Cryptosporidium parvum antigens induce mouse and human dendritic cells to generate Th1-enhancing cytokines.

Cryptosporidium parvum is an opportunistic intracellular parasite that causes mild to severe diarrhoea, which can be life-threatening in an immunocompromised host. To increase our understanding of the mechanisms that play a role in host immune responses, we investigated the effects of C. parvum antigens on the phenotype of mouse and human dendritic cells (DCs). Cryptosporidium parvum antigens induced DC activation as indicated by upregulation of the maturation marker CD209, as well as by the production of the cytokines interleukin-12 p70, IL-2, IL-1beta, IL-6. In particular, significant increases in the expression of IL-12 p70 were observed from mouse DCs derived from bone marrow in response to solubilized sporozoite antigen and the recombinant cryptosporidial antigens, Cp40 and Cp23. We observed a small but significant increase in IL-18 expression following the exposure to Cp40. We found that the induction of Th1 cytokines was MyD88 dependent (MyD88 knockout mouse DCs were unresponsive). Additionally, both sporozoite preparations (solubilized and live) significantly induced IL-12 production by human monocytic dendritic cells (MoDCs). This finding indicates that solubilized as well as recombinant antigens can induce the maturation of DCs and subsequently initiate an innate immune response.

Use of Trichomonas vaginalis clinical isolates to evaluate correlation of gene expression and metronidazole resistance.

We investigated whether variations in gene expression of enzymes associated with anaerobic resistance of laboratory-derived strains of Trichomonas vaginalis could be detected in a group of 28 clinical isolates with variations in metronidazole sensitivity. We compared isolates by real-time PCR because this method allows for highly sensitive quantification of mRNA and for evaluation of several genes simultaneously. We found that PFOR gene A mRNA levels were highly correlated with PFOR gene B levels, as well as the D subunit of malic enzyme and ferrodoxin. Ferrodoxin mRNA expression was also significantly correlated with that of malic enzyme and hydrogenase. However, when we evaluated relationships between these enzymes and resistance to metronidazole, we found no significant correlations between aerobic or anaerobic in vitro sensitivity to drug and mRNA levels of any of the enzymes tested. Similarly, using a Student's t-test, no significant differences in enzyme mRNA levels were observed between isolates separated by metronidazole resistance or susceptibility. The lack of correlation between gene expression and resistance or susceptibility could be the result of differences in expression at the protein level or because other biochemical pathways or genes are involved in the resistance observed in clinical settings.

Mucosal cytokine and antigen-specific responses to Cryptosporidium parvum in IL-12p40 KO mice.

Studies of cellular immune responses to Cryptosporidium parvum have been limited in part by lack of suitable animal models. IL-12p40(-/-)mice are susceptible to initial infection with C. parvum but recover within 2 weeks, rendering the animals resistant to reinfection. Because the host responses that determine duration and severity of primary infection are not yet understood, we studied the cellular immune response to primary infection with C. parvum in IL-12p40(-/-)mice and also explored possible mechanisms for this response. Female IL-12p40(-/-)mice were inoculated with 10,000 oocysts. Uninfected age-matched mice served as controls. At different time intervals following exposure to oocysts, mice were sacrificed and their intestine, spleen, and mesenteric lymph node tissues were harvested. Cellular immune responses to C. parvum were characterized. Infection of IL-12p40(-/-)mice induced changes in the gene expression of the cytokines IFN-gamma, IL-4, IL-15, IL-18, TNF-alpha and TGF-beta during primary infection. There was also a significant increase in total numbers of lymphocytes and CD19/CD62L-expressing cells in mesenteric lymph nodes. These MLN cells exhibited increased antigen-specific proliferation and cytokine production (IL-6 and IFN-gamma) levels when stimulated in vitro. These observations delineate the cellular immune responses during acute C. parvum infection of the IL-12p40(-/-)mouse model.

Identification of two putative ATP-cassette genes in Encephalitozoon intestinalis.

Currently existing chemotherapeutic compounds are limited and few are effective for treating microsporidiosis. It is possible that resistance of Encephalitozoon to some drugs occurs by efflux mechanisms similar to those previously described for mammalian tumour cells, bacteria or protozoal parasites such as Plasmodium, Leishmania and Entamoeba histolytica. The data in the present study suggest that Encephalitozoon intestinalis contains at least one multidrug resistance gene. We report here two complete sequences EiABC1 and EiABC2, encoding different ATP-binding cassette genes from E. intestinalis, including a P-gp.

