Effects of duration of exposure, co-supplementation with iron and Nippostrongylus brasiliensis infection on accumulation of trichloroacetic acid-insoluble copper in rats given molybdate in drinking water.

Thiolation can convert molybdate (MoO4) into a series of thiomolybdates (MoSxO4-x) in the rumen, terminating in tetrathiomolybdate (MoS4), a potent antagonist of copper absorption and, if absorbed, donor of reactive sulphide in tissues. Systemic exposure to MoS4 increases trichloroacetic acid-insoluble copper (TCAI Cu) concentrations in the plasma of ruminants and induction of TCAI Cu in rats given MoO4 in drinking water would support the hypothesis that rats, like ruminants, can thiolate MoO4. Data on TCAI Cu are presented from two experiments involving MoO4 supplementation that had broader objectives. In experiment 1, plasma Cu concentrations (P Cu) tripled in female rats infected with Nippostrongylus brasiliensis after only 5 days exposure to drinking water containing 70 mg Mo L-1, due largely to an increase in TCAI Cu; activities of erythrocyte superoxide dismutase and plasma caeruloplasmin oxidase (CpOA) were unaffected. Exposure for 45-51 days did not raise P Cu further but TCA-soluble (TCAS) Cu concentrations increased temporarily 5 days post infection (dpi) and weakened the linear relationship between CpOA and TCAS Cu. In experiment 2, infected rats were given less MoO4 (10 mg Mo L-1), with or without iron (Fe, 300 mg L-1), for 67 days and killed 7 or 9 dpi. P Cu was again tripled by MoO4 but co-supplementation with Fe reduced TCAI Cu from 65 ± 8.9 to 36 ± 3.8 µmol L-l. Alone, Fe and MoO4 each reduced TCAS Cu in females and males when values were higher (7 and 9 dpi, respectively). Thiolation probably occurred in the large intestine but was inhibited by precipitation of sulphide as ferrous sulphide. Fe alone may have inhibited caeruloplasmin synthesis during the acute phase response to infection, which impacts thiomolybdate metabolism.

Effects of Molybdate and Tetrathiomolybdate Supplementation of Drinking Water on Immature Rats Infected with Nippostrongylus brasiliensis. 2. Copper Status and Tissue Molybdenum Accretion.

Molybdate (MoO4) and tetrathiomolybdate (MoS4) supplementation of rats via drinking water had opposite effects on the establishment of Nippostrongylus brasiliensis larvae but both induced hypercupraemia, temporarily inhibited activities of superoxide dismutase in liver and duodenum after infection and enlarged the femoral head. Effects of MoO4 and MoS4 on activities of caeruloplasmin oxidase (CpO) in plasma, erythrocyte superoxide dismutase (ESOD) and tissue copper (Cu) and molybdenum (Mo) were compared to test the hypothesis that species lacking a rumen can thiolate MoO4. Three groups of 18 immature Wistar rats were given Mo (70 mg/L as MoO4) or MoS4 (5 mg/L) via drinking water or remained untreated; all received a commercial, cubed diet and 12 from each group were infected with larvae of N. brasiliensis. Rats were killed 7-9 days later and liver, kidney, spleen, heart, muscle (quadriceps), brain and bone (femur) removed for Cu and Mo analysis. Plasma Cu was greatly increased by MoO4 and MoS4, without changing CpO activity, but the effect was more variable with MoO4 and accompanied by a smaller decrease in ESOD. Tissue Cu and Mo were increased by MoS4 in all tissues examined except brain and bone, correlating with plasma Cu and with each other; relationships were strongest in spleen, followed by kidney. MoO4 also increased soft tissue Cu and Mo but increases were generally smaller than those induced by MoS4 and correlations between the two elements and with plasma Cu generally weaker. Since hypercupraemia and correlated increases in liver and kidney Cu and Mo are characteristic of systemic thiomolybdate (TM) exposure, we conclude that MoO4 was partially thiolated to give a different TM profile from that produced by MoS4. The pathophysiological significance of systemic exposure to di- and tri-TM merits investigation in non-ruminants as agents of chelation therapy and in ruminants as agents of short-lived TM toxicity on Mo-rich pastures.

