soil and water samples from the cold himalayan region of kaza and spiti in himachal pradesh . polyphasic characterization of the strain rmv - 1378 t , isolated from cold desert of the himalayas , india , clearly confirmed that the strain belong to the genus actinoalloteichus . physiological and biochemical tests allowed genotypic and phenotypic differentiation of the strain rmv - 1378 t from its closest phylogenetic relative . analysis of 16s rdna sequence revealed that the isolate is very closely related to actinoalloteichus cyanogriseus with similarity of 99 %. however , results of dna - dna hybridization , showed low genomic relatedness with actinoalloteichus cyanogriseus ( 51 %). therefore , we proposed that the isolate be classified as a new species of actinoalloteichus , for which we proposed the name actinoalloteichus spitiensis sp . nov . the strain rmv - 1378 t has been deposited in microbial type culture collection and gene bank ( mtcc ), india under accession number mtcc 6194 t and type strain rmv - 1378 t has also been deposited in japan collection of micro - organisms ( jcm ), japan under accession number jcm 12472 t and german collection of microorganisms and cell cultures ( dsmz ) germany , under accession number dsm 44848 t . the active ingredient isolated from strain rmv - 1378 was characterized based on nuclear magnetic resonance ( nmr ), infrared ( ir ) and mass spectral data . the identified compound was ( e )- 4 - methoxy - 2 , 2 ′- bipyridine - 6 - carbaldehyde oxime . the data corresponded well with the already reported data of caerulomycin a ( divekar et al . 1967 ). following are the major characteristics of the isolated strain actinoalloteichus spitiensis : a ) an actinobacterial strain , rmv - 1378 t , forms branching , non - ragmenting vegetative hyphae and do not produce diffusible pigments . neither aerial mycelium nor spore formation is observed . b ) the g + c content of the dna was 72 . 0 mol %. c ) the strain has chemotaxonomic characteristics typical of the genus actinoalloteichus and is closely related ( 99 . 3 % 16s rrna gene sequence similarity ) to actinoalloteichus cyanogriseus , currently the only actinoalloteichus species with a validly published name . however , the results of dna - dna hybridization experiments showed 51 . 9 % relatedness with the type strain of a . cyanogriseus . d ) on the basis of the above data and the physiological and biochemical distinctiveness of rmv - 1378 t (= mtcc 6194 t = jcm 12472 t = dsm 44848 t , this strain is classified as the type strain of a novel species of actinoalloteichus , for which the name actinoalloteichus spitiensis sp . nov . is accorded . the microbial type culture collection & amp ; gene bank ( mtcc ), a national facility , was established in 1986 and is sponsored jointly by the department of biotechnology ( dbt ), govt . of india and the council of scientific and industrial research ( csir ). this is a well - equipped modern facility housed at the institute of microbial technology ( imtech ), chandigarh . mtcc is an affiliate member of the world federation of culture collection ( wfcc ) and is registered with world data centre for microorganisms ( wdcm : reg . no . 773 ). main objectives of this national facility are to act as a depository , to supply authentic microbial cultures and to provide related services to scientists working in research institutions , universities and industries . on oct . 4 , 2002 mtcc was recognised by wipo ( geneva ) as an international depositary authority [ ida ] in india , for the deposit of microorganisms under the budapest treaty . caerulomycin a is produced in this invention by the controlled fermentation of a microorganism . this microorganism is preferably grown in an aqueous nutrient medium , under aerobic and mesophilic conditions , preferably between 25 ° c . and 35 ° c . at a ph ranging between about 6 . 0 and 8 . 0 . the length of the fermentation typically ranges between 24 h and 168 h , preferably between 24 h and 96 h . a good production can be obtained at 30 ° c . and a ph 7 . 0 to 8 . 5 . the nutrient medium employed should preferably be composed of any suitable nitrogen source such as protein hydrolysates , or protein and / or isolated amino acids , or any ammonium and / or nitrate source ; as source of carbon any assimilable carbohydrate and / or fat , and may also contain salts such as sodium chloride , sodium carbonate , sodium bicarbonate , potassium chloride , magnesium chloride , calcium carbonate , etc . with medium containing glucose 5 . 