Exogenous interleukin-12 (IL-12) exacerbates Cryptosporidium parvum infection in gamma interferon knockout mice.

Experimental infection of BALB/c- or C57BL/6-gamma-interferon-knockout (GKO) mice with Cryptosporidium parvum results in infection in both strains with different outcomes of disease. The BALB/c-GKO mice recover from infection, whereas the C57BL/6-GKO mice succumb to infection in less than 2 weeks. Differences in cytokine mRNA expression suggested that recovery may involve other cytokines. To determine whether the addition of either a Th1 or Th2 cytokine could alter the outcome of infection, we treated GKO mice with either recombinant (r)IL-4 or rIL-12 1 day before infection (DBI) or daily. No effect on the oocyst shedding patterns in either strain nor an increase in survival of the C57BL/6-GKO mice was observed in the rIL-4-treated mice. Whereas one dose of 0.5 microg rIL-12 given 1 DBI had no effect on oocyst shedding, we found that daily doses of rIL-12 administered intraperitoneally exacerbated C. parvum infection in both animal models. Administration of rIL-12 shortened the survival time in the C57BL/6-GKO mice and prevented BALB/c-GKO mice from recovering from infection. Specific proliferation of T cells to cryptosporidial antigen and Th1 and Th2 mRNA cytokine expression was markedly decreased in rIL-12-treated mice. Nitric oxide (NO) may have played a minor role in the decreased proliferation observed since levels of NO present in the splenocyte cultures from rIL-12-treated mice in response to parasite antigen stimulation were higher than those observed in controls. Thus, we propose that resistance to and recovery from C. parvum infections involves a fine balance in the amount and timing of Th1 and Th2 cytokines.

Human T and B cell immunoreactivity to a recombinant 23-kDa Cryptosporidium parvum antigen.

Cryptosporidial infection in humans results in parasite-specific IgG, IgM, and IgA antibody responses, but little is known of the cell-mediated immune responses to cryptosporidial antigens. In a convenience sample of 35 Haitian residents, there was a high level of cryptosporidial exposure (>90%) as determined by immunoblot reactivity of serum against cryptosporidial antigens. An attempt was made to determine if there was a relationship between antibody and T cell-mediated responses to recombinant Cp23 antigen and how this correlated with reactivity to crude sporozoite antigen preparations (SAg). T cell reactivity was greater against SAg (57%) than to Cp23 (34.3%) as measured by [3H]thymidine incorporation. Proliferative responses to Cp23 were significantly correlated with SAg responses. By enzyme-linked immunosorbent assay, most persons had IgG responses to both SAg (91.4%) and to recombinant Cp23 (88.5%). Antibody responses were greater among persons who exhibited T cell responses to SAg and Cp23. This study demonstrates that recombinant Cp23 antigen could be a useful antigen for detection of both antibody and cell-mediated responses in epidemiologic studies.

A 23-kDa recombinant antigen of Cryptosporidium parvum induces a cellular immune response on in vitro stimulated spleen and mesenteric lymph node cells from infected mice.

In the present study, we focused on a 23-kDa antigen, Cp23, which has been shown to be a major target of humoral immune responses in Cryptosporidium parvum infections and is present in both the sporozoite and merozoite stages. Recombinant Cp23 antigen was shown to stimulate a specific proliferative response by splenocytes and mesenteric lymph node cells from infected interferon gamma knockout BALB/c mice. Cp23 stimulation also induced TNF-alpha, IL-2, and IL-5 mRNA production by spleen cells from infected animals. In contrast, IL-12 mRNA was decreased by Cp23 stimulation compared with unstimulated splenocytes. These data suggest that, as with humoral responses, Cp23 is an important target of cellular immune responses in experimental C. parvum infections. The potential role of this antigen in conferring protective immunity is also discussed.

Cytokine expression and specific lymphocyte proliferation in two strains of Cryptosporidium parvum-infected gamma-interferon knockout mice.

