neurocysticercosis is the most common parasitic disease of the central nervous system leading to seizures worldwide .
humans develop cysticercosis when they ingest eggs of the tapeworm taenia solium   usually found in fecal - contaminated water or food .
neurocysticercosis ( ncc ) is endemic to many parts of the world [ 37 ] and is becoming an increasingly important cause of seizures in the united states due to immigration from mexico and central and south america [ 8 , 9 ] .  
seizures in ncc most commonly arise as a result of the granulomatous responses to dead cysts in the brain .
the granulomatous response is associated with production of several cytokines including t helper 1 ( th1 ) cytokines such as interferon gamma ( ifn- ) , interleukin-2 ( il-2 ) , and interleukin-12 ( il-12 ) . 

t. crassiceps   infection in mice is an experimental model for t. solium   cysticercosis in man [ 1115 ] .
intraperitoneal inoculation with 10 cysts of t. crassiceps   results in the entire peritoneal cavity of the mouse demonstrating granulomatous inflammation within 36 months .
similar to human infection , minimal granulomatous inflammation is found surrounding live parasites ; rather , granulomatous inflammation is initiated when the parasite dies .
the mediators contributing to development of granulomas around the dead parasite and production of proinflammatory cytokines are not completely understood .
we previously detected substance p ( sp ) protein within granulomas associated with t. crassiceps   infection [ 16 , 17 ] .
we also demonstrated that levels of il-2 , ifn- , il-4 , and il-10 protein were significantly higher in granulomas from infected wt mice than granulomas from infected spp - knockout or the sp - receptor ( neurokinin 1 , nk1 ) nk1-knockout mice [ 16 , 17 ] .
in addition , we detected mrna for il-1 , il-1 , il-1 receptor antagonist , and tnf- in all granulomas derived from infected wt mice .
however , corresponding proteins levels were not assessed nor was the contribution of sp signaling to their mrna and protein production and to granuloma formation .  
the current studies were aimed at determining if sp and nk1 contributed to granuloma development and/or to production of il-1 , tnf- , and il-6 in cysticercosis .
sp stimulates production of proinflammatory cytokines such as of il-1 , il-6 , and tnf- by human peripheral mononuclear cells , bronchial cells , and astrocytes [ 1929 ]   sp also contributes to inflammatory processes associated with other infectious diseases .
sp injection induced recruitment of leukocytes into the pleural cavity of mice and into the skin of humans     [ 1929 ] and stimulated the migration of human fibroblasts and peripheral blood lymphocytes in studies using modified boyden chambers or micropore filter analysis , respectively [ 1929 ] . in the current studies , we determined granuloma size and measured levels of il-1 , tnf- , and il-6 protein within granuloma obtained from t. crassiceps - infected wt and mice deficient in spp or nk1 .
these studies indicate that sp signaling contributes to granuloma development and proinflammatory cytokine production in t. crassiceps   infection and suggests a potential role for this mediator in human cysticercosis .
female mice were infected by intraperitoneal inoculation with 10 cysts of the orf strain of t. crassiceps , as described in [ 16 , 17 ] .
three months following infection , the mice were euthanatized by cervical dislocation under anesthesia using a combination anesthetic sacrifice rodent cocktail ketamine , 25  mg / kg , acepromazine 0.8  mg / kg , xylazine 5  mg / kg intraperitoneally , at a dose of 0.50.7  ml / kg intramuscularly .
granulomas associated with dying cysts were removed from the peritoneal cavity of each of the infected mice that were euthanized .
three groups of mice were included in the experiments : ( 1 ) wild type c57bl/6 mice ; ( 2 ) preprotachykinin or spp - knockout mice ( jackson laborotories , maine , usa , bred > 10 generations onto the c57bl/6 background ) ; ( 3 ) nk1-knockout mice provided by dr .
joel weinstock , tufts new england medical center , boston , usa , bred > 10 generations onto the c57bl/6 background .
three to 8 infected mice from each of the 3 groups were used for this study ; 415 granulomas per mouse group were used for this study .
granulomas associated with parasites were identified visually , removed from the peritoneal cavity , and either used for quantifying cytokine proteins by elisa or used for size determinations .
intact granuloma was obtained from t. crassiceps - infected wt mice ( 2 granulomas ) , spp - knockout mice ( 4 granulomas ) , and nk1-knockout mice ( 3 granulomas ) , fixed with 4% paraformaldehyde , paraffin imbedded and completely sectioned by microtome into 7 micron sections .
the area of granuloma within each section was measured using image j software ( nih ) .
the volume of granuloma within each section was calculated by multiplying the area times 7 microns and the volume of granuloma within each section totaled to give the total granuloma volume .
cytokine protein levels were determined in 1215 granulomas derived from t. crassiceps   infected wt mice , 46 granulomas derived from t. crassiceps   infected spp - knockout mice , and 9 - 10 granulomas derived from t. crassiceps - infected nk1-knockout mice .