4 g , yeast extract 4 . 8 g , malt extract 8 . 5 g , caco 3 3 . 0 g , distilled water 1000 ml , ph 7 . 2 and incubation temperature of 28 ° c ., good production of caerulomycin a occurs . it must be appreciated that the above - mentioned medium is merely an example of a medium suitable for the production of caerulomycin a by strain of actinoalloteichus spitiensis sp . nov . it is believed that a wide range of nutrient media may be substituted for the one disclosed herein , with good growth and production resulting therefrom . all cultures and fermentations must be conducted in sterile media and conditions . to start fermentation , it is necessary to seed it with an inoculum grown in a medium similar to the one already described for the fermentation . the percentage of inoculum typically needed ranges between 1 and 10 %; 10 % being typically preferred . isolation and purification of the caerulomycin a produced by fermentation is typically conducted using a combination of extraction and chromatographic techniques . a preferred sequence of steps is as follows : extract the filtrate broth with an immiscible solvent such as ethyl acetate . combine these extracts and concentrate to dryness in vacuo . dilute the residue extract with nacl 10 %/ methanol ( 1 : 1 ) and partition it with an immiscible solvent such as hexane which is capable of removing the lipids . remove the active materials from the aqueous alcohol fraction by partitioning with an appropriate solvent such as ethyl acetate . the recovered solvent phases constitute crude caerulomycin a . further separation and purification of caerulomycin a from the crude extract can be affected by the use of the proper combination of chromatographic techniques , including , for example , column chromatography ( cc ), high performance flash chromatography , preparative medium pressure liquid chromatography ( mplc ) and thin layer chromatography ( tlc ). fractionation may be guided by immunosuppressive activities . on the basis of detailed analysis of their various spectral characteristics , the pure compound was identified as caerulomycin a ( see 1 h nmr , 13 c nmr and mass spectra reproduced in fig1 , 3 and 4 , respectively ). in an embodiment of the present invention caerulomycin a suppressed the activity of mitogen - stimulated t and b - lymphocytes . the lymphocytes were stimulated with t cell mitogen concanavalin a ( con a ) and b cell mitogen lipopolysaccharide ( lps ) and different doses of caerulomycin a ( 0 . 0003 - 0 . 1 μg / ml ). as compared to the cells cultured with mitogens alone , caerulomycin a induced significant decrease in mitogen - induced proliferation . in another embodiment of the present invention caerulomycin a also showed in vitro suppression of mlr reaction , naïve cd4 + t cells , antigen specific cd4 + t cells , and th1 and th2 cells . caerulomycin a activity was compared with a well - known immunosuppressive drug cyclosporin a ( csa ). it was observed that there was a dose dependent inhibition in mlr by both the drugs . interestingly , caerulomycin a was effective in inhibiting similar amount of proliferation using a 10 fold lesser dose than csa . in still another embodiment of the present invention , caerulomycin a also inhibited in vivo proliferation of the antigen specific t cells . antigen ( ovalbumin : 100 μg / ml ) was emulsified in freund &# 39 ; s complete adjuvant and was injected intraperitoneally in different groups ( 5 mice / group ). different groups of ovalbumin - primed animals were daily - injected different doses of caerulomycin a ( 25 , 50 , 75 , 100 μg / 100 μl / mice ). after seven days , mice were sacrificed , splenocytes from each group were separately pooled , and in vitro proliferation was monitored . in yet another embodiment of the present invention , caerulomycin a significantly suppressed the secretion of il - 4 and ifn - γ . in another embodiment of the present invention , caerulomycin a induces immunosuppression by down regulating cd28 and upregulating ctla - 4 expression . it is established that ctla - 4 delivers immunosuppressive signals . in contrast , cd28 conveys activation signals to t cells . it was observed that caerulomycin a not only significantly enhanced the expression but also the percentage of ctla - 4 / cd4 positive t cells and decreased