Inoculation Of Mice Research Articles

F. novicida-Infected A. castellanii Does Not Enhance Bacterial Virulence in Mice.

Francisella tularensis is a facultative intracellular bacterium that causes tularemia in humans and animals. Epidemiology of tularemia worldwide is often associated with water-borne transmission, which includes mosquitoes and amoebae as the potential host reservoirs of the bacteria in water environment. In vitro studies showed intracellular replication of F. tularensis within Acanthamoeba castellanii and Hartmanella vermiformis cells. While infection of amoeba by Legionella pneumophila has been shown to enhance infectivity of L. pneumophila the role of F. tularensis-infected protozoa in the pathogenesis of tularemia is not known. We used 6 h coculture of A. castellanii and F. novicida for investigation of the effect of inhaled amoeba on the pathogenesis of tularemia on in vivo model. Balb/c mice were infected intratracheally with F. novicida or with F. novicida-infected A. castellanii. Surprisingly, infection with F. novicida-infected A. castellanii did not lead to bronchopneumonia in Balb/c mice, and Francisella did not disseminate into the liver and spleen. Upon inhalation, F. novicida infects a variety of host cells, though neutrophils are the predominant cells early during infection in the lung infiltrates of pulmonary tularemia. The numbers of neutrophils in the lungs of Balb/c mice were significantly lower in the infection of mice with F. novicida-infected A. castellanii in comparison to group of mice infected only with F. novicida. These results demonstrate that following inoculation of mice with F. novicida-infected A. castellanii, mice did not develop tularemia.

Dietary Regulation of the Gut Microbiota Engineered by a Minimal Defined Bacterial Consortium.

We have recently reported that Altered Schaedler Flora (ASF) can be used to durably engineer the gut microbiota to reduce ammonia production as an effective modality to reduce morbidity and mortality in the setting of liver injury. Here we investigated the effects of a low protein diet on ASF colonization and its ability to engineer the microbiota. Initially, ASF inoculation was similar between mice fed a normal protein diet or low protein diet, but the outgrowth of gut microbiota differed over the ensuing month. Notable was the inability of the dominant Parabacteroides ASF taxon to exclude other taxa belonging to the Bacteroidetes phylum in the setting of a low protein diet. Instead, a poorly classified yet highly represented Bacteroidetes family, S24-7, returned within 4 weeks of inoculation in mice fed a low protein diet, demonstrating a reduction in ASF resilience in response to dietary stress. Nevertheless, fecal ammonia levels remained significantly lower than those observed in mice on the same low protein diet that received a transplant of normal feces. No deleterious effects were observed in host physiology due to ASF inoculation into mice on a low protein diet. In total, these results demonstrate that low protein diet can have a pronounced effect on engineering the gut microbiota but modulation of ammonia is preserved.

CARD9 impacts colitis by altering gut microbiota metabolism of tryptophan into aryl hydrocarbon receptor ligands.

Complex interactions between the host and the gut microbiota govern intestinal homeostasis but remain poorly understood. Here we reveal a relationship between gut microbiota and caspase recruitment domain family member 9 (CARD9), a susceptibility gene for inflammatory bowel disease (IBD) that functions in the immune response against microorganisms. CARD9 promotes recovery from colitis by promoting interleukin (IL)-22 production, and Card9(-/-) mice are more susceptible to colitis. The microbiota is altered in Card9(-/-) mice, and transfer of the microbiota from Card9(-/-) to wild-type, germ-free recipients increases their susceptibility to colitis. The microbiota from Card9(-/-) mice fails to metabolize tryptophan into metabolites that act as aryl hydrocarbon receptor (AHR) ligands. Intestinal inflammation is attenuated after inoculation of mice with three Lactobacillus strains capable of metabolizing tryptophan or by treatment with an AHR agonist. Reduced production of AHR ligands is also observed in the microbiota from individuals with IBD, particularly in those with CARD9 risk alleles associated with IBD. Our findings reveal that host genes affect the composition and function of the gut microbiota, altering the production of microbial metabolites and intestinal inflammation.

Netrin-1 Interrupts Amyloid-β Amplification, Increases sAβPPα in vitro and in vivo, and Improves Cognition in a Mouse Model of Alzheimer's Disease.

