miR-181d targets BCL2 to regulate HCT-8 cell apoptosis and parasite burden in response to Cryptosporidium parvum infection via the intrinsic apoptosis pathway.

Cryptosporidium parvum is an important zoonotic pathogen that is studied worldwide. MicroRNAs (miRNAs) act as post-transcriptional regulators and may play a key role in modulating host epithelial responses following Cryptosporidium infection. Our previous study has shown that C. parvum downregulates the expression of miR-181d through the p50-dependent TLRs/NF-κB pathway. However, the mechanism by which miR-181d regulates host cells in response to C. parvum infection remains unclear. The present study found that miR-181d downregulation inhibited cell apoptosis and increased parasite burden in HCT-8 cells after C. parvum infection. Bioinformatics analysis and luciferase reporter assays have shown that BCL2 was a target gene for miR-181d. Moreover, BCL2 overexpression and miR-181d downregulation had similar results. To further investigate the mechanism by which miR-181d regulated HCT-8 cell apoptosis during C. parvum infection, the expression of molecules involved in the intrinsic apoptosis pathway was detected. Bax, caspase-9, and caspase-3 expression was decreased at 4, 8, 12, and 24 hpi and upregulated at 36 and 48 hpi. Interfering with the expression of miR-181d or BCL2 significantly affected the expression of molecules in the intrinsic apoptosis pathway. These data indicated that miR-181d targeted BCL2 to regulate HCT-8 cell apoptosis and parasite burden in response to C. parvum infection via the intrinsic apoptosis pathway. These results allowed us to further understand the regulatory mechanisms of host miRNAs during Cryptosporidium infection, and provided a theoretical foundation for the design and development of anti-cryptosporidiosis drugs.

LncRNA XR_001779380 Primes Epithelial Cells for IFN-γ-Mediated Gene Transcription and Facilitates Age-Dependent Intestinal Antimicrobial Defense.

Interferon (IFN) signaling is key to mucosal immunity in the gastrointestinal tract, but cellular regulatory elements that determine interferon gamma (IFN-γ)-mediated antimicrobial defense in intestinal epithelial cells are not fully understood. We report here that a long noncoding RNA (lncRNA), GenBank accession no. XR_001779380, was increased in abundance in murine intestinal epithelial cells following infection by Cryptosporidium, an important opportunistic pathogen in AIDS patients and a common cause of diarrhea in young children. Expression of XR_001779380 in infected intestinal epithelial cells was triggered by TLR4/NF-κB/Cdc42 signaling and epithelial-specific transcription factor Elf3. XR_001779380 primed epithelial cells for IFN-γ-mediated gene transcription through facilitating Stat1/Swi/Snf-associated chromatin remodeling. Interactions between XR_001779380 and Prdm1, which is expressed in neonatal but not adult intestinal epithelium, attenuated Stat1/Swi/Snf-associated chromatin remodeling induced by IFN-γ, contributing to suppression of IFN-γ-mediated epithelial defense in neonatal intestine. Our data demonstrate that XR_001779380 is an important regulator in IFN-γ-mediated gene transcription and age-associated intestinal epithelial antimicrobial defense. IMPORTANCE Epithelial cells along the mucosal surface provide the front line of defense against luminal pathogen infection in the gastrointestinal tract. These epithelial cells represent an integral component of a highly regulated communication network that can transmit essential signals to cells in the underlying intestinal mucosa that, in turn, serve as targets of mucosal immune mediators. LncRNAs are recently identified long noncoding transcripts that can regulate gene transcription through their interactions with other effect molecules. In this study, we demonstrated that lncRNA XR_001779380 was upregulated in murine intestinal epithelial cells following infection by a mucosal protozoan parasite Cryptosporidium. Expression of XR_001779380 in infected cells primed host epithelial cells for IFN-γ-mediated gene transcription, relevant to age-dependent intestinal antimicrobial defense. Our data provide new mechanistic insights into how intestinal epithelial cells orchestrate intestinal mucosal defense against microbial infection.

