Hookworm infection aggravates metabolic disorder in obesity.

Obesity and ancylostomiasis are considered public health problems. Recent studies have shown that infection by intestinal helminths in obese individuals can ameliorate metabolic disorder and improve glucose tolerance by decreasing both insulin resistance and low-intensity inflammation. However, few helminth species have been studied in this context, and some modulation mechanisms still require deeper investigation. Therefore, the present work aimed to investigate the role of experimental infection with Ancylostoma ceylanicum in the modulation of the immune response in an obese experimental model. Four groups of hamsters were used as follows: two groups were submitted to a hyperlipidic and hypercaloric diet capable of inducing obesity, one infected and the other uninfected; and two normonourished control groups, one infected and one uninfected by A. ceylanicum. Biochemical, haematological, parasitological and immunological parameters were evaluated. The results demonstrated that A. ceylanicum infection accentuated weight loss in obese animals compared to normonourished animals. However, obesity reduced the recovery of worms and oviposition of the females, and both infected groups showed decreased levels of haemoglobin, albumin, iron and erythrocytes. Significant relations were observed for pathogenesis in the following cases: infection interfered in lipid metabolism, which increased levels of total cholesterol and triglycerides in the obese group, and caused a decrease in HDL levels in both groups. Obesity led to an increase in glucose levels, and the infection exacerbated this parameter in both the normonourished and obese groups. Inflammation was intensified in obese animals that showed elevated macrophage and neutrophil activation in adipose tissue, enlargement of the spleen and accumulation of lipids in the liver and faeces. Despite the decrease in IFN-γ levels, the infection did not potentiated the expression of the Foxp3, IL-10 and IL-2 transcription factor for any of the infected groups, markers that could positively compensate the host from the damage caused by obesity.

Evaluation of biochemical, hematological and parasitological parameters of protein-deficient hamsters infected with Ancylostoma ceylanicum.

BACKGROUND: Hookworms infect millions of people worldwide and can cause severe clinical symptoms in their hosts. Prospective cohort studies in Brazil show high rates of hookworm reinfection in malnourished children compared to well-nourished children, despite previous treatment. Additionally, soil-transmitted helminth (STH) infections can worsen the nutritional status of affected populations. Therefore, this study aims to clarify the effects of host malnutrition during Ancylostoma ceylanicum infection and how this infection affects host physiological parameters using a hamster model. METHODOLOGY/PRINCIPAL FINDINGS: Hamsters were divided into four experimental groups: normal diet or low-protein diet (also referred to as "malnourished") and A. ceylanicum infection or no infection. More severe pathogenesis was observed in the infected malnourished group, as demonstrated by significant decreases in the hemoglobin concentration, erythrocyte number and packed-cell volume compared to the non-infected malnourished group. Greater numbers of adult parasites and eggs were observed in the malnourished group compared to the control group; however, the oviposition rate was lower in the malnourished group. In general, greater values of total lipids were observed in malnourished animals compared to control animals, including lipids excreted in the stool. CONCLUSIONS: In this work, we have demonstrated that animals fed an isocaloric low-protein diet presented more severe pathogenesis when infected with A. ceylanicum. The increased lipid concentration in the liver and blood is related to the conversion of the excess carbohydrate into fatty acids that increase the concentration of triglycerides in general. Triglycerides were excreted in the feces, indicating that infection associated with malnutrition caused a greater loss of these molecules for this group of animals and confirming the hypothesis that both nutrition and infection are responsible for the malabsorption syndrome. Taken together, the results found in this work confirm the hypothesis that the nutritional condition of the host greatly influences the course of the infection.

Structural conservation of ligand binding reveals a bile acid-like signaling pathway in nematodes.

