Patent Application: US-15990205-A

Abstract:
the present invention relates to the field of immunomodulation . more particularly , the present invention relates to the identification and isolation of a polypeptide derived from trypanosomes that can be used to modulate the immune response in mammals .

Description:
we previously reported that upon interaction with opsonized trypanosoma brucei brucei parasites , the macrophage hybridoma cell line ( 2c11 - 12 ) exhibits a suppressive activity on con a - induced lymph node cell proliferation . additionally , soluble extracts of t b . brucei triggered the 2c11 - 12 cell line to secrete tnf - α and to exert suppressive activities ( 28 ). to identify molecule ( s ) responsible for these immunomodulatory activities , proteins present in t . b . brucei bloodstream lysate were fractionated by sequential ammonium sulfate precipitation . the presence of a macrophage - suppressive capacity was observed in the 35 - 55 % ammonium sulfate fraction . this fraction was further fractionated by high performance liquid chromatography ( hplc ). fig1 , panel a , shows that mainly the hplc fraction 4 from 35 - 55 % ammonium sulfate precipitation triggered the 2c11 - 12 cell line to secrete tnf - α and to inhibit mitogen - induced t - cell proliferation . both activities were abolished by treating the hplc fraction 4 with pronase , indicating the involvement of protein ( s ) in the macrophage - activating capacity . furthermore , this fraction elicited suppressive macrophages in vivo , since peritoneal macrophages from mice treated intraperitoneally ( i . p .) with this fraction inhibited con a - induced t - cell proliferation ( fig1 , panel b ). rabbit polyclonal antibodies generated against hplc fraction 4 inhibited its suppressive activity . to identify gene ( s ) encoding the putative trypanosoma immunomodulatory molecule ( s ), a bloodstream t . b . brucei cdna expression library was screened with polyclonal anti - hplc fraction 4 serum . from the eight positive phage clones isolated , five harbored sequences encoding for a t . b . brucei basal body component ( 38 ). the other three clones had no homology with sequences reported in data banks . cdna from all clones were expressed as gst - fusion proteins and their capacity to elicit suppressive cells inhibiting con a - induced t - cell proliferation was evaluated in vivo . the only clone exerting suppressive activity was selected for further research . the sequence of this clone revealed that the fragment of 1 . 65 - kb represents the 3 ′ end of the gene . the missing 5 ′ end of this cdna was amplified by rt - pcr using a gene - specific internal primer and a miniexon primer common to all trypanosome mrna . the full - length cdna with an open reading frame of 2499 - bp had an atg codon located 256 - bp downstream of the miniexon sequence and thus encoded a theoretical protein of 833 amino acids ( fig2 a ). the deduced protein with a molecular mass of 92 - kda had an isoelectric point of 4 . 75 , 32 cysteine residues and five potential n - glycosylation sites ( fig2 a ). hydropathy analysis suggested that the protein contained at least three highly hydrophobic regions with length to be membrane spanning ( fig2 b ). furthermore computational analysis suggested the presence of an internal signal sequence between amino acids 230 - 290 . a blast search with the deduced amino acid sequence of the newly identified protein revealed no homology with known proteins . this protein was named tsif ( trypanosoma suppressive immunomodulating factor ). southern blot analysis of t b . brucei dna indicated a restriction pattern characteristic of a single copy gene , since digestion with restriction enzymes that cut once in the tsif orf generated fragments of dissimilar sizes . hybridization with genomic dna from various species belonging to the genus trypanosomatidae demonstrated the presence of the tsif gene only in species from the subgenus trypanosoma , suggesting no difference among the species regarding gene number and / or organization . no hybridization signal was observed with t . cruzi , t . congolense , leishmania or crithidia parasites ( fig3 , panel a ). northern blot analysis revealed that tsif was transcribed as a ˜ 2 . 