Neonatal Mouse Gut Metabolites Influence Cryptosporidium parvum Infection in Intestinal Epithelial Cells.

The protozoan parasite Cryptosporidium sp. is a leading cause of diarrheal disease in those with compromised or underdeveloped immune systems, particularly infants and toddlers in resource-poor localities. As an enteric pathogen, Cryptosporidium sp. invades the apical surface of intestinal epithelial cells, where it resides in close proximity to metabolites in the intestinal lumen. However, the effect of gut metabolites on susceptibility to Cryptosporidium infection remains largely unstudied. Here, we first identified which gut metabolites are prevalent in neonatal mice when they are most susceptible to Cryptosporidium parvum infection and then tested the isolated effects of these metabolites on C. parvum invasion and growth in intestinal epithelial cells. Our findings demonstrate that medium or long-chain saturated fatty acids inhibit C. parvum growth, perhaps by negatively affecting the streamlined metabolism in C. parvum, which is unable to synthesize fatty acids. Conversely, long-chain unsaturated fatty acids enhanced C. parvum invasion, possibly by modulating membrane fluidity. Hence, gut metabolites, either from diet or produced by the microbiota, influence C. parvum growth in vitro and may also contribute to the early susceptibility to cryptosporidiosis seen in young animals.IMPORTANCECryptosporidium sp. occupies a unique intracellular niche that exposes the parasite to both host cell contents and the intestinal lumen, including metabolites from the diet and produced by the microbiota. Both dietary and microbial products change over the course of early development and could contribute to the changes seen in susceptibility to cryptosporidiosis in humans and mice. Consistent with this model, we show that the immature gut metabolome influenced the growth of Cryptosporidium parvumin vitro Interestingly, metabolites that significantly altered parasite growth were fatty acids, a class of molecules that Cryptosporidium sp. is unable to synthesize de novo The enhancing effects of polyunsaturated fatty acids and the inhibitory effects of saturated fatty acids presented in this study may provide a framework for future studies into this enteric parasite's interactions with exogenous fatty acids during the initial stages of infection.

Decreased SLC26A3 expression and function in intestinal epithelial cells in response to Cryptosporidium parvum infection.

The protozoan parasite Cryptosporidium parvum (CP) causes cryptosporidiosis, a diarrheal disease worldwide. Infection in immunocompetent hosts typically results in acute, self-limiting, or recurrent diarrhea. However, in immunocompromised individuals infection can cause fulminant diarrhea, extraintestinal manifestations, and death. To date, the mechanisms underlying CP-induced diarrheal pathogenesis are poorly understood. Diarrheal diseases most commonly involve increased secretion and/or decreased absorption of fluid and electrolytes. We and others have previously shown impaired chloride absorption in infectious diarrhea due to dysregulation of SLC26A3 [downregulated in adenoma (DRA)], the human intestinal apical membrane Cl-/HCO3- exchanger protein. However, there are no studies on the effects of CP infection on DRA activity. Therefore, we examined the expression and function of DRA in intestinal epithelial cells in response to CP infection in vitro and in vivo. CP infection (0.5 × 106 oocysts/well in 24-well plates, 24 h) of Caco-2 cell monolayers significantly decreased Cl-/HCO3- exchange activity (measured as DIDS-sensitive 125I uptake) as well as DRA mRNA and protein levels. Substantial downregulation of DRA mRNA and protein was also observed following CP infection ex vivo in mouse enteroid-derived monolayers and in vivo in the ileal and jejunal mucosa of C57BL/6 mice for 24 h. However, at 48 h after infection in vivo, the effects on DRA mRNA and protein were attenuated and at 5 days after infection DRA returned to normal levels. Our results suggest that impaired chloride absorption due to downregulation of DRA could be one of the contributing factors to CP-induced acute, self-limiting diarrhea in immunocompetent hosts.

