Patent Application: US-28408605-A

Abstract:
a polypeptide comprising one or more sequences derived from cdr2 or cdr3 of a trem - 1 protein , characterised by the ability to treat , ameliorate , or lessen the symptoms of sepsis , septic shock or sepsis - like conditions .

Description:
the present invention will now be described with reference to the following non - limiting examples . trem - 1 peptides matching the following criteria were synthesized : i ) highest homology between human and mouse trem - 1 and lowest homology with trem - 2 . ii ) peptides spanning the complementarity determining regions ( cdrs ) of trem - 1 . according to the published crystal structure of trem - 1 , and in analogy with antibodies , these residues are likely to be involved in cognate ligand recognition ( radaev et al . ( 2003 ) structure ( camb ). dec ; 11 ( 12 ): 1527 - 35 & amp ; kelker , et al . ( 2004 ) j mol . biol . sep 24 ; 342 ( 4 ): 1237 - 48 ) ( see fig1 ). one peptide ( p1 ) was designed in the cdr2 region and three peptides ( p2 , p4 and p5 ) in the cdr3 region . a fourth peptide ( p3 ) was designed in the neck region connecting the v - type immunoglobulin ( ig )- like domain ( ig - v ) to the transmembrane domain . no peptide was designed in the cdr1 region due to high sequence homology between trem - 1 and trem - 2 . thus , the following peptides of the trem - 1 protein were ordered from and were synthesized and purified by the protein and peptide chemistry facility , institute of biochemistry , university of lausanne : p1 ( cdr2 67 - 89 ) lvvtqrpftrpsevhmgkftlkh [ seq id no : 3 ] p2 ( cdr3 114 - 136 ) viyhppndpvvlfhpvrlvvtkg [ seq id no : 4 ] p3 ( neck region 168 - 184 ) tttrslpkptavvsspg [ seq id no : 5 ] p4 ( cdr3 103 - 123 ) lqvtdsglyrcviyhppndpv [ seq id no : 6 ] p5 ( cdr3 103 - 119 ): lqvtdsglyrcviyhpp [ seq id no : 7 ] p1sc * ( p1 scrambled seq .) ltpkhgqrsthvtkfrvfepvml [ seq id no : 8 ] p5sc * ( p5 scrambled seq .) tdsrcviglyhpplqvy [ seq id no : 9 ] * this is a control peptide and indeed does not protect in the experiments of this example , the peptides were administered in a volume of 200 μl of the solution molarity indicated . to assess the ability of trem1 - peptides to protect mice from lps - induced endotoxaemia , the inventors administered peptides p1 , p2 , p3 and p5 ( 300 μm ) 1 hour before a lethal dose of lipopolysaccharide ( lps ) ( fig2 ). lethality was monitored over time and compared with animals that had received control injections of vehicle alone . p5 injection confers maximal protection , with 90 % of the animals still alive 7 days after lps injection , as compared with 10 % of control mice ( p & lt ; 0 . 001 ). 60 % of the p1 - treated mice and 50 % of the p2 treated mice survived endotoxaemia as compared with 10 % of control mice ( p & lt ; 0 . 01 and p & lt ; 0 . 05 respectively ). interestingly , all p3 - treated mice died within 4 days after lps injection . these results indicate that peptides containing sequences of the extracellular portion of trem - 1 corresponding to the putative ligand binding site ( cdr2 and cdr3 ) can protect mice from lethal shock . in order to investigate whether trem - 1 peptide treatment could be delayed until after the administration of lps , the inventors injected the peptides at 4 hours after lps injection . only in the case of p1 , this delayed treatment conferred significant protection against a lethal dose of lps ( fig3 ). 80 % of the mice injected with p1 4 hours after lps survived endotoxaemia compared to 60 % of mice treated 1 h before lps and 10 % of mice treated with vehicle alone ( p & lt ; 0 . 001 and p & lt ; 0 . 01 respectively ). thus , p1 is effective even when injected after the outbreak of endotoxaemia . no late death occurred over one week , indicating that p1 did not merely delay the onset of lps lethality , but provided lasting protection . p1 administration conferred maximal protection ( 80 %) when administered at 600 μm ( p & lt ; 0 . 01 ) and the level of protection dropped to 50 % at 300 μm ( p & lt ; 0 . 05 ) and further down to 30 % at 150 μm as compared to 20 % of control mice , indicating a dose dependent effect of p1 ( fig4 ). the inventors then investigated whether p1 protects against septic shock in the “ clp ” model ( cecal ligation and puncture is a widely used experimental model of sepsis ). mice treated with two doses of p1 at 5 and 24 hours after clp were protected from death as compared to control treated mice ( p = 0 . 