9 kb mrna in t b . brucei bloodstream and procyclic forms ( fig3 , panel b ). expression of the complete tsif orf encountered difficulties . however , a protein containing an n - terminal histidine tag and encoding the c - terminal part of tsif was generated ( rc - tsif , amino acids 543 - 833 ). a mab elicited against rc - tsif was used to analyze the cellular localization of native tsif in the parasite . in immunofluorescence studies , this antibody showed uniform staining over the entire bloodstream t . b . brucei surface under conditions where the membrane was not permeabilized ( fig3 , panel c ), suggesting that the native tsif protein is located in the parasite membrane . these results were confirmed by surface - labeling immunoprecipitation . indeed , following incubation of surface - labeled t . b . brucei bloodstream forms lysate with anti - rc - tsif mab , a protein showing an apparent molecular mass of 70 - kda was identified ( fig3 , panel d ). immunoblot analysis using anti - rc - tsif mab revealed a protein with a similar mass in total lysate from 10 7 parasites ( fig3 , panel e ). this size significantly differed from the 92 - kda molecular mass expected from the tsif cdna sequence . the likely interpretation of this discrepancy is that the 70 - kda component was processed from a 92 - kda precursor , as suggested by the detection of both components when trypanosome extracts ( equivalent to 100 times more parasites ) were bound to a deae column , then eluted by salt gradient ( fig3 , panel e ). these data corroborate the computer prediction of a putative internal signal sequence between amino acids 230 - 290 that may result in the synthesis of a mature tsif with an approximate molecular mass of 70 - kda . based on the above information , rtsif comprising the amino acids 280 - 833 and corresponding to the putative mature tsif protein was engineered , using the same procedure as for c - tsif . the macrophage - activating potential of rtsif was tested on thioglycollate - elicited peritoneal macrophages , measuring cytokine and no secretions into the cell culture medium . as shown in fig4 , rtsif exerted high macrophage - activating activity as monitored by secretion of tnf - a and this activity was dose - dependent with a response starting at 0 . 1 μg / ml . besides the production of the pro - inflammatory cytokine tnf - α , the rtsif activated macrophages to produce high levels of the pleiotropic cytokine il - 6 . the induction of these cytokines was paralleled by the up - regulation of no in cell supernatants . low levels of il - 10 were observed in the supernatants of rtsif - activated macrophages ( not shown ). in vitro pre - activation of macrophages with ifn - γ induced higher secretions of cytokines and no upon interaction with rtsif . to ensure that the activation of macrophages did not result from lps contamination , different controls were undertaken : ( i ) degradation of rtsif with pronase eliminated its no and cytokine - inciucing activity ( fig4 ); ( ii ) co - incubation with the lps - inhibitor polymyxin b had no effect on the macrophage - activating potential of rtsif ( fig4 ); ( iii ) the cytokine and no - inducing activities of rtsif were corroborated in the lps - hyporesponsive c3h / hej mouse strain . collectively , these data indicate that rtsif activates macrophages to secrete pro - inflammatory molecules . we investigated whether the strong macrophage - activating potential of rtsif may trigger a suppressive state in vivo . peritoneal cells from mice treated i . p . with rtsif were co - cultured with con a - activated lymph node cells and proliferation was monitored . peritoneal cells from mice injected with rtsif significantly suppressed t - cell proliferation (& gt ; 90 %) as compared with peritoneal cells from pbs - treated mice . polyclonal antibodies elicited against hplc fraction 4 and used to identify tsif in t . b . brucei cdna library inhibited the in vivo suppressive capacity of rtsif ( fig5 , panel a ). additionally , rtsif rendered the macrophage cell line 2c11 - 12 suppressive . previous studies showed macrophages inhibit t - cell activities via diverse mediators including prostaglandins , h 2 o 2 , no and cytokines such as ifn - γ , tgf - β , il - 10 and tnf - α . the contribution of these mediators towards the suppressive activity of rtsif - treated peritoneal cells was evaluated through addition of blocking agents , respectively , indomethacin , catalase , l - nmma and neutralizing anti - ifn - γ , tgf - β , il - 10 , and tnf - α antibodies . neutralizing anti - il - 6 antibody was used as well in view of the induction of il - 6 secretion by rtsif . results indicate that suppression was mainly mediated by no and ifn - γ , since the presence of the inos inhibitor l - nmma and anti - ifn - γ antibodies in the co - cultures completely restored the proliferative capacity of con a - activated lymph node cells . this was somehow surprising since similar levels of no and ifn - γ were observed in co - cultures of lymph node cells and peritoneal cells from rtsif -( 36 ± 10 μm no 2 7900 ± 700 pg / ml ifn - γ ) and pbs - treated mice ( 30 ± 3 μm no 2 , 8500 ± 500 μg / ml ifn - γ ). we envisaged whether the rtsif - elicited immunosuppressive activity involved a cell - cell contact . for that purpose , con a - stimulated lymph node cells and peritoneal cells from mice treated i . p . with rtsif were cultured in different compartments of transwell plates . fig5 , panel c , clearly indicates that suppression only occurred when suppressive cells were in contact with the mitogen - responding population . thus , in addition to ifn - γ and no , other signal ( s ) mediated by membrane - bound molecules are required to inhibit proliferation . in view of the capacity of rtsif to elicit suppressive cells in vivo , we subsequently evaluated the influence of rtsif on antigen - specific immune responses . to this end , two approaches were undertaken . first , ovalbumin ( ova ) was injected intra foot pad in mice that were pre - treated , either with rtsif or with pbs ( control ). one week later , the draining popliteal lymph nodes were re - stimulated in vitro with ova and productions of il - 4 , il - 10 and ifn - γ were evaluated . as shown in fig6 , control mice immunized with ova mounted a strong type ii immune response characterized by secretions of il - 4 and il - 10 , while ova - immunized mice pre - treated with rtsif exhibited significantly lower ova - induced type ii cytokine responses . the levels of the type i cytokine ifn - γ were lightly increased in rtsif - pretreated mice . these results indicate that rtsif modulates antigen - specific type ii immune responses . second , mice were co - immunized intramuscularly with plasmids encoding ova and tsif genes . two weeks after the last immunization , spleen cells were stimulated ex vivo with ova . ova - specific t - cell proliferation and cytokine secretion in cell culture supernatants were evaluated . results indicate that tsif dna co - immunization suppressed ova - specific t - cell proliferation ( fig7 ). moreover , while spleen cells from mice immunized with ova dna secreted high levels of type i cytokine ( ifn - γ ) and marginal levels of type ii cytokines ( il - 4 , il - 10 ), cells from mice co - immunized with ova and tsif dna did not produce ifn - γ in response to ova . the secretion of type ii cytokines was not affected in ova and tsif dna co - immunized animals . together , these data suggest that depending on the way of administration / delivery through the body , tsif can suppress proliferation and type i or type ii cytokine secretion by memory cells specific for trypanosome - unrelated antigens . the ability of distinct rtsif peptides to interact with macrophages was evaluated . to this end , peptides corresponding to the n - terminal ( amino acid 46 - 269 , rd5 ) or the c - terminal ( amino acid 266 - 533 , r06 ) part of rtsif were generated . the macrophage - activating potential of these peptides was tested on thioglycollate - elicited peritoneal macrophages , measuring tnf - α secretion into the cell culture ( fig8 , top panel ). the c - terminal fragment rc6 induced the secretion of similar amounts of tnf - α as rtsif , while the n - terminal fragment rd5 induced a marginal level of the cytokine . moreover , peritoneal cells from mice injected with rc6 exerted a higher suppressive activity on con a - induced lymph node cell proliferation than peritoneal cells from mice injected with rd5 . together , these data suggest that distinct parts of the rtsif protein differentially modulate the activity of immune cells . for instance , the tnf - inducing , as well as the suppression - inducing , potential of rtsif mainly resides in the c - terminal part of the molecule . to test the potential of the single copy tsif gene as target for a sensitive diagnostic pcr assay , several pairs of primers were designed based on the tsif sequence obtained from stock antat 1 . 