Recent studies have shown that inoculation of susceptible mice with amyloid-β (Aβ) peptides accelerates Aβ deposition in the brain, supporting the idea that Aβ may be self-amplifying; however, the exact mechanism is not understood. Here we provide evidence that Aβ may self-amplify, in part, by inhibiting α-secretase ADAM10 (a disintegrin and metalloprotease) cleavage of full-length Aβ precursor protein (FL AβPP) and therefore allow greater β-secretase processing, and that Aβ itself is a substrate for ADAM10. Exposure of primary neuronal cultures from PDAβPP mice to exogenous rat Aβ1-40 resulted in increased de novo human Aβ1-42 production and exposure of cells to Aβ decreased production of ADAM10 cleavage product soluble AβPPα (sAβPPα). In a cell-free assay, Aβ decreased ADAM10 cleavage of the chimeric substrate MBP-AβPPC125 and Aβ itself was apparently cleaved by the enzyme. The axonal guidance and trophic factor netrin-1, however, reduced the Aβ1-40-induced Aβ1-42 increase, increased sAβPPα, and reversed the Aβ-induced sAβPPα decrease in vitro. In vivo, induction of netrin-1 expression in PDAβPPSwe/Ind transgenic mice resulted in reductions in both Aβ1-42 and Aβ1-40, and ICV delivery of netrin-1 to PDAβPPSwe/Ind mice increased sAβPPα, decreased Aβ, and improved working memory. Finally, to support further study of netrin-1's potential as a therapeutic for Alzheimer's disease, pilot gene therapy studies were performed and a netrin mimetic peptide synthesized and tested that, like netrin, can increase sAβPPα and decrease Aβ1-42in vitro. Taken together, these data provide mechanistic insights into Aβ self-amplification and the ability of netrin-1 to disrupt it.

Burkholderia pseudomallei Capsule Exacerbates Respiratory Melioidosis but Does Not Afford Protection against Antimicrobial Signaling or Bacterial Killing in Human Olfactory Ensheathing Cells.

Melioidosis, caused by the bacterium Burkholderia pseudomallei, is an often severe infection that regularly involves respiratory disease following inhalation exposure. Intranasal (i.n.) inoculation of mice represents an experimental approach used to study the contributions of bacterial capsular polysaccharide I (CPS I) to virulence during acute disease. We used aerosol delivery of B. pseudomallei to establish respiratory infection in mice and studied CPS I in the context of innate immune responses. CPS I improved B. pseudomallei survival in vivo and triggered multiple cytokine responses, neutrophil infiltration, and acute inflammatory histopathology in the spleen, liver, nasal-associated lymphoid tissue, and olfactory mucosa (OM). To further explore the role of the OM response to B. pseudomallei infection, we infected human olfactory ensheathing cells (OECs) in vitro and measured bacterial invasion and the cytokine responses induced following infection. Human OECs killed >90% of the B. pseudomallei in a CPS I-independent manner and exhibited an antibacterial cytokine response comprising granulocyte colony-stimulating factor, tumor necrosis factor alpha, and several regulatory cytokines. In-depth genome-wide transcriptomic profiling of the OEC response by RNA-Seq revealed a network of signaling pathways activated in OECs following infection involving a novel group of 378 genes that encode biological pathways controlling cellular movement, inflammation, immunological disease, and molecular transport. This represents the first antimicrobial program to be described in human OECs and establishes the extensive transcriptional defense network accessible in these cells. Collectively, these findings show a role for CPS I in B. pseudomallei survival in vivo following inhalation infection and the antibacterial signaling network that exists in human OM and OECs.

Streptococcus pneumoniae Cell-Wall-Localized Phosphoenolpyruvate Protein Phosphotransferase Can Function as an Adhesin: Identification of Its Host Target Molecules and Evaluation of Its Potential as a Vaccine.

