Towards the Establishment of a Porcine Model to Study Human Amebiasis

BACKGROUND: Entamoeba histolytica is an important parasite of the human intestine. Its life cycle is monoxenous with two stages: (i) the trophozoite, growing in the intestine and (ii) the cyst corresponding to the dissemination stage. The trophozoite in the intestine can live as a commensal leading to asymptomatic infection or as a tissue invasive form producing mucosal ulcers and liver abscesses. There is no animal model mimicking the whole disease cycle. Most of the biological information on E. histolytica has been obtained from trophozoite adapted to axenic culture. The reproduction of intestinal amebiasis in an animal model is difficult while for liver amebiasis there are well-described rodent models. During this study, we worked on the assessment of pigs as a new potential model to study amebiasis. METHODOLOGY/PRINCIPAL FINDINGS: We first co-cultured trophozoites of E. histolytica with porcine colonic fragments and observed a disruption of the mucosal architecture. Then, we showed that outbred pigs can be used to reproduce some lesions associated with human amebiasis. A detailed analysis was performed using a washed closed-jejunal loops model. In loops inoculated with virulent amebas a severe acute ulcerative jejunitis was observed with large hemorrhagic lesions 14 days post-inoculation associated with the presence of the trophozoites in the depth of the mucosa in two out four animals. Furthermore, typical large sized hepatic abscesses were observed in the liver of one animal 7 days post-injection in the portal vein and the liver parenchyma. CONCLUSIONS: The pig model could help with simultaneously studying intestinal and extraintestinal lesion development

Contrasting nutrient-disease relationships: Potassium gradients in barley leaves have opposite effects on two fungal pathogens with different sensitivities to jasmonic acid

Understanding the interactions between mineral nutrition and disease is essential for crop management. Our previous studies with Arabidopsis thaliana demonstrated that potassium (K) deprivation induced the biosynthesis of jasmonate (JA) and increased the plant's resistance to herbivorous insects. Here we addressed the question how tissue K affects the development of fungal pathogens and whether sensitivity of the pathogens to JA could play a role for the K‐disease relationship in barley (Hordeum vulgare cv. Optic). We report that K‐deprived barley plants showed increased leaf concentrations of JA and other oxylipins. Furthermore, a natural tip‐to base K‐concentrations gradient within leaves of K‐sufficient plants was quantitatively mirrored by the transcript levels of JA‐responsive genes. The local leaf tissue K concentrations affected the development of two economically important fungi in opposite ways, showing a positive correlation with powdery mildew (Blumeria graminis) and a negative correlation with leaf scald (Rhynchosporium commune) disease symptoms. B. graminis induced a JA‐response in the plant and was sensitive to methyl‐JA treatment while R. commune initiated no JA‐response and was JA‐insensitive. Our study challenges the view that high K generally improves plant health and suggests that JA‐sensitivity of pathogens could be an important factor determining the exact K‐disease relationship

Adeno-Associated Virus Vector Genomes Persist as Episomal Chromatin in Primate Muscle▿

Recombinant adeno-associated virus (rAAV) vectors are capable of mediating long-term gene expression following administration to skeletal muscle. In rodent muscle, the vector genomes persist in the nucleus in concatemeric episomal forms. Here, we demonstrate with nonhuman primates that rAAV vectors integrate inefficiently into the chromosomes of myocytes and reside predominantly as episomal monomeric and concatemeric circles. The episomal rAAV genomes assimilate into chromatin with a typical nucleosomal pattern. The persistence of the vector genomes and gene expression for years in quiescent tissues suggests that a bona fide chromatin structure is important for episomal maintenance and transgene expression. These findings were obtained from primate muscles transduced with rAAV1 and rAAV8 vectors for up to 22 months after intramuscular delivery of 5 × 1012 viral genomes/kg. Because of this unique context, our data, which provide important insight into in situ vector biology, are highly relevant from a clinical standpoint

Lack of an Immune Response against the Tetracycline-Dependent Transactivator Correlates with Long-Term Doxycycline-Regulated Transgene Expression in Nonhuman Primates after Intramuscular Injection of Recombinant Adeno-Associated Virus