Effects of Chronic Exposure to Molybdate and Tetrathiomolybdate Supplementation of Water on Immature Rats Acutely Infected with Nippostrongylus brasiliensis. 1. Effects on Outcome of Infection and Host Health.

Low molybdate (MoO4) exposure via drinking water in mature rats infected with Nippostrongylus brasiliensis raised liver and plasma copper (Cu) concentrations. The possibility that anthelmintic effects were attributable to conversion of MoO4 to tetrathiomolybdate (MoS4) in a non-ruminant species was investigated by giving three groups of 18 immature rats drinking water containing 70 mg Mo l-1 as MoO4 (group A), 5 mg Mo l-1 as MoS4 (group B) or no supplement (group C), while receiving a commercial cubed diet. After 41 days, 12 rats from each group were inoculated subcutaneously with 2,000 L3-stage N. brasiliensis larvae. Subgroups were killed 7, 8 or 9 days post infection (dpi), when adult worms are normally expelled, and enzyme markers for the inflammatory response to infection were measured in plasma or liver. Male rats given MoS4 prior to infection grew more slowly than those given MoO4. Eight dpi, females given MoS4 had lost more bodyweight than those in group C, while those given MoO4 had gained weight. Mean worm counts at 7 dpi were 160, 65 and 250 ± 30.6 (SE), respectively, in groups C, A and B, and differed significantly from each other (P <0.05) but only rats given MoO4 remained infected 9 dpi (mean worm count 52 ± 16.4): Faecal egg counts followed a broadly similar pattern. Both Mo sources pre-empted increases in liver and duodenal superoxide dismutase activity, induced by infection 7 and 9 dpi, respectively, in group C and enlarged the femur: neither source prevented hypertrophy of the small intestine and a rise in serum mast cell protease concentration caused by infection. Since data for plasma Cu concentration and caeruloplasmin oxidase activity, reported separately, indicated MoO4 was thiolated in vivo, differences between Mo sources may be attributable to differences in the degree of thiolation, extent of thiomolybdate exposure and rates of thiomolybdate degradation at critical times in host or parasite development.

Effects of Raising or Lowering Molybdenum Status on Outcome of Acute Infection with Nippostrongylus brasiliensis in Mature Rats.

Molybdate (Mo+) supplements can suppress or enhance nematode infections in ruminants, depending on exposure level, but there have been no investigations in non-ruminants. Three groups of 16 mature rats were each fed a commercial diet and given Mo+ (10 mg Mo/l), tungstate (a molybdenum [Mo] antagonist) (MoO4, 350 mg W/l) or no supplement (C) via drinking water for 40 days before acute infection with 3,600 Nippostrongylus brasiliensis larvae. Group Mo- also received allopurinol (1 g/l), a molybdenoenzyme inhibitor, from 4 days post infection (dpi). Subgroups of four rats from each group were killed at 7-14 dpi. A group of six rats was left untreated and uninfected and subgroups killed 10 or 12 dpi. Infection reduced intakes of food and water but impacts were greatest in group Mo-. Median worm counts in groups C, Mo- and Mo+ were 900, 941 and 510, respectively, at 7 dpi and 9, 40 and 0 (P = 0.05) at 10 dpi. Median faecal egg counts were consistently lowest in group Mo+. Worm weight was reduced (P <0.05), worm tissue protease increased and superoxide dismutase activities increased in worm (P < 0.01) and host duodenal homogenates (P < 0.01) from group Mo+. In group Mo-, liver Mo concentration decreased, duodenal xanthine oxidoreductase activity (DXOR) became totally inhibited and plasma uric acid was barely detectable at 10 dpi. Plasma mast cell protease activity and duodenal malonyldialdehyde concentrations, markers of inflammation, were increased by nematode infection (P <0.001) but unaffected by water treatments. Liver Mo, liver copper (Cu) and plasma Cu concentrations were increased in group Mo+ and plasma Cu concentration was increased in group Mo- suggesting systemic exposure to partially thiolated MoO4 and WO4. Supplementary MoO4 impaired larval establishment and changed parasite biochemistry without affecting the inflammatory response to infection but may have required partial thiolation to do so. Rats did not rely on DXOR activity to expel N. brasiliensis.