In Streptococcus pneumonia, phosphoenolpyruvate protein phosphotransferase (PtsA) is an intracellular protein of the monosaccharide phosphotransferase systems. Biochemical and immunostaining methods were applied to show that PtsA also localizes to the bacterial cell-wall. Thus, it was suspected that PtsA has functions other than its main cytoplasmic enzymatic role. Indeed, recombinant PtsA and anti-rPtsA antiserum were shown to inhibit adhesion of S. pneumoniae to cultured human lung adenocarcinoma A549 cells. Screening of a combinatorial peptide library expressed in a filamentous phage with rPtsA identified epitopes that were capable of inhibiting S. pneumoniae adhesion to A549 cells. The insert peptides in the phages were sequenced, and homologous sequences were found in human BMPER, multimerin1, protocadherin19, integrinβ4, epsin1 and collagen type VIIα1 proteins, all of which can be found in A549 cells except the latter. Six peptides, synthesized according to the homologous sequences in the human proteins, specifically bound rPtsA in the micromolar range and significantly inhibited pneumococcal adhesion in vitro to lung- and tracheal-derived cell lines. In addition, the tested peptides inhibited lung colonization after intranasal inoculation of mice with S. pneumoniae. Immunization with rPtsA protected the mice against a sublethal intranasal and a lethal intravenous pneumococcal challenge. In addition, mouse anti rPtsA antiserum reduced bacterial virulence in the intravenous inoculation mouse model. These findings showed that the surface-localized PtsA functions as an adhesin, PtsA binding peptides derived from its putative target molecules can be considered for future development of therapeutics, and rPtsA should be regarded as a candidate for vaccine development.

An experimental challenge model of visceral leishmaniasis by Leishmania donovani promastigotes in mice

Although visceral leishmaniasis is a fatal disease in humans and dogs, the use of mouse models is important for obtaining a better understanding of the pathogenesis, immunity, and host–parasite interactions of this disease. Such models are also useful for the evaluation of vaccines and chemotherapies for treatment of visceral leishmaniasis. Here, we present our method of experimental inoculation of mice with Leishmania donovani promastigotes. Nutrient-enriched undefined media may be beneficial for laboratory maintenance of promastigotes for maintaining their virulence or infectivity in mice. With this method, we could preserve the infectivity of promastigote lines recovered from inoculated animals and use these lines for further in vivo experiments. Furthermore, the use of cryopreserved stabilates is highly recommended for the reproducibility of experiments. To assess a newly developed method for determination of parasite burden in infected animal tissues, initial comparison of parasite burden in the liver obtained in the classic Leishman-Donovan units (LDU) with values obtained from the new method is recommended. As an example, the association between parasite burden determined by LDU and real-time PCR assay targeting the leishmanial gp63 gene in the liver of mice is presented.

Retrospective diagnosis of two rabies cases in humans by high throughput sequencing

BackgroundRabies is prevalent in 150 countries and is definitely found in Russian Federation. The average registered incidence of rabies infection among animals in Russia is 3000 cases per year. At least 500,000 cases of animal bites and scratches are registered in the Russia every year, but only 2–4 cases of rabies infection in humans are reported per year. This relatively low incidence of rabies infection among humans is the result of a well-organized program of rabies surveillance and control as well as the readily available vaccination and immunoglobulin therapies. However, physician awareness of rabies infection in patients with acute encephalopathy is low, and some cases of rabies remain undiagnosed. ObjectivesA retrospective study of autopsy materials from patients who died of encephalitis of unknown etiology in the Astrakhan region of Russia in 2003. Study designA broad-range polymerase chain reaction (PCR) analysis followed by high throughput sequencing were used for the diagnosis. ResultsTwo cases of rabies were detected and subsequently confirmed using a fluorescent antibody test, an enzyme-linked immunosorbent assay (ELISA) and a mouse inoculation test. Two strains of rabies virus were isolated and characterized using virological methods. The entire genome of each strain was sequenced.

Potential application of silver nanoparticles to control the infectivity of Rift Valley fever virus in vitro and in vivo

In this work we have tested the potential antiviral activity of silver nanoparticles formulated as Argovit™ against Rift Valley fever virus (RVFV). The antiviral activity of Argovit was tested on Vero cell cultures and in type-I interferon receptor deficient mice (IFNAR −/− mice) by two different approaches: (i) different dilutions of Argovit were added to previously infected cells or administrated to animals infected with a lethal dose of virus; (ii) virus was pre-incubated with different dilutions of Argovit before inoculation in mice or cells. Though the ability of silver nanoparticles to control an ongoing RVFV infection in the conditions tested was limited, the incubation of virus with Argovit before the infection led to a reduction of the infectivity titers both in vitro and in vivo. These results reveal the potential application of silver nanoparticles to control the infectivity of RVFV, which is an important zoonotic pathogen.

Diversity of viruses in Ixodes ricinus, and characterization of a neurotropic strain of Eyach virus.

