MAPK and NF-kB pathways ofthe host . These pathways, although initially activated by exposure to the parasite, become silenced when theinfection is firmly established, rendering the cell unresponsive to further stimulation with LPS . This silencing has been attributed to different virulence factors, including surface phosphoglycans [16,62], the metalloprotease GP63 , and cysteine proteases from L. mexicana . It is possible that inthe absence of Ctsb, one or more of these key signaling pathways are no longer silenced by L. major promastigotes. This would open a range of new questions regarding the involvement of Ctsb in L. majorinfection, e.g., whether Ctsb interacts directly with the parasites, contributing to processing or activation of one or more virulence factors, or whether Ctsb directly interferes with any intermediate of key signaling pathways, such as NF-kB and MAPK. While Ctsb 2/2 BMDC and BMM were able to up- regulate IL-12 inresponse to L. major and LPS, IL-6 was not regulated. IL-6 transcription has been shown to depend on NF-kB . Therefore, we hypothesize that the molecular mechanism behind the involvement of Ctsb 2/2 inthe expression of IL-12 would not be shared by IL-6. Our results suggest that the regulation of IL-12 expression by Ctsb is not NF-kB-dependent, although further work is necessary to confirm this observation and to explore the interaction of Ctsb with other candidate signaling pathways. In addition, finding the location of these interactions would be a key to further understand the mechanisms underlying these processes, e.g., whether proteolytic processing of signaling intermediates takes place inthe cytoplasm, or whether cleavage of transcription factors by Ctsb occurs inthe nuclear space, as described in thyroid carcinoma cells .
from hamsters (6) there was an improvement inthe understanding oftheimmuneresponsein this experimental model. The profile of Th2 cytokines is directly responsible for suppression, but other factors such as adherent cells (17) and macrophage-mediated suppression have been reported to lead to increasing parasite growth and also to be linked to defective antigen presentation (18). TheTh1 cytokine IFN-γ plays a key role inthe control ofinfection with many intracellular pathogens, including Leishmania spp, and is the cytokine primarily responsible for macrophage activation and killing of intracellular parasites (19,20). In hamsters infected with L. (L.) donovani, adherent spleen cells have been shown to be important lymphoproliferative suppressors and to play a role in defective antigen pre- sentation (13). Furthermore, TGF-β produced by adherent antigen-presentingcells from infected hamsters has been implicated in immunosuppression since a high level of TGF-β was observed inthe cell culture supernatant when theLeishmaniaantigen-induced lymphoproliferative response was inhibited (7,8). Inthe present study, we investigated the cytokine profile produced duringthe course of VL by spleen cellsin culture upon stimulation with Con A and
exposed to sand fly bites and saliva inoculation, the salivary components also seem to display an immuneresponseinthe host, leading to antibody production and cell mediated immune activation. However, the specific role of antibodies against sand fly saliva inthe outcome ofLeishmaniainfection is not fully known. Experimental studies, together with clinical data, suggest a potential transmission blocking mechanism. BALB⁄c mice repeatedly exposed to L. longipalpis bits produced high levels of anti-salivary antibodies, with predominance ofthe IgG1 subclass (Belkaid et al., 1988). Total serum IgG from these animals recognized predominantly bands of 45, 44 and 16 kDa, displaying some similarity to human anti-saliva humoral responses, that recognize most ofthe bands. These proteins were also themajor targets ofthe human antibody responsein an endemic area (Barral et al., 2000). As these proteins are widely recognized, they are natural candidates to be used as markers of exposure to L. longipalpis bits. Besides antibody production, an intense cellular infiltrate of neutrophils, eosinophils and macrophages at the site of sand fly bites also occurs. Moreover, mice pre-exposure to P. papatasi SGS or immunized with SP15, a protein present in P. papatasi saliva, were able to block the establishment of L. majorinfection (Andrade et al., 2007). Also in humans, theresponse generated by sand fly saliva seems to exert a direct effect on human antigenpresentingcells. L. longipalpis SGS inhibit the production of IL-10 and tumor necrosis factor (TNF)-α, but induce IL-6, IL-8 and IL-12p40 release by lipopolysaccharide-stimulated monocytes and dendritic cells. Besides cytokine production, sand fly saliva also interfered with the expression of monocytes/macrophages co-stimulatory molecules. In vitro, the saliva effect on human immunecells can be reverted by incubation with anti-SGS antibodies, revealing the importance of a specific humoral response for the neutralization of sand fly saliva harmful effects on the human immune system (Costa et al., 2004).
