Trypanosoma cruzi screening in Texas blood donors, 2008-2012.

Chagas disease is an important emerging disease in Texas that results in cardiomyopathy in about 30% of those infected with the parasite Trypanosoma cruzi. Between the years 2008 and 2012, about 1/6500 blood donors were T. cruzi antibody-confirmed positive. We found older persons and minority populations, particularly Hispanic, at highest risk for screening positive for T. cruzi antibodies during routine blood donation. Zip code analysis determined that T. cruzi is associated with poverty. Chagas disease has a significant disease burden and is a cause of substantial economic losses in Texas.

Neglected infections of poverty in Texas and the rest of the United States: management and treatment options.

In the poorest regions of the United States, especially along the Gulf Coast and in South Texas, are a group of endemic parasitic and related infections known as the neglected infections of poverty. Such infections are characterized by their chronicity, disabling features, and disproportionate impact on the estimated 46 million people who live below the U.S. poverty line. Today more Americans live in poverty than ever before in the half-century that the Census Bureau has been recording poverty rates. In association with that poverty, a group of major neglected infections of poverty have emerged in the United States. Here we describe the major neglected infections of poverty in the United States, with a brief overview of their significant epidemiological features, their links with poverty, and our approaches to their diagnosis, management, and treatment.

Experimental approaches to the development of a recombinant hookworm vaccine.

Hookworm infection is a major parasitic cause of morbidity in the developing nations of the tropics. Development of a genetically engineered vaccine would be a useful tool in the control of this infection in highly endemic areas. Recombinant polypeptides belonging to the Ancylostoma secreted protein (ASP)-1 family have shown promise for reducing hookworm burdens after larval challenge infections in mice. Typically, these polypeptides are expressed in Escherichia coli and administered as an alum precipitate. Vaccine protection is antibody dependent. It is anticipated that a cocktail of different recombinant hookworm antigens may be required in order to effectively prevent heavy hookworm infections and disease. The progress of this work has been hampered by the absence of both a convenient laboratory animal with which to study hookworm infections resembling human infection, as well as the lack of easy availability of native hookworm antigens. In addition, useful human serologic correlates of antihookworm immunity are still poorly defined.

Histochemical alterations of infective third-stage hookworm larvae (L3) in vaccinated mice.

To study the histochemical alterations of hookworm L3 administered in a challenge dose to mice vaccinated previously with the larvae. Male Kunming strain mice vaccinated subcutaneously with 500 living Ancylostoma caninum L3 once every 2 weeks for a total of three immunizations before a final challenge with 500 L3 one week after the final immunization. The abdominal skin with underlying subcutaneous tissue and muscle were removed from the site of percutaneous challenge entry (from 2-3 mice), and fixed in absolute alcohol, cold acetone and 10% neutralized formalin. The tissue sections containing the L3 from the challenge dose were then stained histochemically of glycogen, RNA, DNA alkaline protein, acid mucopolysaccharide, collagen, reticulin, alkaline phosphatase (AKP) and adenosine triphosphatase (ATPase). Skin samples from non-immunized mice that were also subcutaneously inoculated with the L3 served as negative control. The L3 identified in cutaneous sections from vaccinated mice at 6-72 hours post-challenge exhibited reductions in parasite glycogen, alkaline protein, RNA and DNA, as well as reductions in acid mucopolysaccharide, collagen and reticulin contents in the parasite cuticle. There were also reduced enzyme AKP and ATPase activities. In contrast L3, identified in sections from non-immunized mice exhibited a normal histochemical appearance, as did some L3 who survived in vaccinated mice at 7-14 days post-challenge. Vaccination results in hookworm L3 damage which is manifested by reduced histochemical staining for the challenge inoculum of parasites. There is also reduced hydrolytic enzyme activity. The observed changes could reflect either host-mediated parasite structural damage and disintegration or possibly anti-metabolic properties of the host immune response.

Numerous eruptive lesions of panniculitis associated with group A streptococcus bacteremia in an immunocompetent child.

A previously healthy 13-month-old boy developed group A beta-hemolytic streptococcus bacteremia coinciding with numerous eruptive subcutaneous lesions primarily on his extremities. Skin biopsy revealed infectious panniculitis; gram-positive cocci were present within both fat lobules and septa. Molecular genetic analysis of an isolate from the patient's blood revealed an emm type 4 organism displaying the emm chromosomal pattern E that is characteristic of opacity factor-producing strains; the organism also harbored the gene encoding for streptococcal pyrogenic exotoxin C (speC). To our knowledge, this clinical presentation has not yet been described in the spectrum of infections directly caused by group A beta-hemolytic streptococci.

Cutaneous and subcutaneous granulomata formation in mice immunized and challenged with third-stage infective hookworm (Ancylostoma caninum) larvae.

To determine the inflammatory and immunological mechanisms associated with live third-stage (L3) hookworm larval vaccines, mice were immunized either subcutaneously or orally with three doses of 500 L3 of Ancylostoma caninum at 2-week intervals, and then challenged percutaneously (via abdominal skin) with 500 L3. Non-immunized mice served as negative controls. Skin was excised from post-challenge mice at intervals between 6 h and 28 days, and then examined by light microscopy. In non-immunized mice the L3 exhibited no structural damage and infiltrating inflammatory cells were absent from the surrounding tissues. There were no changes in the cutaneous architecture. In contrast, skin recovered from the immunized mice was edematous and exhibited marked inflammatory changes with resultant destruction of the challenge L3. At 6 h post-challenge the L3 exhibited cuticular swelling and damage; the surrounding tissue was infiltrated by polymorphonuclear inflammatory cells. By 24 h granulomata in the dermis, subcutaneous tissues and underlying abdominal muscles were first observed surrounding dead L3. The number of granulomata peaked at 72 h, with the majority distributed in the subcutaneous tissues. Plasma cells predominated in the early granulomata, but by 3-7 days post-challenge foreign body giant cells began to appear. In some cases, intact and presumably living L3 were noted in the abdominal muscles 14-28 days post-challenge, which suggested that protection against larval challenge was not absolute. Granuloma formation appears to be a major component of the post-vaccination murine host immune response against challenge larvae. The observation generates several hypotheses to investigate the mechanisms of protection afforded by living helminth vaccines.