Ticks transmit more pathogens—including bacteria, parasites and viruses—than any other arthropod vector. Although the epidemiological status of many tick-borne bacteria is very well characterized, tick-borne viruses are still relatively under-studied. Recently, several novel tick-borne viruses have been isolated from human febrile illnesses following tick bites, indicating the existence of other potential new and unknown tick-borne viruses. We used high-throughput sequencing to analyse the virome of Ixodes ricinus, the main vector of tick-borne pathogens in Europe. The majority of collected viral sequences were assigned to two potentially novel Nairovirus and Phlebovirus viruses, with prevalence rates ranging from 3.95% to 23.88% in adults and estimated to be between 0.14% and 72.16% in nymphs. These viruses could not be isolated from the brains of inoculated immunocompromised mice, perhaps indicating that they are unable to infect vertebrates. Within the I. ricinus virome, we also identified contigs with >90% identity to the known Eyach virus. Initially isolated in the 1980s, this virus was indirectly associated with human disease, but had never been extensively studied. Eyach virus prevalence varied between 0.07% and 5.26% in ticks from the French Ardennes and Alsace regions. Eyach virus was successfully isolated following intracerebral inoculation of immunocompromised mice with Eyach virus-positive tick extracts. This virus was also able to multiply and persist in the blood of immunocompetent mice inoculated by intraperitoneal injection, and caused brain infections in three of nine juveniles, without any obvious deleterious effects.

Dried Saliva Spots: A Robust Method for Detecting Streptococcus pneumoniae Carriage by PCR.

The earliest studies in the late 19th century on Streptococcus pneumoniae (S. pneumoniae) carriage used saliva as the primary specimen. However, interest in saliva declined after the sensitive mouse inoculation method was replaced by conventional culture, which made isolation of pneumococci from the highly polymicrobial oral cavity virtually impossible. Here, we tested the feasibility of using dried saliva spots (DSS) for studies on pneumococcal carriage. Saliva samples from children and pneumococcus-spiked saliva samples from healthy adults were applied to paper, dried, and stored, with and without desiccant, at temperatures ranging from −20 to 37 °C for up to 35 days. DNA extracted from DSS was tested with quantitative-PCR (qPCR) specifically for S. pneumoniae. When processed immediately after drying, the quantity of pneumococcal DNA detected in spiked DSS from adults matched the levels in freshly spiked raw saliva. Furthermore, pneumococcal DNA was stable in DSS stored with desiccant for up to one month over a broad range of temperatures. There were no differences in the results when spiking saliva with varied pneumococcal strains. The collection of saliva can be a particularly useful in surveillance studies conducted in remote settings, as it does not require trained personnel, and DSS are resilient to various transportation conditions.

Mosquito saliva induced cutaneous events augment Chikungunya virus replication and disease progression

Chikungunya virus (CHIKV) is transmitted when infected mosquito probes the host skin. While probing, mosquito saliva is expectorated into host skin along with virus which contains cocktail of molecules having anti-hemostatic and immunomodulatory properties. As mosquito saliva is a critical factor during natural arboviral infection, therefore we investigated mosquito saliva induced cutaneous events that modulate CHIKV infection. The effect of mosquito saliva on CHIKV infection was examined through inoculation of suckling mice subcutaneously with either CHIKV alone or uninfected mosquito bite followed by CHIKV. Histopathological evaluation of skin revealed infiltration of transmigrated inflammatory cells. Dermal blood vessels were hyperemic and adnexa showed degenerating lesions. Severe hemorrhage was observed in dermis and hypodermis in mosquito bite+CHIKV group compared to CHIKV group. Analysis of cytokines in skin showed significant downregulation of inflammatory genes like TLR-3, IL-2, IFN-γ, TNF-α and IFN-β in mosquito bite+CHIKV group compared to CHIKV group. In contrast, significant upregulation of anti-inflammatory genes like IL-4 and IL-10 was observed. These early events might have been responsible for increased dissemination of CHIKV to serum and peripheral organs as demonstrated through >10-fold higher viremia, antigen localization, cellular infiltration and degenerative changes. Thus mosquito saliva induced early cellular infiltration and associated cytokines augment CHIKV pathogenesis in a mouse model. This mosquito improved CHIKV mouse model simulates the realistic conditions that occur naturally during infected mosquito bite to a host. It will lead to better understanding of CHIKV pathobiology and promote the evaluation of novel medical countermeasures against emerging CHIKV.