the expression and percentage of cd28 / cd4 positive t cells . similar results were noticed in the case of non - cd4 + t cells . in another embodiment of the present invention , caerulomycin a inhibited the secretion of igg1 and igg2a type of antibodies . in yet another embodiment of the present invention , caerulomycin a upregulated b7 - 1 but downregulated b7 - 2 expression on macrophages . many costimulatory molecules are expressed on the surface of apc &# 39 ; s but b7 - 1 and b7 - 2 are the most potent . their interactions with cd28 / ctla - 4 receptors expressed on t cell surfaces are crucial for the proper regulation of t cell activity . we therefore monitored the expression of their ligands b7 - 1 and b7 - 2 . b7 - 1 binds to both ctla4 and cd28 more strongly than does b7 - 2 . however , when the relative interactions are directly compared , b7 - 1 favors binding to ctla - 4 over cd28 by 20 - fold , whereas b7 - 2 only favors ctla - 4 over cd28 by about 8 - fold . in view of the above - mentioned findings , the potent role of caerulomycin a as an imunosupppression agent can be viewed by a mechanism of enhancement of the expression of ctla - 4 on t cells and b7 - 1 on apc . the mlr was performed in 96 - well tissue culture plates , with each well containing 4 × 10 5 balb / c splenocytes ( responder cells ) and 4 × 10 5 γ - irradiated ( 3000r ) c57bl / 6j splenocytes ( stimulator cells ) in 200 μl rpmi / fcs - 10 % medium and with various concentrations of caerulomycin a ( 1 - 0 . 0125 μg / ml ) and cyclosporin a ( 0 . 0125 - 10 μg / ml ). the control cultures consisting of balb / c splenocytes in medium alone , γ - irradiated c57bl / 6j splenocytes and balb / c splenocytes + γ - irradiated c57bl / 6j splenocytes were also kept . after 4 days , the cultures were pulsed with 1 μci of [ 3 h ]- thymidine and harvested 16 h later by a skatron cell harvester . radioactivity incorporated was measured by liquid scintillation counting and data expressed as mean counts per minute ( cpm ). caerulomycin a substantially reduced the mlr reaction . caerulomycin a was ten times more potent than cyclosporin a in suppressing the mlr reaction . no significant level of [ 3 h ]- thymidine incorporation was observed in the control cultures containing cells only , cells cultured with caerulomycin a ( in the absence of either cona or lps or anti - cd3ab or apcs ). in all assays , caerulomycin a worked in a dose dependent manner . caerulomycin a induces immunosuppression of lymphocytes stimulated with t cell and b cell mitogens splenocytes of balb / c mice were cultured ( 5 × 10 4 cells / well ) in 200 μl rpmi / fcs - 10 % medium and stimulated with different concentrations of either cona ( 1 μg / ml and 2 μg / ml ) or lps ( 5 μg / ml and 10 μg / ml ) and caerulomycin a ( 0 . 0003 - 0 . 1 μg / ml ). the control cultures consisting of splenocytes incubated with medium alone , cona and dmso . after 48 h , the cultures were pulsed with 0 . 5 μci of [ 3 h ]- thymidine and harvested 14 h later by automatic cell harvester ( skatron , tranby , norway ). radioactivity incorporated was measured by liquid scintillation counting and data are expressed as mean counts per minute ( cpm ). as compared to the cells cultured with cona alone , caerulomycin a induced significant decrease in the proliferation of cona ( 1 . 0 and 2 . 0 μg / ml ) stimulated cells . we also stimulated splenocytes with lps ( 5 and 10 μg / ml ). interestingly , caerulomycin a could also suppress the proliferation of the cells stimulated with lps ( 5 and 10 μg / ml ). we observed that the doses ( 0 . 05 and 0 . 1 μg / ml ) of caerulomycin a induced potent inhibition in the proliferation . caerulomycin a induces immunosuppression of cd4 + t cells obtained from the antigen - primed animals a single cell suspension of mice splenocytes obtained from ova - cfa primed mice . the red blood cells were depleted by treatment with hemolytic gey &# 39 ; s solution . the adherent cells were removed by plating onto plastic petri plates for 2h at 37 ° c . and 7 % co 2 . the non - adherent cells were loaded ( 1 × 10 6 cells / ml ) onto the nylon wool column and incubated for 90 min at 37 ° c . after the incubation period , warm rpmi was passed into the column to elute t lymphocytes . the t cells were washed with rpmi and incubated with anti - mac3 ( tib - 168 ), anti - ia d ( mkd6 ), anti - dendritic cell ( tib - 227 ), anti - igm and anti - cd8 