Photosensitization of pancreatic cancer cells by cationic alkyl-porphyrins in free form or engrafted into POPC liposomes: The relationship between delivery mode and mechanism of cell death

Cationic porphyrins bearing an alkyl side chain of 14 (2b) or 18 (2d) carbons dramatically inhibit proliferation of pancreatic cancer cells following treatment with light. We have compared two different ways of delivering porphyrin 2d: either in free form or engrafted into palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine liposomes (L-2d). Cell cytometry shows that while free 2d is taken up by pancreatic cancer cells by active (endocytosis) and passive (membrane fusion) transports, L-2d is internalized solely by endocytosis. Confocal microscopy showed that free 2d co-localizes with the cell membrane and lysosomes, whereas L-2d partly co-localizes with lysosomes and ER. It is found that free 2d inhibits the KRAS-Nrf2-GPX4 axis and strongly triggers lipid peroxidation, resulting in cell death by ferroptosis. By contrast, L-2d does not affect the KRAS-Nrf2-GPX4 axis and activates cell death mainly through apoptosis. Overall, our study demonstrates for the first time that cationic alkyl porphyrins, which have a IC50 ~ 23 nM, activate a dual mechanism of cell death, ferroptosis and apoptosis, where the predominant form depends on the delivery mode

Structural and Functional Diversity of Cathelicidins

Cathelicidins are a ubiquitous family of host defence peptides (HDPs) in vertebrate animals. Unlike other HDP families, they are defined by the common and relatively well conserved proregion rather than the mature active peptides, which are highly diverse and conform to at least five different structural groups. They seem to have fol-lowed a rather distinctive evolutionary path in their development. Cathelicidin-derived peptides play a relevant role in defending the host against microbial infection, by displaying both a broad-spectrum, direct antimicrobial activity and the capacity to modulate other host responses to infection and injury. Both types of effect depend on the structural type, which in turn affects the particular mode of action of each peptide. This chapter begins by briefly describing the discovery of cathelicidins before dis-cussing their molecular diversity and con-sidering their evolution. It then considers their expression and processing, the struc-ture-dependence of the distinct modes of action shown by different members, and briefly touches on th

Evaluation of free or anchored antimicrobial peptides as candidates for the prevention of orthopaedic device-related infections

The prevention of implant-associated infection, one the most feared complications in orthopaedic surgery, remains a major clinical challenge and urges development of effective methods to prevent bacterial colonization of implanted devices. Alpha-helical antimicrobial peptides (AMPs) may be promising candidates in this respect due to their potent and broad-spectrum antimicrobial activity, their low tendency to elicit resistance and possible retention of efficacy in the immobilized state. The aim of this study was to evaluate the potential of five different helical AMPs, the cathelicidins BMAP-27 and BMAP-28, their (1-18) fragments and the rationally designed, artificial P19(9/G7) peptide, for the prevention of orthopaedic implant infections. Peptides were effective at micromolar concentrations against 22 Staphylococcus and Streptococcus isolates from orthopaedic infections, while only BMAP-28 and to a lesser extent BMAP-27 were active against Enterococcus faecalis. Peptides in solution showed activities comparable to those of cefazolin and linezolid, on a molar basis, and also a variable capacity to neutralize bacterial lipopolysaccharide, while devoid of adverse effects on MG-63 osteoblast cells at concentrations corresponding to the MIC. The (1-18) BMAP fragments and P19(9/G7) were selected for further examination, based on better selectivity indices, and showed effectiveness in the presence of hyaluronic acid and in synovial fluid, while human serum affected their activity to variable extents, with BMAP-27(1-18) best retaining activity. This peptide was immobilized on streptavidin-resin beads and retained activity against reference Staphylococcus epidermidis and Staphylococcus aureus strains, with negligible toxicity towards osteoblasts, underlining its potential for the development of infection-resistant biomaterials for orthopaedic application