0791 ) although the difference was not statistically significant . 40 % of the mice injected with p1 at 5 days after clp survived compared to 5 % of mice treated with p3 peptide . at 10 days after clp , the treated mice were still alive , indicating that p1 did not merely delay mortality , but provided lasting protection ( fig5 ). trem - 1 peptide p1 inhibits binding of soluble mouse trem - 1 / igg to trem - 1 ligand positive cells among trem - 1 derived peptides tested in clp , peptides p1 , p2 and p5 demonstrate a protective activity . a possible mechanism of action could be the ability of trem - 1 derived peptide to interfere with trem - 1 / trem - 1 ligand interaction . to address this question the inventors performed competition experiments on trem - 1 ligand positive cells : pec ( peritoneal exudate cells ) from clp treated mice . peritoneal exudate cells ( pec ) from mice suffering from a caecal ligation and puncture ( clp )- induced peritonitis were subjected to flow cytometry analysis after incubation with a pe - conjugated anti - human igg1 ( jackson immunoresearch , bar harbor , usa ). competition with trem - 1 peptides was performed by pre - incubating cells with the indicated concentrations of peptides for 45 minutes on ice before adding mtrem - 1 - igg1 . as shown in fig6 , the p1 peptide , derived from the cdr2 region of mtrem - 1 , and the p2 and p5 peptides spanning the cdr3 region inhibit trem - 1 interaction with its ligand in a dose dependent manner . conversely the p3 peptide , derived from the neck region of trem - 1 connecting the igg like portion to the transmembrane domain was ineffective . additional studies on the modulation of the inflammatory response in murine sepsis by trem - 1 peptide p5 ten ml of peripheral blood samples were collected on edta - k from 5 healthy volunteer donors originating from laboratory staff . after dilution in rpmi ( life technologies , grand island , n . y .) v / v , blood was centrifuged for 30 min at room temperature over a ficoll gradient ( amersham pharmacia , uppsala , sweden ) to isolate pbmc . the cells recovered above the gradient were washed and counted . in order to deplete the suspensions of lymphocytes , cells were then plated in 24 - well flat - bottom tissue culture plates ( corning , corning , n . y .) at a concentration of 5 × 10 6 / ml and allowed to adhere during 2 hours at 37 ° c . the resulting lymphocyte suspension was discarded and the adhering monocytic cells were maintained in a 5 % co2 incubator at 37 ° c . in complete medium ( rpmi 1640 , 0 . 1 mm sodium pyruvate , 2 mm penicillin , 50 μg / ml streptomycin ; life technologies ) supplemented with 10 % fcs ( invitrogen , cergy , france ). using the human trem - 1 sequence in gen - bank , accession # af287008 and the mouse trem - 1 sequence # af241219 , a peptide “ p5 ” ( lqvtdsglyrcviyhpp ; [ seq id no : 7 ]; was chemically synthesized as a c - terminally amidated peptide ( pepscan systems , lelystad , the netherlands ). the correct peptide was obtained in greater than 99 % yield and with measured mass of 1961 da versus a calculated mass of 1962 da and was homogeneous after preparative purification , as confirmed by mass spectrometry and analytic reversed phase - high performance liquid chromatography . a peptide “ p5sc ” containing the same amino - acids than p5 but in a different sequence order ( tdsrcviglyhpplqvy ; [ seq id no : 9 ]) was similarly synthesized and served as ‘ control peptide ’. for activation , monocytes were cultured in the presence of e . coli lps ( o111 : b4 , 1 μg / ml , sigma - aldrich , la verpillière , france ). cell viability was assessed by trypan blue exclusion and by measuring lactate dehydrogenase release . in some experiments , this stimulus was given in combination with tnf - α ( 5 to 100 ng / ml , r & amp ; d systems , lille , france ), il - 1β ( 5 to 100 ng / ml , r & amp ; d systems ), rifn - γ ( up to 100 u / ml , r & amp ; d systems ), ril - 10 ( 500 u / ml , r & amp ; d systems ) or up to 100 ng / ml of p5 or control peptide . in order to activate monocytes through trem - 1 , an anti - trem - 1 agonist monoclonal antibody ( r & amp ; d systems ) was added as follows : flat - bottom plates were precoated with 10 μg / ml anti - trem - 1 per well . after thorough washing in phosphate buffered saline ( pbs ), the monocyte suspensions were added at a similar concentration as above . some experiments were performed in the