Giardia duodenalis assemblages in Egyptian children with diarrhea.

Giardia duodenalis is considered the most common cause of parasitic diarrhea worldwide. Genetic studies revealed that at least eight assemblages (A-H) exist. Of these assemblages, A and B are found primarily in human beings and occasionally in animals. The association between clinical symptoms and G. duodenalis assemblages is controversial. The aim of the present study was to determine the assemblages of G. duodenalis prevalent among Egyptian children with diarrhea. Therefore, 96 positive stool samples for Giardia by light microscopy were subjected to multilocus genotyping targeting the triose phosphate isomerase (tpi), ß-giardin (bg), and glutamate dehydrogenase (gdh) genes. Amplified polymerase chain reaction (PCR) products were then purified, sequenced, and aligned with reference strains to determine the assemblages of the Giardia isolates. Out of the 96 microscopically positive stool samples for Giardia, 77 (80 %) were successfully amplified and sequenced at least at one locus. Of these, 21 (27.3 %) were shown to be assemblage A, 54 (70.1 %) assemblage B, while discordant sequence typing results were observed in 2 (2.6 %) samples. AII was the predominant subassemblage of assemblage A, while it was generally difficult to further classify assemblage B. It was concluded that infection with assemblage B was more common than that with assemblage A. No associations between epidemiological information and assemblage were detected, except with age. Although infections with assemblage B were more frequently associated with abdominal pain and acute diarrhea than with assemblage A, the difference was not statistically significant.

Correlation of T cell response and bacterial clearance in human volunteers challenged with Helicobacter pylori revealed by randomised controlled vaccination with Ty21a-based Salmonella vaccines.

BACKGROUND: Helicobacter pylori remains a global health hazard, and vaccination would be ideal for its control. Natural infection appears not to induce protective immunity. Thus, the feasibility of a vaccine for humans is doubtful. METHODS: In two prospective, randomised, double-blind, controlled studies (Paul Ehrlich Institute application nos 0802/02 and 1097/01), live vaccines against H pylori were tested in human volunteers seronegative for, and without evidence of, active H pylori infection. Volunteers (n = 58) were immunised orally with Salmonella enterica serovar Typhi Ty21a expressing H pylori urease or HP0231, or solely with Ty21a, and then challenged with 2x10(5) cagPAI(-) H pylori. Adverse events, infection, humoral, cellular and mucosal immune response were monitored. Gastric biopsies were taken before and after vaccination, and postchallenge. Infection was terminated with antibiotics. RESULTS: Vaccines were well tolerated. Challenge infection induced transient, mild to moderate dyspeptic symptoms, and histological and transcriptional changes in the mucosa known from chronic infection. Vaccines did not show satisfactory protection. However, 13 of 58 volunteers, 8 vaccinees and 5 controls, became breath test negative and either cleared H pylori (5/13) completely or reduced the H pylori burden (8/13). H pylori-specific T helper cells were detected in 9 of these 13 (69%), but only in 6 of 45 (13%) breath test-positive volunteers (p = 0.0002; Fisher exact test). T cells were either vaccine induced or pre-existing, depending on the volunteer. CONCLUSION: Challenge infection offers a controlled model for vaccine testing. Importantly, it revealed evidence for T cell-mediated immunity against H pylori infection in humans.

Expression of helminth genes in Leishmania: an experimental transfection system to test immunological function.

Functional analysis of genes from parasitic helminths requires, at the present time, heterologous expression. We have adapted the well-characterized system of transfection in Leishmania protozoal parasites, as a means of analysing the effect of single filarial genes on the mammalian immune system. For example, testing the function of the Brugia malayi abundant larval transcript (ALT) gene-transfected Leishmania mexicana were found to be significantly more virulent in macrophages in vitro. The course of infection in vivo is also aggravated by expression of the ALT gene. Examples are also given of transgenes which reduced in vitro growth within macrophages, as well as others which exert no effect on the protozoal parasitism. Thus, Leishmania transfection provides a tractable system to analyse helminth gene function within the context of the host immune system.

First Draft Genome Sequence of Balamuthia mandrillaris, the Causative Agent of Amoebic Encephalitis.

The free-living amoeba Balamuthia mandrillaris is a rare but highly lethal agent of amoebic encephalitis in humans and many other mammalian species. Here, we announce the first draft genome sequence of the original 1990 isolate cultured from the brain of a deceased mandrill baboon.

Targeted integration into a rRNA locus results in uniform and high level expression of transgenes in Leishmania amastigotes.

