Optimization of a Microplate Assay for Generating Listeria Monocytogenes, E. Coli O157:H7, and Salmonella Biofilms and Enzymatic Recovery for Enumeration

Biofilms enable the persistence of pathogens in food processing environments. Sanitizing agents are needed that are effective against pathogens entrapped in biofilms that are more difficult to inactivate than planktonic cells that are displaced and found on equipment surfaces. We examined conditions to develop, analyze, and enumerate the enhanced biofilms of three different foodborne pathogens assisted by fluorescence adherence assay and enzymatic detachment. We compared three different isomeric forms of fluorescent substrates that are readily taken up by bacterial cells based on carboxy-fluorescein diacetate (5-CFDA, 5,6-CFDA, 5,6-CFDA, SE). Biofilm-forming strains of Escherichia coli O157:H7 F4546 and Salmonella Montevideo FSIS 051 were identified using a microplate fluorescence assay defined previously for L. monocytogenes. Adherence levels were determined by differences in relative fluorescence units (RFU) as well as recovered bacterial cells. Multiple hydrolytic enzymes were examined for each representative pathogen for the most suitable enzyme for detachment and enumeration to confirm adherence data obtained by fluorescence assay. Cultures were grown overnight in microplates, incubated, washed and replenished with fresh sterile growth medium; this cycle was repeated for seven consecutive days to enrich for robust biofilms. Treatments were performed in triplicate and compared by one-way analysis of variance (ANOVA) to determine significant differences (p < 0.05)

Asialoglycoprotein receptor targeted delivery of doxorubicin nanoparticles for hepatocellular carcinoma

<p>We report asialoglycoprotein receptor (ASGPR)-targeted doxorubicin hydrochloride (Dox) nanoparticles (NPs) for hepatocellular carcinoma (HCC). Polyethylene sebacate (PES)-Gantrez® AN 119 Dox NPs of average size 220 nm with PDI < 0.62 and ∼20% Dox loading were prepared by modified nanoprecipitation. ASGPR ligands, pullulan (Pul), arabinogalactan (AGn), and the combination (Pul-AGn), were anchored by adsorption. Ligand anchoring enabled high liver uptake with a remarkable hepatocyte:nonparenchymal cell ratio of 85:15. Furthermore, Pul-AGn NPs exhibited an additive effect implying incredibly high hepatocyte accumulation. Galactose-mediated competitive inhibition confirmed ASGPR-mediated uptake of ligand-anchored NPs in HepG2 cell lines. Subacute toxicity in rats confirmed the safety of the NP groups. However, histopathological evaluation suggested mild renal toxicity of AGn. Pul NPs revealed sustained reduction in tumor volume in PLC/PRF/5 liver tumor-bearing Nod/Scid mice up to 46 days. Extensive tumor necrosis, reduced collagen content, reduction in the HCC biomarker serum α-fetoprotein (<i>p</i> < 0.05), a mitotic index of 1.135 (day 46), and tumor treated/tumor control (T/C) values of <0.42 signified superior efficacy of Pul NPs. Furthermore, weight gain in the NP groups, and no histopathological alterations indicated that they were well tolerated by the mice. The high efficacy coupled with greater safety portrayed Pul Dox NPs as a promising nanocarrier for improved therapy of HCC.</p

Use of short interfering RNA delivered by cationic liposomes to enable efficient down-regulation of ptpn22 gene in human t lymphocytes

Insulin-dependent diabetes mellitus and thyroid disease are the most common autoimmune endocrine disorders; these are due to target cell destruction by autoreactive T lymphocytes. They occur frequently together. The associated condition is named autoimmune polyglandular syndrome Type 3 variant. The substitutive administration of the deficient hormones is the standard treatment that, however, does not halt the autoimmune process. The incidence of these disorders in on the increase worldwide, therefore identification of innovative immunotherapies, especially aimed to preserve the residual hormone producing cells, is of crucial importance for the quality of life in pediatric patients. Recently, particular interest was generated by the potential pathophysiological role played in several autoimmune conditions by the C1858T PTPN22 (protein tyrosine phosphatase N22 gene) mutation encoding for the R620W lymphoid tyrosine phosphatase variant protein. The PTPN22 encoded Lyp protein is a negative regulator of T cell antigen receptor signaling, acting in concert with C-terminal Src kinase. R620W variant leads to a gain of function mutation with paradoxical reduced T cell activation. The variant has effect on both innate and adaptive immune responses. The goal of this study was to develop novel PTPN22 antisense oligomers and optimize their delivery into Jurkat T cells and peripheral blood lymphocytes by using liposomal carriers. We generated lipoplexes allowing T cell specific knockdown by using antisense strand complementary to the mRNA target site of PTPN22. The results of our study open the pathway to future trials for the treatment of autoimmune diseases based on variant PTPN22 allele selective inhibition using lipoplexes of SiRNA antisense oligomers