Analysis of inflammatory processes in diffuse thickening of human aorta intima

It is generally recognized that accumulation of lipids and immune inflammatory cells is an early sign of atherosclerosis. In the present study, we investigated the relationship between the deposition of lipids, immune inflammatory cell content and expression of HLA-DR molecules, and class II major histocompatibility complex (MHC) (a marker of immune activation) in diffuse intima thickening (DIT). Lipids, including triglycerides, cholesterol esters, free cholesterol, and phospholipids were studied by chromatography and Oil Red O histochemistry, as well as by electron microscopy. Immune inflammatory cells and the expression of HLA-DR were investigated by immunohistochemistry in serial sections of the same tissue samples. It has been shown that the lipids were unevenly distributed in DIT. In the juxtaluminal sublayer, lipids were detected in the cytoplasm of intima cells and the extracellular area. In the juxtamedial musculoelastic sublayer of the intima, lipids were present predominantly along elastic fibers. The positive correlation between the presence of lipids and the expression of HLA-DR was revealed (r = 0.79; p < 0.001). A positive correlation was also found between the deposition of lipids and the number of immune inflammatory cells, although correlation was different for different sublayers of the intima. In particular, the correlation between the deposition of lipids and immune inflammatory cells in the juxtaluminal sublayer of the intima was higher (r = 0.69; p < 0.001) than in the juxtamedial musculoelastic layer (r = 0.28; p < 0.001). These data support the hypothesis that the accumulation of lipids in the intima is a key factor in the initiation of inflammatory reactions. At the preatherosclerotic stage of development of this disease, earlier pathological processes associated with lipid-dependent activation of immune cells occur mainly in the juxtaluminal portion of the intima.     

Effects of fibroin microcarriers on inflammation and regeneration of deep skin wounds in mice

The process of tissue regeneration following damage takes place with direct participation of the immune system. The use of biomaterials as scaffolds to facilitate healing of skin wounds is a new and interesting area of regenerative medicine and biomedical research. In many ways, the regenerative potential of biological material is related to its ability to modulate the inflammatory response. At the same time, all foreign materials, once implanted into a living tissue, to varying degree cause an immune reaction. The modern approach to the development of bioengineered structures for applications in regenerative medicine should be directed toward using the properties of the inflammatory response that improve healing, but do not lead to negative chronic manifestations. In this work, we studied the effect of microcarriers comprised of either fibroin or fibroin supplemented with gelatin on the dynamics of the healing, as well as inflammation, during regeneration of deep skin wounds in mice. We found that subcutaneous administration of microcarriers to the wound area resulted in uniform contraction of the wounds in mice in our experimental model, and microcarrier particles induced the infiltration of immune cells. This was associated with increased expression of proinflammatory cytokines TNF, IL-6, IL-1β, and chemokines CXCL1 and CXCL2, which contributed to full functional recovery of the injured area and the absence of fibrosis as compared to the control group.     

Carboranyl-Chlorin e6 as a Potent Antimicrobial Photosensitizer

Antimicrobial photodynamic inactivation is currently being widely considered as alternative to antibiotic chemotherapy of infective diseases, attracting much attention to design of novel effective photosensitizers. Carboranyl-chlorin-e₆ (the conjugate of chlorin e₆ with carborane), applied here for the first time for antimicrobial photodynamic inactivation, appeared to be much stronger than chlorin e₆ against Gram-positive bacteria, such as Bacillus subtilis, Staphyllococcus aureus and Mycobacterium sp. Confocal fluorescence spectroscopy and membrane leakage experiments indicated that bacteria cell death upon photodynamic treatment with carboranyl-chlorin-e₆ is caused by loss of cell membrane integrity. The enhanced photobactericidal activity was attributed to the increased accumulation of the conjugate by bacterial cells, as evaluated both by centrifugation and fluorescence correlation spectroscopy. Gram-negative bacteria were rather resistant to antimicrobial photodynamic inactivation mediated by carboranyl-chlorin-e₆. Unlike chlorin e₆, the conjugate showed higher (compared to the wild-type strain) dark toxicity with Escherichia coli ΔtolC mutant, deficient in TolC-requiring multidrug efflux transporters.     

Boronated derivatives of chlorin <Emphasis Type="Italic">e</Emphasis><Subscript>6</Subscript> and fluoride-containing porphyrins as penetrating anions: a study using bilayer lipid membranes

Boronated derivatives of porphyrins are studied extensively as promising compounds for boron-neutron capture therapy and photodynamic therapy. Understanding of the mechanism of their permeation across cell membranes is a key step in screening for the most efficient compounds. In the present work, we studied the ability of boronated derivatives of chlorin e ₆ and porphyrins, which are mono-, di-, and tetra-anions, to permeate through planar bilayer lipid membranes (BLM). The translocation rate constants through the hydrophobic part of the lipid bilayer were estimated for monocarborane and its conjugate with chlorin e ₆ by the method of electrical current relaxation. They were similar, 6.6 and 6.8 sec⁻¹, respectively. Conjugates of porphyrins carrying two and four carborane groups were shown to permeate efficiently through a BLM although they carry two charges and four charges, respectively. The rate of permeation of the tetraanion estimated by the BLM current had superlinear dependence on the BLM voltage. Because the resting potential of most mammalian cells is negative inside, it can be concluded that the presence of negatively-charged boronated groups in compounds should hinder the accumulation of the porphyrins in cells.     

