Inflammatory myofibroblastic tumour of appendix.
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Adult↗
Biomedical subjects
R Chandrashekar
Publications and source records attributed to R Chandrashekar.
Screening for T cell-eliciting proteins of Japanese encephalitis virus in a healthy JE-endemic human cohort using recombinant baculovirus-infected insect cell preparations.
The analysis of cell-mediated immune responses in virus-exposed but healthy individuals may contribute to define the features of the T cell response associated with resistance. We report, for the first time, on adaptive T cell responses to 5 largest of the 10 proteins that together constitute 76% of the coding potential of the Japanese encephalitis virus (JEV) genome in a naturally exposed healthy JE-immune human cohort. Fixed and sonified whole cell preparations of insect cells individually expressing recombinant prM, E, NS1, NS3 and NS5 proteins of JEV were used in vitro to stimulate lymphocytes from individuals who had experienced subclinical JEV infections. NS3-specific memory T cells were detected in up to 86% of the JEV-infected cohort whereas prM, E and NS1 each elicited reactions in approximately 45% among individuals tested, suggesting that NS3 is an important target for JEV-specific cell-mediated immune responses. Responses to NS5, the largest viral protein were in contrast the poorest, seen in only 13% of the cohort. Moreover, NS3 stimulated interferon-gamma production in both CD4(+) and CD8(+) T cells indicating that a Th1 immune response to the NS3 protein may be a critical determinant of immune control of JEV infection.
Animals↗
Novel parasite (nematode) transglutaminase--potential drug target.
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Animals↗
Removal of hydrogen peroxide by a 1-cysteine peroxiredoxin enzyme of the filarial parasite Dirofilaria immitis.
Prior studies have shown that filarial nematodes can effectively metabolize hydrogen peroxide in excess of that generated by activated host cells. However, the mechanisms of H2O2 removal by the filarial parasites are unclear. Herein we report the results of studies carried out on the biochemical activity and on immunolocalization of a recombinant peroxiredoxin (Prx) enzyme from the dog filarial parasite Dirofilaria immitis. A full-length cDNA encoding a 1-Cys Prx enzyme from the dog heartworm D. immitis was expressed in Escherichia coli as a recombinant polyhistidine fusion protein (rDiPrx-1). rDiPrx-1 was capable of reducing H2O2 in the presence of dithiothreitol. The apparent kinetic constants determined for DiPrx-1 using H2O2 as a substrate were a Michaelis constant (Km) of 16.28 mM and a maximal velocity (Vmax) of 16 micromol/min(-1). Consistent with the enzyme activity, D. immitis adult worms could detoxify exogenously added H2O2 in vitro. Antibodies to rDiPrx-1 identified a 27-kDa native antigen in parasite extracts and larval and adult excretory-secretory products. The antibodies were used to localize the native antigen to the lateral hypodermal chords of both male and female worms by immunohistochemistry. In addition, labeling was seen in the afibrillar muscle cells in male worms and in some areas of the uterine wall in female worms. Thus, DiPrx-1 is the first parasite Prx to be shown to detoxify exogenously added H2O2 in an in vitro system.
Animals↗
Transglutaminase-catalyzed reactions in the growth, maturation and development of parasitic nematodes.
Parasitic nematodes cause several debilitating diseases in humans and animals. New drugs that are parasite specific and minimally toxic to the host are needed to counter these infections effectively. The identification and inhibition of enzymes that are vital for the growth and survival of parasites offer new approaches for developing effective chemotherapeutic agents. Several enzymes in nematodes fall into this category. Here, Ramaswamy Chandrashekar and Kapil Mehta examine in detail the role of transglutaminase, a protein-crosslinking enzyme, in the normal growth and development of nematodes, with an emphasis on filarial parasites.
Amino Acid Sequence↗
Cloning and preliminary characterization of a novel cuticular antigen from the filarial parasite Dirofilaria immitis.