Dietary protein supplementation results in molecular and cellular changes related to T helper type 2 immunity in the lung and small intestine in lactating rats re-infected with Nippostrongylus brasiliensis.

Acquired immunity to gastrointestinal nematodes reduces during late pregnancy and lactation which is known as periparturient relaxation of immunity (PPRI). Protein supplementation reduces the degree of PPRI in a rat model re-infected with Nippostrongylus brasiliensis, but the underlying molecular mechanisms have yet to be elucidated. Here, we hypothesized that protein supplementation will enhance T helper type 2 immunity (Th2) in the lung and small intestine. Nulliparous Sprague-Dawley rats were given a primary infection of N. brasiliensis prior to mating and restrictedly fed diets with either low protein (LP) or high protein (HP) during pregnancy and lactation. Dams were secondary infected with N. brasiliensis on day 2 post-parturition, and histology and gene expression were analysed for tissue samples collected at days 5, 8 and 11. Genes related to Th2 immunity in the lung, Retnla, Il13 and Mmp12, and in the intestine, Retnlb, were upregulated in HP dams compared to LP dams, which indicates the effect of dietary protein on Th2 immunity. HP dams also had increased splenic CD68+ macrophage populations compared to LP dams following secondary infection, suggesting enhanced immunity at a cellular level. Our data assist to define strategic utilization of nutrient supply in mammals undergoing reproductive and lactational efforts.

Body Protein Reserves Sustain Maternal Performance in Early Lactation but Dietary Protein Is Necessary to Maintain Performance and Immune Responses to Nippostrongylus brasiliensis in Lactating Rats.

Background: It has been shown that dietary protein supplementation during lactation boosts immunity in Nippostrongylus brasiliensis-infected periparturient rats. It is not known whether body protein reserves accumulated during gestation have a similar effect during lactation. Objective: This study aimed to quantify the impact of body protein reserves and dietary protein supplementation on maternal performance and immune responses to N. brasiliensis during lactation. Methods: Multiparous female Sprague-Dawley rats were administered a primary infection of N. brasiliensis before mating and were restriction-fed either 60 g [low-protein diet gestation (Lge)] or 210 g [high-protein diet gestation (Hge)] crude protein (CP) per kilogram of dry matter (DM) until parturition. From parturition onward, dams were restriction-fed either 100 g [low-protein diet lactation (Lla)] or 300 g [high-protein diet lactation (Hla)] CP per kilogram of DM, generating 4 different dietary treatments. A subset of rats was sampled before parturition; postparturition, dams were secondarily infected with N. brasiliensis and samples were collected at days 5 and 11 postparturition. Results: Maternal performance until parturition, as measured by pup weight, was better in Hge rats than in Lge rats [Lge: 4.84 g; Hge: 6.15 g; standard error of the difference (SED): 0.19]. On day 11, pup weights of dams with reduced protein reserves fed protein during lactation (Lge-Hla; 20.28 g) were higher than their counterparts from Hge-Lla dams (17.88 g; SED: 0.92). Worm counts were significantly different between Lge-Lla-fed (253; 95% CI: 124, 382) and Hge-Hla-fed (87; 95% CI: 22, 104) dams on day 11 (P = 0.024). The expression of splenic interleukin 13 (Il13) and arachidonate 15-lipoxygenase (Alox15) was significantly higher (P < 0.05) in Hge-Hla dams compared with Lge-Lla dams on day 5. Conclusions: Although protein reserves were adequate to maintain maternal performance in the early stage of lactation in dams infected with N. brasiliensis, they were not adequate to maintain maternal performance and effective immune responses at later stages. Dietary protein supplementation was required to achieve this.