1 t . brucei ( j . gomez , department of immunology , parasitology and ultrastructure , flemish interuniversity institute for biotechnology , free university brussels ( vub ), paardenstraat 65 , b - 1640 st - genesius - rode , belgium ). in order to optimize the assay in terms of sensitivity and specificity , parameters for both single and nested pcr assays were chosen , based on the length of the fragment to be amplified and the nature of the primers used . pcr amplification was optimized using extracted parasite dna , followed by experiments on normal blood samples spiked with parasite dna . the pcr assay was optimized for t . evansi strain itmas 110297 . in an initial experiment , a comparative pcr was performed on a dilution series of dna samples , made to contain the equivalent of 0 up to 50 , 000 parasite genomes . a single pcr was performed using single pairs of primers ( dtsif a / s + dtsif a / as ; dtsif b / s + dtsif b / as ; dtsif c / s + dtsif c / as ). in parallel , a nested pcr was performed using primer pair tsif op / s + tsif op / as for the outer pcr reaction . the inner pcr was subsequently performed with either of the three above - mentioned primer pairs using 1 / 10 th of the first round pcr product as template . a single pcr resulted in amplification of the expected fragment until 5 pg purified parasite dna was used per reaction . nested pcr increased the sensitivity until 0 . 2 pg per reaction . this was for all three primer sets used . this is equivalent to a genome content of two parasites , assuming that one trypanosome parasite has a dna content of 0 . 1 pg ( 39 ). the sensitivity of primer set dtsif a tended to be slightly higher than that obtained with the two other primer sets since a weak , but clear , fragment was seen upon pcr amplification from 0 . 1 pg dna . however , it cannot be ruled out that a certain ambiguity in the accuracy in serial dilutions in relation to exact numbers of parasites may account for the observed difference in sensitivity . pcr detection of t . evansi in blood samples using tsif and esag7 as target for pcr technology to be used in the diagnosis of trypanosomiasis , it must be adapted for the detection of parasites in samples of whole peripheral blood . to this end , edta - treated whole blood was spiked with different amounts of either purified trypanosomes or dna purified using the commercially available qiaamp blood kit ( qiagen ) or the sds - proteinase k method . the tsif - based nested pcr assay yielded a positive signal down to 50 trypanosomes / ml . however , using nested pcr to amplify conserved multicopy esag7 dna target sequences could further increase the sensitivity to ten parasites / ml blood . overall , the sensitivity of the pcr assay was similar for both dna isolation methods used . attempts to further increase the sensitivity using different sets of conditions were not successful . in order to evaluate the specificity of the tsif - and esag7 - based pcr detection of trypanosoma species , a library containing dna from 37 representative trypanosome stocks was established . in addition , normal blood from mouse and human was tested as well as dna from leishmania species . no cross - reactions with dna of normal blood samples from mouse or human were found upon single pcr amplification with primer sets dtsif a and esag7 , nor were there any cross - reactions with the leishmania parasites . on the other hand , all trypanosomes belonging to the subgenus trypanozoon produced a specific pcr band of the expected size when using the dtsif a primer set , which could , therefore , be useful in the detection of these related parasites . the esag7 primer set scored positive for all trypanosomes belonging to the trypanozoon subgenus and also for t . congolense trt55 , a stock belonging to the subgenus dutonella . in order to assess the efficacy of the tsif a and esag7 - based pcr for the clinical follow - up of patients or animals upon drug treatment , an experimental drug treatment protocol was set up . female f1 mice were inoculated with 10 4 t . b . brucei antat 1 . 