In L. majorinfection, susceptibility is associated with Th2-type response that is characterized by IL-4 production and isotype switch to IgG1. On the other hand, C57BL/6 mice show a resistance proﬁle with IFN-gamma produc- tion and less involvement of humoral immuneresponse. IL-10 plays regulatory functions in both strains. To verify whether the spleen is an important organ for the develop- ment of both Th1 and Th2 immune responses as well as for anti-inﬂammatory responses duringinfection with L. major, levels of IFN-gamma, IL-4, and IL-10 were mea- sured in tissue culture supernatants of spleen (of control mice only) and popliteal lymph node cells after 72 h of stimulation with L. majorantigen. Fig. 3A shows that spleen cells from control C57BL/6 mice produce higher lev- els of IFN-gamma at 4 weeks post-infection. Fig. 3B shows that IL-4 production is higher in BALB/c than in C57BL/6 mice at week 2 post-infection. There was no diﬀerence in IL-10 production by spleen cells between control C57BL/ 6 and BALB/c control mice at any time point examined after infection (Fig. 3C).
Although reporter-derived AGFP mRNA was present in all developmental stages (and in similar levels to the endogenous ADGF-A transcripts), we did not detect any GFP protein expression at any developmental stage under normal growth conditions. This may be because the expression levels were too low to be detectable using the available methodology. Additionally inthe case of heterozygous animals, the reporter was only expressed from one ofthe two alleles, thus lowering the potentially detectable expression to ,50% compared to normal endogenous ADGF-A expression. However, the detectable GFP fluorescence only at the sites of hemocyte aggregation in both AGFP homozygous and heterozygous animals suggests that heterozygosity ofthe reporter was not the reason. We also used a destabilized version of GFP that is reported to undergo degradation within a couple hours . Whilst this allowed us to observe the important dynamic changes in ADGF-A expression, it probably also lowered the overall reporter signal and as a consequence its sensitivity, Figure 5. AGFP expression in hemocytes and melanotic capsules obtained from the late third-instar larvae. (A) Aggregating lamellocytes from the adgf-a mutant larvae without AGFP as a control to account for autofluorescence. (B) Aggregating lamellocytes from AGFP homozygotes showing GFP fluorescence. (C) Non-aggregating plasmatocytes and lamellocytes from AGFP homozygotes that rarely displayed any GFP fluorescence. (D) A clump of plasmatocytes and lamellocytes from AGFP homozygotes exhibiting strong GFP expression. (E) A melanotic capsule from the adgf-a mutant showing only yellowish autofluorescence ofthe melanized region. (F) GFP fluorescence in hemocytes surrounding a melanotic capsule from AGFP homozygotes. (G) A melanotic capsule from the null cactus mutant with AGFP reporter (cactus[E8]/cactus[D13]; AGFP/+) showing GFP expression in surface lamellocytes. (H) A melanizing clump with lamellocytes and plasmatocytes expressing GFP in hop Tum
Previous results from our laboratory and from the literature have implicated the expression of ecto- nucleotidases inthe establishment ofLeishmaniainfection. Inthe present study we evaluated the correlation between ecto-nucleotidasic activity and the infectivity of L. amazonensis promastigotes that were kept in culture for short or extended numbers of passages, a condition that is known to decrease parasite infectivity. We also analyzed theimmuneresponse associated with theinfection by these para- sites. As expected, we found that long-term cultured parasites induce the development of smaller lesions than the short-term cultured counterparts. Interestingly, long-term cultured parasites presented reduced ecto-nucleotidasic activity. In addition, cells recovered from animals infected with long-term cultured parasites produced higher amounts of IFN-␥ and have smaller parasite load, after 8 weeks ofinfection. Furthermore, after 1 week ofinfection, there is increased expression ofthe chemokine CCL2 mRNA in animals infected with short-term cultured parasites. Finally, infectionof peritoneal macrophages by these parasites also shows marked differences. Thus, while short-term cultured parasites are able to infect a greater proportion of macrophages, cells infected by long-term cultured parasites express higher amounts of CXCL10 mRNA, which may activate these cells to kill the parasites. We suggest that the enzymes involved in metabolism of extracellular nucleotides may have an important role ininfection by L. amazonensis, by acting directly in its adhesion to target cells and by modulating host cell chemokine production.
