Characterization of the Anti-Influenza Activity of the Chinese Herbal Plant Paeonia lactiflora

Bai Shao (BS, the root of Paeonia lactiflora Pall.), a common Chinese herb in many recipes used to treat viral infection and liver diseases, is recognized for its ability to nourish menstruation, its Yin convergence, and as an antiperspirant. However, the mechanism and components for its antiviral function remain to be elucidated. In this study, an ethanolic extract of BS was further partitioned into aqueous and organic parts (EAex) for in vitro functional study and in vivo efficacy testing. EAex exhibited an IC₅₀ of 0.016 ± 0.005 mg/mL against influenza virus A/WSN/33 (H1N1), with broad-spectrum inhibitory activity against different strains of human influenza A viruses, including clinical oseltamivir-resistant isolates and an H1N1pdm strain. The synthesis of both viral RNA and protein was profoundly inhibited when the cells were treated with EAex. A time-of-addition assay demonstrated that EAex exerted its antiviral activity at various stages of the virus replication cycle. We addressed its antiviral activity at virus entry and demonstrated that EAex inhibits viral hemagglutination and viral binding to and penetration into host cells. In vivo animal testing showed that 200 mg/kg/d of EAex offered significant protection against viral infection. We conclude that BS possesses antiviral activity and has the potential for development as an anti-influenza agent.     

An ex vivo swine tracheal organ culture for the study of influenza infection

Background  The threat posed by swine influenza viruses with potential to transmit from pig populations to other hosts, including humans, requires the development of new experimental systems to study different aspects of influenza infection. Ex vivo organ culture (EVOC) systems have been successfully used in the study of both human and animal respiratory pathogens. Objectives  We aimed to develop an air interface EVOC using pig tracheas in the study of influenza infection demonstrating that tracheal explants can be effectively maintained in organ culture and support productive influenza infection. Methods  Tracheal explants were maintained in the air interface EVOC system for 7 days. Histological characteristics were analysed with different staining protocols and co‐ordinated ciliary movement on the epithelial surface was evaluated through a bead clearance assay. Explants were infected with a swine H1N1 influenza virus. Influenza infection of epithelial cells was confirmed by immunohistochemistry and viral replication was quantified by plaque assays and real‐time RT‐PCR. Results  Histological analysis and bead clearance assay showed that the tissue architecture of the explants was maintained for up to 7 days, while ciliary movement exhibited a gradual decrease after 4 days. Challenge with swine H1N1 influenza virus showed that the EVOC tracheal system shows histological changes consistent with in vivo influenza infection and supported productive viral replication over multiple cycles of infection. Conclusion  The air interface EVOC system using pig trachea described here constitutes a useful biological tool with a wide range of applications in the study of influenza infection.     

Nucleotide Binding Oligomerization Domain 1 Is an Essential Signal Transducer in Human Epithelial Cells Infected with Helicobacter pylori That Induces the Transepithelial Migration of Neutrophils

Background/Aims

The cytosolic host protein nucleotide binding oligomerization domain 1 (Nod1) has emerged as a key pathogen recognition molecule for innate immune responses in epithelial cells. The purpose of the study was to elucidate the mechanism by which Helicobacter pylori infection leads to transepithelial neutrophil migration in a Nod1-mediated manner.

Methods

Human epithelial cell lines AGS and Caco-2 were grown and infected with H. pylori. Interleukin (IL)-8 mRNA expression and IL-8 secretion were assessed, and nuclear factor κB (NF-κB) activation was determined. Stable transfections of AGS and Caco-2 cells with dominant negative Nod1 were generated. Neutrophil migration across the monolayer was quantified.

Results

Cytotoxin-associated gene pathogenicity island (cagPAI)(+) H. pylori infection upregulated IL-8 mRNA expression and IL-8 secretion in AGS and Caco-2 cells compared with controls. NF-κB activation, IL-8 mRNA expression and IL-8 secretion by cagPAI knockdown strains were reduced compared with those infected with the wild-type strain. NF-κB activation, IL-8 mRNA expression and IL-8 secretion in dominant-negative (DN)-Nod1 stably transfected cells were reduced compared with the controls. The transepithelial migration of neutrophils in DN-Nod1 stably transfected cells was reduced compared with that in controls.

Conclusions

Signaling through Nod1 plays an essential role in neutrophil migration induced by the upregulated NF-κB activation and IL-8 expression in H. pylori-infected human epithelial cells.     