1 itmas 241195 , a pleiomorphic clone that produces chronic infections in mice . treatment with dfmo was initiated three weeks after infection , the period involving cerebral trypanosomiasis , and administration of melarsoprol was initiated one week after dfmo treatment . a control group of five mice that did not receive any drug therapy following infection died of the disease 35 - 38 days post - infection . blood smears were scored positive for the presence of parasites starting at three days after experimental infection . in contrast , specific pcr products could be amplified as early as one day post - infection in blood samples and remained positive during infection although aparasitemic phases occurred . indeed , parasitemia plunged below microscopical detection level during these phases whereas pcr signals remained positive . after three weeks of infection , parasites were also found in the brains , both by microscopic analysis and pcr detection . moreover , balb / c mice injected intraperitoneally with brain homogenates of the infected animals showed positive parasitemia after six days of injection , confirming cerebral disease . pcr and microscopic detection of parasites in the blood from all mice remained positive during the first two days of dmfo treatment . subsequently , blood smears from some mice were negative by microscopic examination and eventually became all negative after eight days of treatment . during this period , all blood samples gave positive pcr signals when using both the tsif a or esag primer sets . as soon as one day after treatment with melarsoprol , tsif a - based pcr signals disappeared totally in blood , spleen , lymph nodes and brains . with the esag primer set , a positive signal was found in the blood and the brain of a mouse after one and two days of melarsoprol treatment , respectively . no trypanosome dna amplification signal was demonstrated beyond this time point in any of the clinical samples collected until 60 days post - treatment . also , the inoculation of homogenates of brain tissue taken 60 days after end of treatment failed to establish infection in susceptible mice , thus confirming successful elimination of the parasites . clinical samples from control mice that only received therapy were also analyzed by tsif a and esag - based pcr and remained negative throughout the whole treatment and follow - up period . tsif modulates a th2 cytokine - mediated airway inflammatory response in ovalbumin ( ova )- sensitized mice allergic asthma , a complex and chronic disease , is characterized by inflammation of the bronchial mucosa , involving activated eosinophils , mast cells , and cd4 + t - cells , as well as airway hyperresponsiveness , reversible bronchoconstriction , and elevated titers of circulating ige . observations from mouse models of allergic respiratory inflammation identified pulmonary cytokines characteristic of the th2 subset of cd4 + t - cells , mainly il - 4 , il - 5 , il - 9 , and il - 13 , as crucial actors in the etiology of the human disease ( yssel and groux , int . arch . allergy immunol . 2000 , 121 : 10 ). yet , the development of therapies targeted at neutralizing specific cytokines is hampered by intercytokine functional redundancy , thus limiting the effectiveness of the treatment ( riffo - vasquez and spina , pharmacol . ther . 2002 , 94 : 185 ). anti - ige therapy has demonstrated limited clinical efficacy , exerting its action by reducing the amount of free ige available to bind to effector cells ( busse et al ., j . allergy clin . immunol . 2001 , 108 : 184 ). in this regard , modulating upstream processes , such as the preferential th2 skewing in responses to inhaled allergen , may represent an alternative to prevent the development of the disease . the present example examines whether rtsif alters the th2 cell - mediated allergic asthma and reduces eosinophilic lung inflammation . recombinant proteins with n - terminal histidine tags corresponding to amino acids 280 - 833 ( rtsif ) are prepared by using the prset / e . coli bl21 ( de3 ) system ( invitrogen ). transformed cells are induced for 5 hours in the presence of 1 mm ( iptg ), then harvested and sonicated . proteins present in inclusion bodies are dissolved in binding buffer ( 8 m urea - 0 . 