ANTIOXIDANT ENZYME ACTIVITY AMONG ORPHANS INFECTED WITH INTESTINAL PARASITES IN PATHUM THANI PROVINCE, THAILAND.

Intestinal parasitic infections can negatively impact growth and nutrition in children. The infections can induce oxidative stress, resulting in a variety of illnesses. We measured antioxidant enzyme levels in orphan children infected with intestinal parasites to investigate the influence of nutritional status on antioxidant enzymes. This cross sectional study was conducted at an orphanage in Thailand. Stool samples were obtained from each subject and examined for intestinal parasites. Anthropometric measurements, complete blood count and biochemical parameters, including serum superoxide dismutase (SOD) and glutathione peroxidase (GPx) levels, were obtained from studied subjects. One hundred twenty-eight children were included in the study. Intestinal parasites were found on microscopic examination of the stools in 36.7% (47/128); 18% (23/128) had a mixed parasite infection. Intestinal protozoa were found in 34.4% of subjects and intestinal helminthes were found in 2.3%. The median GPx level in children infected with intestinal parasites (2.3 ng/ml) was significantly lower than in non-infected children (7.7 ng/ml) (p < 0.05). However, there was no significant difference in SOD levels between the two groups. When comparing GPx levels in children with 1) pathogenic parasites, 2) non-pathogenic parasites and 3) no intestinal parasite infection, GPx levels differed significantly among three groups (2.2 ng/ml, 2.4 ng/ml and 7.7 ng/ml, respectively) (p < 0.05). When separating children by BMI and type of infection, the median SOD level in underweight children infected with pathogenic parasites (107.2 ng/ml) was significantly higher than in underweight children infected with non-pathogenic parasites (68.6 ng/ml) and without intestinal parasite infections (72.2 ng/ml). The present study identified two key findings: low GPx levels in children with intestinal parasitic infections, and the potential impact of malnutrition on some antioxidants.

The silencing of cysteine proteases in Fasciola hepatica newly excysted juveniles using RNA interference reduces gut penetration.

Probing protein function in parasitic flatworms is hampered by the difficulties associated with the development of transgenic approaches. Although RNA interference (RNAi) in schistosomes shows much promise, it has not been reported in other trematodes. Here, we show the successful silencing of the cysteine proteases cathepsin B and L in the infective stage of Fasciola hepatica newly excysted juveniles (NEJs). Silencing resulted in marked reductions in target transcript levels and significant diminution in the encoded proteins in the gut. RNAi of either enzyme in NEJs induced transient, abnormal locomotory phenotypes, and significantly reduced penetration of the rat intestinal wall.

Trichinella spiralis induces de novo expression of group IVC phospholipase A2 in the intestinal epithelium.

Phospholipase A2 (PLA2) enzymes play a central role in the initiation, propagation and resolution of inflammation. Here, we describe de novo expression of group IVC PLA2 (PLA2g4c) within the intestinal epithelium of Trichinella spiralis parasitised mice. This mouse mast cell protease-1 sensitive, calcium-independent PLA2 is not detectable in the jejunal epithelium of uninfected mice but becomes highly expressed within the epithelial compartment within days of nematode establishment. We propose that epithelial PLA2g4c accounts for the increased lysophospholipase activity observed during intestinal nematodiasis and that it plays a major role in the inflammatory response to nematodes.

Gastrointestinal nematode infection increases organophosphate toxicity in rats.

Serum paraoxonase-1 (PON1) is an esterase associated with high-density lipoproteins in plasma and is involved in the detoxification of organophosphates (OP). We have previously reported a significant decrease in serum PON1 activity following Nippostrongylus brasiliensis infection in Wistar rats. In the present study we investigated the effects of decreased serum PON1 activity due to N. brasiliensis infection on acute toxicity induced by chlorpyrifos oxon (CPO) and paraoxon (PO) in rats. CPO and PO were dermally applied at doses of 8 mg/kg and 0.2 mg/kg body weight, respectively, to infected (on day 7 post-infection) and uninfected rats, after which acetylcholinesterase (AChE) activity was measured within the brain, diaphragm, plasma, and red blood cells, 4h after administration as a measure of toxicity. In addition, serum PON1 activity was measured immediately prior to administration of CPO and PO. N. brasiliensis infection significantly increased the degree of inhibition of AChE in the brain and diaphragm after treatment with CPO and PO in association with a significant reduction in PON1 activity. Likewise, similar findings were observed in the blood (plasma and RBCs) ChE activity after treatment with PO, but not CPO. These results indicate that N. brasiliensis infection makes rats more susceptible to CPO and PO toxicity, suggesting that gastrointestinal nematode infection might be a potential factor affecting OP toxicity.