a portion of each granuloma was homogenized in pbs , followed by centrifugation at 16,000  g. total protein in the supernatant was quantitated using the bradford method ( cat no .
il-1 , il-6 , and tnf- protein levels were determined by sandwich elisa ( r&d systems , san diego , california ) as per manufacturer 's instruction .
to begin to examine the contribution of sp signaling to granuloma formation in ncc , we measured granuloma volume in mice with normal and deficient sp signaling .
the volumes of granulomas from taenia crassiceps   infected spp - knockout mice ( 1.8  0.45  mm ) and nk1-knockout mice ( 1.68  0.40  mm ) were reduced by 31% and 36% , respectively , compared to granulomas derived from infected wt mice ( 2.62  0.28  mm ; p < .05 for both ; student 's t - test ; see figure 1 ) .
il-1 is the primary mediator of granuloma formation in the s. mansoni   pulmonary granuloma model .
also , intratracheal injection of agarose beads coupled to recombinant il-1 induced pulmonary granulomas in mice . to determine if decreased production of il-1 in spp- and nk1-knockout mice contributed to reduced granuloma size in these animals , we measured il-1 protein levels in the granulomas derived from each group of mice .
il-1 protein levels in granulomas from t. crassiceps   infected spp - knockout mice ( 216  129  ng / mg total protein ) and nk1-knockout mice ( 101  43  ng / mg total protein ) were reduced by 48% and 76% , respectively , compared to granulomas from infected wt mice ( 418  278  ng / mg total protein ; p < .05 for both ; student 's t - test ; see figure 2 ) .
thus , sp signaling contributes to il-1 production within granulomas formed in response to dying t. crassiceps   cysts .
furthermore , reduced levels of this cytokine likely contributed to reduced granuloma size in spp- and nk1-knockout mice .
tnf- mediates granuloma growth in the s. mansoni pulmonary granuloma model    and is required for granuloma formation in a mouse model of tuberculosis   .
similar to our findings with il-1 , tnf- protein levels in granulomas from t. crassiceps   infected spp - knockout mice ( 96  67  ng / mg total protein ) were reduced by 79% compared to levels in granulomas derived from infected wt mice ( 460  452  ng / mg total protein ; p < .05 ; student 's t - test ; see figure 3 ) .
tnf- protein levels within granulomas from nk1-knockout mice ( 345  153  ng / mg total protein ) were decreased by 25% compared to levels with granulomas of wt mice ; however , this difference did not achieve statistical significance .
thus , sp contributes to tnf - production within granulomas formed in response to dying t. crassiceps   cysts .
furthermore , reduced levels of this cytokine likely contributed to reduced granuloma size in spp- and , perhaps , nk1-knockout mice .
the failure to detect a significant difference in tnf- protein levels between nk1-knockout and wt mice suggests the possibility that sp - mediated increases in tnf- may occur through binding of sp to other members of the nk family , for example , nk2 or nk3 .
il-6 production in human peripheral blood mononuclear cells , bronchial cells , and astrocytes is increased directly by sp through the action of nuclear factor il-6 ( nf - il-6 ) and p38 mapk , as well as indirectly in response to il-1 and tnf- through the activation of nf-b [ 26 , 27 , 34 , 35 ] .
il-6 mediates its acute proinflammatory effects within infected or injured tissues , in part , through upregulation of cxc chemokines , which leads to recruitment of the first wave of inflammatory cells .  
as we expected from the il-1 and tnf- results summarized above , il-6 protein levels in the granulomas derived from infected spp - knockout mice ( 28  30  ng / mg total protein ) and from infected nk1-knockout mice ( 50  16  ng / mg total protein ) were reduced by 79 and 62% , respectively , compared to levels in granulomas from infected wt mice ( 130  153  ng / mg total protein ; p < .05 for both ; student 's t - test ; see figure 4 ) .
thus , sp signaling either directly or indirectly through the actions of il-1 and tnf- contributes to il-6 production within granulomas formed in response to dying t. crassiceps   cysts .
the current studies were performed to determine the contribution of sp and its specific receptor , nk1 , to granuloma development and proinflammatory cytokine production within granulomas arising in mice infected with t. crassiceps .
we demonstrated that the size of granulomas from the t. crassiceps - infected spp - knockout mice and infected nk1-knockout mice were significantly smaller than granulomas from the infected wt mice .
furthermore , proteins levels of il-1 , a key mediator of granuloma formation , were significantly lower within granuloma from spp- and nk1- knockout mice compared to granuloma from mice .  
in addition , compared to granulomas from wt mice , protein levels of tnf- , another key mediator of granuloma formation , were significantly lower in spp - knockout mice and trended in the same direction in nk1-knockout mice .
thus , sp signaling contributes to granuloma formation , in part , through induction of il-1 and tnf- , key mediators of granuloma formation .
