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Funkhouser-Jones et al. recently identified gut metabolites that affected Cryptosporidium growth. A key focus, indole, was shown to inhibit the parasite in vivo and in vitro by decreasing the host mitochondria function and the membrane potential of parasite mitosomes. These findings help clarify the role microflora and metabolites play in host resistance. [ABSTRACT FROM AUTHOR]

Abstract: Cryptosporidium parvum is a protozoan parasite that infects the epithelial cells of the small intestine causing diarrheal illness in humans. While T cells are known to be important in resistance and recovery from infection, little has been characterized as to the phenotypic expression of surface effector and memory markers after infection. We used an acute model of infection (C57BL/6 interleukin-12p40), which develops long-standing resistance to re-infection, to characterize expression of different effector and memory cells. Using flow cytometry, we found that heterogeneous populations were generated after infection, consisting of both CD62Lhigh central memory T cells (TCM) and CD62Llow effector memory T cells (TEM) that were competent to produce the Th type 1 effector cytokine, IFN-γ. Both CD4+ and CD8+ TCM and TEM populations persisted in the absence of infection (up to 60 days post-infection). Additionally, transfer of either CD62LlowCD4+ TEM or CD62LhighCD4+ TCM into naive recipients resulted in a protective response. Taken together, these studies show that distinct subsets of effector and memory CD4+ T cells develop after infection with C. parvum, and mediate protective immunity to re-challenge. [Copyright &y& Elsevier]

Flavonoids, polyphenolic compounds found in plants, have demonstrated activity against several parasites and can augment the efficacy of other drugs by either increasing the uptake or decreasing the efflux of these drugs. We evaluated 11 of these compounds alone or in combination in order to test the hypothesis that flavonoids are effective against Cryptosporidium parvum and Encephalitozoon intestinalis. Using in vitro cell culture assays, HCT-8 cells or E6 cells were infected with C. parvum and E. intestinalis, respectively, and treated with compounds at doses ranging from 1 to 200 μM. We found that six compounds were active against C. parvum. Naringenin and genistein had the greatest activities with EC50 of 15 and 25 μM, respectively. Two compounds, quercetin and apigenin, had activity against E. intestinalis at EC50 of 15 and 50 μM, respectively. The EC50 of trifluralin, a dinitroaniline compound, was decreased significantly when combined with genistein in an in vitro assay, suggesting that compounds may be used alone on in combination with other moderately active drugs to increase efficacy. In addition, induction of apoptosis by these compounds was studied but not observed to be a significant mechanism of action. [ABSTRACT FROM AUTHOR]

Examines the efficacy of glycoconjugated dinitroanilines against Cryptosporidium parvum. Activity of dinitroaniline compound against C. parvum; Water solubility and low vapor pressure of dinitroaniline; Evaluation of glycoconjugates.

The article presents a summary of research presentations on Cryptosporidium at the International Workshops on Opportunistic infections in Lisbon, Portugal from June 20 to 24, 2006. Over 17 papers were presented which focused on chemotherapy, host/parasite interactions, epidemiology, genotyping, and molecular biology.

Summarizes the proceedings of the papers from papers on Cryptosporidium research presented at the VIII International Workshop on Opportunistic Protists in Ohio. Presentation of paper on therapeutic agents with promising anticryptosporidial activities; Epidemiology and environmental methods discussed; Discussion on scientific fronts during the meeting.

