Neuroretinal degeneration in a mouse model of systemic chronic immune activation observed by proteomics.

Blindness or vision loss due to neuroretinal and photoreceptor degeneration affects millions of individuals worldwide. In numerous neurodegenerative diseases, including age-related macular degeneration, dysregulated immune response-mediated retinal degeneration has been found to play a critical role in the disease pathogenesis. To better understand the pathogenic mechanisms underlying the retinal degeneration, we used a mouse model of systemic immune activation where we infected mice with lymphocytic choriomeningitis virus (LCMV) clone 13. Here, we evaluated the effects of LCMV infection and present a comprehensive discovery-based proteomic investigation using tandem mass tag (TMT) labeling and high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS). Changes in protein regulation in the posterior part of the eye, neuroretina, and RPE/choroid were compared to those in the spleen as a secondary lymphoid organ and to the kidney as a non-lymphoid but encapsulated organ at 1, 8, and 28 weeks of infection. Using bioinformatic tools, we found several proteins responsible for maintaining normal tissue homeostasis to be differentially regulated in the neuroretina and the RPE/choroid during the degenerative process. Additionally, in the organs we observed, several important protein pathways contributing to cellular homeostasis and tissue development were perturbed and associated with LCMV-mediated inflammation, promoting disease progression. Our findings suggest that the response to a systemic chronic infection differs between the neuroretina and the RPE/choroid, and the processes induced by chronic systemic infection in the RPE/choroid are not unlike those induced in non-immune-privileged organs such as the kidney and spleen. Overall, our data provide detailed insight into several molecular mechanisms of neuroretinal degeneration and highlight various novel protein pathways that further suggest that the posterior part of the eye is not an isolated immunological entity despite the existence of neuroretinal immune privilege.

Systemic virus infection results in CD8 T cell recruitment to the retina in the absence of local virus infection.

During recent years, evidence has emerged that immune privileged sites such as the CNS and the retina may be more integrated in the systemic response to infection than was previously believed. In line with this, it was recently shown that a systemic acute virus infection leads to infiltration of CD8 T cells in the brains of immunocompetent mice. In this study, we extend these findings to the neurological tissue of the eye, namely the retina. We show that an acute systemic virus infection in mice leads to a transient CD8 T cell infiltration in the retina that is not directed by virus infection inside the retina. CD8 T cells were found throughout the retinal tissue, and had a high expression of CXCR6 and CXCR3, as also reported for tissue residing CD8 T cells in the lung and liver. We also show that the pigment epithelium lining the retina expresses CXCL16 (the ligand for CXCR6) similar to epithelial cells of the lung. Thus, our results suggest that the retina undergoes immune surveillance during a systemic infection, and that this surveillance appears to be directed by mechanisms similar to those described for non-privileged tissues.

Innovative Nonthermal Technologies: Chlorophyllin and Visible Light Significantly Reduce Microbial Load on Basil.

Due to the high amount of biologically active compounds, basil is one of the most popular herbs. However, several outbreaks have been reported in the world due to the consumption of basil contaminated with different food pathogens. The aim of this study is to apply nonthermal and ecologically friendly approach based on photosensitization for microbial control of basil which was naturally contaminated with mesophils and inoculated with thermoresistant food pathogen Listeria monocytogenes 56Ly. The obtained data indicate that soaking the basil in 1.5·10-4 M chlorophyllin (Chl) for 15 min and illumination with light for 15 min at 405 nm significantly reduced total aerobic microorganisms on basil by 1.3 log CFU/g, and thermoresistant L. monocytogenes 56Ly from 6.1 log CFU/g in control to 4.5 log CFU/g in the treated samples. It is important to note that this treatment had no impact on enzymatic activity of polyphenol oxidase and pectinesterase. Results obtained in this study support the idea that photosensitization technique with its high selectivity, antimicrobial efficiency and nonthermal nature can serve in the future for the development of safe nonthermal and environmentally friendly preservation technology for different fruits and vegetables.

Towards better microbial safety of fresh produce: Chlorophyllin-based photosensitization for microbial control of foodborne pathogens on cherry tomatoes.

The aim of this study is to evaluate the antimicrobial efficiency of Chlorophyllin-based photosensitization for microbial control of cherry tomatoes. Chlorophyllin-based photosensitization (1.5 × 10-4 M, 3 J/cm2) significantly (2.4 log) reduced the population of naturally distributed surface attached various mesophilic bacteria (microbiota) on tomatoes. Moreover, the population of thermoresistant strains of food pathogens Bacillus cereus and Listeria monocytogenes inoculated on tomatoes was reduced by 1.5 log and 1.6 log respectively after this treatment. Conventional washing with water reduced the population of Listeria on tomato by 0.6 log and Bacillus by 0.8 log. In comparison, hypochlorite treatment reduced Listeria on tomatoes by 1.4 log and Bacillus by 1.6 log. The regrowth of mesophilic bacteria and thermoresistant Listeria on the surface of tomatoes after photosensitization was delayed for 28 days and 14 days respectively. Moreover, photosensitization did not induce harmful effects on main parameter of nutritional quality of tomatoes, i.e. antioxidant activity of tomatoes remained unchanged (27.5 mM Fe2+/kg). Eventually, this treatment did not induce visible thermal effects in fruit matrix and prolonged the shelf-life of tomatoes by 4 days. In our opinion, chlorophyllin-based photosensitization has a huge potential as alternative to not-chemical food preservation technology, saving water and energy. In addition, fast development of light emitting diodes (LED's) and light sources based on LED technologies make this treatment low cost, environmentally friendly and easy to maintain.

