Browsing by Author "Prema Arasu, Committee Chair"

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    Host Cytokines and Immune Responses in Pregnancy Associated Transmission of Arrested Hookworm Larvae
    (2006-09-30) Trivedi, Shweta; Prema Arasu, Committee Chair; William Miller, Committee Member; Paul Mozdziak, Committee Member; Scott Laster, Committee Member
    Over one billion people worldwide are infected with blood-feeding intestinal hookworms. The life cycle of A. duodenale (humans) and A. caninum (dogs) includes the propensity for L3 to undergo a temporary state of developmental arrest in the host. In female hosts, tissue-arrested L3 reactivate during pregnancy and are transmitted to neonates through milk. During pregnancy, transforming growth factor (TGF)-β2 is upregulated in the mammary gland. Studies in C. elegans show that TGF-β and insulin-like signaling pathways regulate larval arrest and reactivation. We previously utilized an in vitro assay to demonstrate that recombinant human TGF-β stimulates a feeding response in tissue-arrested A. caninum L3. We hypothesize that host expression of TGF-β and pregnancy hormones such as estrogen and prolactin signal arrested L3 to reactivate. To facilitate in vivo analyses, we utilized a mouse model of A. caninum infection. Mice were utilized because L3 do not develop into adults but arrest in somatic tissues, and reactivate during the periparturient period. We investigated TGF-β1, TGF-β2 and IGF-1 transcript and serum cytokine profiles during late pregnancy, early lactation and mid-lactation to correlate levels with transmammary transmission of L3 to nursing pups. An in vitro co-culture system was developed to mimic in vivo conditions and assess effects of TGF-β and, estrogen and prolactin on larval reactivation. A. caninum L3 were co-incubated with skeletal muscle or mammary epithelial cells and larval reactivation was measured. Additionally, immune responses were assessed as by measuring serum and transcript levels of IFN-γ and IL-4 in skeletal muscle, mammary gland and spleen to dissect the effects of pregnancy and A. caninum infection in the mouse. Our findings suggest that host-derived TGF- β1 and IGF-1, but not TGF- β2, are important in reactivation and transmission of arrested A. caninum larvae. Also, a Th2-like response characterized by increased IL-4 transcript levels was observed in skeletal muscle, while a mixed Th1⁄Th2 profile was observed in mammary gland when comparing infection with A. caninum versus pregnancy/lactation in BALB⁄c mice.
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    TGF-beta Ligands from the Hookworm Parasite, Ancylostoma caninum, in Larval Latency and Reactivation
    (2005-12-01) Freitas, Tori Constance; Prema Arasu, Committee Chair
    The blood-feeding hookworm parasites, Necator and Ancylostoma spp., infect over a billion people worldwide. Typically, developmentally arrested infective larvae (iL3) in the environment enter the host and mature into the adult stage in the intestine. With Ancylostoma duodenale, there is an additional aspect to the life cycle whereby the larvae abort normal development and enter yet another state of arrest within the somatic tissues of the host. At opportune times such as pregnancy, these tissue-arrested larvae reactivate and transmit in milk to the newborns. The related dog hookworm, Ancylostoma caninum, is a major parasite of dogs and a model for human hookworms as well as the phenomenon of pregnancy-induced transmission of infection. Transforming growth factor-beta (TGF-beta) signaling regulates developmental arrest and reactivation in the closely related free-living nematode, Caenorhabditis elegans. Using an in vitro feeding assay, we have previously shown that recombinant mammalian TGF-beta can reactivate tissue-arrested A. caninum larvae. This finding led to the hypothesis that up-regulation of TGF-beta during pregnancy resulted in the interaction of host ligand with parasite-encoded receptors and subsequent larval reactivation. To determine the role of parasite-encoded ligands in parasite development as well as the reactivation process, we have cloned and characterized two TGF-beta-like ligands from A. caninum. The first, Ac-tgh-1 is most similar to C. elegans dbl-1, a ligand involved in regulating body size and male tail patterning. At the deduced amino acid level and within the conserved domain, Ac-tgh-1 is 60% identical to dbl-1 and 50% to the mammalian ligand, bmp-2. Based on RT-PCR results, Ac-tgh-1 transcript is present at all stages tested, namely, the embryo, first and second larval stages (L1/L2), iL3, serum stimulated L3 (ssL3), and adult male and female. Using polyclonal antibodies generated in rabbits against recombinant Ac-TGH-1, protein is detected at the iL3, ssL3, and adult stages. This expression pattern matches that of C. elegans dbl-1. The second parasite-encoded ligand, Ac-tgh-2, is most similar to C. elegans daf-7, the TGF-beta-like ligand involved in the arrest and reactivation pathway of the free-living nematode. Within the conserved domain, Ac-tgh-2 shares 60% amino acid identity to C. elegans daf-7 and 28% to mammalian TGF-beta1. Ac-tgh-2 transcript and protein is detected at all stages tested. This expression profile is different from that found with C. elegans daf-7, as daf-7 is not expressed in the arrested state equivalent to the A. caninum iL3. This difference may be unique to parasitic nematodes that undergo an obligate state of developmental arrest. Immunofluorescent studies suggest Ac-TGH-2 is expressed in the anterior chemosensory region of the worm, similar to the expression pattern of the C. elegans DAF-7 protein. Functional assessment of the two ligands was attempted through antibody cross-reactivity studies, an immunization trial and RNA interference trials. Developmental arrest and reactivation is a complex process resulting in metabolic and structural alterations to the developing worm requiring the differential expression of a number of genes. Two studies were conducted to determine the expression profile of arrested versus in vitro reactivated A. caninum L3s using (1) C. elegans cDNA clones in a 'macroarray' format and (2) A. caninum cDNA clones in a microarray format. Both studies showed trends in gene expression similar to that of C. elegans and we also showed that similarity exists between nucleotide sequences of C. elegans and A. caninum enabling the use of selected C. elegans cDNAs to isolate A. caninum homologs. Deciphering the molecular aspects of A. caninum arrest and reactivation through further functional analyses of parasite-encoded signaling molecules such as TGF-beta homologs and genome-wide expression profiles will advance our understanding of the intricate developmental stages of the parasite and its interaction with the host.