Hematopoietic cell-derived RELMα regulates hookworm immunity through effects on macrophages.

Resistin-like molecule α (RELMα) is a highly secreted protein in type 2 (Th2) cytokine-induced inflammation including helminth infection and allergy. In infection with Nippostrongylus brasiliensis (Nb), RELMα dampens Th2 inflammatory responses. RELMα is expressed by immune cells, and by epithelial cells (EC); however, the functional impact of immune versus EC-derived RELMα is unknown. We generated bone marrow (BM) chimeras that were RELMα deficient (RELMα-/- ) in BM or non BM cells and infected them with Nb. Non BM RELMα-/- chimeras had comparable inflammatory responses and parasite burdens to RELMα+/+ mice. In contrast, both RELMα-/- and BM RELMα-/- mice exhibited increased Nb-induced lung and intestinal inflammation, correlated with elevated Th2 cytokines and Nb killing. CD11c+ lung macrophages were the dominant BM-derived source of RELMα and can mediate Nb killing. Therefore, we employed a macrophage-worm co-culture system to investigate whether RELMα regulates macrophage-mediated Nb killing. Compared to RELMα+/+ macrophages, RELMα-/- macrophages exhibited increased binding to Nb and functionally impaired Nb development. Supplementation with recombinant RELMα partially reversed this phenotype. Gene expression analysis revealed that RELMα decreased cell adhesion and Fc receptor signaling pathways, which are associated with macrophage-mediated helminth killing. Collectively, these studies demonstrate that BM-derived RELMα is necessary and sufficient to dampen Nb immune responses, and identify that one mechanism of action of RELMα is through inhibiting macrophage recruitment and interaction with Nb. Our findings suggest that RELMα acts as an immune brake that provides mutually beneficial effects for the host and parasite by limiting tissue damage and delaying parasite expulsion.

Human resistin protects against endotoxic shock by blocking LPS-TLR4 interaction.

Helminths trigger multiple immunomodulatory pathways that can protect from sepsis. Human resistin (hRetn) is an immune cell-derived protein that is highly elevated in helminth infection and sepsis. However, the function of hRetn in sepsis, or whether hRetn influences helminth protection against sepsis, is unknown. Employing hRetn-expressing transgenic mice (hRETNTg+) and recombinant hRetn, we identify a therapeutic function for hRetn in lipopolysaccharide (LPS)-induced septic shock. hRetn promoted helminth-induced immunomodulation, with increased survival of Nippostrongylus brasiliensis (Nb)-infected hRETNTg+ mice after a fatal LPS dose compared with naive mice or Nb-infected hRETNTg- mice. Employing immunoprecipitation assays, hRETNTg+Tlr4-/- mice, and human immune cell culture, we demonstrate that hRetn binds the LPS receptor Toll-like receptor 4 (TLR4) through its N terminal and modulates STAT3 and TBK1 signaling, triggering a switch from proinflammatory to anti-inflammatory responses. Further, we generate hRetn N-terminal peptides that are able to block LPS proinflammatory function. Together, our studies identify a critical role for hRetn in blocking LPS function with important clinical significance in helminth-induced immunomodulation and sepsis.

Selenoprotein Expression in Macrophages Is Critical for Optimal Clearance of Parasitic Helminth Nippostrongylus brasiliensis.