We have described here the cloning and partial characterization of a cDNA encoding a cuticular antigen of Dirofilaria immitis. A 48-h third-stage larval D. immitis cDNA library was immunoscreened with sera raised in mice against third-stage larval cuticles (mouse anti-L3 cuticle antisera). A strongly immunoreactive clone (L3MC4) was isolated. Sequence analysis of L3MC4 showed that it was a partial length cDNA. The missing 5' end of the clone was amplified by PCR from D. immitis adult female first-strand cDNA using the nematode 22-base splice leader sequence and a L3MC4-specific antisense primer. The composite cDNA sequence comprised 616 bases (nDiL3MC4) encoding a full-length protein of 146 amino acids (DiL3MC4). GenBank analysis showed that DiL3MC4 shared some homology to an unknown C. elegans gene product (31%) at the amino acid level. However, there were no related filarial expressed sequence tags in the current GenBank database. Antibodies to recombinant DiL3MC4 (rDiL3MC4) identified a 19-kDa native antigen in the adults and in the L3 and L4 larval stages of D. immitis. In addition, the antibodies bound to the cortical layers of the L3 cuticle, as revealed by immuno-gold electron microscopy. The native protein was not detected in larval and adult excretory-secretory products. Immunoblot analysis showed that serum from a rabbit that was repeatedly injected with a small number of D. immitis third stage larvae reacted with rDiL3MC4. Thus, DiL3MC4 is a novel cuticular antigen of a filarial parasite.
Amino Acid Sequence↗
Acute respiratory failure secondary to severe hypokalaemia.
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Acute Disease↗
Human antibody responses to Wuchereria bancrofti infective larvae.
Human IgG antibody responses to Wuchereria bancrofti third stage infective larvae (L3) surface and somatic antigens were studied by indirect immunofluorescence (IFA) and immunoblot with endemic Egyptian sera (n = 115) with the aim of identifying targets of protective immunity. Human sera variably recognized 14 major bands in L3 by immunoblot. The statistical significance of group differences in antibody prevalence was assessed by the chi-squared test. Children and young adults (aged 10-20 years) tended to have antibodies to more L3 somatic antigens than older adults, with significant differences for bands at 66, 60 and 5 kDa. Infected subjects had more consistent antibody responses to antigens at 55, 50 and 6 kDa than endemic normal subjects with negative serum filarial antigen tests, who are presumed to be uninfected. A 5 kDa antigen was preferentially recognized by the latter group. Antibodies to L3 surface antigens were equally prevalent in uninfected children (75%) and adults (90%) but less prevalent in people with microfilaremia (38%) than in amicrofilaremic subjects with or without filarial antigenemia (81%) (P < 0.001). IFA-positive sera showed significantly enhanced recognition of antigens at 66, 40 and 14 kDa in immunoblots relative to IFA-negative sera. Additional studies are needed to further characterize antigens identified in this study and to establish whether they are indeed targets of protective immunity in humans.
Adolescent↗
Longitudinal studies of skin microfilaria and antibody conversion rates in children living in an endemic focus of onchocerciasis in Nigeria.
We monitored skin microfilarial conversion and antibody (serum) conversion rates to Onchocerca volvulus recombinant antigens (OC3.6 and OC9.3) over a 2-year period, in a cohort of 208 children aged 9-11 years and living in a mesoendemic focus of savannah-type onchocerciasis in Nigeria in order to detect prepatent onchocerciasis in children. Between time point A and time point B (9 months apart) the seroconversion rate was 19.4 per cent, and between time points B and C (15 months apart), a further 31.4 per cent of seronegative children had seroconverted. However, only a third of the seroconverted children became microfilaria-positive. From the time-lag between antibody conversion and skin snip conversion, we estimated that a 9-12 month interval was required for the detection of O. volvulus microfilariae following infection with viable third stage larva (L3s). The predictive value of antibody detection as a measure of skin microfilarial conversion was >90 per cent when the optical density (OD) at 490 nm was >0.80. We therefore propose a strategy for monitoring changes in transmission of onchocerciasis following vector control or chemotherapy through an evaluation of annual antibody seroconversion rates in a standardized sentinel population of children.
