Tumor Invasion of Salmonella enterica Serovar Typhimurium Is Accompanied by Strong Hemorrhage Promoted by TNF-α

BACKGROUND:Several facultative anaerobic bacteria with potential therapeutic abilities are known to preferentially colonize solid tumors after systemic administration. How they efficiently find and invade the tumors is still unclear. However, this is an important issue to be clarified when bacteria should be tailored for application in cancer therapy. METHODOLOGY/PRINCIPAL FINDINGS:We describe the initial events of colonization of an ectopic transplantable tumor by Salmonella enterica serovar Typhimurium. Initially, after intravenous administration, bacteria were found in blood, spleen, and liver. Low numbers were also detected in tumors associated with blood vessels as could be observed by immunohistochemistry. A rapid increase of TNF-alpha in blood was observed at that time, in addition to other pro-inflammatory cytokines. This induced a tremendous influx of blood into the tumors by vascular disruption that could be visualized in H&E stainings and quantified by hemoglobin measurements of tumor homogenate. Most likely, together with the blood, bacteria were flushed into the tumor. In addition, blood influx was followed by necrosis formation, bacterial growth, and infiltration of neutrophilic granulocytes. Depletion of TNF-alpha retarded blood influx and delayed bacterial tumor-colonization. CONCLUSION:Our findings emphasize similarities between Gram-negative tumor-colonizing bacteria and tumor vascular disrupting agents and show the involvement of TNF-alpha in the initial phase of tumor-colonization by bacteria

Detection of Variants in 15 Genes in 87 Unrelated Chinese Patients with Leber Congenital Amaurosis

BACKGROUND: Leber congenital amaurosis (LCA) is the earliest onset and most severe form of hereditary retinal dystrophy. So far, full spectrum of variations in the 15 genes known to cause LCA has not been systemically evaluated in East Asians. Therefore, we performed comprehensive detection of variants in these 15 genes in 87 unrelated Han Chinese patients with LCA. METHODOLOGY/PRINCIPAL FINDINGS: The 51 most frequently mutated exons and introns in the 15 genes were selected for an initial scan using cycle sequencing. All the remaining exons in 11 of the 15 genes were subsequently sequenced. Fifty-three different variants were identified in 44 of the 87 patients (50.6%), involving 78 of the 88 alleles (11 homozygous and 56 heterozygous variants). Of the 53 variants, 35 (66%) were novel pathogenic mutations. In these Chinese patients, variants in GUCY2D are the most common cause of LCA (16.1% cases), followed by CRB1 (11.5%), RPGRIP1 (8%), RPE65 (5.7%), SPATA7 (4.6%), CEP290 (4.6%), CRX (3.4%), LCA5 (2.3%), MERTK (2.3%), AIPL1 (1.1%), and RDH12 (1.1%). This differs from the variation spectrum described in other populations. An initial scan of 55 of 215 PCR amplicons, including 214 exons and 1 intron, detected 83.3% (65/78) of the mutant alleles ultimately found in these 87 patients. In addition, sequencing only 9 exons would detect over 50% of the identified variants and require less than 5% of the labor and cost of comprehensive sequencing for all exons. CONCLUSIONS/SIGNIFICANCE: Our results suggest that specific difference in the variation spectrum found in LCA patients from the Han Chinese and other populations are related by ethnicity. Sequencing exons in order of decreasing risk is a cost-effective way to identify causative mutations responsible for LCA, especially in the context of genetic counseling for individual patients in a clinical setting

Physiological, biochemical, and ultrastructural characterization of selenium toxicity in cowpea plants

Selenium (Se) is considered a beneficial element for plants; however, in high concentrations, it causes negative effects on plant physiology and development. This study reports the first physiological, nutritional, and ultrastructural description of Se toxicity in cowpea growing under field conditions. Selenium was supplied as a foliar application of sodium selenite at varying concentrations (0, 50, 100, 200, 400, 800, 1200, and 1600 g ha−1). An increased yield was observed with the application of 50 g ha−1 Se. Application of concentrations higher than 50 g ha−1 caused leaf toxicity. Increased lipid peroxidation and hydrogen peroxide concentration and reduced total sugars, sucrose, and carotenoid concentration were observed at highest doses tested (1200 and 1600 g ha−1). Applications of more than 50 g ha−1 Se reduced the phloem diameter, caused chlorosis of the leaf blade with a coalescence of lesions, and caused pink salt deposits to appear. Lesions were observed mainly near the trichomes on the adaxial surface of the leaf blade. An analysis of the element distribution with microprobe X-ray fluorescence spectrometry (μ-XRF) revealed accumulation of Se, calcium (Ca), potassium (K), copper (Cu), and manganese (Mn) near the primary vein and in the necrotic brown areas of the leaf lesions. In contrast, Na was homogeneously distributed in the leaf tissue