A Cell Culture Platform for the Cultivation of Cryptosporidium parvum.

Cryptosporidium is a genus of ubiquitous unicellular parasites belonging to the phylum Apicomplexa. Cryptosporidium species are the second largest cause of childhood diarrhea and are associated with increased morbidity. Accompanying this is the low availability of treatment and lack of vaccines. The major barrier to developing effective treatment is the lack of reliable in vitro culture methods. Recently, our lab has successfully cultivated C. parvum in the esophageal cancer-derived cell line COLO-680N, and has been able to maintain infection for several weeks. The success of this cell line was assessed with a combination of various techniques including fluorescent microscopy and qPCR. In addition, to tackle the issue of long-term oocyst production in vitro, a simple, low-cost bioreactor system using the COLO-680N cell line was established, which produced infectious oocysts for 4 months. This chapter provides details on the methodologies used to culture, maintain, and assess Cryptosporidium infection and propagation in COLO-680N. © 2019 by John Wiley & Sons, Inc.

Induction of Inflammatory Responses in Splenocytes by Exosomes Released from Intestinal Epithelial Cells following Cryptosporidium parvum Infection.

Cryptosporidium, a protozoan parasite that infects the gastrointestinal epithelium and other mucosal surfaces in humans and animals, is an important opportunistic pathogen in AIDS patients and one of the most common enteric pathogens affecting young children in developing regions. This parasite is referred to as a "minimally invasive" mucosal pathogen, and epithelial cells play a central role in activating and orchestrating host immune responses. We previously demonstrated that Cryptosporidium parvum infection stimulates host epithelial cells to release exosomes, and these released exosomes shuttle several antimicrobial peptides to carry out anti-C. parvum activity. In this study, we detected the upregulation of inflammatory genes in the liver and spleen following C. parvum intestinal infection in neonatal mice. Interestingly, exosomes released from intestinal epithelial cells following C. parvum infection could activate the nuclear factor kappa B signaling pathway and trigger inflammatory gene transcription in isolated primary splenocytes. Several epithelial cell-derived proteins and a subset of parasite RNAs were detected in the exosomes released from C. parvum-infected intestinal epithelial cells. Shuttling of these effector molecules, including the high mobility group box 1 protein, was involved in the induction of inflammatory responses in splenocytes induced by the exosomes released from infected cells. Our data indicate that exosomes released from intestinal epithelial cells upon C. parvum infection can activate immune cells by shuttling various effector molecules, a process that may be relevant to host systemic responses to Cryptosporidium infection.

First evidence of cytotoxic effects of human protozoan parasites on zebra mussel (Dreissena polymorpha) haemocytes.

The interaction between human protozoan parasites and the immune cells of bivalves, that can accumulate them, is poorly described. The purpose of this study is to consider the mechanisms of action of some of these protozoa on zebra mussel haemocytes, by evaluating their cytotoxic potential. Haemocytes were exposed to Toxoplasma gondii, Giardia duodenalis or Cryptosporidium parvum (oo)cysts. The results showed a cytotoxic potency of the two largest protozoa on haemocytes and suggested the formation of haemocyte aggregates. Thus, this study reveals the first signs of a haemocyte:protozoan interaction.

Trans-suppression of host CDH3 and LOXL4 genes during Cryptosporidium parvum infection involves nuclear delivery of parasite Cdg7_FLc_1000 RNA.