substance p and its high affinity receptor , nk1 , are known to play an important role in inflammatory responses .
granuloma formation in response to murine schistosomiasis requires binding of sp to its specific receptor .
sp is known to stimulate inflammatory cell infiltration and to induce the production of proinflammatory cytokines such as il-1 , il-6 , and tnf- by human peripheral mononuclear cells , bronchial cells , or astrocytoma cells [ 1929 ] .
our findings extend these observations and indicate that sp signaling contributes to granuloma formation and production of il-1 , tnf- , and il-6 protein within granuloma formed in response to t. crassiceps   infection .
the mechanism by which sp stimulates the production of these cytokines may be by mediating inflammatory cell influx [ 1924 ] .
nerves , endothelial cells , and cells of the immune system produce sp [ 3638 ] .
all of these cells have receptors for sp and are known to respond to sp [ 3840 ] .
sp is known to stimulate influx of lymphocytes , monocytes , macrophages , and other immune cells that produce proinflammatory cytokines such as il-1 , il-6 , and tnf- [ 1929 ] .
although there are various studies on the molecular mechanisms by which sp stimulates the production of il-6 and tnf- , there is limited information on the molecular mechanisms by which sp stimulates il-1 production . a study by martin et al .
these studies demonstrated that treatment with an intracellular calcium chelator blocked sp - induced il-1 production .
other studies have demonstrated that sp induces tnf - alpha and il-6 production through nf kappa b in peritoneal mast cells
. substance p production of tnf- in peritoneal mast cells is also known to be mediated via p38 and jnk map kinases .
also il-6 production in human peripheral blood mononuclear cells , bronchial cells , and astrocytes is increased directly by sp through the action of nuclear factor il-6 ( nf - il-6 ) and p38 mapk [ 26 , 27 , 34 , 35 ] .
preprotachykinin protein is cleaved to form two active neuropeptides , sp and neurokinin a. therefore , sp precursor ( preprotachykinin ) knockout mice produce neither sp nor neurokinin a. consequently , our results in spp - knockout mice can be attributed to a deficiency in either sp or neurokinin a or both . however , since nk1 binds only sp and not neurokinin a and the results in nk1-knockout mice mirror the findings in sp - knockout mice , we are confident in attributing reduced granuloma formation and proinflammatory cytokine production to the absence of sp signaling and not to reduced or absent neurokinin a signaling .   in the current studies
, we demonstrated that the granuloma size and the levels of the proinflammatory cytokine , il-1 , are lower in the infected nk1-knockout mice compared to those of the infected spp - knockout mice . besides sp , peptide hormones such as hemokinin can also bind and activate nk1 at sites of chronic inflammation .
therefore , although the current studies suggest that sp may be an important mediator associated with cytokine production , there may be other peptide hormones like hemokinin that also bind and activate the nk1 receptor that may be associated with granuloma and cytokine production .
therefore , it may be possible that in the nk1-knockout mice , the synergistic lack of activity of both sp and hemokinin may have resulted in lower il-1 levels and granuloma size as compared to spp - knockout mice . granuloma formation by the host in response to agents causing chronic infections is thought to be essential for limiting and eventually clearing infection .
however , recent work in zebra fish infected with mycobacterium marinum   suggests that granuloma formation contributes to early bacterial growth .
intraparenchymal cysts of ncc are thought to die spontaneously and to elicit a granulomatous response that does not in itself contribute to the demise of the cyst .
rather , we have previously demonstrated that early granuloma formed in response to dying cysts contributes to ncc disease manifestations by producing mediators that induce seizures .
the current studies demonstrating that sp signaling contributes to granuloma formation in taenia crassiceps   infection , together with other published observations , suggest the possibility that diminishing granuloma formation in ncc by blocking sp , which contributes to granuloma formation and epileptogenic responses ,   may be beneficial in the treatment of this disease .
 cysticercosis is an infection with larval cysts of the cestode taenia solium . through pathways that are incompletely understood , 
 dying parasites initiate a granulomatous reaction that , in the brain , causes seizures . 
 substance p ( sp ) , a neuropeptide involved in pain - transmission , contributes to inflammation and previously was detected in granulomas associated with dead t. crassiceps cysts . to determine if sp contributes to granuloma formation , we measured granuloma - size and levels of il-1 , tnf- , and il-6 within granulomas in t. crassiceps - infected wild type ( wt ) mice and mice deficient in sp - precursor ( spp ) or the sp - receptor ( neurokinin 1 , nk1 ) . 
 granuloma volumes of infected spp- and nk1-knockout mice were reduced by 31 and 36% , respectively , compared to wt mice ( p < .05 for both ) and produced up to 5-fold less il-1 , tnf- , and il-6 protein . 
 thus , sp signaling contributes to granuloma development and proinflammatory cytokine production in t. crassiceps infection and suggests a potential role for this mediator in human cystercercosis . 