Animals↗
Identification of an asparagine amidohydrolase from the filarial parasite Dirofilaria immitis.
The nematode cuticle is a complex extracellular structure which is secreted by an underlying syncytium of hypodermal cells. Recent studies have demonstrated that the cuticle of parasitic nematodes is a dynamic structure with important absorptive, secretory, and enzymatic activities. In addition, the cuticle serves as a protective barrier against the host. A 48-h third stage larval Dirofilaria immitis cDNA library was immunoscreened with sera raised against larval cuticles. One clone, L3MC4 that reacted strongly with the anti-cuticle antisera was sequenced. The composite cDNA sequence comprises 2073 bp coding for a full-length protein of 590 amino acids. GenBank analysis showed that DiAsp had significant similarity to a Caenorhabditis elegans gene-product (54% identity) and to other asparaginases at the amino acid level. Escherichia coli-expressed recombinant DiAsp (rDiAsp) catalysed the hydrolysis of asparagine to aspartate and ammonia. Antibodies raised against D. immitis larval cuticles reacted with rDiAsp in immunoblots. This is the first report of identification of a cDNA clone encoding an asparaginase enzyme from a parasitic nematode.
Amino Acid Sequence↗
Molecular characterization of a calcium-binding protein from the filarial parasite Dirofilaria immitis.
A full length D. immitis cDNA (nDiCal) encoding a protein with significant similarity to the calreticulin protein family was isolated from a 6-day fourth-stage larval cDNA expression library by immunoscreening, using serum from a rabbit immunized by repeated injection of small numbers of third-stage larvae. nDiCal is 1538 bp long and contains the 21 bp nematode splice leader sequence SL1 at the 5' end. nDiCal encodes for a protein (pDiCal) with a predicted molecular mass of 46 kDa. pDiCal sequence analysis revealed similarities with calreticulin, a protein that typically resides in the endoplasmic reticulum. pDiCal possesses three consensus sequences of the calreticulin family of proteins: a neutral N-terminal region with a putative signal sequence; a proline- and tryptophan-rich P region; and a highly acidic C-terminal region. A 45Ca2+-overlay assay showed that recombinant pDiCal (rDiCal) is a Ca2+-binding protein. Antibodies to rDiCal identified a 56 kDa native antigen in all developmental stages including the excretory-secretory products derived from larvae and adult worms. Localization studies demonstrated the ubiquitous presence of pDiCal with intense expression in the hypodermis and syncitial muscle cells in both male and female adult worms. Labeling was also seen in the developing embryos within the uterus of the female worms. Sera from immune as well as chronically-infected microfilaremic dogs contained antibodies that bind rDiCal. In addition, immunoblot analysis showed that serum from a rabbit immunized with L3 cuticles reacted with rDiCal.
Amino Acid Sequence↗
Molecular cloning of an enzymatically active thioredoxin peroxidase from Onchocerca volvulus.
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Amino Acid Sequence↗
An ERp60-like protein from the filarial parasite Dirofilaria immitis has both transglutaminase and protein disulfide isomerase activity.