Intestinal infection by Cryptosporidium parvum causes significant alterations in the gene expression profile in host epithelial cells. Previous studies demonstrate that a panel of parasite RNA transcripts of low protein-coding potential are delivered into infected host cells and may modulate host gene transcription. Using in vitro models of human intestinal cryptosporidiosis, we report here that trans-suppression of the cadherin 3 (CDH3) and lysyl oxidase like 4 (LOXL4) genes in human intestinal epithelial cells following C. parvum infection involves host delivery of the Cdg7_FLc_1000 RNA, a C. parvum RNA that has been previously demonstrated to be delivered into the nuclei of infected host cells. Downregulation of CDH3 and LOXL4 genes was detected in host epithelial cells following C. parvum infection or in cells expressing the parasite Cdg7_FLc_1000 RNA. Knockdown of Cdg7_FLc_1000 attenuated the trans-suppression of CDH3 and LOXL4 genes in host cells induced by infection. Interestingly, Cdg7_FLc_1000 was detected to be recruited to the promoter regions of both CDH3 and LOXL4 gene loci in host cells following C. parvum infection. Host delivery of Cdg7_FLc_1000 promoted the PH domain zinc finger protein 1 (PRDM1)-mediated H3K9 methylation associated with trans-suppression in the CDH3 gene locus, but not the LOXL4 gene. Therefore, our data suggest that host delivery of Cdg7_FLc_1000 causes CDH3 trans-suppression in human intestinal epithelial cells following C. parvum infection through PRDM1-mediated H3K9 methylation in the CDH3 gene locus, whereas Cdg7_FLc_1000 induces trans-suppression of the host LOXL4 gene through H3K9/H3K27 methylation-independent mechanisms.

Nuclear delivery of parasite Cdg2_FLc_0220 RNA transcript to epithelial cells during Cryptosporidium parvum infection modulates host gene transcription.

Intestinal infection by the zoonotic protozoan, Cryptosporidium parvum, causes significant alterations in the gene expression profile in host epithelial cells. The molecular mechanisms of how C. parvum may modulate host cell gene transcription and the pathological significance of such alterations are largely unclear. Previous studies demonstrate that a panel of parasite RNA transcripts are delivered into infected host cells and may modulate host gene transcription. Using in vitro models of intestinal cryptosporidiosis, in this study, we analyzed the impact of host delivery of C. parvum Cdg2_FLc_0220 RNA transcript on host gene expression profile. We found that alterations in host gene expression profile following C. parvum infection were partially associated with the nuclear delivery of Cdg2_FLc_0220. Specifically, we identified a total of 46 overlapping upregulated genes and 8 overlapping downregulated genes in infected cells and cells transfected with Full-Cdg2_FLc_0220. Trans-suppression of the DAZ interacting zinc finger protein 1 like (DZIP1L) gene, the top overlapping downregulated gene in host cells following C. parvum infection and cells transfected with Full-Cdg2_FLc_0220, was mediated by G9a, independent of PRDM1. Cdg2_FLc_0220-mediated trans-suppression of the DZIP1L gene was independent of H3K9 and H3K27 methylation. Data from this study provide additional evidence that delivery of C. parvum Cdg2_FLc_0220 RNA transcript in infected epithelial cells modulates the transcription of host genes, contributing to the alterations in the gene expression profile in host epithelial cells during C. parvum infection.

Analysis of Parasitic Protozoa at the Single-cell Level using Microfluidic Impedance Cytometry.

At present, there are few technologies which enable the detection, identification and viability analysis of protozoan pathogens including Cryptosporidium and/or Giardia at the single (oo)cyst level. We report the use of Microfluidic Impedance Cytometry (MIC) to characterise the AC electrical (impedance) properties of single parasites and demonstrate rapid discrimination based on viability and species. Specifically, MIC was used to identify live and inactive C. parvum oocysts with over 90% certainty, whilst also detecting damaged and/or excysted oocysts. Furthermore, discrimination of Cryptosporidium parvum, Cryptosporidium muris and Giardia lamblia, with over 92% certainty was achieved. Enumeration and identification of (oo)cysts can be achieved in a few minutes, which offers a reduction in identification time and labour demands when compared to existing detection methods.

Novel Bioengineered Three-Dimensional Human Intestinal Model for Long-Term Infection of Cryptosporidium parvum.