This report describes the construction of a DNA cassette for integration into a genomic small sub-unit rRNA locus of Leishmania mexicana by homologous recombination. Reporter genes encoding beta-galactosidase or green fluorescent protein and the gene conferring hygromycin resistance were integrated downstream of a RNA polymerase I-driven rRNA promoter. To ensure high expression of the marker proteins in the intracellular, amastigote stage, transgene coding sequences were followed by the intergenic region of the L. mexicana cysteine proteinase B 2.8 gene which provides processing signals required for high level expression in this life-cycle stage. Integration of the DNA cassette was also efficiently obtained in L. major. We show that either beta-galactosidase or the green fluorescent protein were abundantly, stably and uniformly expressed in promastigotes and amastigotes of both Leishmania sp. The transgenic lines allow parasite detection at high sensitivity in the tissues of infected mice and will be useful to follow infections in macrophages in culture and in animal hosts.

Recombinant live Salmonella spp. for human vaccination against heterologous pathogens.

Live attenuated Salmonella spp. are promising candidates as oral vaccine delivery systems for heterologous antigens. Clinical trials have demonstrated that this approach is feasible for human vaccinations but further optimisation is necessary to obtain a better efficacy. Here, we discuss how existing clinical and pre-clinical data can be used to guide such optimisation efforts.

How CD4(+) T cells may eliminate extracellular gastric Helicobacter?

Helicobacter pylori is recognised as a causal agent in the pathogenesis of gastritis, gastric and duodenal ulcer disease as well as gastric cancers. Eradication of the bacteria with antibiotics is currently used to treat symptomatic, infected individuals. Theoretically the infection could also be controlled by vaccination. Several immunisation protocols were developed in small animal models and primates in order to validate this approach. Recently making use of mice with defined genetic defects, H. pylori-specific CD4(+) T cells were found to be crucial for protective vaccination. This was unexpected and poses the question of how activation of CD4(+) T cells leads to the elimination of bacteria that reside primarily in the mucin layer behind a barrier of epithelial cells. CD4(+) T cells fulfil their effector function by secreting lymphokines and by engaging specific surface ligands on interacting cells. Here we propose that phagocytes and epithelial cells stimulated either by direct interaction with CD4(+) T cells or by soluble mediators such as cytokines or neuropeptides are the ultimate effector populations in protective immunity induced by vaccination.

Topical treatment with hexadecylphosphocholine (Miltex) efficiently reduces parasite burden in experimental cutaneous leishmaniasis.

Ether-lipids and alkylphosphocholines have been found to have anti-leishmanial activity. Oral treatment with hexadecylphosphocholine (HePC) efficiently reduces parasite burden in murine visceral leishmaniasis. Drugs for the treatment of cutaneous leishmaniasis are most commonly administered parenterally, whereas efficient drugs for topical treatment are not in current use. Here we investigate the efficacy of topical treatment with HePC in mice infected with Leishmania mexicana or L. major, causative agents of cutaneous leishmaniasis in the New and Old World, respectively. BALB/c, CBA/J and C57BL/6 inbred mice do not control infection with L. mexicana because they do not mount an efficient Th1-type anti-parasitic lymphocyte response. In contrast, C57BL/6 mice are resistant to an infection with L. major, developing only transient lesions that heal spontaneously owing to an efficient Th1 response. BALB/c, CBA/J and C57BL/6 mice were infected subcutaneously with L. mexicana amastigotes, causing nodular lesions after 5 months. Topical treatment with HePC (Miltex) was highly effective in reducing parasite burden and healed established lesions. The treatment did not induce a Th1 response in L. mexicana-infected susceptible mice and most of the mice relapsed. In resistant C57BL/6 mice infected subcutaneously with 2 x 10(6) L. major promastigotes at the tail base, nodular lesions developed after 2 weeks. Topical treatment with Miltex reduced the parasite load and the mice healed their lesions much faster than the untreated infected controls. The clinical application of Miltex for treatment of cutaneous leishmaniasis may be highly efficient because humans, similarly to resistant mice, in general do not relapse after healing. Clinical trials should be straightforward considering that Miltex is an approved drug for the treatment of breast cancer metastases.

Detection of Giardia duodenalis assemblage A and B isolates by immunochromatography in stool samples from Rwandan children.