Transglutaminases (TGases; EC 2.3.2.13) are a family of enzymes that catalyze calcium-dependent covalent cross-linking of cellular proteins by establishing epsilon-(gamma-glutamyl)lysine isopeptide bonds. These covalent isopeptide bonds are of great physiological significance because they are highly resistant to proteolysis, denaturants, and reducing agents. Prior studies have demonstrated the presence of isopeptide bonds in the sheath and cuticle of filarial parasites, suggesting an important role for TGase-catalyzed reactions during the growth and development of filarial nematodes. Herein we report the identification and cloning of a cDNA encoding a TGase from the dog heartworm Dirofilaria immitis (DiTG). The DiTG expressed in Escherichia coli (recombinant DiTG) was able to catalyze calcium-dependent cross-linking reactions. The derived amino acid sequence of the DiTG cDNA (pDiTG) predicts a protein of 57.1 kDa and includes an N-terminal hydrophobic signal peptide. The pDiTG has no sequence similarity with any of the known TGases, but it has significant homology to protein disulfide isomerase (PDI) and, particularly, to the PDI-related endoplasmic reticulum protein ERp60, a PDI isoform found in the lumen of endoplasmic reticulum. As predicted from the amino acid sequence homology, recombinant DiTG catalyzed the isomerization of intramolecular disulfide/sulfhydryl bonds in denatured RNase in vitro as effectively as did mammalian PDI. Conversely, purified PDI from bovine liver could catalyze protein cross-linking reactions in a Ca(2+)-dependent manner. This report describes the dual catalytic activity of TGase and PDI in post- and/or cotranslational modification of newly synthesized proteins. These TGase-catalyzed posttranslational modifications may play a pivotal role in the synthesis of new cuticle during the growth and maturation of filarial parasites.
Amino Acid Sequence↗
Molecular cloning, expression and enzymatic activity of a thioredoxin peroxidase from Dirofilaria immitis.
A Dirofilaria immitis cDNA clone encoding a nucleic acid homolog of thioredoxin peroxidase (nDiTPx) was isolated from a fourth-stage larval cDNA library, using serum from dogs vaccinated by chemotherapeutically-abbreviated D. immitis larval infections. The protein encoded by nDiTPx had a predicted molecular mass of 22.1 kDa and the deduced amino acid sequence was homologous to thioredoxin peroxidase-like sequences described in other filarial nematodes, yeast, bacteria and mammals. As is true for other members of this peroxiredoxin family, the nDiTPx-encoded protein had the conserved cysteine near the amino terminus, considered to be essential for enzyme activity. nDiTPx was expressed in E. coli and the resulting recombinant fusion protein was shown to have thioredoxin peroxidase (TPx) activity, by its ability to protect DNA from oxidative-nicking in a metal-catalyzed oxidation system. A polyclonal antibody to the DiTPx fusion protein detected a 22-kDa native protein in D. immitis larval and adult parasite extracts.
Amino Acid Sequence↗
Molecular cloning and characterization of a recombinant Histoplasma capsulatum antigen for antibody-based diagnosis of human histoplasmosis.
Immunological cross-reactivity among fungi has hampered the development of specific serodiagnostic assays for histoplasmosis. We report the molecular cloning and characterization of a Histoplasma capsulatum cDNA (GH17) that encodes an antigen with immunodiagnostic potential. GH17 is an 810-bp cDNA which encodes a protein of 211 amino acid residues. The GH17 sequence has almost no significant homology with other sequences in GenBank. Southern blot analysis suggests that GH17 is confined to a single location in the genomic DNA of H. capsulatum. Immunoblots indicated that the protein product of GH17 (expressed as a 140-kDa beta-galactosidase fusion protein) was recognized by antibodies in 18 of 18 sera from histoplasmosis patients, but not by antibodies in sera from patients or animals infected with other fungi. GH17 was expressed in a prokaryotic expression vector, pPROEX-1, and recombinant protein was purified by preparative electrophoresis. Antibodies raised to this protein bound to a 60-kDa native antigen in immunoblots of H. capsulatum yeast antigen extract. These results suggest that GH17 encodes an H. capsulatum antigen that may be useful for the diagnosis of histoplasmosis in humans.
Amino Acid Sequence↗
Recent advances in diagnosis of filarial infections.
