Non-magnetic shell coating of magnetic nanoparticles as key factor of toxicity for cancer cells in a low frequency alternating magnetic field.

This work is devoted to studying the effects of non-magnetic shell coating on nanoparticles in a low frequency alternating magnetic field (LF AMF) on tumor cells in vitro. Two types of iron oxide nanoparticles with the same magnetic core with and without silica shells were synthesized. Nanoparticles with silica shells significantly decreased the viability of PC3 cancer cells in a low frequency alternating magnetic field according to the cytotoxicity test, unlike uncoated nanoparticles. We showed that cell death results from the intracellular membrane integrity failure, and the calcium ions concentration increase with the subsequent necrosis. Transmission electron microscopy images showed that the uncoated silica nanoparticles are primarily found in an aggregated form in cells. We believe that uncoated nanoparticles lose their colloidal stability in an acidic endosomal environment after internalization into the cell due to surface etching and the formation of aggregates. As a result, they encounter high endosomal macromolecular viscosity and become unable to rotate efficiently. We assume that effective rotation of nanoparticles causes cell death. In turn, silica shell coating increases nanoparticles stability, preventing aggregation in endosomes. Thus, we propose that the colloidal stability of magnetic nanoparticles inside cells is one of the key factors for effective magneto-mechanical actuation.

Advances and Challenges of Nanoparticle-Based Macrophage Reprogramming for Cancer Immunotherapy.

Despite the progress of modern medicine, oncological diseases are still among the most common causes of death of adult populations in developed countries. The current therapeutic approaches are imperfect, and the high mortality of oncological patients under treatment, the lack of personalized strategies, and severe side effects arising as a result of treatment force seeking new approaches to therapy of malignant tumors. During the last decade, cancer immunotherapy, an approach that relies on activation of the host antitumor immune response, has been actively developing. Cancer immunotherapy is the most promising trend in contemporary fundamental and practical oncology, and restoration of the pathologically altered tumor microenvironment is one of its key tasks, in particular, the reprogramming of tumor macrophages from the immunosuppressive M2-phenotype into the proinflammatory M1-phenotype is pivotal for eliciting antitumor response. This review describes the current knowledge about macrophage classification, mechanisms of their polarization, their role in formation of the tumor microenvironment, and strategies for changing the functional activity of M2-macrophages, as well as problems of targeted delivery of immunostimulatory signals to tumor macrophages using nanoparticles.

Preparation and Testing of Cells Expressing Fluorescent Proteins for Intravital Imaging of Tumor Microenvironment.

Intravital microscopy is widely used for in vivo studies of the mechanisms of carcinogenesis and response to antitumor therapy. For visualization of tumor cells in vivo, cell lines expressing fluorescent proteins are needed. Expression of exogenous proteins can affect cell growth rate and their tumorigenic potential. Therefore, comprehensive analysis of the morphofunctional properties of transduced cells is required for creating appropriate models of tumor microenvironment. In the present study, six lines of mouse tumor cells expressing green and red fluorescent proteins were derived. Analysis of cells morphology, growth kinetics, and response to chemotherapy in vitro revealed no significant differences between wild-type and transduced cell lines. Introduction of fluorescent proteins into the genome of 4T1 (murine breast cancer) and B16-F10 (murine melanoma) cells did not affect tumor growth rate after subcutaneous implantation to mice, while both CT26-GFP and CT26-RFP cells (murine colon cancer) were rejected starting from day 8 after implantation. Elucidation of the mechanisms underlying CT26-GFP/RFP rejection is required to modify transduction technique for creating the models of tumor microenvironment accessible for in vivo visualization. Transduced 4T1 and B16-F10 cell lines can be used for intravital microscopic imaging of tumor cells, neoplastic vasculature, and leukocyte subpopulations.

Synthesis and characterization of bacteriochlorin loaded magnetic nanoparticles (MNP) for personalized MRI guided photosensitizers delivery to tumor.