presence of protease inhibitors ( pmsf and protease cocktail inhibitor ; invitrogen ). cell - free supernatants were assayed for the production of tnf - α and il - 1β by elisa according to the recommendations of the manufacturer ( bd biosciences , san diego , usa ). to address the effect of p5 on nf - κb activity in monocytes , an elisa - based assay was performed ( bd mercury ™ transfactor kit , bd biosciences ). monocytes were cultured for 24 hours in the presence of e . coli lps ( o111 : b4 , 1 μg / ml ), and / or an agonist anti - trem - 1 monoclonal antibody ( 10 μg / ml ), and / or p5 ( 100 ng / ml ). whole - cell extracts were then prepared and levels of nf - κb p50 and p65 were determined according to the recommendations of the manufacturer . all experiments were performed in triplicate and data are expressed as means ( sem ). primary monocytes suspensions were cultured as described above . the cells were treated with e . coli lps ( o111 : b4 , 1 μg / ml ) for 24 hours at 37 ° c . cell - conditioned medium was submitted to western - blotting using an anti - trem - 1 monoclonal antibody ( r & amp ; d systems ) in order to confirm the presence of 27 kda material recognized by anti - trem - 1 . soluble trem - 1 levels were measured by assessing the optical intensity of bands on immunodots by means of a reflectance scanner and the quantity one quantitation software ( bio - rad , cergy , france ) as reported elsewhere ( 18 ). soluble trem - 1 concentration from each sample was determined by comparing the optical densities of the samples with reference to standard curves generated with purified trem - 1 . all measurements were performed in triplicate . the sensitivity of this technique allows the detection of strem - 1 levels as low as 5 μg / ml . total mrna was extracted from primary monocytes cultured in the presence of lps using a trizol reagent ( invitrogen ), and reverse transcribed using superscript rt ii ( invitrogen ) to generate cdna . rt - pcr conditions then used for all reactions were 94 ° c ., 30s / 65 ° c ., 30s / 68 ° c ., 1 min for 30 cycles . amplification was performed with 2 . 5 mm mgcl2 , 0 . 2 mm dntp , 2 . 0 u taq polymerase , and 20 pm 5 ′ and 3 ′ oligonucleotide primers ( proligos , paris , france ). the sequences of the 5 ′ and 3 ′ primer pairs used were the following : for trem - 1 ( 17 ) ttgtctcagaactccgagctgc ; [ seq id no : 10 ] and gagacatcggcagttgacttgg ; [ seq id no : 11 ] for trem - 1sv ( 19 ) ggacggagagatgcccaagacc ; [ seq id no : 12 ] and accagccaggagaatgacaatg ; [ seq id no : 13 ] for β - actin ( used as housekeeping amplicon ) ggacgacatggagaagatctgg ; [ seq id no : 14 ] and atagtaatgtcacgcacgatttcc ; [ seq id no : 15 ] pcr products were run on agarose gels and visualized by ethidium bromide staining . after approval by the local ethical committee , male balb / c mice ( 20 to 23 g ) were randomly grouped and treated with e . coli lps intraperitoneally ( i . p .) in combination with p5 ( in 500 μl normal saline ) or control vector before or after lps challenge . in some experiments , 5 μg of an anti trem - 1 monoclonal antibody was administered i . p . one hour after lps injection . the viability of mice was examined every hour , or animals were sacrificed at regular intervals . serum samples were collected by cardiac puncture and assayed for tnf - α and il - 1β by elisa ( bd biosciences ), and for strem - 1 levels by immunodot . male balb / c mice ( 7 to 9 weeks , 20 to 23 g ) were anaesthetized by i . p . administration of ketamine and xylazine in 0 . 2 ml sterile pyrogen - free saline . the caecum was exposed through a 1 . 0 cm abdominal midline incision and subjected to a ligation of the distal half followed by two punctures with a g21 needle . a small amount of stool was expelled from the punctures to ensure patency . the caecum was replaced into the peritoneal cavity and the abdominal incision closed in two layers . after surgery all mice were injected s . c . with 0 . 5 ml of physiologic saline solution for fluid resuscitation and s . c . every 12 h with 1 . 25 mg ( i . e . 