⁶⁸Ga-NOTA-UBI-29-41 as a PET Tracer for Detection of Bacterial Infection.

The cationic peptide (68)Ga-NOTA-UBI-29-41 was synthesized and characterized. Biodistribution and PET/CT examinations were performed for evaluation of its biologic behavior. Differentiation of infection from sterile inflammation was investigated using microbiology methods at the sites of bacterial infections. Labeling of UBI-29-41 conjugated with NOTA with (68)Ga was optimized at 20°C-100°C and pH 3.5-5.5. Radiochemical purity, stability up to 260 min, and binding to serum proteins were determined. In vitro binding to Staphylococcus aureus was evaluated from 9.14 × 10(7) to 1.17 × 10(10) cfu/mL. Of 3 groups of Mus musculus Swiss male mice, the first was inoculated intramuscularly with 1.2 × 10(8) cfu of S. aureus to provoke infection, and the second, with 1.2 × 10(8) cfu of heat shock-treated S. aureus to generate sterile inflammation. The third mouse was not treated and served as a control. After 24 h, (68)Ga-NOTA-UBI-29-41 was administrated intravenously, and biodistribution was performed at 30, 60, and 120 min. PET/CT dynamic studies (120 min) were acquired. Sinograms were reconstructed using 3D maximum-likelihood expectation maximization and analyzed with software. Infected or inflamed muscles were dissected, homogenized, and cultured in tryptic soy agar medium. Recovered S. aureus was calculated as cfu/g. (68)Ga-NOTA-UBI-29-41 showed high renal excretion (83.2% ± 7.3%) of injected dose and rapid blood clearance. More than 95% was bound in vitro to 5 × 10(9) cfu/mL. A significantly higher (P< 0.05) accumulation of (68)Ga-NOTA-UBI-29-41 was observed at sites of S. aureus inoculation in infected mice (ratio of target to nontarget, 5.0 at 60 min and 4.1 at 120 min) compared with animals with inflammation (ratio of target to nontarget, 1.6 at 60 min and 1.2 at 120 min). The difference in uptake of (68)Ga-NOTA-UBI-29-41 in the infected muscles compared with the inflamed muscles was clearly observed in the PET/CT images and positively correlated with the degree of infection.

Emerging evidence of the role of gut microbiota in the development of allergic diseases

The purpose is to review recent studies examining the role of gut microbiota in allergic diseases and asthma. Work in experimental models gives further evidence that a disturbed gut microbiota influences the propensity to develop allergic manifestations, and that changing the gut microbiota by dietary means (high fiber/acetate or prebiotics) in pregnancy may reduce the risk of allergic airways disease and food allergy in the offspring, respectively. The gut microbiome in established allergic disease and prior to disease onset has also been assessed in clinical trials. One study demonstrated a strong association between high abundance of Faecalibacterium prausnitzii and decreased levels of butyrate and propionate, and established eczema. Lower relative abundance of Ruminococcaceae appears to be implicated in food sensitization and to precede the development of atopic eczema. Decreased relative abundance of Lachnospira, Veillonella, Faecalibacterium, and Rothia in early infancy was reported to be associated with increased asthma risk. Inoculation of germ-free mice with these genera decreased airway inflammation in their offspring thereby proposing a causal role of bacteria in preventing allergic airways disease. Gut microbiome research is an actively developing field. Although candidate bacterial taxa have been reported it still remains unclear which bacteria (or other microbes), in which numbers and combinations, and when during the gut colonization process may prevent allergic diseases and asthma. There is still a call for standardized approaches that will enable direct comparison of different studies.

Dynamics of Parasitaemia in &amp;lt;i&amp;gt;Trypanosoma evansi&amp;lt;/i&amp;gt; Infection in Donkeys and the Efficacies of Buparvaquone and Isometamidium chloride Treatments

