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Hookworms, parasitic nematodes that infect nearly one billion people worldwide, are a major cause of anemia and malnutrition. We hypothesize that hookworms actively manipulate the host immune response through the production of specific molecules designed to facilitate infection by larval stages and adult worm survival within the intestine. A full-length cDNA encoding a secreted orthologue of the human cytokine, Macrophage Migration Inhibitory Factor (MIF) has been cloned from the hookworm Ancylostoma ceylanicum. Elucidation of the three-dimensional crystal structure of recombinant AceMIF (rAceMIF) revealed an overall structural homology with significant differences in the tautomerase sites of the human and hookworm proteins. The relative bioactivities of human and hookworm MIF proteins were compared using in vitro assays of tautomerase activity, macrophage migration, and binding to MIF receptor CD74. The activity of rAceMIF was not inhibited by the ligand ISO-1, which was previously determined to be an inhibitor of the catalytic site of human MIF. These data define unique immunological, structural, and functional characteristics of AceMIF, thereby establishing the potential for selectively inhibiting the hookworm cytokine as a means of reducing parasite survival and disease pathogenesis.Hookworms are bloodfeeding intestinal nematodes that currently infect more than 700 million people in developing countries (1). The hookworm life cycle begins when eggs excreted in the feces of an infected individual hatch in soil and undergo successive molts to the infectious L 3 stage. After contacting the skin of a permissive host, larvae migrate to the pulmonary vasculature, traverse alveolar capillaries, ascend the respiratory tree and are swallowed. Hookworms molt to the adult stage in the intestine, where they attach to the mucosal surface and feed on blood and tissue. Chronic blood and serum protein loss attributable to hookworm infection is associated with anemia, malnutrition, and growth/developmental delay, resulting in the loss of tens of millions of disability adjusted life-years annually (2).There is no clear evidence of sterile immunity in humans following naturally acquired infection, suggesting that hookworms may modulate the host immune response, perhaps during tissue migration, and/or while attached to the intestinal mucosa. The fact that adult hookworms can survive within a single human host for many years (3) further suggests that these worms are capable of evading or dampening host immune responses that might kill parasites and/or trigger expulsion. Of the immunomodulatory activities that have been identified from the hookworms Ancylostoma or Necator, none has yet been shown to play a definitive role in the pathogenesis of infection or intestinal disease (4 -7).Macrophage migration inhibitory factor (MIF) 4 is a pro-inflammatory cytokine first identified as a product of activated T cells, and subsequently demonstrated to have diverse biological functions (8). Mammalian MIF inhibits the random migration of ma...

The developmentally arrested third stage infective larva of hookworms resumes development upon entry into the definitive host. This transition to parasitism can be modeled in vitro by stimulating infective larvae with a low molecular weight ultrafiltrate of host serum together with methylated glutathione analogues. When stimulated to resume development in vitro, activated larvae of the hookworm Ancylostoma caninum released a 42-kDa protein, termed Ancylostoma-secreted protein (ASP). ASP was the major protein released by activated hookworm larvae. Degenerate oligonucleotide primers, based on a partial internal amino acid sequence of the protein, were used together with flanking vector sequence primers to amplify a fragment from a third stage larval cDNA library by polymerase chain reaction. The fragment was used as a probe to isolate a longer clone from the larval cDNA library. The full-length ASP cDNA was found to encode a 424-amino acid protein with homology to the antigen 5/antigen 3 family of proteins from hymenopteran venoms and a family of cysteine-rich secretory proteins. ASP was expressed in bacterial cells, and a polyclonal antiserum against purified recombinant ASP was produced. The antiserum, which was demonstrated to be specific for ASP, was used as a probe to measure the kinetics of ASP release by hookworm larvae. ASP is released within 30 min of stimulation, with the majority released by 4 h. Low levels of ASP were released continuously following activation, but only if the stimuli were present in the incubation medium. The compound 4,7-phenanthroline, previously shown to inhibit larval activation, also inhibited release of ASP. The specific, rapid release of ASP by activated infective larvae suggests that this molecule occupies a critical and central role in the transition from the external environment to parasitism.The early events of the hookworm infectious process are poorly understood, especially at the molecular and biochemical level. A better understanding of the molecules released by invading larvae, and the host's responses to them, would allow for the rational design of immuno-and chemotherapeutic intervention strategies. However, the inability to culture hookworms beyond the third-stage larvae (L 3 ) 1 in vitro, together with the difficulties associated with the isolation of sufficient parasitic stages for molecular and biochemical studies, has, until recently, hampered investigations of the critical first steps in the establishment of the parasitic relationship.The initial events of the infectious process can now be modeled in vitro, using the resumption of feeding as a marker for the transition from the free-living L 3 to the developing parasitic L 3 (1-3). When free-living L 3 of the canine hookworm Ancylostoma caninum are stimulated to resume feeding in vitro, they release several molecules into the culture medium. Recently, a zinc metalloprotease activity has been reported from activated A. caninum L 3 ES products (4). Here we report the isolation, cloning, and expression of a 40-kDa...