Bile acid-like molecules named dafachronic acids (DAs) control the dauer formation program in Caenorhabditis elegans through the nuclear receptor DAF-12. This mechanism is conserved in parasitic nematodes to regulate their dauer-like infective larval stage, and as such, the DAF-12 ligand binding domain has been identified as an important therapeutic target in human parasitic hookworm species that infect more than 600 million people worldwide. Here, we report two x-ray crystal structures of the hookworm Ancylostoma ceylanicum DAF-12 ligand binding domain in complex with DA and cholestenoic acid (a bile acid-like metabolite), respectively. Structure analysis and functional studies reveal key residues responsible for species-specific ligand responses of DAF-12. Furthermore, DA binds to DAF-12 mechanistically and is structurally similar to bile acids binding to the mammalian bile acid receptor farnesoid X receptor. Activation of DAF-12 by cholestenoic acid and the cholestenoic acid complex structure suggest that bile acid-like signaling pathways have been conserved in nematodes and mammals. Together, these results reveal the molecular mechanism for the interplay between parasite and host, provide a structural framework for DAF-12 as a promising target in treating nematode parasitism, and provide insight into the evolution of gut parasite hormone-signaling pathways.

On being the right size: the impact of population size and stochastic effects on the evolution of drug resistance in hospitals and the community.

The evolution of drug resistant bacteria is a severe public health problem, both in hospitals and in the community. Currently, some countries aim at concentrating highly specialized services in large hospitals in order to improve patient outcomes. Emergent resistant strains often originate in health care facilities, but it is unknown to what extent hospital size affects resistance evolution and the resulting spillover of hospital-associated pathogens to the community. We used two published datasets from the US and Ireland to investigate the effects of hospital size and controlled for several confounders such as antimicrobial usage, sampling frequency, mortality, disinfection and length of stay. The proportion of patients acquiring both sensitive and resistant infections in a hospital strongly correlated with hospital size. Moreover, we observe the same pattern for both the percentage of resistant infections and the increase of hospital-acquired infections over time. One interpretation of this pattern is that chance effects in small hospitals impede the spread of drug-resistance. To investigate to what extent the size distribution of hospitals can directly affect the prevalence of antibiotic resistance, we use a stochastic epidemiological model describing the spread of drug resistance in a hospital setting as well as the interaction between one or several hospitals and the community. We show that the level of drug resistance typically increases with population size: In small hospitals chance effects cause large fluctuations in pathogen population size or even extinctions, both of which impede the acquisition and spread of drug resistance. Finally, we show that indirect transmission via environmental reservoirs can reduce the effect of hospital size because the slow turnover in the environment can prevent extinction of resistant strains. This implies that reducing environmental transmission is especially important in small hospitals, because such a reduction not only reduces overall transmission but might also facilitate the extinction of resistant strains. Overall, our study shows that the distribution of hospital sizes is a crucial factor for the spread of drug resistance.

Characterisation of hookworm heat shock factor binding protein (HSB-1) during heat shock and larval activation.

When hookworm infective L3s infect their mammalian host, they undergo a temperature shift from that of the ambient environment to that of their endothermic host. Additionally, L3s living in the environment can be exposed to temperature extremes associated with weather fluctuations. The heat shock response (HSR) is a conserved response to heat shock and other stress that involves the expression of protective heat shock proteins (HSPs). The HSR is controlled by heat shock factor-1 (HSF-1), a conserved transcription factor that binds to a heat shock element in the promoter of HSPs, causing their expression. HSF-1 is negatively regulated in part by a HSF binding protein (HSB-1) that binds to and removes HSF-1 trimers bound to HSP gene promoters, resulting in attenuation of the HSR. Herein we describe an HSB-1 orthologue, Ac-HSB-1, from the hookworm Ancylostoma caninum. The Ac-hsb-1 cDNA encodes a 79 amino acid protein that is 71% identical to the Caenorhabditis elegans HSB-1, and is predicted to share the characteristic coiled-coil structural motif comprised of two interacting alpha helices. Recombinant Ac-HSB-1 immunoprecipitated Ce-HSF-1 expressed in mammalian cells that had been heat shocked for 1h at 42°C, but not from cells incubated at 37°C, indicating that HSB-1 only bound to the active DNA binding form of HSF-1. Expression of Ac-hsb-1 transcripts decreased following 1h of heat shock, but increased when L3s were incubated at 37°C for 1h. Activation of hookworm L3s induces a five-sixfold increase in Ac-hsb-1 expression that peaks at 12h, coincident with L3 feeding, but that subsequently decreases to two-threefold above control at 24h. Recombinant Ac-HSB-1 immunoprecipitates greater amounts of 70 and 40kDa proteins from extracts of activated L3s than from non-activated L3s. We propose that an increase in Ac-hsb-1 levels early in activation allows feeding to resume, but that a subsequent decrease in expression permits a HSR that protects non-developing L3s at host-like temperatures. Further investigations of the HSR will clarify the role of HSB-1 and HSF-1 in hookworm infection.