nophils from eosinophilic donors exhibit metabolic, morphologic, and functional changes indicative that they have been “activated” in vivo. Ongoing studies continue to provide evidence for this cytokine “activation” of eosinophils. While the eosinophil-active growth factor cytokines contrib- ute to the process of eosinophil “activation”, these cytokines alone do not elicit all measures of eosin- ophil activation, such as enhanced expression of FcεRI (Gounni et al. 1994a) or CD40 (Ohkawara et al. 1996) found on eosinophils from allergic sub- jects. Other cytokines or tissue or extracellular matrix derived activating stimuli are likely to be involved as well in augmenting specific functional capabilities of eosinophils (Sedgwick et al. 1995). Allergen-induced recruitment of eosinophils into lung tissues is correlated with roles of CD4 + T cells, presumably Th2 cells, and cytokines released by such T cells (Iwamoto et al. 1993, Van Oosterhout et al. 1993, Foster et al. 1996). In hu- mans, IL-5 and to a lesser extent GM-CSF were the predominant eosinophil survival-promoting cytokines inantigen-induced pulmonary late-phase reactions (Ohnishi et al. 1993). The accumulation of eosinophils in tissues, as in chronic asthma or following acute antigen challenges inthe lungs, correlates with measures of local T cell activation (Wardlaw & Kay 1992). For instance, increases in activated T lymphocytes, eosinophils, and cytokine mRNA expression for IL-5 and GM-CSF have been documented in bronchial biopsies after allergen inhalation challenge in atopic asthmatics (Bentley et al. 1993). Thus, there has been an in- creasing recognition that eosinophil accumulation and enhanced effector functions at tissue sites of allergic reactions may be intimately related to lym- phocyte activation, especially by nominally Th2- like lymphocytes elaborating cytokines, including IL-5 and GM-CSF, that prolong the viability and enhance the effector responses of mature eosino- phils.
Together our data suggests that the antibodies present inthe pleural fluid might probably be due to the passive diffusion from the plasma rather than the local production as reported previously  . However studies that employed single or multiple, purified or recombinant antigens support the concept ofthe local antibody production in TP pleuritis [21, 26] . Such studies employing purified antigens have to be carried out particularly in an endemic area to ascertain whether there is local antibody production against specific antigens of mycobacteria. Thus our study demonstrates that the local humoral anti-mycobacterial response does not differ extensively from the systemic humoral immuneresponsein tuberculous pleuritis.
T he human neurocysticercosis (NC) is caused by the presence ofthe larval form of Taenia solium inthe central nervous system, after the consumption of water or food contaminated with parasite eggs. he prevalence of NC is related to socioeconomic and cultural factors, representing an important public health problem in countries with deicient sanitary conditions, and in industrialized countries receiving immigrants from epidemic areas. he disease is one ofthe most severe parasite infections afecting the central nervous system, with complex biological parasite-host interactions due to the occurrence of diferent parasite antigens in diferent stages of evolution, and individual genetic variations interfering with the host response, impairing the understanding ofthe dynamics of parasite survival and host defense mechanisms 1 .
38 Figure 8: miR-106b-5p expression is upregulated during Mtb mono-infectionof macrophages. RNA from non-infected, Msm and Mtb infected Mϕ was extracted and quantified by real-time reverse transcriptase PCR at 1, 4 and 24 h postinfection. (A) Relative quantification of miRNA expression depicted as a heat map with hierarchical clustering performed by Exiqon. Each row represents a miRNA and each column a sample. Sample colour: pink - Mtb 24 h; orange - Mtb 4 h; yellow - Msm 4 h; black - Msm 24 h; green - non-infected cells (Ni) red - Mtb 1 h; blue - Msm 1 h. Colour scale: red – expression above the mean, blue - expression bellow the mean. (B) Specific quantification of miR-106b-5p expression. Values are relative to Ni and are the average of biological triplicates. Error bars show the standard error and the respective significance between samples is shown above (**p <0.01).
