Epidemiology and strain characteristics of Echinococcus granulosus in the Benghazi area of eastern Libya

The incidence of surgically confirmed cystic echinococcosis in eastern Libya was estimated to be at least 4.2 cases/100,000, with significantly more female cases than male. The prevalences of infection with Echinococcus granulosus among 1087 sheep, 881 goats, 428 camels and 614 cattle from the same region, determined postmortem in abattoirs, were 20%, 3.4%, 13.6% and 11%, respectively. Infection in the livestock was age-dependent and, generally, the female animals were more often infected than the male. The measurements of rostellar hooks on protoscoleces collected from sheep and cattle were similar but significantly different from the corresponding measurements of parasites of human or camel origin. However, when a portion of the cytochrome c-oxidase subunit I (coxl) gene from each of 30 protoscolex samples from Libya (12 from cattle, three from humans, five from camels and 10 from sheep) was sequenced, the sequences were all found to be identical to that published for the common sheep strain of E. granulosus.

Canine gastrointestinal parasitic zoonoses in India

Although well recognized and studied in developed countries, canine parasitic zoonoses pose a lowly prioritized public health problem in developing countries such as India, where conditions are conducive for transmission. A study of the most recent parasite survey determining prevalence and epidemiology of canine parasitic zoonoses among tea-growing communities of northeast India demonstrated the endemicity of the problem. This particular study serves as a model using conventional, as well as molecular parasitological, tools to provide novel insights into the role of dogs as mechanical transmitters of human parasites such as Ascaris and Trichuris, and discusses the risks dogs pose with regards to zoonotic transmission of hookworms and Giardia.

Limitations of HLA-transgenic mice in presentation of HLA-restricted cytotoxic T-cell epitopes from endogenously processed human papillomavirus type 16 E7 protein

We investigated the use of mice transgenic for human leucocyte antigen (HLA) A*0201 antigen-binding domains to test vaccines composed of defined HLA A*0201-restricted cytotoxic T-lymphocyte (CTL) epitopes of human papillomavirus (HPV) type 16 E7 oncoprotein. HPV is detected in >90% of cervical carcinomas. HPV16 E7 oncoprotein transforms cells of the uterine cervix and functions as a tumour-associated antigen to which immunotherapeutic strategies may be directed. We report that although the HLA A*0201 E7 epitope peptides function both to prime for E7 CTL responses, and to sensitize target cells for E7-directed CTL killing in situations where antigen processing is not required, the epitopes are not processed out of either endogenously expressed or immunization-introduced E7, by the mouse antigen-processing and presentation machinery. Thus (1) CTL induced by HLA A*0201 peptide immunization killed E7 peptide-pulsed target cells, but did not kill target cells expressing whole E7; (2) immunization with whole E7 protein did not elicit CTL directed to HLA A*0201-restricted E7 CTL epitopes; (3) HLA A*0201-restricted CTL epitopes expressed in the context of a DNA polytope vaccine did not activate E7-specific T cells either in 'conventional' HLA A*0201 transgenic (A2.1K(b) ) mice, or in HHD transgenic mice in which expression of endogenous H-2 class 1 is precluded; and (4) HLA A*0201 E7 peptide epitope immunization was incapable of preventing the growth of an HLA A*0201- and E7-expressing tumour. There are generic implications for the universal applicability of HLA-class 1 transgenic mice for studies of human CTL epitope presentation in murine models of human infectious disease where recognition of endogenously processed antigen is necessary. There are also specific implications for the use of HLA A2 transgenic mice for the development of E7-based therapeutic vaccines for cervical cancer.

Hookworm aspartic protease, Na-APR-2, cleaves human hemoglobin and serum proteins in a host-specific fashion

Hookworms are voracious blood-feeders. The cloning and functional expression of an aspartic protease, Na-APR-2, from the human hookworm Necator americanus are described here. Na-APR-2 is more similar to a family of nematode-specific, aspartic proteases than it is to cathepsin D or pepsin, and the term nemepsins for members of this family of nematode-specific hydrolases is proposed. Na-apr-2 mRNA was detected in blood-feeding, developmental stages only of N. americanus, and the protease was expressed in the intestinal lumen, amphids, and excretory glands. Recombinant Na-APR-2 cleaved human hemoglobin (Hb) and serum proteins almost twice as efficiently as the orthologous substrates from the nonpermissive dog host. Moreover, only 25% of the Na-APR-2 cleavage sites within human Hb were shared with those generated by the related N. americanus cathepsin D, Na-APR-1. Antiserum against Na-APR-2 inhibited migration of 50% of third-stage N. americanus larvae through skin, which suggests that aspartic proteases might be effective vaccines against human hookworm disease.