50 μg / g ) of imipenem . the animals were randomly grouped and treated with normal saline ( n = 14 ), the control peptide ( n = 14 , 100 μg ) or p5 ( 100 μg ) in a single injection at h0 ( n = 18 ), h + 4 ( n = 18 ) or h + 24 ( n = 18 ). the last group of mice ( n = 18 ) was treated with repeated injections of p5 ( 100 μg ) at h + 4 , h + 8 and h + 24 . all treatments were diluted into 500 μl of normal saline and administered i . p . the inventors next sought to determine the effect of various doses of p5 . for this purpose , mice ( n = 15 per group ) were treated with a single injection of normal saline or 10 μg , 20 μg , 50 μg 100 μg or 200 μg of p5 at h0 after the clp and monitored for survival . five additional animals per group were killed under anaesthesia 24 hours after clp for the determination of bacterial count and cytokines levels . peritoneal lavage fluid was obtained using 2 ml rpmi 1640 ( life technologies ) and blood was collected by cardiac puncture . concentrations of tnf - α and il - 1β in the serum were determined by elisa ( bd biosciences ). for the assessment of bacterial counts , blood and peritoneal lavage fluid were plated in serial log dilutions on tryptic soy supplemented with 5 % sheep blood agar plates . after plating , tryptic soy agar plates were incubated at 37 ° c . aerobically for 24 hours , and anaerobically for 48 hours . results are expressed as cfu per ml of blood and cfu per mouse for the peritoneal lavage . serum strem - 1 and cytokines levels were expressed as mean (± sd ). the protection against lps lethality by p5 was assessed by comparison of survival curves using the log - rank test . all statistical analyses were completed with statview software ( abacus concepts , berkeley calif .) and a two - tailed p & lt ; 0 . 05 was considered significant . a soluble form of trem - 1 is released from cultured human monocytes after stimulation with e . coli lps to identify the potential release of strem - 1 in vitro , the inventors stimulated human monocytes with lps and analyzed the conditioned culture medium by sds - page . lps stimulation induced the appearance of a 27 - kda protein in a time - dependent manner ( fig7 a ). western blotting analysis revealed that this protein was specifically recognized by a monoclonal antibody directed against the extra - cellular domain of trem - 1 ( fig7 a ). cell viability was unaffected at lps concentrations that induced the presence of strem - 1 in conditioned medium , indicating that trem - 1 release was not due to cell death . similarly , treatment of monocytes with protease inhibitors did not affect trem - 1 release ( fig7 a ). trem - 1 mrna levels were increased upon lps treatment ( fig7 b ) whereas trem - 1sv mrna levels remained undetectable . this suggests that trem - 1 release is likely to be linked to an increased transcription of the gene and unrelated to trem - 1 sv expression . stimulation of monocytes for 16 hours with tnf - α ( 5 to 100 ng / ml ) or il - 1β ( 5 to 100 ng / ml ) induced very small trem - 1 release in a cytokine dose - dependent manner . stimulation with ifn - γ did not induce trem - 1 release , even at concentrations of up to 100 u / ml . significant tnf - α and il - 1β production was observed in the supernatant of monocytes cultured with lps . tnf - α and il - 1β production was even higher for cells cultured with both trem - 1 mab and lps as compared with those cultured with mab or lps alone ( fig8 a ). the inducible release of pro - inflammatory cytokines was significantly lower after lps stimulation when the medium was supplemented with p5 or il - 10 . p5 reduced , in a concentration - dependent manner , the tnf - α and il - 1β production from cells cultured with lps or with lps and mab and simultaneously increased the release of strem - 1 from cells cultured with lps . the control peptide displayed no action on cytokines or strem - 1 release ( data not shown ). in striking contrast , il - 10 totally inhibited the release of both trem - 1 and inflammatory cytokines ( fig8 a ). both lps and trem - 1 mab induced a strong activation of monocytic nf - κb p50 and p65 and combined administration of lps and trem - 1 mab lead to a synergistic effect . p5 inhibited the nf - κb activation induced by the engagement of trem - 1 but did not alter the effect of lps ( fig8 b ). in order to determine whether strem - 1 was released systemically during endotoxemia in mice , the inventors measured serum strem - 1 levels after lps administration . serum strem - 1 was readily detectable 1 hour after administration of an ld 50 dose of lps and was maintained at peak plateau levels from 4 to 6 hours after lps treatment ( fig9 ). mice treated by a single dose of p5 60 min before a lethal dose ( ld 100 ) of lps were prevented from death in a dose - dependent manner ( fig1 a ). in order to investigate whether