HYPOTHESIS: Hydrophobic bacteriochlorin based photosensitizer (PS) can be effectively immobilized on MNP covered by human serum albumin (HSA). PS loading into MNP protein shell allows solubilizing PS in water solution without altering its photodynamic activity. MNP@PS can serve as diagnostic tool for tracking PS delivery to tumor tissues by MRI. EXPERIMENTS: Immobilization on MNP-HSA-PEG was performed by adding PS solution in organic solvents with further purification. MNP@PS were characterized by DLS, HAADF STEM and AFM. Absorbance and fluorescence measurements were used to assess PS photophysical properties before and after immobilization. MNP@PS internalization into CT26 cells was investigated by confocal microscopy in vitro and MRI/IVIS were used for tracking MNP@PS delivery to tumors in vivo. FINDINGS: MNP@PS complexes were stable in water solution and retained PS photophysical activity. The length of side chain affected MNP@PS size, loading capacity and cell internalization. In vitro testing demonstrated MNP@PS delivery to cancer cells followed by photoinduced toxicity. In vivo studies confirmed that as-synthetized complexes can be used for MRI tracking over drug accumulation in tumors.

[QUANTITATIVE DNA EVALUATION OF THE HIGH CARCINOGENIC RISK OF HUMAN PAPILLOMA VIRUSES AND HUMAN HERPES VIRUSES IN MALES WITH FERTILITY DISORDERS].

Infertility is an actual medical and social problem. In 50% of couples it is associated with the male factor and in more than 50% of cases the etiology of the infertility remains insufficiently understood. The goal of this work was to study the prevalence and to perform quantitative analysis of the human herpes viruses (HHV) and high carcinogenic risk papilloma viruses (HR HPV) in males with infertility, as well as to assess the impact of these infections on sperm parameters. Ejaculate samples obtained from 196 males fall into 3 groups. Group 1 included men with the infertility of unknown etiology (n = 112); group 2, patients who had female partners with the history of spontaneous abortion (n = 63); group 3 (control), healthy men (n = 21). HHV and HR HPV DNA in the ejaculates were detected in a total of 42/196 (21.4%) males: in 31 and 11 patients in groups 1 and 2, respectively (p > 0.05) and in none of healthy males. HHV were detected in 24/42; HR HPV, in 18/42 males (p > 0.05) without significant difference between the groups. Among HR HPV genotypes of the clade A9 in ejaculate were more frequent (14/18, p = 0.04). Comparative analysis of the sperm parameters showed that in the ejaculates of the infected patients sperm motility as well as the number of morphologically normal cells were significantly reduced compared with the healthy men. The quantification of the viral DNA revealed that in 31% of the male ejaculates the viral load was high: > 3 Ig10/100000 cells. Conclusion. The detection of HHV and HR HPV in the ejaculate is associated with male infertility. Quantification of the viral DNA in the ejaculate is a useful indicator for monitoring viral infections in infertility and for decision to start therapy.

[Interaction of herpesviruses with mature human spermatozoa in the model system in vitro].

The DNA of human herpesviruses (HHV), including the herpes simplex virus (HSV) and cytomegalovirus (CMV), is often identified in ejaculates of patients with urogenital diseases and infertility. At least a part of viral DNA is associated with cell fraction of ejaculate. However, it remains unclear how the semen is infected by the virus. It can be located in gametes or be capable of infecting mature germ cells, including motile sperm cells. In order to resolve this issue, interactions of the CMV and HSV with human sperm cells were studied using an original optimized model of the herpesviral infection of male gametes in vitro. The analysis of the immunofluorescent staining of gametes for viral antigens has shown that CMV infected 2% gametes, while HSV infected 17.26 ± 2.58% gametes. The fraction of progressively motile sperm cells contained 13.99 ± 4.64% infected cells. Localization of HSV was studied by the confocal microscopy. Sometimes, viral gB protein was found on sperm cell membrane. In addition, optical scanning of other cells has shown the intracellular localization of the viral proteins. In the majority of spermatozoa, the viral proteins were observed in the head and neck. In some cells, they were located in the middle piece or, rarely, in the equatorial segment. In general, after in vitro infection HSV antigens were located in the same areas of the sperm cells as in ejaculates from infected patients. According to DNA-DNA hybridization in situ, gametes containing HSV DNA accounted for 16.94 ± 5.28%, which is consistent with the results obtained in the immunofluorescence assay. It can be concluded that mature male gametes are infected by HHV in the genital tract, where the virus binds to the sperm cell membrane and enters the cell. Interaction of HHV with progressively motile sperm cells implies a vertical viral transmission upon fertilization and points to the necessity of testing ejaculate for herpesviruses infections.

[Herpes viruses and male infertility--is there any relationship?].