abs for 45 min at 4 ° c . the cells were washed with rpmi and incubated with baby rabbit complement for 30 min at 37 ° c . the cells were washed three times with rpmi and used for the proliferation assay . the purity of the cells was analyzed by flow - cytometry of the cells stained with anti - cd3 and cd4 abs . cd4 + t cells were purified from the ova - fca injected mice and stimulated with antigen - pulsed and γ - irradiated splenocytes . as compared to antigen stimulated cd4 + t cells , addition of caerulomycin a in the cultures significantly suppressed the proliferation . the proliferation of th2 clones was measured using cells harvested on 7 - 9 days after stimulation with antigen pulsed splenocytes . the dead cells were removed by ficoll - histopaque . th2 clones ( 5 × 10 4 cells / well ) were either stimulated with anti - cd3 ab ( 0 . 1 μg / ml and 0 . 5 μg / ml ) or incubated with γ - irradiated ( 3000 r ) syngeneic splenocytes ( 5 × 10 5 cells / well ) and conalbumin ( 100 μg / ml ) in 200 μl rpmi / fcs - 10 % medium and with various concentrations of caerulomycin a ( 0 . 00625 - 0 . 1 μg / ml ). the control cultures consisting of th2 cells incubated with conalbumin alone and γ - irradiated syngeneic splenocytes ( no antigen ) were also kept . the cultures were kept in a flat bottom 96 well microtitre plate and the cells were incubated at 37 ° c . and 7 % co 2 . after 48 h , the cultures were pulsed with 0 . 5 μci of [ 3 h ]- thymidine and harvested 16 h later . radioactivity incorporated was measured by liquid scintillation counting and data are expressed as mean counts per minute ( cpm ). caerulomycin a was added into the cultures of th2 cells stimulated either with anti - cd3 ab or conalbumin pulsed and γ - irradiated splenocytes . as observed in the case of mitogen stimulated lymphocytes and antigen specific t cells , caerulomycin a also substantially restricted the proliferation of th2 clones in both the stimulatory conditions . th1 cells ( 1 × 10 4 cells / well ) were stimulated with anti - cd3 ab ( 10 μg / ml ) and different doses caerulomycin a . after 24 h , [ 3 h ]- thymidine was added , and its incorporation was measured 8 h later . caerulomycin a also inhibited the proliferation of th1 cells ( 3do . 54 . 8 ). this feature was observed irrespective of whether th1 cells were stimulated with anti - cd3 ab . ova ( 2 mg / ml ) was dissolved in pbs ( 0 . 01 m , ph 7 . 2 ) and emulsified in freund &# 39 ; s complete adjuvant ( fca ). emulsion ( 100 μl ) was then injected intraperitoneally in a group consisting of 5 balb / c mice . the control group was injected with pbs alone . after seven days , mice were sacrificed and splenocytes were pooled and used for proliferation and cytokine assays . splenocytes ( 5 × 10 5 cells / well ) were cultured with ova ( 200 μg / ml ) in 200 μl rpmi / fcs - 10 % medium and with various concentrations of caerulomycin a ( 0 . 0003 - 0 . 1 μg / ml ). the control cultures consisting of splenocytes incubated with different concentrations of caerulomycin a ( no ova ), ova and medium were also kept . the culture supernatants were collected after 48 h and cytokines were measured by elisa . it was observed that caerulomycin a ( 0 . 05 - 0 . 1 μg / ml ) significantly suppressed the secretion of ifn - γ by ova - specific t cells . caerulomycin a failed to induce any change in the production of il - 10 . it was also interesting to notice that caerulomycin a suppressed the release of il - 4 by d10g4 . 1 th2 clones stimulated either with anti - cd3 ab ( 0 . 1 and 0 . 5 μg / ml ) or conalbumin pulsed apc . caerulomycin a inhibits the in vivo proliferation of antigen specific t cells ova ( 2 mg / ml ) was dissolved in pbs ( 0 . 01 m , ph 7 . 2 ) and emulsified in freund &# 39 ; s complete adjuvant ( fca ). emulsion ( 100 μl ) was then injected intraperitoneally ( i . p .) in 7 groups , comprising 5 balb / c mice in each set . four groups of animals were injected intraperitoneally daily with caerulomycin a ( 25 , 50 , 75 , 100 μg / 100 μl / mice ). the control groups were immunized intraperitoneally with 100 μl each of pbs and ethanol - pbs . after seven days , mice were sacrificed and splenocytes were isolated and pooled for in vitro proliferation . the cells isolated from the antigen - primed animals that were injected . caerulomycin a showed substantial inhibition in the proliferation as compared to the animals that were not administered drug . th2 clones ( 5 × 10 4 / well ) were stimulated with anti - cd3 ab ( 0 . 