15 m nacl - 50 mm tris - hcl ph 8 . 0 ) and purified by using ni - nitrilotriacetic acid ( nta ) resin ( qiagen ). columns are washed with 50 - column volumes of buffer containing decreasing concentrations of urea ( 8 - 6 - 2 m in 0 . 5 m nacl - 50 mm tris - hcl ph 6 . 5 ). bound proteins were eluted ( 0 . 5 m imidazole - 2 m urea - 0 . 1 m nacl - 50 mm tris - hcl ph 6 . 5 ) and additionally purified by hplc superdex 75 column ( pharmacia ) in pbs . endotoxin contaminations , determined using limulus amoebocyte lysate ( lal ) assay ( biowhittaker ), were below 20 units / mg protein . the amount of rtsif produced as such is in the order of 1 to 5 mg and takes about two weeks . balb / c mice are sensitized by three intraperitoneal injections of 10 μg ova adsorbed to 1 mg al ( oh ) 3 on days 0 , 7 and 14 . on day 21 , sensitized mice ( five per experimental group ) are challenged with 10 μg ova admixed or not with 5 μg rtsif in 30 μl pbs via the intranasal route . a third group of sensitized animals is treated with rtsif one day before ova challenge ( i . e . at day 20 ). the fourth experimental group consists of sensitized animals treated with rtsif at day 21 but not challenged with ova . the last group consists of non - sensitized mice challenged with ova . bronchoalveolar ravages ( bal ) are performed 48 hours after the last challenge and cytospin preparations are stained with may - grünwald giemsa as described in sehra et al . ( j . immunol . 2003 , 171 : 2080 ). cytokine production is estimated by stimulating bal cells ( 10 6 / ml , in triplicate ) with 1 μg / ml anti - cd3 ( clone 2c11 ) and 1 μg / ml anti - cd28 ( clone 37 . 51 ) monoclonal antibody . twenty - four hours later , il - 4 , il - 5 , il - 13 , il - 10 , il - 12 p70 , tnf and ifn - γ are determined by cytokine - specific elisa . trypanosome bloodstream forms were cultivated in rats or mice starting from a cryostabilate kept on liquid nitrogen . trypanosoma theileri was cultured in vitro as described by verloo et al . ( 40 ). blood specimens were collected by heart puncture on edta . visual inspection of parasites in the blood samples was performed by microscopic observation of at least 20 fields at 400 × magnification . the parasitemia was estimated by the matching method ( 41 ). for the optimization of the pcr - based diagnosis , we used t . evansi antat 3 . 1 itmas 270274c , a pleiomorphic clone kindly provided by n . van meirvenne ( prince leopold institute of tropical medicine , antwerp , belgium ). this strain had been derived from trypanosomes isolated from blood of the hydrochoerus hydrochoeris in south america in 1969 and produces a chronic infection in mice . the organism was maintained by serial passages in mice . trypanosomes were purified from infected mouse blood by deae - 52 anion - exchange column chromatography using phosphate - saline - glucose ( psg ) solution as eluting buffer ( 42 ). purified trypanosomes were kept at − 80 ° c . dna from trypanosome cells was purified by the sodium dodecyl sulphate ( sds )- proteinase k method ( gross - bellard et al ., 1973to find = 43 ). serial dilutions were prepared in 10 mmtris - hcl , ph 8 . 0 and used as template for pcr amplification . dna from total blood treated with edta was isolated using two methods : sds - proteinase k lysis method : dna from 100 μl fresh total blood was extracted in 500 μl blood lysis buffer ( 10 mm tris - hcl ph 7 . 6 , 10 mm edta , 0 . 1 m nacl , 0 . 5 % sds , 300 μg / ml proteinase k ) for 2 hours at 55 ° c . samples were subsequently extracted twice with a mixture of phenol , chloroform , and isoamylalcohol ( 25 : 24 : 1 ). sodium acetate was added to give a final concentration of 0 . 