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Hookworm infection is a major cause of iron deficiency anemia and malnutrition in developing countries. The Ancylostoma ceylanicum Kunitz-type inhibitor (AceKI) is a 7.9-kDa broad-spectrum inhibitor of trypsin, chymotrypsin, and pancreatic elastase that has previously been isolated from adult hookworms. Site-directed mutagenesis of the predicted P1 inhibitory reactive site amino acid confirmed the role of Met 26 in mediating inhibition of the three target serine proteases. By using reverse transcription-PCR, it was demonstrated that the level of AceKI gene expression increased following activation of third-stage larvae with serum and that the highest level of expression was reached in the adult stage of the parasite. Immunohistochemistry studies performed with polyclonal immunoglobulin G raised against recombinant AceKI showed that the inhibitor localized to the subcuticle of the adult hookworm, suggesting that it has a potential in vivo role in neutralizing intestinal proteases at the surface of the parasite. Immunization with recombinant AceKI was shown to confer partial protection against hookworm-associated growth delay without a measurable effect on anemia. Taken together, the data suggest that AceKI plays a role in the pathogenesis of hookworm-associated malnutrition and growth delay, perhaps through inhibition of nutrient absorption in infected hosts.Hookworm infection remains a major global health problem, and over one billion people are reportedly infected in developing countries (9, 14). Hookworms, which are bloodfeeding intestinal nematodes, are a major cause of iron deficiency anemia and malnutrition (15,20,(59)(60)(61)64). While the anemia is presumably due to the cumulative effect of chronic intestinal blood loss, the molecular mechanisms underlying the pathogenesis of hookworm malnutrition remain unknown. Although it has been suggested that hookworm malnutrition and growth delay occur secondary to chronic iron deficiency, particularly in children, evidence from prior clinical studies suggests that hookworm infection is also associated with various degrees of intestinal malabsorption (18,35,54,57,62). It has been hypothesized that this hookworm malabsorption syndrome might occur secondary to mucosal inflammation triggered by the adult worm attached to the intestinal epithelium or might be a result of secretion of parasite inhibitors of host digestive enzymes (18).As part of a series of ongoing studies aimed at characterizing adult hookworm secretory proteins, a cDNA corresponding to the gene encoding a putative Kunitz-type serine protease inhibitor was previously identified from adult Ancylostoma ceylanicum RNA by using a PCR-based approach (48). The A. ceylanicum Kunitz-type inhibitor (AceKI) cDNA was expressed in Escherichia coli, and the recombinant protein was found to inhibit the pancreatic enzymes chymotrypsin, pancreatic elastase, and trypsin in vitro, with equilibrium inhibitory dissociation constant (K i ) values ranging from picomolar levels to low nanomolar levels. The native AceKI prote...