Cryptosporidium spp. are apicomplexan parasites of global importance that cause human diarrheal disease. In vitro culture models that may be used to study this parasite and that have physiological relevance to in vivo infection remain suboptimal. Thus, the pathogenesis of cryptosporidiosis remains poorly characterized, and interventions for the disease are limited. In this study, we evaluated the potential of a novel bioengineered three-dimensional (3D) human intestinal tissue model (which we developed previously) to support long-term infection by Cryptosporidium parvum Infection was assessed by immunofluorescence assays and confocal and scanning electron microscopy and quantified by quantitative reverse transcription-PCR. We found that C. parvum infected and developed in this tissue model for at least 17 days, the extent of the study time used in the present study. Contents from infected scaffolds could be transferred to fresh scaffolds to establish new infections for at least three rounds. Asexual and sexual stages and the formation of new oocysts were observed during the course of infection. Additionally, we observed ablation, blunting, or distortion of microvilli in infected epithelial cells. Ultimately, a 3D model system capable of supporting continuous Cryptosporidium infection will be a useful tool for the study of host-parasite interactions, identification of putative drug targets, screening of potential interventions, and propagation of genetically modified parasites.

The Cryptosporidium parvum C-Type Lectin CpClec Mediates Infection of Intestinal Epithelial Cells via Interactions with Sulfated Proteoglycans.

The apicomplexan parasite Cryptosporidium causes significant diarrheal disease worldwide. Effective anticryptosporidial agents are lacking, in part because the molecular mechanisms underlying Cryptosporidium-host cell interactions are poorly understood. Previously, we identified and characterized a novel Cryptosporidium parvum C-type lectin domain-containing mucin-like glycoprotein, CpClec. In this study, we evaluated the mechanisms underlying interactions of CpClec with intestinal epithelial cells by using an Fc-tagged recombinant protein. CpClec-Fc displayed Ca(2+)-dependent, saturable binding to HCT-8 and Caco-2 cells and competitively inhibited C. parvum attachment to and infection of HCT-8 cells. Binding of CpClec-Fc was specifically inhibited by sulfated glycosaminoglycans, particularly heparin and heparan sulfate. Binding was reduced after the removal of heparan sulfate and following the inhibition of glycosaminoglycan synthesis or sulfation in HCT-8 cells. Like CpClec-Fc binding, C. parvum attachment to and infection of HCT-8 cells were inhibited by glycosaminoglycans and were reduced after heparan sulfate removal or inhibition of glycosaminoglycan synthesis or sulfation. Lastly, CpClec-Fc binding and C. parvum sporozoite attachment were significantly decreased in CHO cell mutants defective in glycosaminoglycan synthesis. Together, these results indicate that CpClec is a novel C-type lectin that mediates C. parvum attachment and infection via Ca(2+)-dependent binding to sulfated proteoglycans on intestinal epithelial cells.

Assessing protozoan risks for surface drinking water supplies in Nova Scotia, Canada.

Protozoa, such as Cryptosporidium parvum and Giardia lamblia, pose a human health risk when present in drinking water. To minimize health risks, the Nova Scotia Treatment Standards for surface water and groundwater under the direct influence of surface water require a 3-log reduction for Giardia cysts and Cryptosporidium oocysts. This study determined the protozoan risk of municipal surface source waters in Nova Scotia, through the use of a pre-screening risk analysis of water supplies, followed by subsequent water quality analysis of the seven highest risk supplies. The water supplies were monitored monthly for 1 year to obtain baseline data that could be used for a quantitative microbial risk assessment (QMRA). The QMRA model outcomes were compared to the Health Canada health target of 10(-6) disability-adjusted life years/person/year. QMRA modeling shows that the treatment facilities meet the required log reductions and disability-adjusted life year target standards under current conditions. Furthermore, based on the results of this work, Nova Scotia should maintain the current 3-log reduction standard for Giardia cysts and Cryptosporidium oocysts. The results of this study show that a pre-screening step can help to inform water sources that are particularly vulnerable to protozoan contamination, which can lead to more focused, cost-effective sampling, and monitoring programs.