The protozoan protein kinase calcium-dependent protein kinase 1 (CDPK1) has emerged as a potential therapeutic target for the treatment of cryptosporidiosis. A focused screen of known kinase inhibitors identified a pyridopyrimidinone as a new chemotype of Cryptosporidium parvum (Cp) CDPK1 inhibitors. Structural comparison of Cp CDPK1 to two representative human kinases, RIPK2 and Src, revealed differences in the positioning of the αC-helix that was used in the design of a potent pyridopyrimidinone-based Cp CDPK1 inhibitor 7 (a.k.a. UH15–16, IC 50 = 10 nM), which blocked the growth of three C. parvum strains (EC 50 = 12–40 nM) as well as C. hominis (EC 50 = 85 nM) in HCT-8 host cells. Pharmacokinetic and tissue distribution analyses indicated that 7 had low systemic exposure after oral administration, but high gastrointestinal concentration, as well as good Caco-2 cell permeability. Finally, 7 demonstrated partial efficacy in an IL-12 knock-out mouse model of acute cryptosporidiosis. [Display omitted] • Identified pyridopyrimidinones as a new class of Cp CDPK1 inhibitors. • Optimized compound 7 demonstrated potent CDPK1 inhibition and improved selectivity. • Molecular modeling provided new insights for CDPK1 inhibitor design. • Compound 7 blocked C. parvum and C. hominis growth inhibition in HCT-8 host cells. • Compound 7 was partially efficacious in an acute mouse model of cryptosporidiosis. [ABSTRACT FROM AUTHOR]

Highlights: [•] Depletion of DCs in vivo increases susceptibility to Cryptosporidium parvum infection. [•] Adoptive transfer of stimulated DCs reduces C. parvum infection levels. [•] Reduction in infection correlates to an increase in IFN-γ in T cell. [•] DCs pulsed with sporozoites and co-cultured with CD4+ and CD8+ T cells produced higher IFN-γ levels. [Copyright &y& Elsevier]

Abstract: In a previous study, we showed that the Cryptosporidium parvum ATP half-transporter CpABC4 (cgd1_1350) transcript was up-regulated in response to drug treatment with paromomycin and cyclosporine A in an in vitro infection model. CpABC4 may be directly or indirectly involved in the metabolic interactions between host and parasite in response to drug treatment and/or be involved in the intrinsic resistance to chemotherapy. In order to characterize the catalytic site of this transporter, an extended region of the nucleotide-binding domain of CpABC4 (H6-1350NBD) was expressed and purified as an N-terminal hexahistidine-tagged protein in E. coli. The presence of a single tryptophan residue enabled the intrinsic fluorescence to be monitored in response to binding of different compounds. A dose-dependent quenching of the domain''s intrinsic fluorescence was observed with its natural substrate, ATP and the fluorescent analogue TNP-ATP. A similar effect was observed with progesterone as well as the flavonoids quercetin and silibinin, previously shown to inhibit parasite development in a cell-based assay. The purified domain also exhibited ATPase activity in the nanomolar range, which further confirmed correct folding and activity of the recombinant domain. The H6-1350NBD serves as a tool to test and design stereospecific inhibitors of the catalytic site, as well as other compounds that bind elsewhere in the domain that may indirectly interact with the catalytic site of the NBD of the CpABC4 transporter. [Copyright &y& Elsevier]

Abstract: The contribution of cytokines IL-12, IL-18, IL-23, and IFN-γ, and Stat1 signaling molecules involved in Th1 responses associated with host resistance to Cryptosporidium parvum infection was investigated in adult IL-12p40−/−mice. Host resistance to C. parvum infection was assessed in different mouse strains lacking IL-12, IL-18, and IL-23 genes. We found that as in IL-12p40−/− mice (which lack both IL-12 and IL-23), IL-12p35−/− mice (which lack IL-12) and IL-18 deficient mice were also susceptible to infection with C. parvum. Varied levels of resistance were observed when mice were treated with cytokines like IL-18, IL-23 and IFN-γ. Mice treated with IL-12, as expected, were completely resistant to infection until day 5 post infection, and had significantly decreased (85%) parasite loads at peak infection (day 7), whereas rIL-23 had a lesser effect, decreasing parasite load by approximately 45%. Interestingly, IL-18 appears to play a significant role in initial immune response, even in the absence of IL-12, since treatment with IL-18 in IL-12p40−/− knockout mice decreased parasite load by approximately 70%. In addition, the establishment of C. parvum infection in mice lacking the Stat1 gene demonstrated the involvement of this pathway in resolution of infection. These observations indicate a strong requirement for Th1 response in the development of immunity to C. parvum in the adult IL-12p40−/− mice, information that will be essential to further investigate the immune responses during infections and in the development of potential vaccine candidates. [Copyright &y& Elsevier]

Compares in vitro cell culture and a mouse assay for measuring ineffectivity of genotype 2 isolates of Cryptosporidium (C.) parvum. Detection of oocyst doses; Properties of C. parvum isolates; Exposure of oocyst to disinfectants.