Acetylcholine receptor subunit genes from Ancylostoma caninum: altered transcription patterns associated with pyrantel resistance.

The molecular mechanism of resistance to nicotinic agonist anthelmintics such as pyrantel and levamisole in nematodes of medical and veterinary significance is poorly understood. The identification of pyrantel-resistant isolates of the canine hookworm, Ancylostoma caninum, provides an opportunity to explore, at a molecular level, the mechanism of cholinergic resistance in a species that is a model for the human hookworms. Here we describe the cloning of three A. caninum genes orthologous to components of the pyrantel-sensitive nicotinic acetylcholine receptor in Caenorhabditis elegans (UNC-29, -38, -63). Analysis of mRNA levels by quantitative PCR was also performed on these genes, plus an additional three nicotinic acetylcholine receptor subunit genes thought not to be constituents of the pyrantel-sensitive receptor, for which a partial sequence was obtained. Gene sequences and mRNA levels were compared between two isolates of A. caninum showing either high- or low-level resistance to pyrantel (as shown previously by in vivo efficacy and in vitro comparative studies). While no polymorphisms of likely significance between the two A. caninum isolates were observed, quantitative analysis of transcription revealed significantly lower levels for the three putative pyrantel receptor subunits (AAR-29, -38 and -63) in the highly pyrantel-resistant isolate compared with the isolate with low-level resistance. In contrast, transcription of the three subunits thought not to constitute the pyrantel receptor (AAR-8, -15 and -19) was either not significantly different between the two isolates, or slightly higher in the highly-resistant isolate. This data suggests that reduced transcription of the mRNA coding for nicotinic acetylcholine receptor subunits that form the pyrantel-sensitive receptors may be a component of the pyrantel resistance mechanism in A. caninum.

Dietary iron content mediates hookworm pathogenesis in vivo.

Hookworm infection is associated with growth delay and iron deficiency anemia in developing countries. A series of experiments were designed in order to test the hypothesis that host dietary iron restriction mediates susceptibility to hookworm infection using the hamster model of Ancylostoma ceylanicum. Animals were maintained on diets containing either 10 ppm iron (iron restricted) or 200 ppm iron (standard/high iron), followed by infection with A. ceylanicum third-stage larvae. Infected animals fed the standard diet exhibited statistically significant growth delay and reduced blood hemoglobin levels compared to uninfected controls on day 20 postinfection. In contrast, no statistically significant differences in weight or hemoglobin concentration were observed between infected and uninfected animals fed the iron-restricted diet. Moreover, iron-restricted animals were observed to have reduced intestinal worm burdens on day 10 and day 20 postinfection compared to those of animals maintained on the standard/high-iron diet. In a subsequent study, animals equilibrated on diets containing a range of iron levels (10 ppm, 40 ppm, 100 ppm, or 200 ppm) were infected with A. ceylanicum and followed for evidence of hookworm disease. Infected animals from the intermediate-dietary iron (40- and 100-ppm) groups exhibited greater weight loss and anemia than those in the low (10-ppm)- or high (200-ppm)-iron diet groups. Mortality was also significantly higher in the intermediate-dietary-iron groups. These data suggest that severe dietary iron restriction impairs hookworm development in vivo but that moderate iron restriction enhances host susceptibility to severe disease.