We evaluated the performance of an immunochromatographic assay (ICA) in comparison with light microscopy and PCR for the detection of Giardia duodenalis in stool samples from 558 Rwandan children. The association of infection with clinical symptoms was similar for the three diagnostic tools. The ICA equally detected parasites of assemblages A and B and was more sensitive than light microscopy (50.4 versus 29.5% of PCR-positive samples considered true positive; p <0.0001). Hence, the ICA shows superior sensitivity compared with microscopy but still misses half of the G. duodenalis infections detected by PCR in this hyperendemic area.

Visceral leishmaniasis: immunology and prospects for a vaccine.

Human visceral leishmaniasis (HVL) is the most severe clinical form of a spectrum of neglected tropical diseases caused by protozoan parasites of the genus Leishmania. Caused mainly by L. donovani and L. infantum/chagasi, HVL accounts for more than 50 000 deaths every year. Drug therapy is available but costly, and resistance against several drug classes has evolved. Here, we review our current understanding of the immunology of HVL and approaches to and the status of vaccine development against this disease.

Helicobacter pylori infection in anterior uveitis.

BACKGROUND: Despite intensive research, the etiology of acute anterior uveitis (AAU) remains poorly defined. Infection with gram-negative bacteria such as Yersinia, Salmonella, Shigella, and Chlamydia have already been suggested as a possible trigger event for AAU. Helicobacter pylori is also a gram-negative bacterium, shares the lipopolysaccharides, but did not attract the attention of many ophthalmologists until recently. Having in mind the relatively high incidence of H. pylori infection in the population, we propose that H. pylori may also be a trigger factor for AAU. PATIENTS AND METHODS: The presence of anti-H. pylori antibodies in matching serum and aqueous humor samples of 15 idiopathic AAU patients was determined using a commercial Western blot assay. Control serum and aqueous humor were obtained from five patients undergoing cataract surgery. RESULTS: Six out of 15 AAU patients (40%) were serum-positive for H. pylori, and half of these (n = 3) also had anti-H. pylori antibodies in the aqueous humor. All five aqueous humor and sera controls tested negative for H. pylori infection. CONCLUSION: These are the first results demonstrating anti-H. pylori antibodies in the aqueous humor of AAU patients. Further studies are needed to demonstrate whether this antibody is indeed locally produced. Our data may provide first evidence for a causative link between H. pylori infection and AAU.

Recurrent cutaneous leishmaniasis: a role for persistent parasites?

Leishmaniosis is, with increasing frequency, reported as an opportunistic infection of immunosuppressed individuals. Re-activation of persistent parasites may be responsible for the disease in a number of these patients. Here, Toni Aebischer reviews some of the evidence for the implication of persistent Leishmania infections in recurrent disease with the emphasis on cutaneous leishmoniasis in humans and in the mouse model. The data suggest that parasite persistence is a common feature in Leishmania infections. The availability of on excellent laboratory model provides on opportunity to study this phenomenon in detail. The findings of these analyses are likely to be important for the identification of people at risk of developing recurrent disease and for the assessment of new therapies for relapsing leishmaniasis and might also have implications for the design of a future anti-Leishmania vaccine.

Antigen presentation by macrophages harboring intravesicular pathogens.

Resting macrophages can be host cells for the replication of several protozoan parasites and bacteria. Upon activation, infected cells mobilize potent microbicidal mechanisms that eliminate the intracellular pathogen. This transition from a resting to an activated state is mediated by the interaction with specific T cells that recognize pathogen-derived peptides complexed to major histocompatibility complex (MHC) molecules at the surface of host cells. In this review, Peter Overath and Toni Aebischer discuss antigen presentation in infected macrophages from a cell biological point of view, a perspective that has important implications for the design of subunit vaccines.

Intravenous injection of irradiated Leishmania major into susceptible BALB/c mice: immunization or protective tolerance.

It is well established that BALB/c mice can be protected from fatal infection with Leishmania major by prophylactic intravenous (i.v.) immunization with irradiated parasites. Protection is critically dependent on the route of injection with i.v. injection being protective and subcutaneous injection not protective. We used this BALB/c-L. major model system to investigate this phenomenon. We analyzed quantitatively the parasite-specific, CD4+ T cell mediated immune responses by limiting dilution. Subcutaneous vaccination resulted in priming of CD4+ precursor T cells, whereas i.v. vaccination was ineffectual. Moreover, i.v. injection prevented the increase in the number of specific precursor cells induced by infection of normal mice during the first weeks post-challenge with virulent parasites. We show here that this was not due to the elimination of the virulent challenge parasites as a result of immunity nor to inefficient antigen presentation of the irradiated organisms after i.v. injection. The data presented here suggest that i.v. injection results in tolerization rather than immunization. Tolerization as a mechanism of host protection is consistent with earlier observations that transient immunosuppression results in cure of L. major infection in BALB/c mice. Transfer of antigen presenting cells (APC) isolated from spleens of mice injected previously with irradiated parasites mimicked to some extent the effect of i.v. immunization with irradiated parasites. The possible involvement of these APC in decreasing the parasite-specific T cell response is discussed.