The plasticity of macrophages is evident in helminthic parasite infections, providing protection from inflammation. Previously we demonstrated that the micronutrient selenium induces a phenotypic switch in macrophage activation from a classically activated (pro-inflammatory; M1/CAM) toward an alternatively activated (anti-inflammatory; M2/AAM) phenotype, where cyclooxygenase (COX)-dependent cyclopentenone prostaglandin J2 (15d-PGJ2) plays a key role. Here, we hypothesize that dietary selenium modulates macrophage polarization toward an AAM phenotype to assist in the increasing clearance of adult Nippostrongylus brasiliensis, a gastrointestinal nematode parasite. Mice on a selenium-adequate (0.08 ppm) diet significantly augmented intestinal AAM presence while decreasing adult worms and fecal egg production when compared with infection of mice on selenium-deficient (<0.01 ppm) diet. Further increase in dietary selenium to supraphysiological levels (0.4 ppm) had very little or no impact on worm expulsion. Normal adult worm clearance and enhanced AAM marker expression were observed in the selenium-supplemented Trsp(fl/fl)Cre(WT) mice that express selenoproteins driven by tRNA(Sec) (Trsp), whereas N. brasiliensis-infected Trsp(fl/fl)Cre(LysM) selenium-supplemented mice showed a decreased clearance, with lowered intestinal expression of several AAM markers. Inhibition of the COX pathway with indomethacin resulted in delayed worm expulsion in selenium-adequate mice. This was rescued with 15d-PGJ2, which partially recapitulated the effect of selenium supplementation on fecal egg output in addition to increasing markers of AAMs in the small intestine. Antagonism of PPARγ blocked the effect of selenium. These results suggest that optimal expression of selenoproteins and selenium-dependent production of COX-derived endogenous prostanoids, such as Δ(12)-PGJ2 and 15d-PGJ2, may regulate AAM activation to enhance anti-helminthic parasite responses.

The effect of tribendimidine and its metabolites against Necator americanus in golden hamsters and Nippostrongylus braziliensis in rats.

The antinematode effect of tribendimidine (TBD) and its metabolites has been studied. A total of 107 hamsters were each infected with 250 Necator americanus third stage infective larvae (NaL3) for 25 days. In the first test, 75 hamsters were divided equally into 15 groups for determination of ED(50) and ED(90.) Among them, five groups were treated orally with TBD or its metabolite, p-(1-dimethylamino ethylimino)aniline (aminoamidine, deacylated amidantel, BAY d 9216, dADT), at single doses of 1, 2, 4, 8, and 16 mg/kg. The remaining five groups were administered with acetylated dADT (AdADT) at single oral doses of 8, 12, 18, 24, and 30 mg/kg. In the second test, 20 hamsters were equally divided into four groups. Two groups were treated intramuscularly with TBD and dADT at a single dose of 16 mg/kg, while in the remaining two groups, single intramuscular dose of AdADT 15 or 30 mg/kg was administered. In the third test, two groups of six hamsters were treated orally with terephthalaldehyde (TPAL) and terephthalic acid (TPAC) at a single dose of 1,000 mg/kg. Other 85 rats, each infected with 300 Nippostrongylus braziliensis third stage infective larvae (NbL3), were used in three tests. For determination of ED(50) and ED(90) in the first test, five groups of five rats were treated orally with TBD or dADT at single doses of 3.0, 4.2, 5.9, 8.2, and 11.5 mg/kg or 2.0, 2.9, 4.2, 6.1, and 8.8 mg/kg, respectively. In the second test, three groups of eight to nine rats were treated orally with TBD at a single 8.4-mg/kg dose (ED(90)) and AdADT 100 or 200 mg/kg, respectively. In the third test, two groups of four rats were treated orally with TPAL and TPAC at a single dose of 1,000 mg/kg. Twenty-four to 48 h post-treatment, all the feces of each hamster and rat were collected for recovery of worms expelled from the feces. Following this period, all of the animals were sacrificed, and the adult hookworm or N. braziliensis from small intestine and large intestine were recovered and counted for calculation of worm burden reduction. The results showed that the ED(50) and ED(90) for TBD, dADT, and AdADT determined in treatment of N. americanus-infected hamsters were 1.849 and 13.598, 3.922 and 54.354, as well as 20.966 and 51.633 mg/kg, respectively. In intramuscular administration of TBD and dADT at single dose of 16 mg/kg or AdADT 30 mg/kg, similar worm burden reductions of 71.4-76.3% were observed. Two other metabolites, i.e., TPAL and TPAC, exhibited no effect against N. americanus. The ED(50) and ED(90) for TBD and dADT determined in treatment of rats infected with N. braziliensis were 3.234 and 8.435, as well as 2.345 and 5.104 mg/kg. Oral administration of AdADT at a higher single dose of 100 or 200 mg/kg resulted in worm burden reductions of 11.9-46.3%, which was significantly lower than 84.5% of worm burden reduction obtained from rats treated with TBD 8.4 mg/kg. The results indicate that in oral administration, TBD exhibits slightly better effect against N. americanus in hamsters than dADT, but AdADT possesses less effect; TBD, dADT, and AdADT show promising effect in intramuscular treatment of N. americanus-infected hamsters; the effect of oral dADT against N. braziliensis in rats is somewhat better than TBD, while AdADT endorses poor effect; and TPAL and TPAC are ineffective metabolites of TBD against both species of nematodes.