p5 treatment could be delayed until after the administration of lps , the inventors injected p5 beginning 4 or 6 hours after lps injection . this delayed treatment up to 4 hours conferred significant protection against a ld 100 dose of lps ( fig1 b ). no late death occurred over one week , indicating that p5 did not merely delay the onset of lps lethality , but provided lasting protection . control mice all developed lethargy , piloerection , and diarrhoea before death . by contrast , p5 - treated mice remained well groomed and active , had no diarrhoea , and were lively . to clarify the mechanism by which p5 protected mice from lps lethality , the inventors determined the serum levels of tnf - α , il - 1β and strem - 1 of endotoxemic mice at 2 and 4 hours . as compared to controls , pre - treatment by 100 μg of p5 reduced cytokines levels by 30 % and increased strem - 1 levels by 2 fold as shown in table 4 : to further highlight the role of trem - 1 engagement in lps - mediated mortality , mice were treated with agonist anti - trem - 1 mab in combination with the administration of an ld 50 dose of lps . this induced a significant increase in mortality rate from 50 % to 100 % ( fig1 c ). to investigate the role of p5 in a more relevant model of septic shock , the inventors performed clp experiments ( fig1 a ). the control groups comprised mice injected with normal saline or with the control peptide . in this model of polymicrobial sepsis , p5 still conferred a significant protection against lethality even when administered as late as 24 hours after the onset of sepsis . interestingly , repeated injections of p5 had the more favourable effect on survival ( p & lt ; 0 . 01 ). there was a dose response effect of p5 on survival ( fig1 b ) and cytokine production ( table 5 ). p5 had no effect on bacterial clearance ( fig1 ). sepsis exemplifies a complex clinical syndrome that results from a harmful or damaging host response to severe infection . sepsis develops when the initial , appropriate host response to systemic infection becomes amplified , and then dysregulated ( 4 , 5 ). neutrophils and monocyte / macrophages exposed to lps , for instance , are activated and release such pro - inflammatory cytokines as tnf - α and il - 1β . excessive production of these cytokines is widely believed to contribute to the multi - organ failure that is seen in septic patients ( 20 - 23 ). trem - 1 is a recently identified molecule involved in monocytic activation and inflammatory response ( 12 , 14 ). it belongs to a family related to nk cell receptors that activate downstream signalling events . the expression of trem - 1 on pnns and monocytes / macrophages has been shown to be inducible by lps ( 16 , 17 ). as described herein , the inventors demonstrate that a soluble form of trem - 1 was released from cultured human monocytes after stimulation with e . coli lps . such a soluble form was also detectable in the serum of endotoxemic mice as early as 1 hour after lps challenge . this is consistent with the implication of trem - 1 in the very early phases of the innate immune response to infection ( 14 , 15 , 24 ). the mechanism by which strem - 1 is released is not clearly elucidated but seems to be related to an increased transcription of the trem - 1 gene . nevertheless , although incubation with a protease inhibitor cocktail does not alter the strem - 1 release , cleavage of the surface trem - 1 from the membrane cannot be totally excluded . interestingly , stimulation of human monocytes with such pro - inflammatory cytokines as tnf - α , il - 1β or ifn - γ induced very small strem - 1 release unless lps was added as a co - stimulus . the expression of an alternative mrna trem - 1 splice variant ( trem - 1sv ) has been detected in monocytes that might translate into a soluble receptor ( 18 ) upon stimulation with cell wall fraction of mycobacterium bovis bcg but not lps ( 25 ). this was confirmed in this study as i ) lps did not increase the level of mrna trem - 1sv in monocytes and ii ) only a 27 - kda protein was released by monocytes upon lps stimulation and not the 17 . 