Preferential usage of V alpha 4 and V beta 10 T cell receptor genes by lymphocytic choriomeningitis virus glycoprotein-specific H-2Db-restricted cytotoxic T cells.

Correlations between the T cell receptor (TcR) V gene usage and the specificity of T cells have been primarily described for major histocompatibility complex (MHC) class II-restricted helper T cell responses. In the present study the TcR genes expressed by MHC class I-restricted murine cytotoxic T cells (CTL) specific for a major epitope of the lymphocytic choriomeningitis virus (LCMV), LCMV-GP2(275-289), were investigated. The TcR primary structure of an LCMV-GP2(275-289) specific H-2Db-restricted CTL clone has been determined. It uses a member of the V alpha 4 family joined to J alpha AN14.4 for the alpha chain and V beta 10 rearranged to D beta 2.1 and J beta 2.4 for its beta chain. Four other independent LCMV-GP2(275-289) specific H-2Db-restricted CTL clones also expressed V alpha 4 and V beta 10 gene elements. Furthermore, V alpha 4 and V beta 10 were preferentially expressed by polyclonal CTL of C57BL/6 origin specific for LCMV. These results suggest that both TcR V alpha and V beta regions are important for the recognition of the LCMV-GP2(275-289) epitope on H-2Db molecules.

Proteophosphoglycan, a major secreted product of intracellular Leishmania mexicana amastigotes, is a poor B-cell antigen and does not elicit a specific conventional CD4+ T-cell response.

Secreted and surface-exposed antigens of intracellular pathogens are thought to provide target structures for detection by the host immune system. The major secreted product of intracellular Leishmania mexicana amastigotes, a proteophosphoglycan (aPPG), is known to contribute to the establishment of the parasitophorous vacuole and is able to activate complement. aPPG belongs to a novel class of serine- and threonine-rich Leishmania proteins that are extensively modified by phosphodiester-linked phosphooligosaccharides and terminal mannooligosaccharides. Here we show that mice chronically infected with L. mexicana generally do not produce antibodies or Th cells specific for aPPG. Similarly, antibody titers are very low in mice vaccinated with aPPG, and specific CD4+ T cells are undetectable. Comparative analyses of other Leishmania glycoconjugates indicate that L. mexicana-specific carbohydrate structures are poorly immunogenic in mice and that the proteophosphoglycan aPPG behaved immunologically like a carbohydrate. The latter observation is explained by the lack of induction of aPPG-specific CD4+ T cells. In contrast, recombinant aPPG peptides stimulate CD4+ T-cell responses and high titers of specific antibodies are found in the sera of mice vaccinated with these peptides. Native aPPG is highly resistant to proteinases and apparently cannot be degraded by macrophages. It is concluded that conventional CD4+ T cells against the polypeptide backbone of aPPG are not induced because the molecule resists antigen processing due to its extensive and complex carbohydrate modification. The complex glycan chains of aPPG, which exhibit important biological functions for the parasite, may therefore also have evolved to evade detection by the immune system of the host organism.

Phagocytosis of Leishmania mexicana amastigotes by macrophages leads to a sustained suppression of IL-12 production.

Healing of leishmaniases is dependent on activation of parasitized macrophages (Mphi) by IFN-gamma, which is secreted by Leishmania-specific Th1 cells. IL-12 enhances IFN-gamma production by Th1 cells and is crucial for cure. The host cells of Leishmania sp., Mphi, are a main source of IL-12 in vivo. We report that infection of quiescent murine Mphi with L. mexicana or L. major amastigotes does not induce IL-12 production. Moreover, infection suppresses IL-12 secretion by Mphi activated by LPS, by CD40 cross-linking or cognate interaction with Th1 cells. IL-12 secretion is also suppressed in Mphi activated after phagocytosis of latex beads. Suppression is independent of engagement of CR3 or FcgammaR during phagocytosis, is not mediated by IL-10 and does not alter steady state IL-12p40 mRNA levels. In addition, suppression of IL-12 secretion does not depend on Mphi activation concurrent to infection. In contrast, NO production was not inhibited. Thus, Mphi effector functions are differentially affected and this may be a general effect of phagocytosis of non-activating particles. The possible implications of this effect on the infection are discussed.