5 μg / ml ). caerulomycin a ( 0 . 05 μg / ml ) was added either at the initiation of the cultures ( time 0 ) or at various time points ( 4 h - 48 h ) after 48 h of the cultures , [ 3 h ]- thymidine was added , and its incorporation was measured 12 h later . caerulomycin a showed maximum inhibitory ( 77 - 90 %) effect on th2 cells if added before 16 h of the initiation of the cultures . the inhibitory effect of caerulomycin a was retained in the cultures when added even after 42 h ( 56 %). however , the response was lesser as compared to when the drug was added before 16 h . thus , indicating that caerulomycin a not only exerts its inhibitory effect on the activation events but also on the later stages of cell division . cells were seeded at 5 × 10 4 cells / well in flat - bottom 96 - well plates . the various stimuli and caerulomycin a were added at the initiation of the cultures . after 48 h , [ 3 h ]- thymidine ( 1 μci / well ) was added , and its incorporation was measured 12 h later . it was observed that caerulomycin a significantly inhibited the proliferation of calcium dependent pathway . cd28 and ctla - 4 expression was detected on the surface of the resting and cona activated cd4 + t cells by flow cytometric analysis . briefly , the splenocytes ( 3 × 10 6 cells / well ) were activated with ova ( 200 μg / ml ) or cona ( 1 μg / ml and 2 μg / ml ) and were incubated in the presence of different concentrations of caerulomycin a ( 0 . 0125 - 0 . 45 μg / ml ). the cultures were harvested after 24 , 48 , 72 and 96 h and cells were stained for the expression of cd4 , cd28 and ctla - 4 . further , cultures were also set where caerulomycin a was added after 24 h and 48 h of the initiation of cultures and the cells were stained after incubating further for 24 h . the cells were harvested and 3 - color staining was done using pe ( phycoerythrin ) conjugated anti - cd4 ab , fitc ( fluorescein isothiocyanate ) conjugated anti - ctla - 4 , and cy ( cy - chrome ) conjugated cd28 abs . the cells from each suspension were acquired on cellquest software of facscan ( becton dickinson , mountain view , calif .). debris in the cell suspension was excluded from the analysis by suitable gating that allowed the collection of data only from these light scattering events ( i . e . cells ) of a size consistent with lymphocytes . the analysis for the mean fluorescence intensity ( mfi ) was done on histograms where abcissa and ordinate denote log fluorescence and relative cell count , respectively . since we observed caerulomycin a mediated immunosuppression , therefore it became quite necessary to look into the mechanism of its action . it is very well established that ctla - 4 delivers immunosuppressive signals to t cells and cd28 delivers stimulatory signals for the proliferation . we therefore became curious to monitor the expression of ctla - 4 and cd28 on cd4 + t cells . it was interesting to observe that caerulomycin a not only significantly enhanced the expression but also the percentage of ctla - 4 positive cells . in contrast , it decreased the expression and percentage of cd28 . similar results were noticed in the case of non cd4 + t cells . thus , indicating that caerulomycin a suppresses the proliferation and cytokine secretion by cd4 + t cells by enhancing the expression of ctla - 4 and inhibiting the expression of cd28 . further , it was observed that caerulomycin a did not exhibit any significant change either in the expression or percentage change in lfa - 1 positive cells . the expression of cd69 was detected on the surface of thymocytes in the presence of caerulomycin a . thymocytes were obtained from 3 - 4 weeks old balb / c mice . thymocytes ( 4 × 10 6 cells / well ) were stimulated with 1 μg / ml and 5 μg / ml of cona for 24 h in the presence and absence of caerulomycin a at 37 ° c . the cells were stained with pe labeled anti - mouse cd69 ab . the stained cells were acquired on a facscan as mentioned in the case of cd28 / ctla - 4 . it is known that cd69 is an activation marker on t cells and thymocytes . interestingly , after addition of caerulomycin a there was no change in the expression of cd69 on the thymocytes . caerulomycin a modulates the expression of co - stimulatory molecules b7 - 1 , b7 - 2 and cd40 on macrophages the expression of b7 - 1 and b7 - 2 was detected on the surface of peritoneal macrophages incubated with different concentrations of caerulomycin a ( 0 . 