A relationship between the herpesviral infections and male reproductive health is of importance to both theoretical and practical medicine. The review contains the data on the frequency of herpes virus identification in sperm, the effect of the viruses on structure and function of male germ cells, potential vertical transmission of the herpes viruses with male gametes, and experimental models of study the effects of herpes viruses on spermatogenesis. From the analysis of these data it can be concluded that: 1) identification of herpes virus in sperm is associated with reduced fertility; 2) herpes simplex virus has a negative effect on spermatogenesis, which manifests itself in a decreased proliferative activity of spermatogonia, meiosis block and enhanced apoptosis of germ cells; 3) herpes viruses can be found intracellularly in male gametes; and 4) the analysis of the markers of widespread herpes viruses (HSV, CMV) should be included in examination of men attending infertility clinics.

[Effect of herpes simplex virus on spermatogenesis].

To investigate the effect of Herpes Simplex virus (HSV) on spermatogenesis, HSV in ejaculate was detected by a rapid cultural method in 268 infertile males and 47 healthy ones. The number of mobile spermatozoa in HSV infected samples was less than in non-infected samples (21 mln/mlversus 40 mln/ml, p = 0.0001). The relative number of morphologically normal gametes was 13% versus 19% (p = 0.002), respectively. The quantitative karyological test discovered that males with HSV-infected ejaculate have more degenerating sex cells while in high virus contamination (more than 10 virus particles in 1 ml) the number of spermatides and spermatocytes of the 1 order at diploten stage is low. Organic testicular culture was used for more detailed study of pathogenetic mechanisms of HSV impact on spermatogenesis. Testicular explants infection was associated with reduction in the number of spermatogones, spermatocytes and spermatides on culturing week 2. The above findings reveal some pathogenetic mechanisms underling fertility disorders in males with HSV infection: a gametotoxic effect of the virus reducing populations of spermatogones, spermatocytes and spermatide; affected mobility and morphological characteristics of spermatozoa. Detection of the mechanisms of HSV action on spermatogenesis opens a perspective of antivirus drug administration in combined treatment of male infertility.

[Possibilities of using long-wave irradiation in the near-infrared and red spectral ranges for visualization of eyeball structures].

The proposed study technique makes it possible to non-invasively visualize the structures of the anterior and posterior portions of the eyeball in the red and near-infrared spectral ranges of irradiation. Further study of the capacities of the technique and its introduction into clinical practice should be made.

[Herpes simplex virus and cytomegalovirus in male ejaculate: herpes simplex virus is more frequently encountered in idiopathic infertility and correlates with the reduction in sperm parameters].

The present investigation was undertaken to study the detection rate of herpes simplex virus (HSV) and cytomegalovirus (CMV) in the ejaculates of males with infertility and to evaluate the impact of virus infection on the major parameters of sperm. Ejaculates from 808 patients were studied. As compared with apparently healthy individuals, the coupled males with primary infertility were found to have HSV more frequently in both the whole ejaculate (31% versus 17%; p = 0.049) and the fraction of actively motile spermatozoa (30% versus 8%; p = 0.016). Ejaculate HSV detection directly correlated with the reduced amount of actively motile spermatozoa (p = 0.0001) and the smaller proportion of morphologically normal forms of germ cells (p = 0.002). CMV was found to have no impact on the motility and morphology of spermatozoids in the ejaculate. Both HSV and CMV in the male ejaculate were significantly more frequently detectable in winter months. The findings lead to the conclusion that HSV is one of the factors for male infertility and can negatively affect the results of assisted reproductive technologies.

[Localization of human cytomegalovirus immediate-early protein IE-1 pp72 in the juxtanuclear inclusion at the late stage of infection].

Immediate-early protein IE-1 pp72 is one of the most abundant proteins at the early stage of human cytomegalovirus infection and has a number of intranuclear activities. This paper gives immunocytochemical and ultrastructural data on IE-1 pp72 accumulation in the juxtanuclear inclusion at the late stage of low-multiplicity infection. Detection of a new localization site infers that this protein may participate in the final steps of virus morphogenesis and play a functional role in the pathogenesis of cytomegalovirus infection.

[Application of confocal microscopy to the use of a study of the morphogenesis of human immunodeficiency virus type 1].

Confocal microscopy is widely used in different fields of biology. By using the reports on the study of the morphogenesis, diagnosis, drug therapy of HIV-1 as an example, the authors consider the procedures of confocal microscopy, which have found wide application in virology.

[Ischemic type of diabetic maculopathy]. / Ishemicheskaia forma diabeticheskoi makulopatii.

The pathogenesis of diabetic maculopathy is discussed. The possibility of different diagnostic methodic (OCT, FAG and etc.) is shown. Positive results of selective coagulation of pigment epithelium are achieved.