Antifungal activity of cathelicidin peptides against planktonic and biofilm cultures of Candida species isolated from vaginal infections

Vulvovaginal candidiasis (VVC) is a frequent gynecological condition caused by Candida albicans and afew non-albicans Candida spp. It has a significant impact on the quality of life of the affected womenalso due to a considerable incidence of recurrent infections that are difficult to treat. The formation offungal biofilm may contribute to the problematic management of recurrent VVC due to the intrinsicresistance of sessile cells to the currently available antifungals. Thus, alternative approaches for theprevention and control of biofilm-related infections are urgently needed. In this regard, the cationicantimicrobial peptides (AMPs) of the innate immunity are potential candidates for the development ofnovel antimicrobials as many of them display activity against biofilm formed by various microbial species.In the present study, we investigated the in vitro antifungal activities of the cathelicidin peptides LL-37and BMAP-28 against pathogenic Candida spp. also including C. albicans, isolated from vaginal infections,and against C. albicans SC5314 as a reference strain. The antimicrobial activity was evaluated againstplanktonic and biofilm-grown Candida cells by using microdilution susceptibility and XTT [2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide] reduction assays and, in the case ofestablished biofilms, also by CFU enumeration and fluorescence microscopy. BMAP-28 was effectiveagainst planktonically grown yeasts in standard medium (MIC range, 2\u201332 M), and against isolatesof C. albicans and Candida krusei in synthetic vaginal simulated fluid (MIC range 8\u201332 M, dependingon the pH of the medium). Established 48-h old biofilms formed by C. albicans SC5314 and C. albicansand C. krusei isolates were 70\u201390% inhibited within 24 h incubation with 16 M BMAP-28. As shownby propidium dye uptake and CFU enumeration, BMAP-28 at 32 M killed sessile C. albicans SC5314by membrane permeabilization with a faster killing kinetics compared to 32 M miconazole (80\u201385%reduced biofilm viability in 90 min vs 48 h). In addition, BMAP-28 at 16 M prevented Candida biofilmformation on polystyrene and medical grade silicone surfaces by causing a >90% reduction in the viabilityof planktonic cells in 30 min. LL-37 was overall less effective than BMAP-28 against planktonic Candidaspp. (MIC range 4\u2013 6564 M), and was ineffective against established Candida biofilms. However, LL-37 at64 M prevented Candida biofilm development by inhibiting cell adhesion to polystyrene and siliconesurfaces. Finally, Candida adhesion was strongly inhibited when silicone was pre-coated with a layerof BMAP-28 or LL-37, encouraging further studies for the development of peptide-based antimicrobialcoatings.\ua9 2015 Elsevier Inc. All rights reserve

Effect of the formulation and structure of monoglyceride-based gels on the viability of probiotic Lactobacillus rhamnosus upon in vitro digestion

This research was conducted to evaluate the potential use of saturated monoglyceride (MG)-based gels in the protection of probiotics upon in vitro digestion. For this purpose, a Lactobacillus rhamnosus strain was inoculated into binary and ternary systems, containing MGs, a water phase composed of an aqueous solution at controlled pH or UHT skimmed milk, and in ternary gels, sunflower oil. Gel structure characterization was initially performed just after preparation and after 14 days of storage at 4 \ub0C by rheological, mechanical, thermal, and microscopy analyses. Afterwards, probiotic viability upon in vitro digestion was evaluated. The results highlighted that all freshly prepared samples showed good capability to protect L. rhamnosus with the exception of the binary system containing milk. However, the digestion of samples after 14 days of storage showed that the ternary system containing skimmed milk exhibited the best protection performance ensuring a L. rhamnosus viability of almost 106 CFU g-1 at the end of the gastrointestinal passage. Confocal microscopy results demonstrated that bacterial cells were located prevalently within the aqueous domain near the monoglycerides and protein aggregates. Under these conditions, they can simultaneously achieve physical protection and find nutrients to survive environmental stresses. These findings suggest that MG-based gels can be proposed as efficient carriers of probiotic bacteria not only during food processing and storage but also upon digestion