05 - 0 . 1 μg / ml ). peritoneal macrophages were harvested from balb / c mice inoculated 4 days previously with 2 - 3 ml of thioglycolate . the cells were washed with bss . the macrophages were obtained by adhering for 1 h at 37 ° c . on plastic petri dishes followed by washing several times in cold bss . the macrophages ( 1 × 10 6 ) were stimulated with 10 μg / ml of lps for 24 , 48 and 72 h in the presence and absence of caerulomycin a ( 0 . 05 - 0 . 1 μg / ml ) at 37 ° c . and 7 % co 2 . then the cells were harvested and stained with their respective antibodies . briefly , the cells were centrifuged ( 1200 × g , 4 ° c ., 5 min ) and the supernatant was aspirated . the cells were washed 3 × with 1 % bsa , 0 . 1 % sodium azide in pbs . in the first step , the cells ( 1 × 10 6 ) were incubated with biotinylated anti - mouse b7 - 1 , cd40 and i - a d ( 1 μg / 100 μl ) abs for 45 min at 4 ° c . in the next step cells were stained with streptavidin fitc ( 0 . 5 μg / 100 μl ) or pe conjugated anti - mouse b7 - 2 ( 0 . 5 μg / 100 μl ) abs and incubated for 45 min . usual steps of washing were followed at each step . finally the cells were washed five times and fixed in paraformaldehyde . the stained cells were acquired on a facscan as mentioned for cd28 / ctla - 4 . since the antigen presenting cells express b7 - 1 and b7 - 2 , which are the ligands of ctla - 4 and cd28 . interestingly , caerulomycin a enhanced the expression of b7 - 1 and decreased the display of b7 - 2 molecule . in contrast , it increased the expression of b7 - 2 but no major change was observed in the case of b7 - 1 expression on j774 we also evaluated the role of caerulomycin a on the expression of another co stimulatory molecule cd40 . slight increase in the expression of cd40 was observed after 72 h . caerulomycin a down regulates the expression of ia d on macrophages the expression of ia d was detected on the surface of peritoneal macrophages incubated with different concentrations of caerulomycin a ( 0 . 05 - 0 . 1 μg / ml ). peritoneal macrophages were harvested from balb / c mice inoculated 4 days previously with 2 - 3 ml of thioglycolate . the cells were washed with bss . the macrophages were obtained by adhering for 1 h at 37 ° c . on plastic petri dishes followed by washing several times in cold bss . the macrophages ( 1 × 10 6 ) were stimulated with 10 μg / ml of lps for 24 , 48 and 72 h in the presence and absence of caerulomycin a ( 0 . 05 - 0 . 1 μg / ml ) at 37 ° c . and 7 % co 2 . then the cells were harvested and stained with their respective antibodies . briefly , the cells were centrifuged ( 1200 × g , 4 ° c ., 5 min ) and the supernatant was aspirated . the cells were washed 3 × with 1 % bsa , 0 . 1 % sodium azide in pbs . in the first step , the cells ( 1 × 10 6 ) were incubated with biotinylated anti - mouse i - a d ( 1 μg / 100 μl ) abs for 45 min at 4 ° c . in the next step cells were stained with streptavidin fitc ( 0 . 5 μg / 100 μl ) and incubated for 45 min . usual steps of washing were followed at each step . finally the cells were washed five times and fixed in paraformaldehyde . the stained cells were acquired on a facscan as mentioned for cd28 / ctla - 4 . it is of interest to mention here that caerulomycin a down regulated the expression of ia d . this may indicate that caerulomycin a may inhibit the processing and presentation of antigen by apc . ovalbumin ( 3 mg / ml ) was dissolved in pbs ( ph 7 . 2 ) and emulsified in freund &# 39 ; s complete adjuvant ( fca ). emulsion ( 100 μl ) was then injected intraperitoneally ( i . p .) in different groups , comprising 5 balb / c mice in each set . four groups of animals were injected intraperitoneally daily with caerulomycin a ( 25 , 50 , 75 , 100 μg / 100 μl / mice ). the control groups were immunized intraperitoneally with ethanol - pbs . after seven days , mice were sacrificed and splenocytes were pooled and used for proliferation assays . splenocytes ( 2 × 10 5 cells / well ) were cultured with ova ( 100 μg / ml ) in 200 μl rpmiifcs - 10 % medium and with various concentrations of caerulomycin a ( 0 . 1 - 0 . 