Entamoeba histolytica activates host cell caspases during contact-dependent cell killing.

Entamoeba histolytica is a human intestinal parasite that causes amoebic colitis as well as liver abscesses. Host tissues are damaged through a three-step process involving adherence, contact-dependent cytolysis, and phagocytosis. These three processes all contribute to the pathogenicity of this parasite. Adherence is provided by the Gal/GalNAc adherence lectin. Host cells are lysed in a contact-dependent fashion. There is evidence that suggests that this contact-dependent killing involves the induction of the host cell's apoptotic machinery. Phagocytosis can then occur, consistent with metazoan apoptotic clearance.

Modulation of porcine biotransformation enzymes by anthelmintic therapy with fenbendazole and flubendazole.

Fenbendazole (FEN) and flubendazole (FLU) are benzimidazole anthelmintics often used in pig management for the control of nematodoses. The in vivo study presented here was designed to test the influence of FLU and FEN on cytochrome P4501A and other cytochrome P450 (CYP) isoforms, UDP-glucuronosyl transferase and several carbonyl reducing enzymes. The results indicated that FEN (in a single therapeutic dose as well as in repeated therapeutic doses) caused significant induction of pig CYP1A, while FLU did not show an inductive effect towards this isoform. Some of the other hepatic and intestinal biotransformation enzymes that were assayed were moderately influenced by FEN or FLU. Strong CYP1A induction following FEN therapy in pigs may negatively affect the efficacy and pharmacokinetics of FEN itself or other simultaneously or consecutively administered drugs. From the perspective of biotransformation enzyme modulation, FLU would appear to be a more convenient anthelmintic therapy of pigs than FEN.

Angiostrongylus cantonensis cathepsin B-like protease (Ac-cathB-1) is involved in host gut penetration.

Although the global spread of the emerging zoonosis, human angiostrongyliasis, has attracted increasing attention, understanding of specific gene function has been impeded by the inaccessibility of genetic manipulation of the pathogen nematode causing this disease, Angiostrongylus cantonensis. Many parasitic proteases play key roles in host-parasite interactions, but those of A. cantonensis are always expressed as the inactive form in prokaryotic expression systems, thereby impeding functional studies. Hence, a lentiviral system that drives secreted expression of target genes fused to a Myc-His tag was used to obtain recombinant Ac-cathB-1 with biological activity. Although this class of proteases was always reported to function in nutrition and immune evasion in parasitic nematodes, recombinant Ac-cathB-1 was capable of hydrolysis of fibronectin and laminin as well as the extracellular matrix of IEC-6 monolayer, so that the intercellular space of the IEC-6 monolayer increased 5.15 times as compared to the control, while the shape of the adherent cells partly rounded up. This suggests a probable role for this protease in intestinal epithelial penetration. The inhibition of Ac-cathB-1 enzymatic activity with antiserum partly suppressed larval penetration ability in the isolated intestine. Thus, an effective system for heterologous expression of parasite proteases is presented for studying gene function in A. cantonensis; and Ac-cathB-1 was related to larval penetration ability in the host small intestine.

Signal transduction events in human peripheral blood mononuclear cells stimulated by Schistosoma mansoni antigens.

Activation of protein tyrosine kinases (PTKs) is a common step of T cell stimulation. However, the relationship between PTKs and activation of peripheral blood mononuclear cells (PBMC) from intestinal chronic schistosomiasis patients has not been explored yet. In this study, we investigated the participation of Lck and ZAP-70 protein tyrosine kinases (PTKs), as well as PLC-gamma1 and Shc proteins in PBMC activation by Schistosoma mansoni antigens. PBMC were stimulated with SEA (soluble egg antigen) or SWAP (soluble worm preparation), lysed, precipitated with specific antibodies and the level of tyrosine phosphorylation evaluated. Our results show that Lck and Shc were phosphorylated upon stimulation of the cells with SWAP, as well as with SEA. However, the phosphorylation level was more pronounced in SWAP than in SEA-stimulated cells. Phosphorylation of ZAP-70 was observed only in SWAP stimulated cells. Additionally, PLC-gamma1 phosphorylation was not observed in PBMC stimulated with SEA. Together, these results indicate that SEA and SWAP induce PBMC proliferation through distinct intracellular signaling pathways. Moreover, the weaker response of PBMC to SEA compared to SWAP stimulation suggests down-regulation of cells from intestinal chronic schistosomiasis patients to SEA, which may occur during immunomodulation to S. mansoni response.