Bovine TLR2 and TLR4 mediate Cryptosporidium parvum recognition in bovine intestinal epithelial cells.

Cryptosporidium parvum (C. parvum) is an intestinal parasite that causes diarrhea in neonatal calves. It results in significant morbidity of neonatal calves and economic losses for producers worldwide. Innate resistance against C. parvum is thought to depend on engagement of pattern recognition receptors. However, the role of innate responses to C. parvum has not been elucidated in bovine. The aim of this study was to evaluate the role of TLRs in host-cell responses during C. parvum infection of cultured bovine intestinal epithelial cells. The expressions of TLRs in bovine intestinal epithelial cells were detected by qRT-PCR. To determine which, if any, TLRs may play a role in the response of bovine intestinal epithelial cells to C. parvum, the cells were stimulated with C. parvum and the expression of TLRs were tested by qRT-PCR. The expression of NF-κB was detected by western blotting. Further analyses were carried out in bovine TLRs transfected HEK293 cells and by TLRs-DN transfected bovine intestinal epithelial cells. The results showed that bovine intestinal epithelial cells expressed all known TLRs. The expression of TLR2 and TLR4 were up-regulated when bovine intestinal epithelial cells were treated with C. parvum. Meanwhile, C. parvum induced IL-8 production in TLR2 or TLR4/MD-2 transfected HEK293 cells. Moreover, C. parvum induced NF-κB activation and cytokine expression in bovine intestinal epithelial cells. The induction of NF-κB activation and cytokine expression by C. parvum were reduced in TLR2-DN and TLR4-DN transfected cells. The results showed that bovine intestinal epithelial cells expressed all known TLRs, and bovine intestinal epithelial cells recognized and responded to C. parvum via TLR2 and TLR4.

CCL20 Displays Antimicrobial Activity Against Cryptosporidium parvum, but Its Expression Is Reduced During Infection in the Intestine of Neonatal Mice.

CCL20 is a chemokine with antimicrobial activity. We investigated its expression and role during neonatal cryptosporidiosis, a worldwide protozoan enteric disease leading to severe diarrhea. Surprisingly, during infection by Cryptosporidium parvum, CCL20 production by the intestine of neonatal mice is reduced by a mechanism independent both of the enteric flora and of interferon γ, a key cytokine for the resolution of this infection. However, oral administration of recombinant CCL20 to neonatal mice significantly reduced the parasite load by a mechanism that was independent of immune cell recruitment and occurred instead by direct cytolytic activity on free stages of the parasite. MiR21 functionally targets CCL20 and is upregulated during the infection, thus contributing to the downregulation of the chemokine. Our findings demonstrate for the first time the direct antiparasitic activity of CCL20 against an enteric protozoan and its downregulation during C. parvum infection, which is detrimental to parasite clearance.

Electrical impedance sensor for quantitative monitoring of infection processes on HCT-8 cells by the waterborne parasite Cryptosporidium.

Cryptosporidium is the main origin of worldwide waterborne epidemic outbreaks caused by protozoan parasites. Its resilience to water chemical treatments and the absence of therapy led to consider it as a reference pathogen to assess water quality and as a possible bioterrorism agent. We here show that an electrical impedance-based device is able to get insights on Cryptosporidium development on a cell culture and to quantify sample infectivity. HCT-8 cells were grown to confluency on Interdigitated Microelectrode Arrays (IMA's) during 76h and then infected by Cryptosporidium parvum during 60h. The impedimetric response was measured at frequencies ranging from 100Hz to 1MHz and a 7min sampling period. As the infection progresses the impedance signal shows a reproducible distinct succession of peaks at 12h post infection (PI), 23h PI and 31h PI and local minima at 9h PI, 19h PI and 28h PI. An equivalent circuit modeling-based approach indicates that these features are mostly originated from paracellular pathway modifications due to host-parasite interactions. Furthermore, our data present for the first time a real-time monitoring of early parasitic stage development with alternating zoite and meront predominances, observed respectively at peaks and local minima in the impedimetric signal. Finally, by quantifying the magnitude of the impedimetric response, we demonstrate this device can also be used as an infectivity sensor as early as 12h PI thus being at least 6 times faster than other state of the art techniques.