In vitro fluorescence assay to study the folding of K<Subscript>v</Subscript> ion channels

A fluorescence assay to check the folding of potassium K_v channels expressed in vitro has been developed. For this aim, the fluorescently labeled channel blocker, recombinant agitoxin of yellow scorpion was employed. The level of expression of various K_v channels in vitro has been tested. It has been demonstrated that K_v2 channels form clusters on the cell surface, which are not associated with actin filaments. On the other hand, K_v10 channels form larger clusters, which are associated with actin, indicating the principal differences in the organization of cytoplasmic domains of K_v2 and K_v10 channels.     

Novel Photosensitizers Trigger Rapid Death of Malignant Human Cells and Rodent Tumor Transplants via Lipid Photodamage and Membrane Permeabilization

Background

Apoptotic cascades may frequently be impaired in tumor cells; therefore, the approaches to circumvent these obstacles emerge as important therapeutic modalities.

Methodology/Principal Findings

Our novel derivatives of chlorin e₆, that is, its amide (compound 2) and boronated amide (compound 5) evoked no dark toxicity and demonstrated a significantly higher photosensitizing efficacy than chlorin e₆ against transplanted aggressive tumors such as B16 melanoma and M-1 sarcoma. Compound 5 showed superior therapeutic potency. Illumination with red light of mammalian tumor cells loaded with 0.1 µM of 5 caused rapid (within the initial minutes) necrosis as determined by propidium iodide staining. The laser confocal microscopy-assisted analysis of cell death revealed the following order of events: prior to illumination, 5 accumulated in Golgi cysternae, endoplasmic reticulum and in some (but not all) lysosomes. In response to light, the reactive oxygen species burst was concomitant with the drop of mitochondrial transmembrane electric potential, the dramatic changes of mitochondrial shape and the loss of integrity of mitochondria and lysosomes. Within 3–4 min post illumination, the plasma membrane became permeable for propidium iodide. Compounds 2 and 5 were one order of magnitude more potent than chlorin e₆ in photodamage of artificial liposomes monitored in a dye release assay. The latter effect depended on the content of non-saturated lipids; in liposomes consisting of saturated lipids no photodamage was detectable. The increased therapeutic efficacy of 5 compared with 2 was attributed to a striking difference in the ability of these photosensitizers to permeate through hydrophobic membrane interior as evidenced by measurements of voltage jump-induced relaxation of transmembrane current on planar lipid bilayers.

Conclusions/Significance

The multimembrane photodestruction and cell necrosis induced by photoactivation of 2 and 5 are directly associated with membrane permeabilization caused by lipid photodamage.     

The model of resting forms of micobacteria for testing of chemodrugs for latent forms of tuberculosis

The new model of obtaining of ovoid resting forms of Mycobacterium smegmatis, which are morphologically different from vegetative (rod-like) cells, was developed. Ovoid forms were characterized by a drastically decreased level of metabolic activity, an increased stability to heat or antibiotics treatment, and also by prolonged (more than 2 months) storage time preserving colony-forming ability. Obtained resting forms of micobacteria may be used in test-systems for checking efficiency of new medical agents against latent forms of tuberculosis and determination of role of the genes in entering dormant state.     

Radioprotective role of cyanobacterial phycobilisomes

Cyanobacteria are thought to be responsible for pioneering dioxygen production and the so-called “Great Oxygenation Event” that determined the formation of the ozone layer and the ionosphere restricting ionizing radiation levels reaching our planet, which increased biological diversity but also abolished the necessity of radioprotection. We speculated that ancient protection mechanisms could still be present in cyanobacteria and studied the effect of ionizing radiation and space flight during the Foton-M4 mission on Synechocystis sp. PCC6803. Spectral and functional characteristics of photosynthetic membranes revealed numerous similarities of the effects of α-particles and space flight, which both interrupted excitation energy transfer from phycobilisomes to the photosystems and significantly reduced the concentration of phycobiliproteins. Although photosynthetic activity was severely suppressed, the effect was reversible, and the cells could rapidly recover from the stress. We suggest that the actual existence and the uncoupling of phycobilisomes may play a specific role not only in photo-, but also in radioprotection, which could be crucial for the early evolution of Life on Earth.     

Photodynamic activity of the boronated chlorin e6 amide in artificial and cellular membranes

Photodynamic tumor-destroying activity of the boronated chlorin e6 derivative BACE (chlorin e6 13(1)-N-{2-[N-(1-carba-closo-dodecaboran-1-yl)methyl]aminoethyl}amide-15(2), 17(3)-dimethyl ester), previously described in Moisenovich et al. (2010) PLoS ONE 5(9) e12717, was shown here to be enormously higher than that of unsubstituted chlorin e6, being supported by the data on much higher photocytotoxicity of BACE in M-1 sarcoma cell culture. To validate membrane damaging effect as the basis of the enhanced tumoricidal activity, BACE was compared with unsubstituted chlorin e6 in the potency to photosensitize dye leakage from liposomes, transbilayer lipid flip-flop, inactivation of gramicidin A ionic channels in planar lipid membranes and erythrocyte hemolysis. In all the models comprising artificial and cellular membranes, the photodynamic effect of BACE exceeded that of chlorin e6. BACE substantially differed from chlorin e6 in the affinity to liposomes and erythrocytes, as monitored by fluorescence spectroscopy, flow cytometry and centrifugation. The results support the key role of membrane binding in the photodynamic effect of the boronated chlorin e6 amide.