0003 μg / ml ). the control cultures consisting of splenocytes incubated with different concentrations of caerulomycin a ( no ova ), ova and medium were also kept . after 72 h , the cultures were pulsed with 1 μci of [ 3 h ]- thymidine and harvested 14 h later . radioactivity incorporated was measured by liquid scintillation counting and data are expressed as mean counts per minute ( cpm ). caerulomycin a inhibits the in vivo proliferation of antigen specific t cells . the mice were administered with different doses of caerulomycin a for 7 days ( fig1 ). the animals were sacrificed and splenocytes were cultured in vitro with antigen and different concentrations of caerulomycin a ( 0 . 00625 - 0 . 10 μg / ml ). interestingly , cells isolated from the animals injected with all the four concentrations ( 25 - 100 μg / mice / day ) of caerulomycin a showed substantial inhibition in the proliferation as compared to the animals that were not administered with drug ( fig1 ). the decrease in the proliferation was observed in a dose dependent manner . nearly complete inhibition was observed when cells were incubated with 0 . 10 μg / ml of caerulomycin a . in another set of experiments , the cells were isolated from the animals primed with antigen and later on administered either pbs - ethanol or caerulomycin a ( 25 μg / mice / day ) for 7 days ( fig2 ). the cells isolated from the mice injected with caerulomycin a showed significant level of retardation in the growth as compared to the cells isolated from the animals immunized with pbs - ethanol ( fig2 ). the following examples are given by way of illustration of the present invention and therefore should not be construed to limit the scope of the present invention . prepare a seed culture inoculating test tubes with 5 ml of a medium having the following composition : glucose 5 . 4 g , yeast extract 4 . 8 g , malt extract 8 . 5 g , caco 3 3 . 0 g per liter in distilled water . adjust ph to 7 . 2 , sterilize the broth and after cooling add a frozen culture of actinoalloteichus spitiensis sp . nov . cultivate the bacterium at 28 ° c . for 30 hours with orbital agitation at 200 rpm . inoculate aseptically with 7 . 5 ml of the above culture in a shaking flask of 1000 ml capacity with 100 ml of sterile culture medium as defined below : glucose 5 . 4 g , yeast extract 4 . 8 g , malt extract 8 . 5 g , caco 3 3 . 0 g per liter in distilled water . adjust ph to 7 . 0 with an alkali solution . cultivate the bacterium at 28 ° c . for 30 hours with orbital agitation at 200 rpm . sterilize at 122 ° c . for 30 minutes a fermenter of 7 liters capacity with 5 liters of the production medium described as below : glucose 5 . 4 g , yeast extract 4 . 8 g , malt extract 8 . 5 g , caco 3 3 . 0 g per liter in distilled water . inoculate the fermenter with 500 ml of the second stage inoculum . incubate the fermentation culture at 28 ° c . with an agitation of 220 rpm with 1v / v / m aeration . after completion of the cultivation , remove the solids by centrifugation . extract the supernatant portion ( 4 . 8 liters ) twice with 2 . 5 liters of ethyl acetate . desiccate the combined organic phases with sodium sulfate , filter and concentrate to dryness under vacuum . dissolve the crude residue in 80 ml of water : methanol ( 1 : 1 ) which was defatted by partitioning twice with 50 ml of hexane . extract the water / alcohol fraction twice with 50 ml of ethyl acetate . concentrate the organic solvent in an evaporator yielding a crude residue containing caerulomycin a . dissolve the crude residue in 5 ml acetone and chromatograph on silica gel ( 32 - 63 μm ) by a high performance flash chromatography system ( horizon hpfc system , biotage , usa ) using a mixture of toluene / acetone 75 : 25 as the eluting solvent . combine similar fractions on the basis of tlc analysis , using chloroform - methanol - 20 % aqueous ammonia 95 : 4 : 1 as the mobile phase . examples establishing the role of caerulomycin a [ isolated from a novel species of actinoalloteichus ] in inducing immunosuppression based on the following : splenocytes of balb / c mice were stimulated with either cona ( 1 μg / ml and 2 μg / ml ) or lps ( 5 μg / ml and 10 μg / ml ) and were cultured with different concentrations of caerulomycin a ( 0 . 0003 - 0 . 1 μg / ml ). after 48 h , the cultures were pulsed with 0 . 