Effects of nematode infection on sensitivity to intestinal distension: role of tachykinin NK2 receptors.

Distension of the rat intestine causes a depressor response which is predictive of nociception. This study investigated the effects of previous infection with Nippostrongylus (N.) brasiliensis on the sensitivity to intestinal distension and the role of tachykinin NK2 receptors. The tachykinin NK2 receptor antagonist, SR48968 (S)-N-methyl-N[4-(4-acetylamino-4-phenylpiperidino)-2-(3,4-dichloropheny l)butyl]benzamide) inhibited the nociceptive response (ED50 = 0.7 mg/kg) in control rats. In post-N. brasiliensis-infected rats sensitivity to intestinal distension was increased which was accompanied by an increase in the apparent potency value of SR48968 (ED50 = 0.1 mg/kg). The hypersensitivity was limited to areas of hypermastocytosis. It is concluded that the post-inflammatory changes that occur in post-infected rats increase visceral sensitivity and the apparent potency of tachykinin NK2 receptor antagonists.

Neuronal nitric oxide synthase activity is increased during granulomatous inflammation in the colon and caecum of pigs infected with Schistosoma japonicum.

Neuronal nitric oxide is a non-adrenergic non-cholinergic neurotransmitter in the enteric nervous system and plays a role in a variety of enteropathies including Crohn's and Chagas' diseases, ulcerative colitis, diabetes, atrophy and hypertrophy. The content of neuronal nitric oxide synthase (nNOS) in the colon and the caecum from pigs infected with Schistosoma japonicum was studied using immunohistochemical and histochemical staining for nNOS and nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-diaphorase), respectively. In the infected pigs, lightly, moderately and less severely inflamed tissues showed increased nNOS and NADPH-diaphorase activities in nerve cell bodies and nerve fibres in the enteric plexuses compared to control pigs. There was a significant increase in the nerve cell body density of nNOS immunoreactive nerve cell bodies in the inner submucous plexus, outer submucous plexus and in the myenteric plexus. More intensely stained nerve cell bodies and varicosities were observed in tissue from prenatally infected and prenatally infected, postnatally re-infected pigs compared to postnatally infected pigs. However, the latter showed the highest numerical density of nNOS immunoreactive nerve cell bodies. Marked increases were seen in the inner submucous plexus followed by myenteric plexus, inner circular muscle, outer submucous plexus and mucous plexus. However, in very severe inflamed tissues, the number and staining intensity of nerve cell bodies and nerve fibre varicosities were reduced in plexuses located in the lesions with the inner submucous and mucous plexuses being the most affected. There was no staining in the nervous tissue within the eosinophilic cell abscesses and productive granulomas. The apparent alterations in the activities of enzymes responsible for the generation of nitric oxide (NO) show possible alterations in the NO mediated non-adrenergic non-cholinergic reflexes in the enteric nervous tissue. These alterations might contribute to impaired intestinal motility and absorption, and other pathophysiological conditions seen during S. japonicum infections.

The response of the intestinal epithelium in B10.A mice to infection with Trichinella spiralis.

Previous studies on intestinal trichinosis have dealt mainly with areas other than the intestinal epithelium. Since the epithelium is now known to be the parasite's habitat, its response to infection is important. Infection with Trichinella spiralis in immunologically slow-responding B10.A mice was associated with crypt hyperplasia and villus atrophy. With similar infection levels in both primary and challenge infections, there was no difference in the maximal degree of atrophy or hyperplasia between the 2 groups. However, challenged mice underwent these mucosal changes in about half the time. Expulsion of worms always occurred during regeneration of the intestinal epithelium suggesting that the host's defense mechanism of altering the kinetics of the epithelium was not the prime factor causing expulsion. Pulse labelling of enterocytes with [3H] thymidine showed that there was no significant increase in the relative size of the proliferation zone. This indicates that the crypt cell output was not altered by this parasite. Atrophy of the villus was analysed with respect to its 3-dimensional shape. There was a decrease in both height and width of the villus but not thickness. Thus, there is a real decrease in the size of the enterocyte population per villus. Histochemical staining of the enterocyte brush border by an alkaline phosphatase method showed that (1) hyperplastic crypts have an enlarged maturation zone and (2) the villus epithelium is composed entirely of mature cells. The distribution of the nematode population was compared to these changes in the intestine. Trichinella spiralis showed a marked anteriad (distal to proximal) migration prior to expulsion. Thus, utilizing a novel approach to study intestinal trichinosis, the response of the mucosal epithelium has been characterized.

