Duckweed (Lemna minor) as a Model Plant System for the Study of Human Microbial Pathogenesis

BACKGROUND: Plant infection models provide certain advantages over animal models in the study of pathogenesis. However, current plant models face some limitations, e.g., plant and pathogen cannot co-culture in a contained environment. Development of such a plant model is needed to better illustrate host-pathogen interactions. METHODOLOGY/PRINCIPAL FINDINGS: We describe a novel model plant system for the study of human pathogenic bacterial infection on a large scale. This system was initiated by co-cultivation of axenic duckweed (Lemna minor) plants with pathogenic bacteria in 24-well polystyrene cell culture plate. Pathogenesis of bacteria to duckweed was demonstrated with Pseudomonas aeruginosa and Staphylococcus aureus as two model pathogens. P. aeruginosa PAO1 caused severe detriment to duckweed as judged from inhibition to frond multiplication and chlorophyll formation. Using a GFP-marked PAO1 strain, we demonstrated that bacteria colonized on both fronds and roots and formed biofilms. Virulence of PAO1 to duckweed was attenuated in its quorum sensing (QS) mutants and in recombinant strains overexpressing the QS quenching enzymes. RN4220, a virulent strain of S. aureus, caused severe toxicity to duckweed while an avirulent strain showed little effect. Using this system for antimicrobial chemical selection, green tea polyphenols exhibited inhibitory activity against S. aureus virulence. This system was further confirmed to be effective as a pathogenesis model using a number of pathogenic bacterial species. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that duckweed can be used as a fast, inexpensive and reproducible model plant system for the study of host-pathogen interactions, could serve as an alternative choice for the study of some virulence factors, and could also potentially be used in large-scale screening for the discovery of antimicrobial chemicals

Tirofiban for Stroke without Large or Medium-Sized Vessel Occlusion

The effects of the glycoprotein IIb/IIIa receptor inhibitor tirofiban in patients with acute ischemic stroke but who have no evidence of complete occlusion of large or medium-sized vessels have not been extensively studied. In a multicenter trial in China, we enrolled patients with ischemic stroke without occlusion of large or medium-sized vessels and with a National Institutes of Health Stroke Scale score of 5 or more and at least one moderately to severely weak limb. Eligible patients had any of four clinical presentations: ineligible for thrombolysis or thrombectomy and within 24 hours after the patient was last known to be well; progression of stroke symptoms 24 to 96 hours after onset; early neurologic deterioration after thrombolysis; or thrombolysis with no improvement at 4 to 24 hours. Patients were assigned to receive intravenous tirofiban (plus oral placebo) or oral aspirin (100 mg per day, plus intravenous placebo) for 2 days; all patients then received oral aspirin until day 90. The primary efficacy end point was an excellent outcome, defined as a score of 0 or 1 on the modified Rankin scale (range, 0 [no symptoms] to 6 [death]) at 90 days. Secondary end points included functional independence at 90 days and a quality-of-life score. The primary safety end points were death and symptomatic intracranial hemorrhage. A total of 606 patients were assigned to the tirofiban group and 571 to the aspirin group. Most patients had small infarctions that were presumed to be atherosclerotic. The percentage of patients with a score of 0 or 1 on the modified Rankin scale at 90 days was 29.1% with tirofiban and 22.2% with aspirin (adjusted risk ratio, 1.26; 95% confidence interval, 1.04 to 1.53, P = 0.02). Results for secondary end points were generally not consistent with the results of the primary analysis. Mortality was similar in the two groups. The incidence of symptomatic intracranial hemorrhage was 1.0% in the tirofiban group and 0% in the aspirin group. In this trial involving heterogeneous groups of patients with stroke of recent onset or progression of stroke symptoms and nonoccluded large and medium-sized cerebral vessels, intravenous tirofiban was associated with a greater likelihood of an excellent outcome than low-dose aspirin. Incidences of intracranial hemorrhages were low but slightly higher with tirofiban

Generation of Th1 and Th17 polarized cells in <i>Lrrk2</i> (-/-) (“KO”) mice is moderately less vigorous than in their WT controls.

<p>Suspensions of pooled spleen cells of mice of the two groups, 14 days post immunization with IRBP, were examined by flow cytometry for intracellular staining with antibodies against IFN-γ or IL-17, the signature cytokines for Th1 and Th17, respectively. Data of two individual experiments.</p

Similar levels of Treg cells expressing FoxP3 in spleens of <i>Lrrk2</i> (-/-) (“KO”) and their WT controls.