We previously documented persistent regulation of erythropoietin (Epo) secretion in mice after a single intramuscular (i.m.) injection of a recombinant adeno-associated virus (rAAV) vector harboring both the tetracycline-dependent transactivator (rtTA) and the Epo cDNA (D. Bohl, A. Salvetti, P. Moullier, and J. M. Heard, Blood 92:1512-1517, 1998). Using the same vector harboring the cynomolgus macaque Epo cDNA instead, the present study evaluated the ability of the tetracycline-regulatable (tetR) system to establish long-term transgene regulation in nonhuman primates. The vector was administered i.m., after which 5-day induction pulses were performed monthly for up to 13 months by using doxycycline (DOX), a tetracycline analog. We show that initial inductions were successful in all individuals and that there was a tight regulation and a rapid deinduction pattern upon DOX withdrawal. For one macaque, regulation of Epo secretion was maintained during the entire experimental period; for the five remaining macaques, secreted Epo became indistinguishable from endogenous Epo upon repeated DOX inductions. We investigated the mechanism involved and showed that, except in the animal in which secretion persisted, delayed humoral and cellular immune responses were directed against the rtTA transactivator protein associated with the reduction of vector DNA in transduced muscles. This study provides some evidence that, when the immune system is not mobilized against the rtTA transactivator, the tetR-regulatable system is able to support long-term transgene regulation in the context of an rAAV in nonhuman primates. In addition, our results suggest potential improvements for vector design

Systemic delivery of allogenic muscle stem cells induces long-term muscle repair and clinical efficacy in duchenne muscular dystrophy dogs.

Muscle Stem = MuStemInternational audienceDuchenne muscular dystrophy (DMD) is a genetic progressive muscle disease resulting from the lack of dystrophin and without effective treatment. Adult stem cell populations have given new impetus to cell-based therapy of neuromuscular diseases. One of them, muscle-derived stem cells, isolated based on delayed adhesion properties, contributes to injured muscle repair. However, these data were collected in dystrophic mice that exhibit a relatively mild tissue phenotype and clinical features of DMD patients. Here, we characterized canine delayed adherent stem cells and investigated the efficacy of their systemic delivery in the clinically relevant DMD animal model to assess potential therapeutic application in humans. Delayed adherent stem cells, named MuStem cells (muscle stem cells), were isolated from healthy dog muscle using a preplating technique. In vitro, MuStem cells displayed a large expansion capacity, an ability to proliferate in suspension, and a multilineage differentiation potential. Phenotypically, they corresponded to early myogenic progenitors and uncommitted cells. When injected in immunosuppressed dystrophic dogs, they contributed to myofiber regeneration, satellite cell replenishment, and dystrophin expression. Importantly, their systemic delivery resulted in long-term dystrophin expression, muscle damage course limitation with an increased regeneration activity and an interstitial expansion restriction, and persisting stabilization of the dog's clinical status. These results demonstrate that MuStem cells could provide an attractive therapeutic avenue for DMD patients

Representative histology of tissues from swine infected with <i>Entamoeba histolytica</i> in isolated intestinal loop or after injection in the portal vein and the liver.

<p>(A–F except B showing mock jejunum) In jejunum, edema (*) and hemorrhages (arrowheads) were present in mucosa and musculosa. A few amebas (arrows), observable in HES and PAS staining, were observed in crypts of Lieberkühn (C, D), blood vessel (E) and hemorrhagic area (F). (G–I) In liver, some necrotic foci (n) were randomly scattered through the parenchyma (p). Necrotic center of these foci attracted many neutrophils and were surrounded by mononuclear cells and some fibrous tissue at the periphery. (A, B, D, E) 14 days, (C, F) 4 days, and (G–I) 7 days after inoculation.</p

Experimental inoculation of porcine gut and gut loops.

<p><b>A) Injection sites of </b><b><i>Entamoeba histolytica</i></b><b> in the pig digestive tract.</b> A total of 1–3 10⁶ virulent trophozoites in 10 ml TY-1-S-33 medium were injected at three different locations (cecum, ileocecal junction and proximal colon). Four animals were used. <b>B)</b><b>Schematic representation of jejunal loops</b>. Three loops were surgically created in a 2–4 m section of the pig jejunum. Proximal loop was inoculated with 1 10⁶ virulent trophozoites in 10 ml TY-1-S-33 medium while distal loops were inoculated only with 10 ml medium. Besides isolated loops intestinal flow from duodenum to jejunum and ileum was maintained in the gut via an end-to-end anastomosis. Blood and lymph flow to the loops were preserved. A total of 9 pigs were used for gut loops.</p