A multi-enzyme cascade of hemoglobin proteolysis in the intestine of blood-feeding hookworms.

Blood-feeding pathogens digest hemoglobin (Hb) as a source of nutrition, but little is known about this process in multicellular parasites. The intestinal brush border membrane of the canine hookworm, Ancylostoma caninum, contains aspartic proteases (APR-1), cysteine proteases (CP-2), and metalloproteases (MEP-1), the first of which is known to digest Hb. We now show that Hb is degraded by a multi-enzyme, synergistic cascade of proteolysis. Recombinant APR-1 and CP-2, but not MEP-1, digested native Hb and denatured globin. MEP-1, however, did cleave globin fragments that had undergone prior digestion by APR-1 and CP-2. Proteolytic cleavage sites within the Hb alpha and beta chains were determined for the three enzymes, identifying a total of 131 cleavage sites. By scanning synthetic combinatorial peptide libraries with each enzyme, we compared the preferred residues cleaved in the libraries with the known cleavage sites within Hb. The semi-ordered pathway of Hb digestion described here is surprisingly similar to that used by Plasmodium to digest Hb and provides a potential mechanism by which these hemoglobinases are efficacious vaccines in animal models of hookworm infection.

Liver plasma membrane-bound enzymes and lipids in golden hamsters infected with Ancylostoma ceylanicum.

Infection of golden hamsters with Ancylostoma ceylanicum caused significant decrease in body weight, liver weight and the protein content of liver plasma membrane. Significant inhibition of liver plasma membrane enzymes activities-5'Nucleotidase, gamma-glutamyl transpeptidase, NADHK3Fe (CN)6 reductase, Na+/K(+)-ATPase, CA(2+)-ATPase and Mg(2+)-ATPase was observed in infected animals compared to corresponding controls. Sialic acid content and phospholipid/cholesterol ratio in liver plasma membrane of the infected group were significantly reduced. The study suggests that changes in both the structural and functional organization of membrane may be a biochemical basis of the hepatotoxic effects.

Hookworm infections and human iron metabolism.

Ancylostoma duodenale and Necator americanus are extremely common species of soil-transmitted helminth which flourish where poverty and malnutrition prevail. Hookworms contribute significantly to iron-deficiency anaemia, which remains one of the world's major nutritional problems, through the feeding activities of intestinal stages leading to chronic blood loss into the gut. In this article, a mathematical model is proposed to explain how human iron metabolism may respond to hookworm infection of varying intensity. The model draws attention to the importance of the regulation of stored iron levels in the process. The results from the model are presented for the effects of hookworm infection on the iron metabolism of a healthy adult male. Calculations are also presented in which the effects of hookworms on the iron metabolism of a non-pregnant woman are compared with those of a pregnant woman. Use of the model may help develop a better understanding of the pathology of hookworm disease.

Hepatic microsomal cytochrome P450 system during experimental hookworm infection.

Experimental infection of golden hamsters with the hookworm, Ancylostoma ceylanicum, caused a profound decline in the hepatic microsomal cytochrome P450 content. Concomitant decrease was also noticed in aminopyrine N-demethylase and benzo[a]pyrene hydroxylase activities. However, aniline hydroxylase activity was only marginally elevated during the infection. Microsomal markers, viz., cytochrome b5, NADH-cytochrome-c reductase, and glucose-6-phosphatase, were not significantly altered. Hepatic tissue exhibited an accumulation of lipids, especially phospholipids, triglycerides, and cholesterol, resulting in fatty necrosis around the central vein region. Isolated hepatic microsomes showed a decrease in phosphatidylcholine content. Impairment in hepatic mixed function oxidase (MFO) activities was further confirmed by prolongation in hexobarbital sleeping time and zoxazolamine-induced paralysis. The hepatic MFO system of A. ceylanicum-infected hamsters responded qualitatively and quantitatively in a manner similar to that of control hamsters, upon stimulation with selective chemical inducers like phenobarbitone and 3-methylcholanthrene. Kinetic and in vitro substrate binding studies revealed that for aminopyrine the substrate affinity and the maximum enzyme activity (Vmax) were decreased, while for aniline the binding affinity was decreased and the binding capacity was enhanced. Results indicate specific/selective impairment of the hepatic microsomal cytochrome P450 system during hookworm infection and may have many practical implications in toxicology and pharmacology.