Persistence of virulent Leishmania major in murine cutaneous leishmaniasis: a possible hazard for the host.

The persistence of Leishmania major parasites in mice resistant to infection was investigated by the polymerase chain reaction and in vitro culture methods. Parasite-specific DNA was detected in the lymph nodes, spleens, bone marrow, and livers of C57BL/6 mice 1 year after their recovery from infection. Live parasites were also recovered from these tissues (except liver tissues) and were used to establish in vitro isolates. Pulsed-field gel electrophoresis, Southern blotting, and Western blot (immunoblot) analyses showed that these isolates retained the karyotype and the phenotype of the original inoculum, including the levels of expression of gp63 and lipophosphoglycan, the two major surface molecules of Leishmania species. More importantly, these isolates were virulent and induced fatal disease when injected into susceptible BALB/c mice. Persistence was shown to be a more general phenomenon, since several different strains of mice which were resistant to L. major infection also harbored persistent parasites. The implications for the etiology of human leishmaniasis in immunocompromised individuals such as AIDS patients are discussed.

Resistance of BALB/c mice to Leishmania major infection is associated with a decrease in the precursor frequency of antigen-specific CD4+ cells secreting interleukin-4.

BALB/c mice are highly susceptible to infection with the protozoan parasite Leishmania major and develop a chronic fatal disease. They can, however, be manipulated to resist disease and this has been shown to correlate with increased expression of IFN-gamma mRNA and the absence of IL-4 mRNA in the draining lymph nodes and spleens of these animals. Here we show that anti-IL-4 or anti-CD4 treatment of BALB/c mice resulted in a reduction in the size of the lesion and in the number of parasites in the draining lymph nodes compared with untreated mice. The precursor frequency of CD4+ T cells proliferating in response to Leishmania antigens in vitro in the treated animals was not significantly different from untreated animals. Analysis of the lymphokines secreted by the clonal progeny of these cells showed that the precursor frequency of IL-4 secreting clones was at least 10-fold lower in animals treated with either mAb. However, there was no reciprocal increase in the precursor frequencies of IFN-gamma secreting clones. Comparisons of the total number of precursors of specific CD4+ cells secreting IFN-gamma showed that anti-CD4-treated animals, which are resistant to disease, had considerably fewer for the first 6 weeks than untreated mice with chronic disease. Protection of BALB/c mice was therefore associated with a reduction in the numbers of precursors of cells secreting IL-4 without a concomitant increase in the number of precursors of IFN-gamma secreting cells.

Subunit vaccination of mice against new world cutaneous leishmaniasis: comparison of three proteins expressed in amastigotes and six adjuvants.

A mixture of well-defined recombinant antigens together with an adjuvant that preferentially stimulates specific gamma interferon (IFN-gamma)-secreting helper type 1 CD4(+) T cells (Th1 cells) presents a rational option for a vaccine against leishmaniasis. The potential of this approach was investigated in murine infections with Leishmania mexicana, which are characterized by the absence of a parasite-specific Th1 response and uncontrolled parasite proliferation. A mixture of three antigens (glycoprotein 63, cysteine proteinases, and a membrane-bound acid phosphatase), which are all expressed in amastigotes, the mammalian stage of the parasite, were used for the immunization of C57BL/6 mice in combination with six adjuvants (interleukin 12 [IL-12], Detox, 4'-monophosphoryl lipid A, QS-21, Mycobacterium bovis BCG, and Corynebacterium parvum). All six vaccine formulations containing the mixture of recombinant antigens were protective against challenge infections with promastigotes, the insect stage of the parasite, in that mice controlled and healed infections but developed transient and, in certain cases, accentuated disease. The most effective adjuvants were IL-12 followed by Detox. Further studies using these two adjuvants showed that a similar protective effect was observed with a mixture of the corresponding native proteins, and mice which had controlled the infection showed a preponderance of IFN-gamma-secreting CD4(+) T cells in the lymph nodes draining the lesion. Using the recombinant proteins individually, it is shown that the relatively abundant cysteine proteinases and glycoprotein 63, but not the acid phosphatase, are able to elicit a protective response. The results are discussed in comparison to previous studies with subunit vaccines and with respect to cell biological aspects of antigen presentation in Leishmania-infected macrophages.