Investigates the establishment of secondary response and long-term immunity in the interleukin-12 knockout model of Cryptosporidium parvum infection. Role of interferon in the development of immunity; Protection from re-infection with C. parvum; Reduction in some metabolic antigens in Eimeria tenella.

Cryptosporidium spp. are opportunistic protozoan parasites that infect epithelial cells in the intestinal tract and cause a flu-like diarrheal illness. Innate immunity is key to limiting the expansion of parasitic stages early in infection. One mechanism in which it does this is through the generation of early cytokines, such as IL-18. The processing and secretion of mature IL-18 (and IL-1β) is mediated by caspase-1 which is activated within an inflammasome following the engagement of inflammasome-initiating sensors. We examined how the absence of caspase-1 and caspase-11, the adapter protein Asc, and other inflammasome components affects susceptibility to cryptosporidial infection by these and other key cytokines in the gut. We found that Casp-11 −/− Casp-1 −/− knockout mice have increased susceptibility to Cryptosporidium parvum infection as demonstrated by the 35-fold higher oocyst production (at peak infection) compared to wild-type mice. Susceptibility correlated with a lack of IL-18 in caspase-1 and caspase1/11 knockout mice, whereas IL-18 is significantly elevated in wildtype mice. IL-1β was not generated in any significant amount following infection nor was any increased susceptibility observed in IL-1β knockout mice. We also show that the adapter protein Asc is important to susceptibility, and that the caspase-1 canonical inflammasome signaling pathway is the dominant pathway in C. parvum resistance. [ABSTRACT FROM AUTHOR]

Background: Cryptosporidiosis affects all human populations, but can be much more severe or life-threatening in children and individuals with weak or weakened immune systems. However, current options to treat cryptosporidiosis are limited.Methods: An in vitro phenotypic screening assay was employed to screen 1200 existing drugs for their anticryptosporidial activity and to determine the inhibitory kinetics of top hits. Selected top hits were further evaluated in mice. The action of the lead compound vorinostat on the parasite histone deacetylase (HDAC) was biochemically validated.Results: Fifteen compounds exhibited anticryptosporidial activity at nanomolar level in vitro. Among them, the histone deacetylase (HDAC) inhibitor vorinostat retained outstanding efficacy in vitro (half maximal effective concentration, EC50 = 203 nM) and in an interleukin 12 knockout mouse model (50% inhibition dose = 7.5 mg/kg). Vorinostat was effective on various parasite developmental stages and could irreversibly kill the parasite. Vorinostat was highly effective against the parasite native HDAC enzymes (half maximal inhibitory concentration, IC50 = 90.0 nM) and a recombinant Cryptosporidium parvum HDAC (the inhibitor constant, Ki = 123.0 nM).Conclusions: These findings suggest the potential for repurposing of vorinostat to treat cryptosporidiosis, and imply that the parasite HDAC can be explored for developing more selective anticryptosporidial therapeutics. [ABSTRACT FROM AUTHOR]

[Display omitted] BMS906024, a γ-secretase inhibitor that blocks Notch signaling, was previously shown to inhibit Cryptosporidium parvum growth in vitro. A structure–activity relationship (SAR) analysis of BMS906024 reported herein demonstrates the importance of the stereochemistry of the C-3 benzodiazepine and the succinyl β-substituent. However, concomitant removal of the succinyl α-substituent and switching the primary amide with secondary amides was tolerated. For example, 32 (SH287) inhibited C. parvum growth in HCT-8 host cells with an EC 50 = 6.4 nM and an EC 90 = 16 nM; however, blocking C. parvum growth with BMS906024 derivatives was correlative with inhibition of Notch signaling, highlighting that additional SAR analysis will be needed to separate these two activities. [ABSTRACT FROM AUTHOR]