Anthelmintic efficacy of extracts of Spigelia anthelmia Linn on experimental Nippostrongylus braziliensis in rats.

Spigelia anthelmia Linn is used as a herb and is a common annual weed of cultivation in open re-growths, on unused land in towns as well as on road sides. The plant can grow to approximately 30 cm in height. The aim of this study was to screen extracts of Spigelia anthelmia for their anthelmintic activity against an experimental Nippostrongylus braziliensis infection in rats. Acute oral toxicity occurred at a dose of 1,140 mg/kg, while anthelmintic trials against Nippostrongylus braziliensis in rats using the aqueous fraction showed a progressive decrease in worm count with increasing dose (10, 13, 16, 20 and 25 mg per kg body weight) (p < 0.05). At 25 mg per kg body weight, the worm count was significantly lower than that at 10 mg per kg body weight (p < 0.05).

Anthelmintic efficacy of extracts of Spigelia anthelmia Linn on experimental Nippostrongylus braziliensis in rats

Spigelia anthelmia Linn is used as a herb and is a common annual weed of cultivation in open re-growths, on unused land in towns as well as on road sides. The plant can grow to approximately 30 cm in height. The aim of this study was to screen extracts of Spigelia anthelmia for their anthelmintic activity against an experimental Nippostrongylus braziliensis infection in rats. Acute oral toxicity occurred at a dose of 1,140 mg/kg, while anthelmintic trials against Nippostrongylus braziliensis in rats using the aqueous fraction showed a progressive decrease in worm count with increasing dose (10, 13, 16, 20 and 25 mg per kg body weight) (p < 0.05). At 25 mg per kg body weight, the worm count was significantly lower than that at 10 mg per kg body weight (p < 0.05).

[Activity of the anthelmintic agent trichlorophen on the models of human helminthiasis]. / Aktivnost' otechestvennogo antigel'mintika trikhlofena na modeliakh gel'mintozov cheloveka.

Trials of trichlorophen have shown its high efficacy on models of cestode infections: hymenolepiasis (at the adult and cysticercoid stages of development on three types of animals: outbred albino mice, albino rats and golden hamsters), preimaginal echinococciasis alveolaris, larval alveolar echinococciasis (at the early stage of development of the parasite in experiments on cotton rats). The high nematodical activity of trichlorophen was first found on models of trichocephaliasis in DBA/2y mice, nippostrongyloidiasis (in in vitro experiments), and aspiculuriasis in outbred mice. The agent proved to be ineffective at the tissue developmental stage of Hymenolepsis nana (H. nana), the dwarf tapeworm, in albino mice, during experimental opisthorchiasis in golden hamsters. It showed a low efficacy in treating trichinosis in outbred albino mice. Unlike carbamatebenzimidazoles, trichlorophen was inactive at the tissue stage of H. nana; it exerted no effects on the eggs of a dwarf tapeworm in trichinosis. Trichlorophen was also inactive in treating experimental opisthorchiasis in golden hamsters.