functional maturation of inflammatory DCs. The inhibition of iNOS expression could be reproduced in vitro, since stimulating pioglitazone pre-treated BM-DCs with heat-inactivated Ca (Figure S4B) led to similar reductions in iNOS expression. Strikingly, the reduced early cell infiltration in kidneys resulted inthe absence ofthe late detrimental neutrophil influx (Figure 6D). Hence, pioglitazone treatment improves kidney damage by diminishing local inflammation, which was evident by reduced serum urea levels at day 5 (Figure 6E). Although there was a general decrease inthe magnitude of inflammation in drug-treated animals, the measurements for IL-6 and Ccl2 in blood and kidney (Figure S4C) did not reach statistical significance due to high variability between mice. Nevertheless, pioglitazone pre-treatment significantly re- duced CCL2 and CCL7 release from Ca-stimulated BM-DCs in a dose-dependent manner (Figure 6F), whereas production of KC and MIP-2 was not influenced by the treatment (Figure S4D). A cytotoxic effect of pioglitazone was excluded by live-dead staining ofcells after drug treatment (Figure S4E). Interestingly, in addition to monocyte-attracting chemokines, pioglitazone also decreased the initial release of IFN-b by BM-DCs (Figure 6F). This observation prompted us to investigate whether the reduced release of CCL2/CCL7 results from the impaired IFN-b production. Therefore, we pre-treated Ifnar1 2/2 BM-DCs with pioglitazone and examined if the remaining release of chemokines from these cells was further decreased by the drug. Pioglitazone treatment was able to even further suppress the production of CCL2 in IFNAR1-deficient cells (Figure S4F). Therefore, the suppressive function on chemokine expression must be a direct effect ofthe PPAR-c-mediated transcription inhibition of those genes , without involving secondary IFN-b signaling.
The above results suggest that the individual CD4+ T cells that secrete anti-viral CCR5 ligands might be selectively protected from infection by R5 viruses. This ‘self-protection’ hypothesis predicts that the synthesis of anti-viral CCR5 ligands and viral proteins at the single cell level should tend toward mutual exclusivity for R5 viruses but not for X4 viruses in our culture system. Since we have to add blocking anti-beta chemokine antibodies to establish infections with R5 viruses inthe SEB stimulated cultures; it is difficult to test this hypothesis using this potent antigen as the readout. In preliminary studies, we found that using weaker antigenic stimuli, such as allogeneic MDDC, permits modest replication of R5 viruses, which allowed us to test the hypothesis inthe absence of blocking anti- b -chemokine antibodies. The study shown in Figure 4 was carried out essentially the same as that shown in Figure 1 except that allogeneic MDDC were used in lieu of SEB and autologous MDDC as antigen to elicit the primary CD4+ T cell response. Single cell analyses were carried out on day 7 of culture by intracellular staining and flow cytometry using fluorescent monoclonal antibodies to CCL4 and p24 as markers for b-chemokine and HIV-1 infection, respectively. As shown in Figure 4a, there was less apparent coincident staining for CCL4 and p24 in cultures infected with R5 viruses (upper panel) as compared with X4 viruses (lower panel). R5/X4 viruses were not evaluated in this series of studies. The apparent differences in coincident CCL4 and p24 staining were evaluated for statistical significance by calculating the ratios of CCL4 +
Autoimmune diseases result from disorders intheimmune system that erroneously recognizes self antigens. Autoreactive Bcells can arise inthe bone marrow or inthe periphery and, if not properly in- hibited or eliminated, autoimmune diseases may develop. In fact, the pathogenic roles ofBcellsin autoimmunity can be due to several mechanisms that include autoantibody production and immu- ne complex formation, cytokine synthesis, che- mokine-mediated functions, antigen presentati- on, T cell activation and ectopic lymphogenesis. These effects are part ofthe process of antibody formation and their exact roles in human autoim- munity are in fact unclear. Nevertheless, there is a strong correlation between autoimmune diseases and autoantibody production: in Rheumatoid Arthritis (RA), rheumatoid factors (RF) against the Fc portion of Ig are produced and form immune complexes that are deposited inthe joints; 182,183