Auranofin is an FDA-approved gold-containing compound used for the treatment of rheumatoid arthritis. Recent reports of antimicrobial activity against protozoa and bacteria indicates auranofin targets the reductive enzyme thioredoxin reductase (TrxR). We evaluated auranofin as well as 5 auranofin analogs containing N-heterocyclic carbenes (instead of the triethylphosphane present in auranofin) and 5 gold-carbene controls for their ability to inhibit or kill Helicobacter pylori in vitro. Auranofin completely inhibited bacterial growth at 1.2 µM. Purified H. pylori TrxR was inhibited by auranofin in a cell-free assay (IC50 ~88 nM). The most active gold(I)-N-heterocyclic carbene compounds exhibited MICs comparable to auranofin against H. pylori (2 µM) while also exhibiting lower toxicities for human embryonic kidney cells (HEK-293T cells). Median toxic concentrations (TC50) were 13-20 fold higher compared to auranofin indicating they were less cytotoxic. The N-heterocyclic carbene analogs maybe well tolerated, but further evaluation is needed in vivo. Finally, auranofin was synergistic with the antibiotic amoxicillin suggesting that targeting both the reductive enzyme TrxR and cell wall synthesis may be effective against H. pylori infections. [ABSTRACT FROM AUTHOR]

IL-18 is known to play a key role limiting Cryptosporidium parvum infection. In this study, we show that IL-18 depletion in SCID mice significantly exacerbates C. parvum infection, whereas, treatment with recombinant IL-18 (r IL-18), significantly decreases the parasite load, as compared to controls. Increases in serum IFN-γ levels as well as the up-regulation of the antimicrobial peptides, cathelicidin antimicrobial peptide and beta defensin 3 ( Defb3) were observed in the intestinal mucosa of mice treated with r IL-18. In addition, C. parvum infection significantly increased m RNA expression levels (> 50 fold) of the alpha defensins, Defa3 and 5, respectively. Interestingly, we also found a decrease in m RNA expression of IL-33 (a recently identified cytokine in the same family as IL-18) in the small intestinal tissue from mice treated with r IL-18. In comparison, the respective genes were induced by IL-18 depletion. Our findings suggest that IL-18 can mediate its protective effects via different routes such as IFN-γ induction or by directly stimulating intestinal epithelial cells to increase antimicrobial activity. [ABSTRACT FROM AUTHOR]

Background. Cryptosporidium is emerging as 1 of the 4 leading diarrheal pathogens in children in developing countries. Its infections in patients with AIDS can be fatal, whereas fully effective treatments are unavailable. The major goal of this study is to explore parasite fatty acyl-coenzyme A synthetase (ACS) as a novel drug target.Methods. A colorimetric assay was developed to evaluate biochemical features and inhibitory kinetics of Cryptosporidium parvum ACSs using recombinant proteins. Anticryptosporidial efficacies of the ACS inhibitor triacsin C were evaluated both in vitro and in vivo.Results. Cryptosporidium ACSs displayed substrate preference toward long-chain fatty acids. The activity of parasite ACSs could be specifically inhibited by triacsin C with the inhibition constant Ki in the nanomolar range. Triacsin C was highly effective against C. parvum growth in vitro (median inhibitory concentration, 136 nmol/L). Most importantly, triacsin C effectively reduced parasite oocyst production up to 88.1% with no apparent toxicity when administered to Cryptosporidium-infected interleukin 12 knockout mice at 8–15 mg/kg/d for 1 week.Conclusions. The findings of this study not only validated Cryptosporidium ACS (and related acyl-[acyl-carrier-protein]-ligases) as pharmacological targets but also indicate that triacsin C and analogues can be explored as potential new therapeutics against the virtually untreatable cryptosporidial infection in immunocompromised patients. [ABSTRACT FROM AUTHOR]