Cloning and characterization of Ancylostoma-secreted protein. A novel protein associated with the transition to parasitism by infective hookworm larvae.

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.

Anticoagulant repertoire of the hookworm Ancylostoma caninum.

Hookworms are hematophagous nematodes that infect a wide range of mammalian hosts, including humans. There has been speculation for nearly a century as to the identity of the anticoagulant substances) used by these organisms to subvert host hemostasis. Using molecular cloning, we describe a family of potent small protein (75-84 amino acids) anticoagulants from the hookworm Ancylostoma caninum termed AcAP (A. caninum anticoagulant protein). Two recombinant AcAP members (AcAP5 and AcAP6) directly inhibited the catalytic activity of blood coagulation factor Xa (fXa), while a third form (AcAPc2) predominantly inhibited the catalytic activity of a complex composed of blood coagulation factor VIIa and tissue factor (fVIIa/TF). The inhibition of fVIIa/TF was by a unique mechanism that required the initial formation of a binary complex of the inhibitor with fXa at a site on the enzyme that is distinct from the catalytic center (exo-site). The sequence of AcAPc2 as well as the utilization of an exo-site on fXa distinguishes this inhibitor from the mammalian anticoagulant TFPI (tissue factor pathway inhibitor), which is functionally equivalent with respect to fXa-dependent inhibition of fIIa/TF. The relative sequence positions of the reactive site residues determined for AcAP5 with the homologous regions in AcAP6 and AcAPc2 as well as the pattern of 10 cysteine residues present in each of the inhibitors suggest that the AcAPs are distantly related to the family of small protein serine protease inhibitors found in the nonhematophagous nematode Ascaris lumbricoides var. suum.

Ancylostoma caninum anticoagulant peptide: a hookworm-derived inhibitor of human coagulation factor Xa.

Human hookworm infection is a major cause of gastrointestinal blood loss and iron deficiency anemia, affecting up to one billion people in the developing world. These soil-transmitted helminths cause blood loss during attachment to the intestinal mucosa by lacerating capillaries and ingesting extravasated blood. We have isolated the major anticoagulant used by adult worms to facilitate feeding and exacerbate intestinal blood loss. This 8.7-kDa peptide, named the Ancylostoma caninum anticoagulant peptide (AcAP), was purified by using a combination of ion-exchange chromatography, gel-filtration chromatography, and reverse-phase HPLC. N-terminal sequencing of AcAP reveals no homology to any previously identified anticoagulant or protease inhibitor. Single-stage chromogenic assays reveal that AcAP is a highly potent and specific inhibitor of human coagulation, with an intrinsic K*i for the inhibition of free factor Xa of 323.5 pM. In plasma-based clotting time assays, AcAP was more effective at prolonging the prothrombin time than both recombinant hirudin and tick anticoagulant peptide. These data suggest that AcAP, a specific inhibitor of factor Xa, is one of the most potent naturally occurring anticoagulants described to date.

Molecular pathobiology of hookworm infection.

Within the past 2 years, progress has been made in the identification, isolation, and cDNA cloning of several macromolecules from hookworms. While the predicted amino acid sequences of some cDNAs resemble those from other nematodes, such as Caenorhabditis elegans, other cDNAs are unique to hookworms. Studies are under way to evaluate the function of these recombinant hookworm polypeptides with respect to the biology of hookworms in experimental animal models. The recombinant polypeptides are also under evaluation as vaccine targets and as natural products used in the treatment of human cardiovascular and autoimmune diseases.

Weight loss associated with an endotoxin-induced mediator from peritoneal macrophages: the role of cachectin (tumor necrosis factor).

Endotoxin-induced cells of the reticuloendothelial system were shown to produce mediator(s) that evoke a state of cachexia in recipient animals. The factor(s) responsible were assayed in endotoxin-resistant (C3H/HeJ) mice, which were injected with dialyzed conditioned medium obtained from lipopolysaccharide-induced peritoneal macrophages. The mice exhibited weight loss and anorexia, and they died if sufficient quantities of medium were administered. The syndrome was reversible if injections were discontinued. Endotoxin alone did not produce this effect, and no gross pathologic lesions were discernable in the treated animals. In this model system, cachexia appears to result from the action of soluble macromolecules produced by activated macrophages in vitro. Cachectin (murine tumor necrosis factor) is thought to play a central role in this phenomenon.

Secretion of a proteolytic anticoagulant by Ancylostoma hookworms.

Hookworms of the genus Ancylostoma secrete an anticoagulant that both inhibits the clotting of human plasma and promotes fibrin clot dissolution. This anticoagulant activity is attributable to a 36,000 dalton proteolytic enzyme. The protease can degrade fibrinogen into five smaller polypeptides that intrinsically have anticoagulating properties, covert plasminogen to a mini-plasminogen-like molecule, and hydrolyze a synthetic peptide substrate with specificity for elastolytic enzymes. It is hypothesized that the parasite uses this enzyme to prevent blood clotting while feeding on villous capillaries.