A distinct family of acetylcholinesterases is secreted by Nippostrongylus brasiliensis.

A third variant of acetylcholinesterase (AChE A) secreted by the parasitic nematode Nippostrongylus brasiliensis has been isolated which shows 63-64% identity to AChE B and AChE C, with a truncated carboxyl terminus and a short internal insertion relative to AChEs from other species. Three of the fourteen aromatic residues which line the active site gorge in Torpedo AChE are substituted by non-aromatic residues (Y70T, W279D and F288M). All three enzymes have 8 cysteine residues in conserved positions, including 6 which have been implicated in disulphide bonds in other AChEs. Phylogenetic analysis suggests that these enzymes form a distinct group which evolved after speciation and are most closely related to ACE-2 of Caenorhabditis elegans. Recombinant AChE A secreted by Pichia pastoris was monomeric and hydrophilic, with a substrate preference for acetylthiocholine and negligible activity against butyrylthiocholine. A model structure of AChE A built from the coordinates of the Torpedo californica AChE suggests that W345 (F331 in Torpedo) limits the docking of butyrylcholine. This model is consistent with mutational analysis of the nematode enzymes. Expression of AChE A is regulated at the transcriptional level independently of the other 2 secreted variants, with maximal expression by fourth stage larvae and young adult worms. These enzymes thus appear to represent an unusual family of AChEs with conserved structural features which operate outside the normal boundaries of known functions in regulation of endogenous neurotransmitter activity.

Activity of the cyclic depsipeptide emodepside (BAY 44-4400) against larval and adult stages of nematodes in rodents and the influence on worm survival.

The present investigations deal with the activity of the cyclic depsipeptide emodepside (BAY 44-4400) against larval and adult stages of three rodent nematodes. While emodepside acts strongly against the adult stages of the rat nematodes Nippostrongylus brasiliensis and Strongyloides ratti, as well as against the mouse nematode Heligmosomoides polygyrus, its actions against the larval stages of these nematodes vary according to the species. Thus, emodepside is highly effective against the lung and intestine larval stages of N. brasiliensis and S. ratti. By contrast. the larval stages of H. polygyrus in the intestine are only partly affected by higher emodepside dosages.

Use of L4 larvae of Nippostrongylus brasiliensis for the in vivo screening of anthelmintic drugs.

Five groups of Wistar rats were subcutaneously infected with 5,000 L3 stage larvae of Nippostrongylus brasiliensis. Four groups were dosed orally with 5, 10, 15, and 20 mg/kg body weight of albendazole at 5 h postinfection, and one group served as an untreated control. The animals were euthanized and the intestines were dissected out 72 h postinfection and were incubated on a Baermann's apparatus to recover L4 stage larvae. No larvae were recovered from the 20 mg/kg albendazole-treated group, and means of 15, 33, and 175 L4 larvae were recovered from the 15, 10, and 5 mg/kg albendazole-treated groups, respectively. Levamisole and fenbendazole were also tested using the same procedure. Means of 100 and 1,887 L4 larvae were obtained from the 50 and 10 mg/kg of levamisole-treated groups respectively; while, 191 and 583 L4 larvae were recovered from rats treated with 50 and 10 mg/kg of fenbendazole, respectively. These results indicate that the L4 stage of N. brasiliensis could be useful for in vivo screening of new nematocide drugs.

Depletion of eosinophils in mice through the use of antibodies specific for C-C chemokine receptor 3 (CCR3).