Highlights: [•] Sera from Cryptosporidium parvum-infected individuals bound to multivalent Tn antigen glycopeptides. [•] Glycopeptides of C. parvum antigens Cp17 and Cp40 were synthesised with different Tn valency. [•] Varying lengths of repeating serine residues (Cp40) were synthesised. [•] The site of glycosylation had a pronounced effect on reactivity. [•] Sera from individuals infected with Toxoplasma or Plasmodium recognised C. parvum Tn glycopeptides. [ABSTRACT FROM AUTHOR]

Abstract: Cryptosporidium parvum (Cp) is a potential biowarfare agent and major cause of diarrhea and malnutrition. This protozoan parasite relies on inosine 5′-monophosphate dehydrogenase (IMPDH) for the production of guanine nucleotides. A CpIMPDH-selective N-aryl-3,4-dihydro-3-methyl-4-oxo-1-phthalazineacetamide inhibitor was previously identified in a high throughput screening campaign. Herein we report a structure–activity relationship study for the phthalazinone-based series that resulted in the discovery of benzofuranamide analogs that exhibit low nanomolar inhibition of CpIMPDH. In addition, the antiparasitic activity of select analogs in a Toxoplasma gondii model of C. parvum infection is also presented. [Copyright &y& Elsevier]

Abstract: The Cryptosporidium parvum acidic ribosomal protein P2 (CpP2) is an important immunodominant marker in C. parvum infection. In this study, the CpP2 antigen was evaluated as a vaccine candidate using a DNA vaccine model in adult C57BL/6 IL-12 knockout (KO) mice, which are susceptible to C. parvum infection. Our data show that subcutaneous immunization in the ear with DNA encoding CpP2 (CpP2-DNA) cloned into the pUMVC4b vector induced a significant anti-CpP2 IgG antibody response that was predominantly of the IgG1 isotype. Compared to control KO mice immunized with plasmid alone, CpP2-immunized mice demonstrated specific in vitro spleen cell proliferation as well as enhanced IFN-γ production to recombinant CpP2. Further, parasite loads in CpP2 DNA-immunized mice were compared to control mice challenged with C. parvum oocysts. Although a trend in reduction of infection was observed in the CpP2 DNA-immunized mice, differences between groups were not statistically significant. These results suggest that a DNA vaccine encoding the C. parvum P2 antigen is able to provide an effective means of eliciting humoral and cellular responses and has the potential to generate protective immunity against C. parvum infection but may require using alternative vectors or adjuvant to generate a more potent and balanced response. [Copyright &y& Elsevier]

The article highlights the 9th International Workshops on Opportunistic Protists which focus on AIDS-related protistan pathogens. The author offers information about the keynote speakers, organizers, and the topic discussed during the workshops. They have also presented the themes and overviews of every workshop.

In the present study, the gene expression of three multidrug resistance (MDR) and resistance-associated protein (MRP) transport proteins or efflux pumps was characterized and the phenotypic evidence for such pumps was demonstrated in cultured Madin–Darby canine kidney (MDCK) cells. A gradient for the fluorescent probe calcein was established between parasite and host cell suggestive of a parasite extrusion pump at the parasite–host interface. This gradient was decreased in a glucose-free medium containing 2-deoxyglucose or 3-O-methylglucose, by probenecid, and by the isoflavonoid, narigenin, suggesting that the calcein extrusion was energy-dependent and involved an MRP-like pump. While neither MDR or MRP inhibiters significantly affected transcript levels of any of the ABC transporters, transcript levels of the Cryptosporidium parvum ABC protein (CpABC1), an MRP transporter, were consistently expressed 4 logs higher than either CpABC3 or CpABC2, suggesting a prominent role in the intracellular stages of the parasite. [Copyright &y& Elsevier]