Plant flotillins are required for infection by nitrogen-fixing bacteria

To establish compatible rhizobial-legume symbioses, plant roots support bacterial infection via host-derived infection threads (ITs). Here, we report the requirement of plant flotillin-like genes (FLOTs) in Sinorhizobium meliloti infection of its host legume Medicago truncatula. Flotillins in other organisms have roles in viral pathogenesis, endocytosis, and membrane shaping. We identified seven FLOT genes in the M. truncatula genome and show that two, FLOT2 and FLOT4, are strongly up-regulated during early symbiotic events. This up-regulation depends on bacterial Nod Factor and the plant''s ability to perceive Nod Factor. Microscopy data suggest that M. truncatula FLOT2 and FLOT4 localize to membrane microdomains. Upon rhizobial inoculation, FLOT4 uniquely becomes localized to the tips of elongating root hairs. Silencing FLOT2 and FLOT4 gene expression reveals a nonredundant requirement for both genes in IT initiation and nodule formation. FLOT4 is uniquely required for IT elongation, and FLOT4 localizes to IT membranes. This work reveals a critical role for plant flotillins in symbiotic bacterial infection.     

Hepatocellular Carcinoma Related to Schistosoma mansoni Infection: Case Series and Literature Review

Background and Aims: Schistosomiasis is a major chronic disease of humans in endemic regions, and infected individuals may develop a spectrum of pathology, including hepatic fibrosis, hepatosplenomegaly, and portal hypertension. Hepatocellular carcinoma (HCC) is considered the fifth most common cancer in the world, and there is limited and controversial evidence suggesting that Schistosoma mansoni infection may be a possible risk factor for HCC. The aim of this study was to report a case series of patients with HCC and S. mansoni infection and to conduct a literature review on the topic. Methods: From January 2002 to January 2015, an institutional database was screened retrospectively to identify patients with HCC and S. mansoni infection at a single center in the Department of Gastroenterology of University of São Paulo School of Medicine and Hospital das Clínicas, Brazil. Results: Seven cases were included. The mean age of patients was 62.1±10.3 years; six (85.7%) were male and one (14.3%) was female. All cases had positive epidemiology, coming from endemic areas of S. mansoni infection in Brazil, and four (57.1%) had previous complications (upper gastrointestinal bleeding) related to portal hypertension or surgery intervention (splenectomy) performed more than 10 years before the HCC diagnosis. Nontumoral portal vein thrombosis was identified in five (71.4%) patients. All patients had negative serology for HCV, and four (57.1%) had positivity of HBVcore antibodies without evidence of viral replication. According to BCLC staging, one (14.3%) patient was BCLC A and received TACE instead of RFA because HCC size was >30 mm; three (42.8%) BCLC B patients received sorafenib instead of local regional treatment due to the presence of nontumoral TPV. During follow-up, all patients developed tumoral progression and died. Conclusions: It remains unclear if S. mansoni infection alone has carcinogenic potential. The available literature indicates that S. Mansoni, in the presence of HBV and HCV infections, likely acts as a cofactor for the hepatic lesion and potentiates injury.     

Development of loop-mediated isothermal amplification (LAMP) for detection of Theileria equi

Several approaches have been developed for diagnosis of Theileria equi infection in horses and donkeys but all of them have limitations in practice. Due to numerous strengths including easy operation, cheapness and high sensitivity and specificity, LAMP has been already extensively used for surveillance of a number of diseases. We here set up a LAMP assay based on 18S rRNA gene for T. equi diagnosis. The approach was specific enough to differentiate T. equi from other evolutionary-related protozoa. Moreover, it was sensitive enough that LAMP was capable of detecting as much low as 10 copy target gene and 1 pg/μl blood genomic DNA. It was further demonstrated that LAMP was much more sensitive than canonical blood smear and comparable to PCR using test and field samples. The present results support an idea that LAMP developed in this study is reliable, reproducible and highly sensitive and specific, being a potential to be globally used for surveillance of T. equi infection in the field.     

Biofilm and Helicobacter pylori:From environment to human host

Helicobacter pylori(H.pylori)is a Gram negative pathogen that selectively colonizes the human gastric epithelium.Over 50%of the world population is infected with H.pylori reaching up to 90%of infected individuals in developing countries.Nonetheless the increased impact upon public health care,its reservoir and the transmission pathway of the species has not been clearly established yet.Molecular studies allowed the detection of H.pylori in various aquatic environments,even forming biofilm in tap water distribution systems in several countries,suggesting a role of water as a possible reservoir of the pathogen.The persistence of human infection with H.pylori and the resistance of clinical isolates to commonly used antibiotics in eradication therapy have been related to the genetic variability of the species and its ability to develop biofilm,demonstrated both in vivo and in vitro experiments.Thus,during the last years,experimental work with this pathogen has been focused in the search for biofilm inhibitors and biofilm destabilizing agents.However,only two anti-H.pylori biofilm disrupting agents have been successfully used:Curcumin-a natural dye-and N-acetyl cysteine-a mucolytic agent used in respiratory diseases.The main goal of this review was to discuss the evidences available in the literature supporting the ability of H.pylori to form biofilm upon various surfaces in aquatic environments,both in vivo and in vitro.The results published and our own observations suggest that the ability of H.pylori to form biofilm may be important for surviving under stress conditions or in the spread of the infection among humans,mainly through natural water sources and water distribution systems.     