The c-Myb oncoprotein

The c-myb gene is the cellular homologue of the v-myb oncogenes carried by the avian leukaemia viruses AMV and E26. It encodes a transcription factor (c-Myb), as does each of the viral oncogenes, which recognises the core DNA sequence C/T-A-A-C-G/T-G via a repeated helix-turn-helix-like motif. c-myb is expressed in immature haemopoietic cells, as well as immature cells of the gastro-intestinal epithelium and is down-regulated with differentiation. Enforced expression of activated or even normal forms of Myb can transform haemopoietic cells, most often of the myeloid lineage, in vitro and in vivo. Although many genes have been identified which are likely to be regulated by c-Myb, the critical target genes involved in Myb's transforming activity are not known. Together with data showing increased c-myb expression in colonic tumours, these observations raise the possibility that c-myb may play a role in human malignant disease. (C) 1998 Elsevier Science Ltd. All rights reserved.

Further studies on an intermediate host murine model showing that a primary Echinococcus granulosus infection is protective against subsequent oncospheral challenge

We describe the use of a murine model to evaluate resistance against subsequent challenge following a primary infection with oncospheres of Echinococcus granulosus. Mice (Kunming strain) were infected with hatched oncospheres of Echinococcus granulosus; 21 days later a second challenge was given by a different route of infection. A primary infection by intraperitoneal (i.p.) injection stimulated 100 and 90.5% protection in terms of reduced cyst numbers against a secondary infection given subcutaneously (s.c.) or intravenously (i.v.) respectively. A primary infection given s.c. followed by i.p. or i.v. challenge resulted in 84.0 and 100% protection, respectively. Intravenous infection followed by i.p. or s.c. challenge resulted in 98.5 and 69.4% protection, respectively. With the i.v. route of infection, almost all resultant cysts were present in the lungs. The data show that a primary infection with oncospheres can induce total or a high degree of protection against a subsequent challenge and confirms that natural (concomitant) immunity can be stimulated in the intermediate host as the result of a primary infection. This may explain the decline in hydatid infection in sheep older than 2 years in hyper-endemic areas such as those found in Xingjiang, China. These older sheep may have been earlier infected and have subsequently self-cured, with the primary infection stimulating an immune response that protects the intermediate host animals from further infection. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.

Effect of Fusobacterium nucleatum on the T and B cell responses to Porphyromonas gingivalis in a mouse model

T cell cytokine profiles and specific serum antibody levels in five groups of BALB/c mice immunized with saline alone, viable Fusobacterium nucleatum ATCC 25586, viable Porphyromonas gingivalis ATCC 33277, F. nucleatum followed by P. gingivalis and P. gingivalis followed by F nucleatum were determined. Splenic CD4 and CD8 cells were examined for intracytoplasmic interleukin (IL)-4, interferon (IFN)-gamma and IL-10 by dual colour flow cytometry and the levels of serum anti-F. nucleatum and anti-P. gingivalis antibodies determined by an ELISA. Both Th1 and Th2 responses were demonstrated by all groups, and while there were slightly lower percentages of cytokine positive T cells in mice injected with F. nucleatum alone compared with the other groups immunized with bacteria., F nucleatum had no effect on the T cell production of cytokines induced by P gingivalis in the two groups immunized with both organisms. However, the percentages of cytokine positive CD8 cells were generally significantly higher than those of the CD4 cells. Mice immunized with F nucleatum alone had high levels of serum anti-E nucleatum antibodies with very low levels of P. gingivalis antibodies, whereas mice injected with P gingivalis alone produced anti-P. gingivalis antibodies predominantly. Although the levels of anti-E nucleatum antibodies in mice injected with E nucleatum followed by P. gingivalis were the same as in mice immunized with F nucleatum alone, antibody levels to P. gingivalis were very low. In contrast, mice injected with P. gingivalis followed by F nucleatum produced equal levels of both anti-P. gingivalis and anti-F nucleatum antibodies, although at lower levels than the other three groups immunized with bacteria, respectively. Anti-Actinobacillus actitiomycetemcomitans, Bacteroides forsythus and Prevotella intermedia serum antibody levels were also determined and found to be negligible. In conclusion, F nucleatum immunization does not affect the splenic T cell cytokine response to P. gingivalis. However, F nucleatum immunization prior to that of P. gingivalis almost completely inhibited the production of anti-P gingivalis antibodies while P. gingivalis injection before F. nucleatum demonstrated a partial inhibitory effect by P. gingivalis on antibody production to F. nucleatum. The significance of these results with respect to human periodontal disease is difficult to determine. However, they may explain in part differing responses to P. gingivalis in different individuals who may or may not have had prior exposure to F. nucleatum. Finally, the results suggested that P. gingivalis and F. nucleatum do not induce the production of cross-reactive antibodies to other oral microorganisms.

Towards a taxonomic revision of the genus Echinococcus

Echinococcus remains a significant public health problem worldwide and, in several regions, the aetiological agents of cystic hydatid disease/echinococcosis are extending their range. The taxonomy of Echinococcus has been a controversial issue for decades, but the outcome of recent molecular epidemiological studies has served to reinforce proposals made ten years ago to revise the taxonomy of Echinococcus. A formal nomenclature is essential for effective communication, and provides the stability that underpins epidemiological investigations. It will also serve to recognize the contribution of early taxonomists.