Differences in the immune response between 2 strains of interferon-gamma knockout mice (BALB/c-GKO and C57BL/6-GKO) infected with Cryptosporidium parvum were examined because the course of infection among these 2 strains is markedly different. Infection of the BALB/c-GKO with C. parvum (2 X 10(6) oocysts/mouse) resulted in slight weight loss, oocyst shedding, and recovery from infection by 2 wk postinfection (PI). Infection with 100 oocysts in the C57BL/6-GKO mice resulted in significant weight loss, oocyst shedding, and death by day 10 PI. Splenocytes from infected mice were able to proliferate in a dose-dependent manner to soluble C. parvum-sporozoite antigen (SAg). In vitro stimulation with SAg resulted in an increase in interleukin (IL)-2, IL-4, IL-5, and tumor necrosis factor-alpha mRNA cytokine expression from splenocytes of infected BALB/cGKO mice. In contrast, only IL-5 mRNA expression was increased in the splenocytes from C. parvum-infected C57BL/6-GKO mice. Phenotypic analysis indicated no significant differences in the splenic cell populations. Previous studies indicated that susceptibility to C. parvum is dependent on CD4+ T cells and interferon-gamma production. The present study indicates that although both of these strains of knockout mice become infected with C. parvum, resolution of infection may be in part dependent on the expression of Th2 cytokines.

Cryptosporidium andersoni n. sp. (Apicomplexa: Cryptosporiidae) from cattle, Bos taurus.

A new species of Cryptosporidium is described from the feces of domestic cattle, Bos taurus. Oocysts are structurally similar to those of Cryptosporidium muris described from mice but are larger than those of Cryptosporidium parvum. Oocysts of the new species are ellipsoidal, lack sporocysts, and measure 7.4 x 5.5 microm (range, 6.0-8.1 by 5.0-6.5 microm). The length to width ratio is 1.35 (range, 1.07-1.50). The colorless oocyst wall is < 1 microm thick, lacks a micropyle, and possesses a longitudinal suture at one pole. A polar granule is absent, whereas an oocyst residuum is present. Oocysts were passed fully sporulated and are not infectious to outbred, inbred immunocompetent or immunodeficient mice, chickens or goats. Recent molecular analyses of the rDNA 18S and ITS1 regions and heat-shock protein 70 (HSP-70) genes demonstrate this species to be distinct from C. muris infecting rodents. Based on transmission studies and molecular data, we consider the large form of Cryptosporidium infecting the abomasum of cattle to be a new species and have proposed the name Cryptosporidium andersoni n. sp. for this parasite.

Lipoprotein lipase, a key role in atherosclerosis?

Lipoprotein lipase (LPL) plays a central role in lipid metabolism and transport by catalysing the hydrolysis of triacylglycerol-rich lipoproteins. The importance of LPL expressed by the adipose tissue and muscles in the provision of non-esterified fatty acids and 2-monoacylglycerol for tissue utilisation is well established. However, recent studies on LPL expressed by cells of the vascular wall, particularly macrophages, have identified additional actions of the enzyme that contribute to the promotion of foam cell formation and atherosclerosis. This review deals with the role of LPL in atherosclerosis, and its regulation by mediators that are known to be present in the lesion.

Characterization of an alpha-tubulin gene of Cryptosporidium parvum.

A gene encoding an alpha-tubulin of Cryptosporidium parvum was isolated and characterized. It had no introns, and encoded a 441-amino acid protein whose predicted ORF represented a typical alpha-tubulin protein with a MW of 50.5 kDa. This tubulin had an amino acid sequence similarity with Apicomplexa Plasmodium falciparum and Toxoplasma gondii higher than 88% and shared a number of conserved motifs.

Susceptibility differences to Cryptosporidium parvum infection in two strains of gamma interferon knockout mice.