<p>Spleens from mice of the two groups were collected 14 days post immunization, pooled, and their cells were examined by flow cytometry for expression of FoxP3. <b>A</b>. A representative experiment. <b>B</b>. Mean +/- SEM of proportions of FoxP3 cells in spleens of mice of the two groups from five individual experiments.</p

Spleen cells of <i>Lrrk2</i> (-/-) (“KO”) mice immunized with IRBP secrete lower levels of IFN-γand IL-17 than their WT controls.

<p><b>A and C.</b> Levels of IFN-γ and IL-17 measured by ELISA in supernatants of spleen cells of five individual experiments, collected 14 days following immunization and cultured with IRBP at 10 μg/ml for 48 hrs. <b>B and D</b>. Summaries of the averaged and normalized data of the two cytokines in the five individual experiments, as detailed in the Materials and Methods section.</p

Minute differences seen in the profiles of cytokines/chemokines released by peritoneal macrophages of the <i>Lrrk2</i> (-/-) (“KO”) and their WT controls, cultured with LPS (0.5 μg/ml) or CpG (40 μg/ml) for 48 hrs.

<p>The levels of the released products were measured by a bead-based multi-plex screening assay. The assay measured secretion of 14 analytes, as detailed in the Materials and Methods section, but molecules with undetectable levels were not included in the figure. The figures show combined data collected in two individual experiments and combined for presentation.</p

Immune responses to BSA by <i>Lrrk2</i> (-/-) mice are lower than those of their WT controls.

<p>Mice of the two lines were immunized with BSA and tested for DTH skin response with BSA (<b>A</b>) and secretion of IFN-γ (<b>B</b>) and of IL-17 (<b>C</b>) by spleen cells incubated with BSA. Data of a representative experiment; similar trends were found in a repeated experiment.</p

Leucine-Rich Repeat Kinase 2 (<i>Lrrk2</i>) Deficiency Diminishes the Development of Experimental Autoimmune Uveitis (EAU) and the Adaptive Immune Response

<div><p>Background</p><p>Mutations in <i>LRRK2</i> are related to certain forms of Parkinson’s disease and, possibly, to the pathogenesis of Crohn’s disease. In both these diseases inflammatory processes participate in the pathogenic process. LRRK2 is expressed in lymphoid cells and, interestingly, <i>Lrrk2</i> (-/-) mice were reported to develop more severe experimental colitis than their wild type (WT) controls. Here, we examined the possible involvement of LRRK2 in the pathogenesis of experimental autoimmune uveitis (EAU), an animal model for human uveitis, by testing <i>Lrrk2</i> (-/-) mice for their capacity to develop this experimental eye disease and related immune responses.</p><p>Methods</p><p><i>Lrrk2</i> (-/-) mice and their WT controls (C57Bl/6) were immunized with interphotoreceptor retinoid-binding protein (IRBP) and compared for their development of EAU, delayed type hypersensitivity (DTH) by skin tests, production of cytokines in culture, and expression of interferon (IFN)-γ, interleukin (IL)-17 and FoxP3 by spleen cells, using flow cytometry. Peritoneal macrophages were examined for their production of cytokines/chemokines in culture following stimulation with LPS or the oligodeoxynucleotide CpG. The <i>Lrrk2</i> (-/-) and WT mice were also compared for their response to bovine serum albumin (BSA).</p><p>Results</p><p>The <i>Lrrk2</i> (-/-) mice developed lower levels of EAU, DTH responses and cytokine production by lymphocytes than did their WT controls. Intracellular expression of IFN-γ and IL-17, by spleen cells, and secretion of cytokines/chemokines by activated peritoneal macrophages of Lrrk2 (-/-) mice trended toward diminished levels, although variabilities were noted. The expression levels of FoxP3 by Lrrk2 (-/-) spleen cells, however, were similar to those seen in WT controls. Consistent with their low response to IRBP, <i>Lrrk2</i> (-/-) mice responded to BSA less vigorously than their WT controls.</p><p>Conclusions</p><p><i>Lrrk2</i> deficiency in mice diminished the development of EAU and the related adaptive immune responses to IRBP as compared to the WT controls.</p></div