Relatedness of Helicobacter pylori populations to gastric carcinogenesis

Helicobacter pylori (H. pylori) is a Gram-negative bacterium that infects half of the human population. The infection is associated with chronic inflammation of the gastric mucosa and peptic ulcers. It is also a major risk factor for gastric cancer. Phylogenetic analysis of global strains reveals there are seven populations of H. pylori, including hpAfrica1, hpAfrica2, hpEastAsia, hpEurope, hpNEAfrica, hpAsia2 and hpSahul. These populations are consistent with their geographical origins, and possibly result from geographical separation of the bacterium leading to reduced bacterial recombination in some populations. For each population, H. pylori has evolved to possess genomic contents distinguishable from others. The hpEurope population is distinct in that it has the largest genome of 1.65 mbp on average, and the highest number of coding sequences. This confers its competitive advantage over other populations but at the cost of a lower infection rate. The large genomic size could be a cause of the frequent occurrence of the deletion of the cag pathogenicity island in H. pylori strains from hpEurope. The incidence of gastric cancer varies among different geographical regions. This can be attributed in part to different rates of infection of H. pylori. Recent studies found that different populations of H. pylori vary in their carcinogenic potential and contribute to the variation in incidence of gastric cancer among geographical regions. This could be related to the ancestral origin of H. pylori. Further studies are indicated to investigate the bacterial factors contributing to differential virulence and their influence on the clinical features in infected individuals.     

What is the role of small rodents in the transmission cycle of Trypanosoma cruzi and Trypanosoma evansi (Kinetoplastida Trypanosomatidae)? A study case in the Brazilian Pantanal

Determining the reservoir hosts for parasites is crucial for designing control measures, but it is often difficult to identify the role that each host species plays in maintaining the cycle of infection in the wild. One way to identify potential maintenance hosts is to estimate key parameters associated with transmission and pathogenicity. Here we assess the potential for three native rodent species of the Brazilian Pantanal (Clyomys laticeps, Thrichomys pachyurus and Oecomys mamorae) to act as reservoir or maintenance hosts of Trypanosoma evansi, an important parasite of domestic livestock. By analyzing blood parameters of naturally infected wild-caught rodents of these species, we compared their levels of parasitemia and anemia due to T. evansi infection with literature values for other host species infected by this parasite. We also analyzed levels of these blood parameters relative to infection by Trypanosoma cruzi, the causative agent of Chagas disease in humans, for which wild rodents are already thought to be important reservoir species. All three species showed low impacts of the two trypanosomes on their blood parameters compared to other species, suggesting that they experience a low virulence of trypanosome infection under natural conditions in the Pantanal and might act as maintenance hosts of trypanosome infections. The low parasitemia of trypanosome infections suggests that these rodents play a secondary role in the transmission cycle compared to other species, especially compared to the capybara (Hydrochaeris hydrochaeris) which also experiences low pathogenicity due to infection despite much higher levels of parasitemia.     

NS1’ Protein Expression in the JaOArS982 Strain of Japanese Encephalitis Virus Does Not Enhance Virulence in Mice

Using a mouse model, we previously demonstrated that subcutaneous infection with the JaTH160 strain of Japanese encephalitis virus (JEV) causes significantly higher virulence and stronger virus propagation in the brain compared with that of the JaOArS982 strain. We also showed that the JaTH160 strain, but not JaOArS982, expresses the NS1’ protein and that NS1’ enhances JEV production in avian cells and embryonated chicken eggs. In this study, we examined whether NS1’ expression affects virulence in mice infected with the JaOArS982 and JaTH160 strains using the corresponding recombinant viruses S982-IC and JaTH-IC. Expression of the NS1’ protein in S982-IC diminished the mortality in mice, whereas S982-IC viruses without NS1’ caused 40–60% mortality. However, the viral loads in the brains of these mice were not significantly different despite the dvariation in NS1’ expression. JaTH-IC viruses depleted of the NS1’ protein exhibited high mortality levels, similar to those of the virus expressing NS1’. Previous studies showed that the NS1’ protein plays a role in the enhanced virulence of the JEV SA14 strain in mice. However, our current data suggest that NS1’ protein expression in S982-IC reduces, rather than enhances, the mortality in mice. Thus, the effect of NS1’ on pathogenicity in vivo may vary among virus strains. Our data also suggest that the reduced mortality resulting from NS1’ expression in S982-IC is not simply due to viral replication in the brains. Further investigation is needed to uncover the mechanism by which NS1’ affects pathogenicity in JEV-infected animals.     