Differences in susceptibility to cryptosporidial infections were investigated between 2 strains of gamma interferon knockout (GKO) mice. Male C57BL/6J-Ifg and BALB/c-Ifg (GKO) mice, ages 8-10 wk, were inoculated with infectious oocysts at various doses. C57BL/6J-Ifg mice developed overwhelming infections and died 9-12 days after infection. Low inoculum doses (1 x 10(3)) did not increase the survival time significantly. The infection intensity in C57BL/6J-Ifg mice inoculated with 1 x 10(5) oocysts/mouse increased markedly on day 4 postinfection (PI) and continued to increase significantly over the next 6-7 days. Most of the C57BL/6J-Ifg mice exceeded 15% weight loss and died by day 10 PI. In contrast, BALB/c-Ifg mice developed moderate infections from which they recovered. The average parasite load in the BALB/c-Ifg mice was 100 times lower than in C57BL/6J-Ifg mice. Mice survived until termination of the experiment (39 days) even when 1 x 10(6) oocysts per mouse were used for inoculation. BALB/c-Ifg mice did not exhibit significant weight loss (or difference in stool consistency). These 2 mouse strains make excellent models for studying differences in recovering and nonrecovering immune mechanisms.

Synthesis and evaluation of dinitroanilines for treatment of cryptosporidiosis.

The efficacy of a series of dinitroaniline herbicide derivatives for the treatment of Cryptosporidium parvum infections has been studied. The lead compounds oryzalin (compound 1) and trifluralin (compound 2) have low water solubility (<3 ppm) which was alleged to be a major contributor to their poor pharmacokinetic availability. Derivatives of compounds 1 and 2 were synthesized. In these derivatives the functionality at the C-1 amine position or the C-4 position was substituted with groups with various hydrophilicities to determine if a direct relation existed between water solubility and overall activity. The chlorinated precursors of these derivatives were also examined and were found to be less active in the C. parvum assays, a result in direct contrast to earlier work with Leishmania. Enhanced water solubility alone did not overcome the drug availability problem; however, several candidates with similar activities but with toxicities lower than those of the lead compounds were produced.

In-vitro activities of paromomycin and lasalocid evaluated in combination against Cryptosporidium parvum.

Using a chemiluminescence immunoassay, paromomycin and lasalocid were shown to inhibit Cryptosporidium parvum growth in Madin-Darby canine kidney cells in a concentration-dependent manner. The median effective concentrations (EC50s) for paromomycin and lasalocid were 1184 mg/L and 0.4 mg/L, respectively. Neither drug was cytotoxic to host cells at concentrations up to five times their EC50s. Drug combination studies were conducted and the resulting data were analysed by the median-effect principle and combination index method. Statistically significant synergy was observed when combinations of paromomycin and lasalocid were used at ratios of 5000:1 and 2500:1. A possible mechanism for synergy is discussed.

Characterization of experimental Cryptosporidium parvum infection in IFN-gamma knockout mice.

Severe cryptosporidial infections were produced in gamma interferon (IFN-gamma) knockout mice. Mean oocyst shedding increased from 332 to 30,717 oocysts/100 microliters of faecal suspension between day 4 and 9 after administration of 1 x 10(5) oocysts/mouse. No significant differences in oocyst shedding were observed in mice after being inoculated with 1 x 10(5), 1 x 10(4) or 1 x 10(3) oocysts/mouse (P > 0.05). Infected mouse weights decreased an average 3-4 g before death or euthanization. Histological studies revealed heavy parasite colonization in small intestinal epithelium (approximately 250 organisms/high-power field at x 400). Mesenteric lymph nodes in infected mice were markedly enlarged compared to controls (P < 0.05). Both CD4+ and CD8+ T cell populations increased in spleens of infected mice while the B cell population increased in mesenteric lymph nodes from infected mice. No significant proliferation was observed when pooled lymphocytes from infected mice were exposed to C. parvum antigens in vitro. Addition of recombinant mouse IFN-gamma did not restore antigen responsiveness. While lymphoproliferative responses to specific antigen were not significant in the short period following infection, this mouse model provides unique features to study the characteristics of acute infection and the immune response against C. parvum.

Isolation of the gene coding for elongation factor-1alpha in Cryptosporidium parvum.

A gene encoding for Cryptosporidium parvum (C. parvum) elongation factor 1alpha (EF-1alpha) was isolated and sequenced from a cDNA expression library. The recombinant protein cross-reacted with a monoclonal antibody that was raised to a sporozoite cell surface antigen. The gene encoded a 435 amino acid protein with a predicted molecular weight of 48.1 kDa. The predicted C. parvum EF-1alpha protein sequence showed extensive homology with the EF-1alpha proteins of other eukaryotic organisms and included three conserved sequence motifs implicated in GTP binding.