Functional expression and characterization of Eehinococcus granulosus thioredoxin peroxidase suggests a role in protection against oxidative damage

A full-length cDNA sequence coding for Echinococcus granulosus thioredoxin peroxidase (EgTPx) was isolated from a sheep strain protoscolex cDNA library by immunoscreening using a pool of sera from mice infected with oncospheres. EgTPx expressed as a fusion protein with glutathione S-transferase (GST) exhibited significant thiol-dependent peroxidase activity that protected plasmid DNA from damage by metal-catalyzed oxidation (MCO) in vitro. Furthermore, the suggested antioxidant role for EgTPx was reinforced in an in vivo assay, whereby its expression in BL21 bacterial cells markedly increased the tolerance and survival of the cells to high concentrations of H2O2 compared with controls. Immunolocalization studies revealed that EgTPx was specifically expressed in all tissues of the protoscolex and brood capsules. Higher intensity of labelling was detected in many, but not all, calcareous corpuscle cells in protoscoleces. The purified recombinant EgTPx protein was used to screen sera from heavily infected mice and patients with confirmed hydatid infection. Only a portion of the sera reacted positively with the EgTPx-GST fusion protein in Western blots, suggesting that EgTPx may form antibody-antigen complexes or that responses to the EgTPx antigen may be immunologically regulated. Recombinant EgTPx may prove useful for the screening of specific inhibitors that could serve as new drugs for treatment of hydatid disease. Moreover, given that TPx from different parasitic phyla were phylogenetically distant from host TPx molecules, the development of antiparasite TPx inhibitors that do not react with host TPx might be feasible. (C) 2003 Elsevier B.V. All rights reserved.

Identification and analysis of novel genes expressed in the mouse embryonic facial primordia

Craniofacial anomalies are a common feature of human congenital dysmorphology syndromes, suggesting that genes expressed in the developing face are likely to play a wider role in embryonic development. To facilitate the identification of genes involved in embryogenesis, we previously constructed an enriched cDNA library by subtracting adult mouse liver cDNA from that of embryonic day (E)10.5 mouse pharyngeal arch cDNA. From this library, 273 unique clones were sequenced and known proteins binned into functional categories in order to assess enrichment of the library (1). We have now selected 31 novel and poorly characterised genes from this library and present bioinformatic analysis to predict proteins encoded by these genes, and to detect evolutionary conservation. Of these genes 61% (19/31) showed restricted expression in the developing embryo, and a subset of these was chosen for further in silico characterisation as well as experimental determination of subcellular localisation based on transient transfection of predicted full-length coding sequences into mammalian cell lines. Where a human orthologue of these genes was detected, chromosomal localisation was determined relative to known loci for human congenital disease.

Identification of membrane-bound and secreted proteins from Echinococcus granulosus by signal sequence trap

The signal sequence trap technique was applied to identify genes coding for secreted and membrane bound proteins from Echinococcus granulosus, the etiologic agent of cystic hydatid disease. An E. granulosus protoscolex cDNA library was constructed in the AP-PST vector such that randomly primed cDNAs were fused with a placental alkaline phosphatase reporter gene lacking its endogenous signal peptide. E. granulosus cDNAs encoding a functional signal peptide were selected by their ability to rescue secretion of alkaline phosphatase by COS-7 cells that had been transfected with the cDNA library. Eighteen positive clones were identified and sequenced. Their deduced amino acid sequences showed significant similarity with amino acid transporters, Krebs cycle intermediates transporters, presenilins and vacuolar protein sorter proteins. Other cDNAs encoded secreted proteins without homologues. Three sequences were transcribed antisense to E. granulosus expressed sequence tags. All the mRNAs were expressed in protoscoleces and adult worms, but some of them were not found in oncospheres. The putative E. granulosus secreted and membrane bound proteins identified are likely to play important roles in the metabolism, development and survival in the host and represent potential targets for diagnosis, drugs and vaccines against E. granulosus. (c) 2005 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Correlation of T-cell immune response with spontaneous resolution and subsequent relapse of Hodgkin's lymphoma

To our knowledge, there has been no report of spontaneous regression in a non-immunocompromised adult with classical Hodgkin's lymphoma (HL) in the absence of chemotherapy. We describe spontaneous regression and subsequent relapse of Epstein - Barr virus (EBV)-positive HL in an otherwise healthy male adult. The clinical course was associated with an increase in regulatory T-cell markers within the peripheral blood and diseased lymph node at the time of relapse and with a concomitant reduction in cellular immunity against relevant EBV latent membrane protein tumor-associated antigens. Our findings are in keeping with previous observations that implicate impaired cellular immunity in the immunopathogenesis of EBV-positive HL.