Measurement of Elastic Modulus and Vickers Hardness of Surround Bone Implant Using Dynamic Microindentation - Parameters Definition

The clinical performance of dental implants is strongly defined by biomechanical principles. The aim of this study was to quantify the Vicker's hardness (VHN) and elastic modulus (E) surround bone to dental implant in different regions, and to discuss the parameters of dynamic microindantion test. Ten cylindrical implants with morse taper interface (Titamax CM, Neodent; 3.5 mm diameter and 7 mm a height) were inserted in rabbit tibia. The mechanical properties were analyzed using microhardness dynamic indenter with 200 mN load and 15 s penetration time. Seven continuous indentations were made distancing 0.08 mm between each other perpendicularly to the implant-bone interface towards the external surface, at the limit of low (Lp) and high implant profile (Hp). Data were analyzed by Student's t-test (a=0.05) to compare the E and VHN values obtained on both regions. Mean and standard deviation of E (GPa) were: Lp. 16.6 ± 1.7, Hp. 17.0 ± 2.5 and VHN (N/mm2): Lp. 12.6 ± 40.8, Hp. 120.1 ± 43.7. No statistical difference was found between bone mechanical properties of high and low profile of the surround bone to implant, demonstrating that the bone characterization homogeneously is pertinent. Dynamic microindantion method proved to be highly useful in the characterization of the individual peri-implant bone tissue.

O desempenho clínico de implantes dentais é fortemente definido por princípios biomecânicos. Este trabalho objetivou quantificar a Dureza Vickers (VHN) e módulo de elasticidade (E) do osso periimplantar e discutir parâmetros metodológicos de ensaio dinâmico de indentação. Foram utilizados 10 implantes de corpo cilíndrico com interface cone morse, (Titamax CM; Neodent, Curitiba, PR, Brasil), diâmetro de 3.5 mm e altura de 7 mm inseridos em tíbia de coelho recém obtidas após abate dos animais. As propriedades mecânicas foram analisadas usando penetrador dinâmico de microdureza Vickers (CSM Micro-Hardness Tester; CSM Instruments, Peseux, Switzerland) com carga de 200 mN e tempo de penetração de 15s. Foram feitas 7 indentações no osso cortical na base da rosca (Br) e na ponta da rosca (Pr) na direção perpendicular ao implante, com distância entre elas de 0,08 mm perpendicular a interface osso implante em direção a superfície esterna. Os dados foram analisados por meio de teste t-Student (P<0,05). O valores médios e desvio padrão de E (GPa) foram: Br. 16,6 ± 1,7A; Pr. 17,0 ± 2,5A e VHN (N/mm2): Br. 125,6 ± 40,8A; Pr. 120,1 ± 43,7A. Não houve diferença significativa entre as propriedades mecânicas avaliadas no osso na base e na ponta da rosca do implante, demonstrando que a caracterização desta estrutura de forma homogênea em análises computacionais é pertinente. O método de indentação dinâmica mostrou ser altamente útil na caracterização individualizada do tecido ósseo periimplantar.

A new in vitro model using small intestinal epithelial cells to enhance infection of Cryptosporidium parvum.