5 - kda variant . although its natural ligand has not been identified ( 13 , 14 ), engagement of trem - 1 on monocytes with an agonist monoclonal antibody resulted in a further enhancement of pro - inflammatory cytokines production , while p5 induced a decrease of these syntheses in a concentration - dependent manner , and il - 10 completely suppressed it . inflammatory cytokines , and especially tnf - α , are considered to be deleterious , yet they also possess beneficial effects in sepsis ( 5 ) as shown by the fatal issue of peritonitis in animals with impaired tnf - α responses ( 9 - 11 ). moreover , in clinical trials , the inhibition of tnf - α increased mortality ( 8 ). finally , the role of tnf - α in the clearance of infection has been highlighted by the finding that sepsis is a frequent complication in rheumatoid arthritis patients treated with tnf - α antagonists ( 26 ). the mechanism by which p5 modulates cytokine production is not yet clear . p5 comprises the complementary determining region ( cdr )- 3 and the ‘ f ’ β strand of the extra - cellular domain of trem - 1 . the latter contains a tyrosine residue mediating dimerization . radaev et al postulated that trem - 1 captures its ligand with its cdr - equivalent loop regions ( 27 ). p5 could thus impair trem - 1 dimerization and / or compete with the natural ligand of trem - 1 . moreover , the increase of strem - 1 release from monocytes mediated by p5 could prevent the engagement of membrane trem - 1 , strem - 1 acting as a decoy receptor , as in the tnf - α system ( 28 , 29 ). activation of the transcription factor nf - κb is a critical step in monocyte inflammatory cytokine production after exposure to bacterial stimuli such as lps ( 30 , 31 ). among the various nf - κb / rel dimers , the p65 / p50 heterodimer is the prototypical form of lps - inducible nf - κb in monocytes ( 32 ). p5 abolishes the p65 / p50 nf - κb over - activation induced by the engagement of trem - 1 . this might at least partially explain the effects of p5 on cytokine production and the protection from lethality shown here to occur when the peptide was injected one hour before lps - induced septic shock , or even up to 4 hours after . endotoxemia is simple to achieve experimentally , but imperfectly suited to reproduce human sepsis , while polymicrobial sepsis induced by clp is a more complex but better model , including the use of fluid resuscitation and antibiotics . the latter was thus also used in this study , and confirmed the dose - dependent protection provided by p5 , even when administered as late as 24 hours after the onset of sepsis . the favourable effect of p5 was however unrelated to an enhanced bacterial clearance . one difficulty in the use of immunomodulatory therapies is that it is not possible to predict the development of sepsis , and , thus , patients receiving those treatments frequently already have well - established sepsis ( 6 ). since p5 appeared to be effective even when injected after the outbreak of sepsis , it could thus constitute a realistic treatment ( 24 , 33 ). by contrast , engagement of trem - 1 by an agonist anti - trem - 1 monoclonal antibody mediated a dramatic increase of mortality rate in lps - challenged mice : this further underscores the detrimental effect of trem - 1 engagement during septic shock . experimental septic shock reproduces human sepsis only in part . indeed , our group recently showed that significant levels of strem - 1 were released in the serum of critically ill patients with sepsis patients ( 34 ), the highest levels being observed in patients who survived . this is consistent with our experimental findings indicating that the more important strem - 1 release , the more favourable is the outcome , and thus sustains , at least theoretically , the potential value of soluble trem peptides as post - onset sepsis therapy . trem - 1 appears to be a crucial player in the immediate immune response triggered by infection . in the early phase of infection , neutrophils and monocytes initiate the inflammatory response owing to the engagement of pattern recognition receptors by microbial products ( 3 , 4 ). at the same time , bacterial products induce the up - regulation and the release of strem - 1 . upon recognition of an unknown ligand , trem - 1 activates signalling pathways which amplify these inflammatory responses , notably in monocytes / macrophages . the modulation of trem - 1 signalling reduces , although without complete inhibition , cytokine production and protects septic animals from hyper - responsiveness and death . modulation of trem - 1 engagement with such a peptide as p5 might be a suitable therapeutic tool for the treatment of sepsis , particularly because it seems to be active even after the onset of sepsis following infectious aggression . haemodynamic studies in lps treated and septic rats treated with p1 and p5 the role of trem - 1 peptides in further models of septic shock , was investigated by performing lps and clp ( caecal ligation and puncture ) experiments in rats . animals were randomly grouped ( n = 10 - 20 ) and treated with escherichia coli lps ( o111 : b4 , sigma - aldrich , lyon , france ) i . p . in combination with the trem - 1 or scrambled peptides . the procedure has been described in details elsewhere ( see mansart , a . et al . shock 19 : 38 - 44 ( 2003 )). briefly , rats ( n = 6 - 10 per group ) were anesthetized by i . p . administration of ketamine ( 150 mg / kg ). the caecum was exposed through a 3 . 0 - cm abdominal midline incision and subjected to a ligation of the distal half followed by two punctures with a g21 needle . a small amount of stool was expelled from the punctures to ensure potency . the caecum was replaced into the peritoneal cavity and the abdominal incision closed in two layers . after surgery , all rats were injected s . c . with 50 ml / kg of normal saline solution for fluid resuscitation . trem - 1 or scrambled peptides were then administered as above . immediately after lps administration as well as 16 hours after clp , arterial bp ( systolic , diastolic , and mean ), heart rate , abdominal aortic blood flow , and mesenteric blood flow were recorded using a procedure described elsewhere ( see mansart , a . et al . shock 19 : 38 - 44 ( 2003 )). briefly , the left carotid artery and the left jugular vein were cannulated with pe - 50 tubing . arterial bp was continuously monitored by a pressure transducer and an amplifier - recorder system ( iox emka technologies , paris , france ). perivascular probes ( transonic systems , ithaca , n . y .) wrapped up the upper abdominal aorta and mesenteric artery , allowed to monitor their respective flows by means of a flowmeter ( transonic systems ). after the last measurement ( 4 th hour during lps experiments and 24 th hour after clp ), animals were sacrificed by an overdose of sodium thiopental i . v . blood was sequentially withdrawn from the left carotid artery . arterial lactate concentrations and blood gases analyses were performed on an automatic blood gas analyser ( abl 735 , radiometer , copenhagen , denmark ). concentrations of tnf - α and il - 1β in the plasma were determined by an elisa test ( biosource , nivelles , belgium ) according to the recommendations of the manufacturer . plasmatic concentrations of nitrates / nitrites were measured using the griess reaction ( r & amp ; d systems , abingdon , uk ). results are expressed as mean ± sd . between - group comparisons were performed using student &# 39 ; t tests . all statistical analyses were completed with statview software ( abacus concepts , calif .) and a two - tailed p & lt ; 0 . 05 was considered significant . following lps administration , arterial pressures , aortic and mesenteric blood flows dropped rapidly in control animals ( scrambled peptides treated rats ) while the heart rate remained unchanged ( table 6 ). the decrease of arterial pressures and aortic blood flow was delayed until the second hour in trem - 1 peptide treated animals with significantly higher values by that time than in control animals . there was no difference between p1 and p5 treated groups . by contrast , none of these two peptides had any effect on the decrease of the mesenteric blood flow ( table 6 ). arterial ph remained constant over time until the fourth hour after lps injection where it severely dropped in the control group only ( table 6 ). the significant arterial lactate level elevation present in control animals after the third hour was abolished by the trem - 1 peptides ( table 6 ). there was no difference between p1 and p5 with regard to ph , arterial bicarbonate and lactate concentrations . as expected , a peak of tnf - α plasmatic concentration was induced by lps between 30 minutes and 1 hour after injection followed by a progressive decline thereafter ( fig1 a ). p1 peptide injection had no effect on this production , while p5 attenuated tnf - α production by ˜ 30 %. p1 delayed the il - 1β peak until the third hour after lps injection , but without attenuation . by contrast , p5 strongly reduced il - 1β release ( fig1 b ). nitrite / nitrate concentrations increased rapidly after lps administration in control and p1 treated animals but remained stable upon p5 treatment ( fig1 ). as the