Microbial control of food-related surfaces: Na-Chlorophyllin-based photosensitization.

The aim of this study was to evaluate efficiency of photosensitization as surface sanitation alternative using model systems when food pathogens, their spores and biofilms were attached to the food-related surface (polyolefine). In addition it was important to compare antibacterial efficiency of Na-Chlorophyllin (Na-Chl)-based photosensitization with conventional sanitizers. Obtained results indicate that Bacilluscereus ATCC 12826 and Listeriamonocytogenes ATCC 7644 as well as their thermoresistant strains B.cereus SV90 and L.monocytogenes 56LY were effectively inactivated (7 log) by Na-Chl-based photosensitization in vitro. Inactivation rate of thermoresistant strains was slower. The number of attached to the surface B.cereus ATCC 12826 and L.monocytogenes ATCC 7644 was reduced from 4-4.5 log to 0 log after photosensitization treatment. To achieve adequate inactivation of thermoresistant strains the higher Na-Chl concentration and longer illumination times had to be used. Comparison of different surface decontamination treatments reveal that photosensitization is much more effective against all surface-attached B.cereus and L.monocytogenes strains than washing with water or 200 ppm Na-hypochlorite. It is important to note, that surface-attached B.cereus spores and L.monocytogenes biofilms can be eliminated from it by photosensitization as well. Our data support the idea that Na-Chlorophyllin-based photosensitization has high antibacterial potential which may serve in the future for the development of human and environment friendly, non-thermal surface decontamination technique.

Inactivation of several strains of Listeria monocytogenes attached to the surface of packaging material by Na-Chlorophyllin-based photosensitization.

This study was focused on the possibility to inactivate thermosensitive Listeria monocytogenes ATC(L3)C 7644 and thermoresistant 56 Ly strain by Na-Chlorophyllin (Na-Chl)-based photosensitization in vitro and on the surface of packaging. Comparative analysis of antimicrobial efficiency of photosensitization with conventional surface cleaning was performed. Data indicate that both Listeria strains, after incubation with Na-Chl and following illumination (λ=400nm, 20mWcm(-2)), were inactivated by 7 log in vitro. This treatment cleaned both Listeria strains from packaging surfaces. Comparative analysis indicates that washing with water diminishes pathogens by less than 1 log, 200ppm Na-hypochlorite by 1.7 log, Na-Chl-based photosensitization by 4.5 log. Listeria biofilms were totally removed from the surface by photosensitization at higher photosensitizer concentrations and longer incubation times. In conclusion, both strains of L. monocytogenes can be effectively inactivated by photosensitization in vitro and on the surface of packaging. Listeria biofilms are susceptible to this treatment as well. Comparison of different surface decontamination treatments reveals that photosensitization is much more effective against both Listeria strains than washing with water or 200ppm Na-hypochlorite. Our data support the idea that Na-Chl-based photosensitization is an effective antimicrobial tool which may serve in the future for the development of human and environmentally friendly surface decontamination techniques.

Photosensitization-based inactivation of food pathogen Listeria monocytogenes in vitro and on the surface of packaging material.

The study was focused on the susceptibility of Listeria monocytogenes ATCL3C 7644 cells and biofilms to non-thermal antimicrobial treatment - photosensitization in vitro and after adhesion to the surface of packaging material. L. monocytogenes was incubated with 5-aminolevulinic acid (ALA) (7.5 mM) for 0-2h and illuminated with visible light. The LED-based light source used for the illumination emitted light lambda=400 nm with energy density 20 mW/cm(2). The illumination time varied 0-20 min, and a total light dose reached 0-24 J/cm(2). The obtained data indicate that L. monocytogenes produces endogenous porphyrins after incubation with 7.5mM ALA. Subsequent illumination of cells remarkably inactivates (4 log) them in vitro. Photosensitization diminished population of Listeria cells adhered onto the packaging material by 3.7 log and inactivated bacterial biofilms by 3.1 log. It was shown that antimicrobial efficiency of photosensitization depended on the illumination time, incubation with ALA time as well as on the used ALA concentration. In conclusion, cells and biofilms of L. monocytogenes ATCL3C 7644 can be effectively inactivated by ALA-based photosensitization in the solution as well as adhered onto the surface of packaging material. Obtained data support the idea, that photosensitization as non-thermal and effective antimicrobial treatment has potential to develop into environmentally safe, surface decontamination technique.