We have generated rat monoclonal antibodies specific for the mouse eotaxin receptor, C-C chemokine receptor 3 (CCR3). Several anti-CCR3 mAbs proved to be useful for in vivo depletion of CCR3-expressing cells and immunofluorescent staining. In vivo CCR3 mAbs of the IgG2b isotype substantially depleted blood eosinophil levels in Nippostrongyus brasiliensis-infected mice. Repeated anti-CCR3 mAb treatment in these mice significantly reduced tissue eosinophilia in the lung tissue and bronchoalveolar lavage fluid. Flow cytometry revealed that mCCR3 was expressed on eosinophils but not on stem cells, dendritic cells, or cells from the thymus, lymph node, or spleen of normal mice. Unlike human Th2 cells, mouse Th2 cells did not express detectable levels of CCR3 nor did they give a measurable response to eotaxin. None of the mAbs were antagonists or agonists of CCR3 calcium mobilization. To our knowledge, the antibodies described here are the first mAbs reported to be specific for mouse eosinophils and to be readily applicable for the detection, isolation, and in vivo depletion of eosinophils.

Cloning, expression, and properties of a nonneuronal secreted acetylcholinesterase from the parasitic nematode Nippostrongylus brasiliensis.

We have isolated a full-length cDNA encoding an acetylcholinesterase secreted by the nematode parasite Nippostrongylus brasiliensis. The predicted protein is truncated in comparison with acetylcholinesterases from other organisms such that the carboxyl terminus aligns closely to the end of the catalytic domain of the vertebrate enzymes. The residues in the catalytic triad are conserved, as are the six cysteines which form the three intramolecular disulfide bonds. Three of the fourteen aromatic residues which line the active site gorge in the Torpedo enzyme are substituted by nonaromatic residues, corresponding to Tyr-70 (Thr), Trp-279 (Asn), and Phe-288 (Met). High level expression was obtained via secretion from Pichia pastoris. The purified enzyme behaved as a monomeric hydrophilic species. Although of invertebrate origin and possessing the above substitutions in the active site gorge residues, the enzyme efficiently hydrolyzed acetylthiocholine and showed minimal activity against butyrylthiocholine. It displayed excess substrate inhibition with acetylthiocholine at concentrations over 2. 5 mM and was highly sensitive to both active site and "peripheral" site inhibitors. Northern blot analysis indicated a progressive increase in mRNA for AChE B in parasites isolated from 6 days postinfection.

[Approaches to the combined therapy of helminthiases with leukinferon and etiotropic preparations]. / Podkhody k kombinirovannoi terapii gel'mintozov leikinferonom i étiotropnymi preparatami.

The results of therapy with anthelmintics in combination with leukinferon show either higher efficiency of anthelmintics (medamin) or their same efficiency (albendazole). The outcomes of combined treatment of infected animals with anthelmintics and leukinferon are different and associated with the time of leukinferon administration as to the tissue stages of parasitic development, with the host's sensitivity and susceptibility to helminth.

[The effect of leukinferon on the invasive process in experimental helminthiases]. / Vliianie leikinferona na invazionnyi protsess pri éksperimental'nykh gel'mintozakh.

Two models of helminthiases (nippostrongyliasis and trichocephaliasis) were used to reveal the therapeutical activity of leukinferon (LF). In experimental nippostrongyliasis, the efficacy of LF was similar to that in infected young rats and mice whose sensitivity and resistance to infections were different. In the model of trichocephaliasis, the therapeutical effect of leukinferon was noticeably higher in C57BL/6 mice resistant to T. muris than in sensitive mice strains. In intact animals, LF brought about similar lymphocytic prestimulation depending on their sensitivity to infection. The infection itself also caused increased LF levels comparable to those observed during LF administration to intact animals. LF-induced differences in cell prestimulation were especially great as infection progressed in animals whose sensitivity to host helminths was opposite. The levels of splenocytic LF was higher in resistant hosts (non-inbred mice with nippostrongyliasis and C57BL/6 mice with trichocephaliasis) that under the similar experimental conditions in animals (rats with nippostrongyliasis and DBA/2j mice with trichocephaliasis sensitive to host helminths).