Improved diagnostic methods for human filariasis are needed to facilitate surveillance activities, to monitor control efforts and to evaluate new drugs and vaccines. Currently, diagnosis of filarial infections largely depends upon detection of worms themselves, principally of microfilariae in blood or skin. In many infected people with lymphatic filariae, microfilariae (MF) are not detectable in blood, and removal of skin snips for detection of microfilariae in onchocerciasis seems a rather primitive technique. In addition, because the clinical manifestations of filariae vary greatly between individuals, an ideal diagnostic test would not only reveal individuals that are infected or have been exposed to infection, but would also differentiate between various clinical manifestations that the lymphatic-dwelling parasites, in particular, induce in the infected population. This is important because the pathological reactions induced following treatment with diethylcarbamazine vary with the clinical picture induced by the lymphatic filariae. They are certainly a major problem in onchocerciasis. Recent advances in biotechnology have started revolutionizing the diagnosis of filarial parasites not only in the host but also in their vectors. Monoclonal antibodies have been developed that are specific for detection of circulating antigens in lymphatic filariasis. Species-specific DNA probes have been developed for Brugia malayi, Wuchereria bancrofti, Onchocerca volvulus, and Loa loa. Diagnostic antigens have been obtained by cloning parasite DNA that codes for proteins recognized by infected individuals with only certain species of filariae. Recombinant antigens (rAgs) are available today which detect prepatent infections in onchocerciasis. Several laboratories developing new diagnostic tests for filariasis are currently evaluating these tests in the field with the collaboration of parasitologists, epidemiologists, and vector biologists.
Animals↗
Use of recombinant Onchocerca volvulus antigens for diagnosis and surveillance of human onchocerciasis.
The diagnostic value of ELISAs based on recombinant Onchocerca volvulus antigens OC 3.6 and OC 9.3 was evaluated with sera from endemic areas in West Africa, Guatemala and Ecuador. IgG assays were slightly more sensitive than those that detected IgG4, and the antigen combination was significantly more sensitive than either antigen alone (OC 3.6, 93%; OC 9.3, 84%, combined 98%). These assays were also evaluated with sera from 2 villages in the Onchocerciasis Control Programme area of West Africa including one village (Pendie) with recent recrudescence of infection and one (Niarba) where transmission had been interrupted for 15 years by vector control. The OC 3.6 IgG antibody assay was sensitive for new infections and exposure in Pendie; 24/24 (100%) of people with positive skin snips and 15/74 (20%) of sera from MF negative people had IgG antibodies to this antigen. In addition, antibodies to OC 3.6 often preceded the onset of skin snip positivity in Pendie. In contrast, IgG antibodies to OC 3.6 and OC 9.3 were rarely seen in children born during the 15 years since transmission was interrupted by vector control in Niarba. These encouraging results suggest that antibody assays based on OC 3.6 and OC 9.3 may be valuable tools for surveillance of onchocerciasis and also for monitoring the efficacy of control programmes.
Adolescent↗
Parasite antigenemia without microfilaremia in bancroftian filariasis.
The term "endemic normal" in the context of filariasis refers to people who are amicrofilaremic and free of clinical signs or symptoms of filariasis despite regular exposure to the parasite. Some sera from endemic normals contain soluble Wuchereria bancrofti antigens that are detectable by enzyme-linked immunosorbent assay. We now report evidence that filarial antigenemia in these people is not an artifact and that it is indicative of active W. bancrofti infection. Filarial antigenemia was first detected within one month of the onset of microfilarial patency in experimentally infected primates. Human sera from antigen-positive endemic normals contained the same filarial antigens (by Western blot) as sera from people with microfilaremia. Sera from antigen-positive endemic normals also contained significantly higher levels of immunoglobulin G4 antibodies to native and recombinant filarial antigens than sera from antigen-negative controls matched for age and sex. The epidemiology of filarial antigenemia in endemic normals was studied with sera from a population-based study of filariasis in an Egyptian village with a microfilaria prevalence of 29%. Seventeen percent of endemic normals had antigenemia, and this group comprised 11% of the total village sample. Filarial antigenemia was significantly more common in endemic normals more than 30 years of age than in younger people. These results suggest that amicrofilaremic and asymptomatic W. bancrofti infections are relatively common in endemic areas. Additional studies are needed to determine the clinical significance, prognosis, and optimal management of such infections.
Adolescent↗