Tight junction disruption: Helicobacter pylori and dysregulation of the gastric mucosal barrier

Long-term chronic infection with Helicobacter pylori (H. pylori) is a risk factor for gastric cancer development. In the multi-step process that leads to gastric cancer, tight junction dysfunction is thought to occur and serve as a risk factor by permitting the permeation of luminal contents across an otherwise tight mucosa. Mechanisms that regulate tight junction function and structure in the normal stomach, or dysfunction in the infected stomach, however, are largely unknown. Although conventional tight junction components are expressed in gastric epithelial cells, claudins regulate paracellular permeability and are likely the target of inflammation or H. pylori itself. There are 27 different claudin molecules, each with unique properties that render the mucosa an intact barrier that is permselective in a way that is consistent with cell physiology. Understanding the architecture of tight junctions in the normal stomach and then changes that occur during infection is important but challenging, because most of the reports that catalog claudin expression in gastric cancer pathogenesis are contradictory. Furthermore, the role of H. pylori virulence factors, such as cytotoxin-associated gene A and vacoulating cytotoxin, in regulating tight junction dysfunction during infection is inconsistent in different gastric cell lines and in vivo, likely because non-gastric epithelial cell cultures were initially used to unravel the details of their effects on the stomach. Hampering further study, as well, is the relative lack of cultured cell models that have tight junction claudins that are consistent with native tissues. This summary will review the current state of knowledge about gastric tight junctions, normally and in H. pylori infection, and make predictions about the consequences of claudin reorganization during H. pylori infection.     

Von Willebrand factor-binding protein is a hysteretic conformational activator of prothrombin

Von Willebrand factor-binding protein (VWbp), secreted by Staphylococcus aureus, displays secondary structural homology to the 3-helix bundle, D1 and D2 domains of staphylocoagulase (SC), a potent conformational activator of the blood coagulation zymogen, prothrombin (ProT). In contrast to the classical proteolytic activation mechanism of trypsinogen-like serine proteinase zymogens, insertion of the first 2 residues of SC into the NH₂-terminal binding cleft on ProT (molecular sexuality) induces rapid conformational activation of the catalytic site. Based on plasma-clotting assays, the target zymogen for VWbp may be ProT, but this has not been verified, and the mechanism of ProT activation is unknown. We demonstrate that VWbp activates ProT conformationally in a mechanism requiring its Val¹-Val² residues. By contrast to SC, full time-course kinetic studies of ProT activation by VWbp demonstrate that it activates ProT by a substrate-dependent, hysteretic kinetic mechanism. VWbp binds weakly to ProT (K_D 2.5 μM) to form an inactive complex, which is activated through a slow conformational change by tripeptide chromogenic substrates and its specific physiological substrate, identified here as fibrinogen (Fbg). This mechanism increases the specificity of ProT activation by delaying it in a slow reversible process, with full activation requiring binding of Fbg through an exosite expressed on the activated ProT*·VWbp complex. The results suggest that this unique mechanism regulates pathological fibrin (Fbn) deposition to VWF-rich areas during S. aureus endocarditis.     

Compatibility of Ugandan Schistosoma mansoni isolates with Biomphalaria snail species from Lake Albert and Lake Victoria

In order to investigate the capacity of being intermediate host for Schistosoma mansoni, the Ugandan F₁ generation of Biomphalaria snail species that were laboratory-bred from parent populations originally collected from either Lake Victoria or Lake Albert was challenged with sympatric and non-sympatric S. mansoni isolates. After a prepatent period of 20 days, a daily 10-hourly snail shedding for cercariae was done to determine the infection rate, cercarial production per hour and survival period of infected snails. The study suggests that when parasite strains from a different geographical origin is used for infection, survival of infected snails increase, leading to an increased transmission potential. Although earlier literature had indicated that the Lake Victoria Biomphalaria sudanica is refractory to S. mansoni, we showed that all Ugandan Biomphalaria spp., including B. sudanica from all locations, were highly susceptible to the S. mansoni isolates. Thus if B. choanomphala, which is an efficient intermediate host in Lake Victoria, is given an opportunity to occupy Lake Albert, it will most likely be compatible with the Albertine S. mansoni parasites. Equally, if B. stanleyi, currently restricted to Lake Albert invades Lake Victoria, it is likely to act as an efficient intermediate host. Future work should concentrate on intraspecific population-level differences in compatibility.