TLR2 activation by Porphyromonas gingivalis requires both PPAD activity and fimbriae

Porphyromonas gingivalis, a keystone oral pathogen implicated in development and progression of periodontitis, may also contribute to the pathogenicity of diseases such as arthritis, atherosclerosis, and Alzheimer’s. P. gingivalis is a master manipulator of host immune responses due to production of a large variety of virulence factors. Among these, P. gingivalis peptidilarginine deiminase (PPAD), an enzyme unique to P. gingivalis, converts C-terminal Arg residues in bacterium- and host-derived proteins and peptides into citrulline. PPAD contributes to stimulation of proinflammatory responses in host cells and is essential for activation of the prostaglandin E2 (PGE2) synthesis pathway in gingival fibroblasts. Since P. gingivalis is recognized mainly by Toll-like receptor-2 (TLR2), we investigated the effects of PPAD activity on TLR2-dependent host cell responses to P. gingivalis, as well as to outer membrane vesicles (OMVs) and fimbriae produced by this organism. Using reporter cell lines, we found that PPAD activity was required for TLR2 activation by P. gingivalis cells and OMVs. We also found that fimbriae, an established TLR2 ligand, from wild-type ATCC 33277 (but not from its isogenic PPAD mutant) enhanced the proinflammatory responses of host cells. Furthermore, only fimbriae from wild-type ATCC 33277, but not from the PPAD-deficient strains, induced cytokine production and stimulated expression of genes within the PGE2 synthesis pathway in human gingival fibroblasts via activation of the NF-ĸB and MAP kinase-dependent signaling pathways. Analysis of ten clinical isolates revealed that type I FimA is preferable for TLR2 signaling enhancement. In conclusion, the data strongly suggest that both PPAD activity and fimbriae are important for TLR2-dependent cell responses to P. gingivalis infection

In vitro toxicity studies of biodegradable, polyelectrolyte nanocapsules

Alicja Karabasz,1 Krzysztof Szczepanowicz,2 Agnieszka Cierniak,1,3 Joanna Bereta,1 Monika Bzowska1 1Department of Cell Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland; 2Jerzy Haber Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, Kraków, Poland; 3Department of Biochemistry, Faculty of Medicine and Health Sciences, Andrzej Frycz Modrzewski Kraków University, Kraków, Poland Background: Toxicity of nanomaterials is one of the most important factors limiting their medical application. Evaluation of in vitro nanotoxicity allows for the identification and elimination of most of the toxic materials prior to animal testing. The current knowledge of the possible side effects of biodegradable nanomaterials, such as liposomes and polymeric organic nanoparticles, is limited. Previously, we developed a potential drug delivery system in the form of nanocapsules with polyelectrolyte, biodegradable shells consisting of poly-L-lysine and poly-L-glutamic acid (PGA), formed by the layer-by-layer adsorption technique. Methods: Hemolysis assay, viability tests, flow cytometry analysis of vascular cell adhesion molecule-1 expression on endothelium, analysis of nitric oxide production, measurement of intracellular reactive oxygen species levels, detection of antioxidant enzyme activity, and analysis of DNA damage with comet assay were performed to study the in vitro toxicity of nanocapsules. Results: In this work, we present the results of an in vitro analysis of toxicity of five-layer positively charged poly-L-lysine–terminated nanocapsules (NC5), six-layer negatively charged PGA-terminated nanocapsules (NC6) and five-layer PEGylated nanocapsules (NC5-PEG). PGA and polyethylene glycol (PEG) were used as two different “stealth” polymers. Of all the polyelectrolyte nanocapsules tested for blood compatibility, only cationic NC5 showed acute toxicity toward blood cells, expressed as hemolysis and aggregation. Neither NC6 nor NC5-PEG had proinflammatory activity evaluated through changes in the expression of NF-κB–dependent genes, iNOS and vascular cell adhesion molecule-1, induced oxidative stress, or promoted DNA damage in various cells. Conclusion: Our studies clearly indicate that PGA-coated (negatively charged) and PEGylated polyelectrolyte nanocapsules do not show in vitro toxicity, and their potential as a drug delivery system may be safely studied in vivo. Keywords: polyelectrolyte nanocapsules, layer-by-layer, nanotoxicity, oxidative stress, genotoxicit

Transforming growth factor-β and epidermal growth factor modulate basal and interleukin-6-induced amino acid uptake and acute phase protein synthesis in cultured rat hepatocytes

AbstractRat hepatocytes cultured for 2 days with interleukin-6 show increased synthesis of acute phase proteins and enhanced accumulation of 14C-labelled α-aminoisobutyric acid. Transforming growth factor-β1 (0.1–10 ng/ml) inhibits whereas epidermal growth factor (1–100 ng/ml) enhances both basal and interleukin-6-induced amino acid uptake by rat hepatocytes with only a slight alteration of acute phase protein synthesis