The aim of this study was to evaluate the efficacies of Buparvaquone (an anti-theilerial drug) and Isometamidium chloride (a standard trypanocide) in the treatment of experimental Trypanosoma evansi infection in donkeys. Out of a total of 24 donkeys, 6 were assigned to each of the experimental groups A1, A2, A3 and B at random. About 2.0x106 T. evansi parasite in 2 mL inoculum was used to infect each of the donkeys in groups A1, A2 and A3 through jugular vein while group B remained as Un-infected control. On day 28 post-infection, animals in groups A2 and A3 were treated with Isometamidium chloride and Buparvaquone respectively. Parasitaemia levels were evaluated post-infection and post-treatment using Haematocrit Centrifugation Technique and supported with mice inoculation tests to ascertain effectiveness of treatments. Group means of parasitaemia were presented on graphs. Result showed that parasites were first detectable from peripheral blood of the infected animals from day 3 and by day 4 post-infection all animals were parasitaemic and the levels fluctuated in subsequent days. The infected-untreated group of animals was aparasitaemic on day 100 post-infection onward using haematocrit centrifugation technique test but mice inoculation test detected relapse with death of all mice used due to heavy parasitemia. Similarly, the buparvaquone-treated group was aparasitemic on day 49 post-treatment (corresponding to day 77 post-infection) but mice inoculation test detected relapse of parasitaemia in the group and all mice used also died. Isometamidium-treated animals were negative for parasitaemia for 50 days on mice inoculation test but later on day 100 post-treatment, showed 60% relapse with parasiaemia level of 1+ in the mice used and they did not die within 48 days of observation. In conclusion, experimental Trypanosoma evansi infection in donkeys have pre-patent period of 3-4 days, parasitaemia pattern is undulating and infected-untreated animals became subclinical carriers from day 100 post-infection. The parasites in untreated, carrier animals maintained their virulence and pathogenicity. Buparvaquone on the other hand, depressed parasitaemia but the parasites were virulent and pathogenic to mice. Isometamidium chloride treatment resulted in incomplete clearance of parasitaemia due to Trypanosoma evansi and the relapsed parasites were avirulent and apathogenic. This is also the first report on the trial of buparvaquone for anti-Trypanosoma evansi effect in donkeys.

Rabies lyssavirus Isolates from Brazilian Different Reservoirs Species Present Distinct Pattern of Propagation in N2a Cell

Background: Rabies cell culture infection test was developed for the isolation of Rabies lyssavirus and as an alternative for the mouse inoculation test. However, tissue culture for street rabies strains produces low viral titer. Here, we assessed the quantity of brain tissue for successful viral isolation toward increased virus titer in effective way. Methods: Brain tissue isolates from different reservoirs species of Brazil were harvested in different concentration and inoculated in mouse neuroblastoma cells (N2a). These isolates were measured infectious viral titer and cell viability followed by consecutive passages in N2a cells. Results: Inoculum containing were prominent Rabies lyssavirus due to higher viral titer and not significantly dead cell. After consecutive passages in N2a cells Rabies lyssavirus variant maintained by vampire bat had remarkable adaptation to the culture system, while isolates from marmoset presents distinct pattern of propagation in N2a cell when compared with other groups. Conclusion: Based on these results, the isolation followed by viral replication assay may be used in isolates from different reservoirs which enable an effective amplification of the wild type virus strains.

Phosphoenolpyruvate Phosphotransferase System Components Modulate Gene Transcription and Virulence of Borrelia burgdorferi

The phosphoenolpyruvate phosphotransferase system (PEP-PTS) and adenylate cyclase (AC) IV (encoded by BB0723 [cyaB]) are well conserved in different species of Borrelia. However, the functional roles of PEP-PTS and AC in the infectious cycle of Borrelia have not been characterized previously. We examined 12 PEP-PTS transporter component mutants by needle inoculation of mice to assess their ability to cause mouse infection. Transposon mutants with mutations in the EIIBC components (ptsG) (BB0645, thought to be involved in glucose-specific transport) were unable to cause infection in mice, while all other tested PEP-PTS mutants retained infectivity. Infectivity was partially restored in an in trans-complemented strain of the ptsG mutant. While the ptsG mutant survived normally in unfed as well as fed ticks, it was unable to cause infection in mice by tick transmission, suggesting that the function of ptsG is essential to establish infection by either needle inoculation or tick transmission. In Gram-negative organisms, the regulatory effects of the PEP-PTS are mediated by adenylate cyclase and cyclic AMP (cAMP) levels. A recombinant protein encoded by B. burgdorferi BB0723 (a putative cyaB homolog) was shown to have adenylate cyclase activity in vitro; however, mutants with mutations in this gene were fully infectious in the tick-mouse infection cycle, indicating that its function is not required in this process. By transcriptome analysis, we demonstrated that the ptsG gene may directly or indirectly modulate gene expression of Borrelia burgdorferi. Overall, the PEP-PTS glucose transporter PtsG appears to play important roles in the pathogenesis of B. burgdorferi that extend beyond its transport functions.