severity of the inventors &# 39 ; model was at its highest 16 to 20 hours after the completion of the clp , the inventors chose to investigate animals by the 16 th hour . importantly , there were no deaths before this time point . although all animals were fluid resuscitated , none received antibiotics in order to strictly consider the role of the peptides . there was a dramatic decline in arterial pressure in the control animals over time , and by h24 systolic , diastolic and mean arterial pressures were 58 ± 7 mmhg , 25 ± 4 mmhg and 38 ± 2 mmhg respectively . this decrease was almost totally abolished with p1 or p5 treatment with no significant difference between h16 and h24 ( fig1 ). there was no difference between p1 and p5 treated rats . trem - 1 peptides also prevented the aortic and mesenteric blood flows decrease observed in control animals ( table 7 ). the protective effect on mesenteric blood flow alterations was even higher under p5 treatment . the relative preservation of blood flows was not related to an increased heart rate , since the latter was rather slower than in control animals ( table 7 ). the progressive metabolic acidosis that developed in control rats was attenuated by the p1 peptide , and almost abrogated by p5 . the same protective trend was observed for arterial lactate elevation with a more pronounced effect of p5 ( table 7 ). both p1 and p5 induced a decrease in tnf - α production , again with a stronger effect of p5 . by h20 , plasmatic tnf - α was almost undetectable under p5 treatment whereas it remained elevated in the other groups of animals ( fig1 ). nitrite / nitrate concentrations were increased in control animals but remained at a low level in both trem - 1 peptides treated groups ( fig1 ). a protective action of both p5 and p1 on hemodynamics was thus observed in septic rats . both arterial pressure and blood flows were preserved , independently of heart rate . moreover , modulation of trem - 1 signalling reduced , although not completely , cytokine production and protected septic animals from hyper - responsiveness . the fact that the cytokine production was not totally inhibited is a crucial point . indeed , although inflammatory cytokines such as tnf - α are considered deleterious , they also display beneficial effects in sepsis as underlined by the fatal issue of peritonitis models in animals with impaired tnf - α responses . the activation of inos observed during septic shock leads to the production of large amount of no that partly explains some of the peripheral vascular disorders ( notably vasodilation and hypotension ). on the myocardium itself , most of the action of no is mediated by an activation of the soluble guanylate - cyclase responsible for the production of cgmp which impairs the effect of cytosolic calcium on contraction . cyclic gmp is also able to stimulate the activity of some phosphodiesterases . the subsequent decrease of intra - cellular camp levels could explain the ability of no to attenuate the effects of beta adrenergic stimulation . the preservation of arterial pressure could therefore be partly explained by a lessened production of no , as reflected by the lower concentrations of plasma nitrite / nitrate in trem - 1 peptides treated animals . the decrease in inflammatory cytokine production could partly explain the effect noted on blood flows . indeed , although the list of potential cytokine mediators of myocardial depression is long , tnf - α and il - 1β have been shown to be good candidates both these latter cytokines depressed myocardial contractility in vitro or ex vivo . moreover , the neutralization or removal of tnf - α or il - 1β from human septic serum partly abrogates the myocardial depressant effect in vitro and in vivo . although p1 and p5 had an identical action on blood flows and arterial pressure during endotoxinemia , their action on cytokine production differed with only a slight effect of p1 on plasma tnf - α and il - 1β concentrations . the protective role of the trem - 1 peptides could therefore be only partly related to their action on cytokine release , or involve redundant pathways . the modulation of the trem - 1 pathway by the use of small synthetic peptides had beneficial effects on haemodynamic parameters during experimental septic shock in rats , along with an attenuation of inflammatory cytokine production . in summary , these data show that the trem - 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