Intestinal mast cells play a central role in innate immuneresponse to bacterial, parasitic, and viral infections by releasing pro-inflammatory cytokines (TNF, IL-6, LTB4) that mediate neutrophil recruitment into infected tissues and bacterial clearance [42,43]. Specifically, it has been shown that mast cell-derived IL-6 is a major mediator of survival from severe infections by enhancing intracellular killing of bacteria by neutrophils . There is a paucity of data regarding influence of weaning age or early life stress on innate mast cell responses to subsequent infections. Given that (1) mast cells play a central role inthe innate immuneresponse to enteric infections by releasing mediators that recruit neutrophils and (2) early weaned pigs exhibited suppressed cytokine production and neutrophil recruitment inthe present study, we investigated whether early weaning stress impacted intestinal mast cell activation in ETEC-challenged pigs. Our studies revealed that while ETEC-induced increases in mast cell numbers and marked degranulation in late-weaned control pigs, mast cell degranulation was profoundly attenuated in early weaned pigs. The precise role of suppressed mast cell activation in disease exacerbation in ETEC-challenged, early weaned pigs in this study is not known; however, it is plausible that this could represent an important mechanism for diminished cytokine responses and neutrophil infiltration and exacerbated clinical disease observed in early weaned pigs. With regards to elevated TNF levels and impaired mast cell degranulation observed in early weaning stressed pigs, Piliponsky et al., (2012) demonstrated in a
During blood feeding of infected phlebotomine females, neu- trophils rapidly migrate to the site ofinfection and phagocy- tize metacyclic promastigotes that are injected together with the insect saliva [8,9]. The role of neutrophils duringinfection is contradictory. These cells have been reported to help eliminate the parasite via the production of ROS and thein- duction of extracellular traps [10,11]. However, they also may play a role in inhibiting macrophage activation, due to their death by apoptosis after infection by the parasite . Neu- trophils also secrete chemokines that recruit monocytes/ macrophages as well as dendritic cells to the site ofinfection [4,13].
says (Valenzuela et al. 1999). The salivary gland homogenate ofthe tick Rhodnius prolixus (Fig. 1d) presents a 19kDa protein named Rhodnius proli- xus aggregation inhibitor 1 (RPAI-1) that inhibits collagen-induced platelet aggregation by binding to ADP (Francischetti et al. 2000), the same effect ob- served by a molecule with similar sequence and structure (pallidipin) isolated from saliva of Tria- toma pallidipennis (Fig. 1d) (Noeske-Jungblut et al. 1994). The deerfly Chrysops spp. saliva (Fig. 1a, b and d) can prevent platelet aggregation induced by ADP, thrombin and collagen, and also inhibits fibrinogen, binding to the glycoprotein IIb/IIIa re- ceptor on platelets (Grevelink et al. 1993). ADP has a key function in hemostasis through induction of platelet aggregation and derives from activated platelets and injured cells (Vargaftig et al. 1981). Thus, it is not surprisingly that the most common molecule involved in inhibition of platelet aggre- gation encountered on the majority of blood feed- ing arthropods seems to be the salivary apyrase en- zyme, that hydrolyses ATP and ADP to AMP and orthophosphate, preventing the effect of ADP on hemostasis. However, at least two different fami- lies of this enzyme exist and both known families require Ca 2+ and/or Mg 2+ for their action. Aedes aegypti (Champagne et al. 1995b), Anopheles (Arca et al. 1999) and Culex mosquitoes (Fig. 1e) (Nasci- mento et al. 2000) present in their saliva apyrases from the same family of 5’-nucleotidases. A novel apyrase enzyme sequence was found recently inthe salivary glands ofthe haematophagous bed bug Cimex lectularius (Valenzuela et al. 1998) and ho- mologous sequences were found inthe sand flies Lutzomia longipalpis (Charlab et al. 1999) and Phle- botomus papatasi (Valenzuela et al. 2001), indi- cating that this family of enzymes is widespread among arthropod species (Fig. 1e). This novel apyrase functions exclusively with Ca 2+ . It is im- portant to show that, inthe sand flies salivary com- ponents analyzed, a salivary 5’- nucleotidase was also found in L. longipalpis but not in P. papatasi (Fig. 1e) (Charlab et al. 1999). Finally, the salivary
depend on a parasite trans-sialidase activity which modifies the cell-surface CD45 and thus initiates a new signalling program independent of ROR t, RORα or AhR for the stimulation of IL-17. Neither IL-6, IL-βγ nor MyD88-associated TLR signalling were relevant for this IL-17 production inBcells . TheB cell activating factor activating factor (BAFF), a member ofthe tumor necrosis superfamily, has been recognized to positively influence proinflammatory cytokine production inBcells and might also be capable to stimulate IL-17 production inBcells. BAFF seems to play a pivotal role in different autoimmune diseases including RA . In RA, increased BAFF levels have been observed to correlate with high levels ofthe rheumatoid factor , with clinical disease activity, and response to treatment in early rheumatoid arthritis . In a clinical phase II trial the blockade of BAFF with a neutralising antibody called Tabalumab provided promising results in patients with active rheumatoid arthritis (RA) . BAFF gene silencing has been shown to lead to a significant reduction of IL-17 and Th17 cells . In addition, had Doreau et al. observed that IL-17 alone, but together with BAFF even with higher efficiency, could promote proliferation and differentiation ofB-cells and prolong their survival [6β]. These findings suggest complex mutual interactions of BAFF and IL-17 on the regulation ofB cell differentiation and function.