Malignant transformation of human gastric epithelium cells via reactive oxygen species production and Wnt/β-catenin pathway activation following 40-week exposure to ochratoxin A

Ochratoxin A (OTA), one of the most abundant food-contaminating mycotoxins, is a possible carcinogenic to humans. We previously demonstrated that OTA treatment induced oxidative damage in human gastric epithelium cells (GES-1) in vitro. In this study, we found that long-term OTA treatment could result in increased proliferation, migration, and invasion abilities of GES-1 cells and induce anchorage-independent growth of cells in soft agar. Inoculation of OTA-treated GES-1 cells resulted in the formation of tumor xenografts in Balb/c nude mice in vivo, confirming that long-term OTA treatment can induce the malignant transformation of GES-1 cells. In addition, we found that long-term OTA treatment induced oxidative stress and activated the Wnt/β-catenin pathway, including the nuclear transition of β-catenin and the upregulation of the downstream molecules of the pathway. Finally, pretreatment with the antioxidant N-acetyl-l-cysteine (NAC) inhibited ROS formation and activation of the Wnt pathway in OTA-transformed GES-1 cells, which decreased the tumor formation abilities of these cells after inoculation in nude mice. These findings suggest that long-term OTA exposure induces the malignant transformation of GES-1 cells via intracellular ROS production and activation of the Wnt/β-catenin signaling pathway.

Antioxidant and hepatoprotective role of gold nanoparticles against murine hepatic schistosomiasis.

In recent years, gold nanoparticles (AuNPs) have become the focus of much attention in biomedical research, especially in the context of nanomedicine, due to their distinctive physicochemical properties. The current study was planned to assess the effect of three dose levels of AuNPs on the gene expression, histology, and oxidative stress status of Schistosoma mansoni-infected mice liver. Inoculation of mice with 100 μL AuNPs at different doses (0.25, 0.5, and 1 mg/kg mice body weight) twice on day 46 and day 49 postinfection reduced the total worm burden, the egg load in the liver, and the granuloma size. AuNPs also appeared to decrease the activities of malondialdehyde and nitric oxide significantly, and increase the level of glutathione compared to the infected untreated group. Concomitantly, AuNPs ameliorated the inflammatory response by decreasing the mRNA expression of interleukin-1β, interleukin-6, tumor necrosis factor-α, interferon-γ, and inducible nitric oxide synthase. These consistent molecular, histopathological, and biochemical data suggest that AuNPs could ameliorate infection-induced damage in the livers of mice. Our results indicated that AuNPs are effective anti-schistosomal and antioxidant agents. Further confirmation of the role of nanogold as an anti-schistosomal agent, as well as its mechanism of action, requires further studies to be undertaken in the future.

Virus isolation in cell culture for confirmatory diagnostic of rabies in bovine specimens

ABSTRACT: This study investigated the suitability of virus isolation (VI) in mouse neuroblastoma cells (N2A) and baby hamster kidney cells (BHK-21) as a confirmatory test for diagnosis of bovine rabies. Fourty-eight brain samples from cattle suspected of rabies were initially submitted to fluorescent antibody test (FAT) and mouse inoculation test (MIT) for routine diagnostic. Subsequently, these specimens were submitted to three protocols of VI in each cell line: a single 24h or 72h passage (T1, T2), or three 48h passages (T3). The FAT and MIT combined detected 32/48 positive samples, from which MIT detected 32 and FAT 31. The average time required for final MIT results was 12.3 days (8 - 21). VI in BHK-21 cells provided definitive, positive results in 100% of the samples in 72h (T2) and in 96.9% after three 48h passages (T3). VI in N2A cells yielded positive results in 100% in 72h (T2) and in 93.7% of samples after three 48h passages (T3). Sensitivity, specificity, positive and negative predictive values were 100% in T2 in N2A and BHK-21 cells, and the Kappa value was excellent in both cells (k=1). A single 24h passage (T1) in both cell lines performed poorly, detecting less than 40% of the positive samples. Taking together, these results indicate that VI in both cell lines, especially in BHK-21 cells that grow faster and are much easier to maintain, does represent an adequate alternative for MIT as a confirmatory test for rabies diagnostic in bovine specimens, yielding reliable results in reduced time.