To better understand and study the infection of the protozoan parasite Cryptosporidium parvum, a more sensitive in vitro assay is required. In vivo, this parasite infects the epithelial cells of the microvilli layer in the small intestine. While cell infection models using colon, kidney, and stomach cells have been studied to understand the infectivity potential of the oocysts, an ideal in vitro model would be readily-available, human-derived, and originating from the small intestine. In this study, we developed a reproducible, quantitative infection model using a non-carcinoma, human small intestinal epithelial cell type, named FHs 74 Int. Our results show that FHs 74 Int cells are productively infected by viable oocysts, and exhibit higher levels of infection susceptibility compared to other cell types. Moreover, infection rate of the sporozoites on the monolayer was found to be comparable or better than other cell types. We furthermore demonstrate that infection can be improved by 65% when pre-treated oocysts are directly inoculated on cells, compared to inoculation of excysted sporozoites on cells. Identification of a better infection model, which captures the preferred site of infection in humans, will facilitate studies on the host pathogenesis mechanisms of this important parasitic human pathogen.

Cryptosporidium parvum-induced ileo-caecal adenocarcinoma and Wnt signaling in a mouse model.

Cryptosporidium species are apicomplexan protozoans that are found worldwide. These parasites constitute a large risk to human and animal health. They cause self-limited diarrhea in immunocompetent hosts and a life-threatening disease in immunocompromised hosts. Interestingly, Cryptosporidium parvum has been related to digestive carcinogenesis in humans. Consistent with a potential tumorigenic role of this parasite, in an original reproducible animal model of chronic cryptosporidiosis based on dexamethasone-treated or untreated adult SCID mice, we formerly reported that C. parvum (strains of animal and human origin) is able to induce digestive adenocarcinoma even in infections induced with very low inoculum. The aim of this study was to further characterize this animal model and to explore metabolic pathways potentially involved in the development of C. parvum-induced ileo-caecal oncogenesis. We searched for alterations in genes or proteins commonly involved in cell cycle, differentiation or cell migration, such as ß-catenin, Apc, E-cadherin, Kras and p53. After infection of animals with C. parvum we demonstrated immunohistochemical abnormal localization of Wnt signaling pathway components and p53. Mutations in the selected loci of studied genes were not found after high-throughput sequencing. Furthermore, alterations in the ultrastructure of adherens junctions of the ileo-caecal neoplastic epithelia of C. parvum-infected mice were recorded using transmission electron microscopy. In conclusion, we found for the first time that the Wnt signaling pathway, and particularly the cytoskeleton network, seems to be pivotal for the development of the C. parvum-induced neoplastic process and cell migration of transformed cells. Furthermore, this model is a valuable tool in understanding the host-pathogen interactions associated with the intricate infection process of this parasite, which is able to modulate host cytoskeleton activities and several host-cell biological processes and remains a significant cause of infection worldwide.

Histone deacetylases and NF-kB signaling coordinate expression of CX3CL1 in epithelial cells in response to microbial challenge by suppressing miR-424 and miR-503.

The NF-kB pathway is key to epithelial immune defense and has been implicated in secretion of antimicrobial peptides, release of cytokines/chemokines to mobilize immune effector cells, and activation of adaptive immunity. The expression of many inflammatory genes following infection involves the remodeling of the chromatin structure. We reported here that histone deacetylases (HDACs) and NF-kB signaling coordinate expression of CX3CL1 in epithelial cells following Cryptosporidium parvum infection. Upregulation of CX3CL1 was detected in cultured human biliary epithelial cells following infection. Expression of miR-424 and miR-503 was downregulated, and was involved in the induction of CX3CL1 in infected cells. C. parvum infection suppressed transcription of the mir-424-503 gene in a NF-kB- and HDAC-dependent manner. Increased promoter recruitment of NF-kB p50 and HDACs, and decreased promoter H3 acetylation associated with the mir-424-503 gene were observed in infected cells. Upregulation of CX3CL1 in biliary epithelial cells and increased infiltration of CX3CR1(+) cells were detected during C. parvum infection in vivo. Induction of CX3CL1 and downregulation of miR-424 and miR-503 were also detected in epithelial cells in response to LPS stimulation. The above results indicate that HDACs and NF-kB signaling coordinate epithelial expression of CX3CL1 to promote mucosal antimicrobial defense through suppression of the mir-424-503 gene.