Loss of spinal motor neurons and alteration of alpha-synuclein immunostaining in MPTP induced Parkinsonism in mice

1-Methyl, 4-phenyl, 1,2,3,6-tetrahydropiridine (MPTP) is a neurotoxin, widely used to produce experimental models of Parkinson Disease in rodents and primates. Although dopaminergic neurons are the most sensitive to MPTP neurotoxicity, different neuronal subtypes are affected. Among these, recent studies indicate that MPTP may produce pathological effects on spinal neurons. In fact, MPTP activates apoptotic proteins within the spinal cord and in particular within the motor neurons, suggesting commonalities between Parkinson Disease and Amyotrophic Lateral Sclerosis. In order to assess this point, in the present study we measured whether MPTP produces motor neurons loss. We chose a dose of MPTP (20. mg/kg. × 3, 2. h apart), which in C57BL/6N mice was able to induce a massive nigrostriatal damage. Since both Parkinson Disease and Amyotrophic Lateral Sclerosis are characterized by altered alpha-synuclein immunostaining, this protein was also evaluated within spinal motor neurons, following MPTP administration. Three different monoclonal antibodies, recognizing distinct epitopes in the sequence of alpha-synuclein were used. Severe dopaminergic cell loss was quantified by stereology within the substantia nigra pars compacta, along with marked decrease of striatal tyrosine hydroxylase densitometry. The same doses of MPTP also caused a significant motor neuron loss in the spinal cord (roughly 30%). Spared motor neurons appeared often dysmorphic and vacuolated and possessed altered alpha-synuclein immunostaining. This latter finding extended to other cell types of the spinal cord. These data indicate that MPTP, apart from being a dopaminergic neurotoxin, produces also motor neuron death, thus bridging experimental Parkinsonism and motor neuron disease. © 2012

loss of spinal motor neurons and alteration of alpha- synuclein subcellular localization in mPtP induced parkinsonism in mice

-methyl, 4-phenyl, 1, 2, 3, 6-tetrahydropiridine (MPTP) is a neurotoxin, widely used to produce experimental models of Parkinson Disease in rodents and primates. Although dopaminergic neurons are the most sensitive to MPTP neurotoxicity, differ- ent neuronal subtypes could be affected. In particular, noradrenergic neurons of the Locus Coeruleus may be involved as well as nigral dopaminergic neurons. Moreover, apart from catecholamine-containing nuclei, recent studies indicate that MPTP may produce pathological effects within the spinal cord. This point deserves compelling interest since it suggests, at experimental level, commonalities between Parkinson Disease and Amyotrophic Lateral Sclerosis. For instance, recent reports demonstrate that MPTP activates apoptotic proteins at the level of spinal cord. However, to our knowledge, none of these studies so far analyzed whether motor neuron loss really occurs following MPTP administration. Therefore, in the present study we evaluat- ed the effect of a robust dose of MPTP (20 mg/Kg X3) in the nigro-striatal system and spinal cord. Along with a severe dopaminergic cells loss within the substantia nigra, quantified by stereology and a marked decrease of striatal catecholoamine fib- ers measured by semi-quantitative densitometry, we found a significant (roughly 30 %) depletion of motor neurons in the lumbar spinal cord of MPTP-treated C57BL/6J mice. At the same level, spared motor neurons often present an altered morphology, being dysmorphic and vacuolated. Furthermore, using four different antibodies (tree monoclonal, one polyclonal), recognizing distinct epitopes in the sequence of alpha- synuclein, we found that alpha-synuclein immunostaining is markedly altered in the spinal cord of MPTP-treated mice. The present data shed new lights on similarities between dopaminergic neu- rons and spinal motor neurons, while suggesting that MPTP might be a neurotoxin diverse from what originally considered