3 m and the nucleic acids were precipitated with two volumes of ethanol . after centrifugation for 15 minutes at 12 , 000 × g , the dna pellets were rinsed with 70 % ethanol and dissolved in 100 μl sterile water . qiaamp blood kit ( qiagen ): this nucleic acid preparation kit combines the selective binding properties of a silica gel - based membrane with the speed of microspin technology . blood samples were treated freshly or after preservation by mixing with an equal volume of stabilizing buffer as - 1 reagent ( qiagen ). this procedure allows storage of blood samples for up to 12 weeks at temperatures up to 37 ° c . dna was extracted from 200 μl blood according to the manufacturer &# 39 ; s recommendations . primer pair tsif op / s ( 5 ′- cagtagccgtcttctccctgaatg - 3 ′)+ tsif op / as ( 5 ′- atgttggtcacgcgcagttccgtg - 3 ′) was used in the outer pcr , yielding a pcr product of 2 . 1 kb . primer pairs dtsif a / s ( 5 ′- gtgagtgttcttgcaacctt cc - 3 ′)+ dtsif a / as ( 5 ′- gaagttgtaacagactgcagcg - 3 ′), dtsif b / s ( 5 ′- gttctagtcgatcgagctca cc - 3 ′)+ dtsif b / as ( 5 ′- agggtgtgcgtcagtgtatacc - 3 ′) and dtsif c / s ( 5 ′- aggtatacactgacgcacaccc - 3 ′)+ dtsif c / as ( 5 ′- taagagcctcggtctttag tgg - 3 ′) were used for inner pcr , yielding pcr products of 314 bp , 328 bp and 268 bp , respectively . esag7 primers were designed from the known sequence of t . brucei rhodesiense cdna , encoding the transferrin - binding protein ( 23 ). primer pair esag7 op / s ( 5 ′- gaggttttggtttgtgttgttg - 3 ′)+ esag7 op / as ( 5 ′- agtatagttgaattcgcttttac - 3 ′) was used in the outer pcr , yielding a pcr product of 1 . 3 kb . primer pair esag7 / s ( 5 ′- acattccagcaggagttggag - 3 ′)+ esag7 / as ( 5 ′- cacgtgaatcctcaattttgt - 3 ′) was used for the inner pcr , yielding a pcr product of 238 bp . initially , the pcr conditions were optimized on serial dilutions of purified parasite dna . dna samples were amplified in a reaction mixture containing 1 × pcr buffer ( 20 mm tris - hcl ph 8 . 4 , 50 mm kcl ), 1 . 5 mm mgcl 2 , 200 μm each of the four dntps , 0 . 5 μm each of the primers and 1 . 25 units of platinum ™ taq dna polymerase ( gibco brl life technologies , merelbeke , belgium ). outer pcr reactions were performed on 10 μl template and 5 μl outer pcr product was subsequently used as template in the inner pcr reaction . five μl of distilled water was added to pcr buffer as a negative amplification control . for tsif - based amplification , samples were incubated at 94 ° c . for 3 minutes as initial denaturation step , followed by 40 cycles of 1 minute at 94 ° c ., 45 seconds at 60 ° c . and 1 minute 30 seconds at 72 ° c ., and a final extension at 72 ° c . for 5 minutes . nested pcr amplification was performed by an initial denaturation at 94 ° c . for 3 minutes , followed by 35 cycles of 1 minute at 94 ° c ., 45 seconds at 60 ° c ., and 1 minute at 72 ° c ., and a final extension at 72 ° c . for 5 minutes . outer pcr for esag7 was performed using the same conditions as for tsif - based amplification with the exception that the annealing of the primers was performed at 55 ° c . a 15 - μl sample of each pcr product was finally tested by agarose gel electrophoresis . experimental infections in mice were performed with t . b . brucei antat 1 . 1e itmas 241195 kindly provided by dr . n . van meirvenne ( itg , belgium ). this pleiomorphic clone has been derived from trypanosomes isolated from blood of the tragelaphus scriptus in uganda in 1966 and produces a chronic infection in mice , allowing them to survive for at least 40 days if untreated . female f1 mice were inoculated intraperitoneally with 10 4 t . b . brucei antat 1 . 1e parasites and blood parasitemia was followed by microscopic analysis and pcr . three weeks after infection , when the central nervous system became infected and blood parasitemia reached about 10 6 parasites / ml blood , the animals were treated for 15 consecutive days with 2 % dfmo in the drinking water . mice consumed an average of 5 ml of 2 % drug solution / animal / day yielding a dose rate of 3 mg / g of body weight / day . one week after initiation of dfmo treatment , melarsoprol was administered intravenously for four consecutive days at a dose of 14 . 4 mg melarsoprol / kg body weight . great care was taken to ensure no perivenous drug leakage to avoid phlebitis . blood samples were taken throughout the infection and after treatment for microscopy and pcr . during therapy and at the end of the follow - 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