5 μci of [ 3 h ]- thymidine and harvested 14 h later by automatic cell harvester . radioactivity incorporated was measured by liquid scintillation counting . antigen ovalbumin ( 100 μg / ml ) emulsified in freund &# 39 ; s complete adjuvant was injected in a group consisting of 5 balb / c mice . the control group was injected with pbs alone . after seven days , mice were sacrificed and splenocytes were pooled and used for proliferation and cytokine assays . splenocytes were cultured with ovalbumin ( 200 μg / ml ) in 200 μl rpmi / fcs - 10 % medium and with various concentrations of caerulomycin a ( 0 . 0003 - 0 . 1 μg / ml ). after 72 h , the cultures were pulsed with 1 μci of [ 3 h ]- thymidine and harvested 14 h later . radioactivity incorporated was measured by liquid scintillation . the culture supernatants were collected after 48 h and cytokines were measured by elisa . immunosuppressive effect of different doses of caerulomycin a was monitored on naïve cd4 + t cells , antigen reactive cd4 + t cells , th2 clone and th1 hybridoma . the cells were incubated with γ - irradiated ( 3000 rads ) and antigen - pulsed splenocytes and various concentrations of caerulomycin a ( 0 . 00625 - 0 . 1 μg / ml ). the cultures were incubated at 37 ° c . and 7 % co 2 . after 72 h , the cultures were pulsed with 0 . 5 μci of [ 3 h ]- thymidine and harvested 14 h later . radioactivity incorporated was measured by liquid scintillation . immunosuppressive effect of caerulomycin a on mlr reaction . the mlr was performed using balb / c splenocytes ( responder cells ) and γ - irradiated c57bl / 6j splenocytes ( stimulator cells ) with various concentrations of caerulomycin a ( 0 . 0125 - 1 μg / ml ) and a positive control of cyclosporin a ( 0 . 0125 - 10 - 82 g / ml ). after 4 days , the cultures were pulsed with 1 μci of [ 3 h ]- thymidine and harvested 16 h later and radioactivity incorporated was measured by liquid scintillation counting . in vivo immunosuppressive effect of caerulomycin a on the proliferation of t cells . ovalbumin was emulsified in freund &# 39 ; s complete adjuvant and injected ( 100 μg / 100 μl ) intraperitoneally in different groups ( 5 mice / group ) of animals . different groups of antigen - primed animals were daily injected with caerulomycin a ( 25 , 50 , 75 , 100 μg / 100 μl / mice ). after seven days , mice were sacrificed and splenocytes were isolated , pooled for in vitro proliferation . caerulomycin a induces immunosuppression by up regulating ctla - 4 and inhibiting cd28 expression on t cells . cd28 and ctla - 4 expression was detected by flowcytometry on the surface of resting and cona activated cd4 + t cells . the splenocytes were activated with either antigen ( ova ) or mitogen ( cona ) and were incubated for different duration ( 24 , 48 , 72 and 96 h ) with caerulomycin a ( 0 . 0125 - 0 . 45 μg / ml ). the cultures were harvested after 24 , 48 , 72 and 96 h and the expression of cd28 and ctla - 4 on cd4 + t cells was evaluated by 3 - color staining using pe ( phycoerythrin ) conjugated anti - cd4 ab , fitc ( fluorescein isothiocyanate ) conjugated anti - ctla - 4 ab and cy ( cy - chrome ) conjugated anti - cd28 ab . similarly , expression of b7 - 1 , b7 - 2 and i - a d was also monitored by flowcytometry on peritoneal macrophages as mentioned in no . 6 . the compound caerulomycin of the general formula 1 , derivatives and pharmaceutically acceptable salts thereof are effective as immunosuppressive agents . ( i ) since cd4 + t cell plays a crucial role in the initiation and regulation of immune responses , inhibition of their activation provides a powerful approach for immunosuppressive therapy . this has been very well documented in the case of many other immunosuppressive drugs and their treatment for autoimmunity ( galvin et al . 1993 , thomson 1991 , crespo - leiro 2003 ). moreover , the role of cd4 + t cells is very well documented in many autoimmune diseases ( abbas et al . 2004 , wraith et al . 2004 ). since caerulomycin a suppressed the proliferation and cytokines ( il - 4 and ifn - γ ) secretion by naïve and antigen specific effector cd4 + t cells , therefore it will be quite promising drug in autoimmune diseases . further , caerulomycin a significantly augmented ctla - 4 and subsequently down - regulated cd28 expression on t cells . we also noticed increased quantity of cd4 + t cells expressing ctla - 4 and decrease in the number of cd28 positive cells . similar results were observed in the case of non - cd4 + t cells . there is a growing appreciation for the concept that lymphocyte responses are suppressed by ctla - 4 mediated inhibitory signals ( krummel et al . 1996 , leibson 2004 ). 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