Alkaline phosphatase activities in intestinal mucosa from calves infected with Cooperia punctata and Eimeria bovis.

Thirty calves 12 weeks of age raised under essentially parasite-free conditions were used to determine the effects of Cooperia punctata and Eimeria bovis (in single and combination infections) on mucosal alkaline phosphatase activities at 8 locations in the small intestine. In experiment 1, 5 calves infected with C punctata 3 weeks previously had highly significant (P less than 0.01) reductions in mucosal alkaline phosphatase activities compared with those in 5 noninfected control calves. These reductions were greatest in the 2 locations of the intestine closest to the pylorus. Infected calves had a mean of 44,356 C punctata adults present. In experiment 2, 5 calves infected with E bovis 2 weeks previously had significant (P less than 0.05) reductions in mucosal alkaline phosphatase activities compared with those in 5 noninfected control calves. These reductions were present in the caudal half of the intestine. Numerous 1st-generation E bovis schizonts were present in the caudal third of the intestine. In experiment 3, 5 calves infected with C punctata and E bovis of the same durations as in experiments 1 and 2 had highly significant (P less than 0.01) reductions in mucosal alkaline phosphatase activities in the cranial half of the intestine compared with those in the controls. These reductions were much larger than in either of the monospecific infections (experiments 1 and 2). A mean of 31,968 adult C punctata were recovered from the infected calves, and numerous E bovis schizonts were observed in the caudal third of the intestine.

[Trypsin and alpha-amylase activities in the pancreas of guinea pigs. II. Effect of per os and intra-intestinal infection with invasive ova of Ascaris suum]. / Aktywnosc trypsyny i alfa-amylazy w trzustce swinek morskich. II. Wplyw zarazenia per os i dojelitowo inwazyjnymi jajami ascaris suum.

The guinea pigs were infected orally or intraintestinally (after laparotomy) with 5000 invasive eggs of Ascaris suum. On the 7th day after infection the invasion was controlled by lung's weight, degree of congestion, and number of larvae in the lungs. The activity of amylase was determined according to Fennel method, trypsin to Anson method. The activities of both enzymes in pancreas of orally infected animals (after laparotomy or not) were lower, and relative weight of lungs of these animals was higher than in control animals. In pancreas of guinea pig with invasive material introduced into its, the activities of amylase and trypsin were higher than in control animals, but only the activity of trypsin differed significantly.

Intestinal infection with Nematospiroides dubius selectively increases lactase expression by mouse jejunal enterocytes.

1. Intestinal structure, lactase (beta-galactosidase; EC 3.2.1.23) activity and alkaline phosphatase activity have been determined in mouse jejunal and ileal tissues before and during infection with the intestinal parasite Nematospiroides dubius. 2. Oral infection with small numbers of N. dubius larvae caused villus height, crypt depth and enterocyte migration rate to increase in the mouse jejunum. None of these effects occurred in ileal tissue. 3. Lactase activity also increased in jejunal, but not ileal, tissue of infected mice. This increase was associated with a doubling of the rate at which activity appeared in the brush-border membrane of enterocytes during migration over the basal regions of jejunal villi. Alkaline phosphatase activity in jejunal tissue remained unchanged in infected mice. 4. Attention is drawn to the fact that this is the first occasion when crypt cell hyperplasia has been found to be positively correlated with an increase in lactase activity and a decrease in cytotoxic/suppressor T-cells. Further work is needed to establish the primary cause of these effects.

Changes in enzyme activities in the small intestine of rabbits during early infection with Trichostrongylus colubriformis.

In rabbits experimentally infected by fourth and fifth stages (L4 and S5) of Trichostrongylus colubriformis, 16 enzymatic activities in the small intestine were compared to control values, by means of a modified Api Zym method. In the proximal part of the gut, the wide depletion in brush border enzyme activities previously recorded with presence of adult T colubriformis was also noticed with L4 and S5. Moreover, a 10 to 23% reduction was observed for enzyme activities linked to lysosomes and endoplasmic reticulum. In the distal region of the small intestine, no difference in enzymatic activities was present between infected and control animals, although an hyperplasia of villi has been previously described in this part of the parasitised gut. Referring to these enzymatic results for the whole small intestine, the hypothesis of a mechanical effect of worms on the intestinal epithelium and/or disturbance in the renewal of epithelial cells is discussed.