[Experience with the use of nemocide (pyrantel pamoate) in nematodiases]. / Opyt primeneniia nemotsida (pirantelia pamoata) pri nematodozakh.

The clinical efficacy of and tolerance to the nemocide pyranthel pamoate produced by "IPCA" (India) and applied to nematodiasis treatment have been investigated. A high efficacy of the drug (94.4-100%) for ascariasis, enterobiasis and ancylostomiasis treatment has been established. The drug is well tolerated by adults and induces slight short-term alterations of hepatic functional activity in 5-10-year-old children.

Biochemical and histopathological alterations in golden hamster during infection with Ancylostoma ceylanicum.

Ancylostoma ceylanicum infection in golden hamsters (Mesocricetus auratus) caused marked biochemical and histopathological derangements. Jejunum, the primary site of infection, showed pronounced alterations compared with liver. Though the biochemical composition of jejunum was not significantly altered, activities of a few lysosomal enzymes were enhanced during hookworm infection. Marked damage to mitochondrial and microsomal membranes was reflected in changes in the activities of the marker enzymes from jejunal tissue. Lipid content, especially phospholipids and neutral lipids of hepatic tissue, exhibited marked elevation. Levels of hexokinase, phosphofructokinase, and lactate dehydrogenase were enhanced in jejunal as well as hepatic tissues, indicating activation of the glycolytic machinery during hookworm infection. A decrease in the levels of mucosal disaccharidases indicated damage to intestinal brush border membranes. However, alkaline phosphatase activity was increased in intestinal mucosa during the infection. Light microscopic examination of jejunal tissue revealed peeling off of the upper epithelial layer, activation of the goblet cells, and thickening of muscularis mucosa. However, hepatic tissue did not show gross alterations, except for slight necrosis in the centrilobular region.

Iron loss and reabsorption in Ancylostoma duodenale infection and bilharzial colonic polyposis.

PIP: Individuals infected with Ancylostoma duodenale may lose up to 6 mg of iron daily and those infected with bilharzial colonic polyposis may lose an average of 3 mg daily; patients suffering with both infections may lose up to 9 mg of iron daily. 1 study has shown that some iron loss to the upper gastrointestinal tract was reabsorbed. A study was undertaken to ascertain whether any iron lost in the lower gastrointestinal tract is reabsorbed and to measure the amount of iron reabsorbed in patients with A. duodenale. 7 Egyptian farmers with A. duodenale and 8 with bilharzial intestinal polyposis but without hookworm infection were given thorough physical and laboratory examinations. Hookworm and Schistosoma mansoni egg counts were performed. All patients were given oral ferrous sulfate before starting the experiment to raise their hemoglobin levels to over 10 gm %. The mean daily blood loss in the 7 patients with heavy hookworm infections was 64.8 ml and the mean iron loss was 18.7; but a mean of 7.7 of this iron was reabsorbed. In the 8 patients with bilharzial polyposis, blood loss averaged 13.1 ml and iron loss, 4.3 mg. In 4 patients, the amount of iron reabsorbed was not significant, in the remaining 4, it reached only 2 mg. The reabsorption of 40% of the iron initially lost in the upper gastrointestinal tract may explain the remarkable tolerance of Egyptian farmers to prolonged hookworm infections. The general iron deficiency anemia prevalent among patients with bilharzial polyposis is due in part to the loss of iron which is not reabsorbed.^ieng