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In a previous study, we observed an increase in the severity of cryptosporidial infection corresponding to decreased levels of short-chain fatty acids (SCFAs). Therefore, we decided to examine the effect of SCFAs on Cryptosporidium growth in human ileocecal adenocarcinoma (HTC-8) cells. HTC-8 cells were infected with 1 × 105 C. parvum oocysts. After 48 h of incubation with selected SCFAs, cells were fixed and labeled with monoclonal antibody directed to all intracellular stages, and the number of parasites was quantitated using a fluorescent microscope. Acetate, butyrate, propionate and valproate significantly inhibited growth, with an EC50 between 4 and 10 mM. Additionally, when combined, butyrate, acetate and propionate showed increased efficacy. Butyrate also inhibited growth when incubated with sporozoites prior to infection of host cell monolayers. In addition, we looked at possible mechanisms of action of inhibition. A combination of C. parvum infection and butyrate treatment led to increases in apoptosis and certain inflammatory cytokines. We conclude that acetate, propionate and butyrate have direct inhibitory activities in host cells against C. parvum, and butyrate can also affect sporozoite infectivity directly. While not preventing infection, SCFAs may help in keeping the infection low or in check. [ABSTRACT FROM AUTHOR]

Research has been conducted on the infectivity of Cryptosporidium parvum. The relationship between cell culture-based and animal-based infectivity of C. parvum has been investigated in determining whether different methods vary in their sensitivity to dose-response curves and infection and the results are reported.

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]

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]

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]

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.

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]

Cryptosporidium spp. are opportunistic protozoan parasites that infect epithelial cells of the small intestine, causing diarrheal illness in humans. Differences in severity may be due to the immunological status of the host, malnutrition or prior exposure but may also be due to differences in the host gut flora. We examined changes in bacterial flora following antibiotic treatment to determine how cryptosporidial infections and gut integrity were affected by alterations in the microbiome. DNA was extracted from fecal and intestinal samples during peak infection. V4 region amplicons were generated and sequenced using 16sRNA on an Illumina MiSeq. Species evenness and richness were estimated using the Shannon diversity index. There was a significant decrease in anaerobes and overgrowth of Enterobacteriaceae in mice treated with cloxacillin. We also examined levels of short-chain fatty acids in fecal samples. There was a significant decrease in acetate, propionate, and butyrate in these same mice. Concurrent with the shift in bacterial infection was a significant increase in severity of cryptosporidial infection and increase in gut permeability. Treatment with other antibiotics significantly altered the microbiome but did not change the infection, suggesting that specific alterations in the host microbiome allow for more favorable growth of the parasite. [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.

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.

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.