response to L. majorinfection (von Stebut et al. 2000). So far it has been given more emphasis in searching peptides that, recognized by the adaptative immune system, will direct theimmuneresponse to theTH1 or TH2 side. Recently, however, it has been shown that a recombinant leishmanial protein, a homologue of eukaryotic ribosomal elongation and initiation factor 4a (LeIF), induces the production of IL-12 and IL-18 by APC cells (Skeiky et al. 1998, Borges et al. 2001). LeIF also directs the adaptative immuneresponse to theTH1 side in BALB/c mice (Skeiky et al. 1998). Moreover, contrary to previously held beliefs, due to IL-12 action, LeIF converts TH2 cell populations from L. (L.) major infected BALB/c mice to IFN-γ producers (Skeiky et al. 1998). These findings expand the classical group ofthe so called pathogen- associated molecular patterns (PAMP), mainly applicable to homopolymers that are integral components of bacterial and fungal cell walls (Borges et al. 2001). The classical PAMP group includes: LPS, peptidoglycan, lipoteichoic acid, mannans, bacterial DNA, double strand RNA, and glucans (Medzhitov & Janeway 2000). The PAMP are recognized inthe host by pattern-recognition receptors (PRR). These receptors are chiefly present in APC cells as, for example, monocytes, macrophages, Bcells, dendritic cells (Medzhitov & Janeway 2000) and neutrophils (Hayashi et al. 2003). Toll-like receptors allow immediate effector cell response upon PAMP recognition. Leishmania, with its characteristic down modulating effects on monocytes/macrophages (see below), seems to avoid or overcome this immediate response. Accor- dingly, L. (L.) major was shown to induce the transcription of IL-1α through an adapter protein, MyD88 (myeloid differentiation factor 88), known to interact with Toll-like receptors (Hawn et al. 2002). Hence, signaling pathways of Toll-like receptors are involved inthe inflammatory response to Leishmaniainfection. On the other hand
ATP, released upon tissue damage and concomitant early inflammatory process, constitutes a danger signal, which initiates several pro-inflammatory responses upon binding to purinergic receptors. In particular it has been shown that ATP, released into the airways during asthmatic airway inflammation, can modulate the function of myeloid dendritic cells thereby triggering and maintaining asthmatic airways inflammation . Indeed, DCs are crucial for asthmatic inflammation because they recruit Th2 lymphocytes to the airway wall and trigger local Th2 effector cytokine production. It was also reported that P2Y 2 receptor exerts
We used immunohistological techniques to charac- terise the cell distribution and quantification of iNOS ex- pression in C57BL/6, BALB/c and IL-4-/- mice follow- ing intradermal inoculation of L. major into the ear. The LSAB method was used on 4-µm-thick paraffin sections of ear using a rabbit polyclonal antibody against iNOS (1:10.000; Chemicon, Temecula, CA). The immunola- beled area was measured and the results are expressed as the iNOS labelled area/mm². This study provides the first documentation ofthein situ antimicrobial function of NO inthe L. major natural model. The expression of iNOS protein inthe lesions of BALB/c was low through- out the course ofinfection, whereas the expression in C57BL/6 and IL-4 -/- mice was very low between 1-4.5 weeks p.i., but significantly increased after 12 weeks p.i (p < 0.01). Also, iNOS expression was significantly in- creased in C57BL/6 mice when compared to BALB/c at 12 weeks p.i (p = 0.0021) (Fig. 3A-D). Moreover, only a few parasites were detected inthe iNOS-immunopositive areas, whereas a large number of parasites was found in iNOS-negative regions, independent ofthe lineage.