The Effect of Therapy Regimen on Antitumor Efficacy of the Nanosomal Doxorubicin against Rat Glioblastoma 101.8.

One of the key problems of glioblastoma treatment is the low effectiveness of chemotherapeutic drugs. Incorporation of doxorubicin into PLGA nanoparticles allows increasing the antitumor effect of the cytostatics against experimental rat glioblastoma 101.8. Animal survival, tumor volume, and oncogene expression in tumor cells were compared after early (days 2, 5, and 8 after tumor implantation) and late (days 8, 11, and 14) start of the therapy. At late start, a significant increase in the expression of oncogenes Gdnf, Pdgfra, and Melk and genes determining the development of multidrug resistance Abcb1b and Mgmt was revealed. At early start of therapy, only the expression of oncogenes Gdnf, Pdgfra, and Melk was enhanced. Early start of treatment prolonged the survival time and increased tumor growth inhibition by 141.4 and 95.7%, respectively, in comparison with the untreated group; these differences were not observed in the group with late start of therapy. The results indicate that the time of initiation of therapy is a critical parameter affecting the antitumor efficacy of DOX-PLGA.

Advantages and disadvantages of treatment of experimental ARDS by M2-polarized RAW 264.7 macrophages.

Innate immunity reactions are core to any immunological process, including systemic inflammation and such extremes as acute respiratory distress syndrome (ARDS) and cytokine storm. Macrophages, the key cells of innate immunity, show high phenotypic plasticity: depending on microenvironmental cues, they can polarize into M1 (classically activated, pro-inflammatory) or M2 (alternatively activated, anti-inflammatory). The anti-inflammatory M2 macrophage polarization-based cell therapies constitute a novel prospective modality. Systemic administration of 'educated' macrophages is intended at their homing in lungs in order to mitigate the pro-inflammatory cytokine production and reduce the risks of 'cytokine storm' and related severe complications. Acute respiratory distress syndrome (ARDS) is the main mortality factor in pneumonia including SARS-CoV-associated cases. This study aimed to evaluate the influence of infusions of RAW 264.7 murine macrophage cell line polarized towards M2 phenotype on the development of LPS-induced ARDS in mouse model. The results indicate that the M2-polarized RAW 264.7 macrophage infusions in the studied model of ARDS promote relocation of lymphocytes from their depots in immune organs to the lungs. In addition, the treatment facilitates expression of M2-polarization markers Arg1, Vegfa and Tgfb and decreases of M1-polarization marker Cd38 in lung tissues, which can indicate the anti-inflammatory response activation. However, treatment of ARDS with M2-polarized macrophages didn't change the neutrophil numbers in the lungs. Moreover, the level of the Arg1 protein in lungs decreased throughtout the treatment with M2 macrophages, which is probably because of the pro-inflammatory microenvironment influence on the polarization of macrophages towards M1. Thus, the chemical polarization of macrophages is unstable and depends on the microenvironment. This adverse effect can be reduced through the use of primary autologous macrophages or some alternative methods of M2 polarization, notably siRNA-mediated.

Morphofunctional Characteristics of Lung Macrophages in Rats with Acute Respiratory Distress Syndrome.

A comprehensive morphofunctional study of the lungs and alveolar macrophages was carried out in Sprague-Dawley rats with acute respiratory distress syndrome (n=10) induced by intratracheal administration of E. coli LPS 0111:B4 in a dose of 15 mg/kg. On the first day after LPS administration, bronchopneumonia was observed in the lungs, the number of macrophages of the bone marrow origin and the number of M1 macrophages with the proinflammatory phenotype in the bronchoalveolar lavage increased, the expression of proinflammatory cytokines increased and the expression of anti-inflammatory cytokines decreased, which was accompanied by an increase in LPS and C-reactive protein in the blood serum. The revealed changes correspond to the development of acute respiratory distress syndrome in humans, and the decrease in the number of macrophages in the lungs and their predominant polarization to the M1-proinflammatory phenotype substantiate the use of cell therapy with reprogrammed M2 macrophages.

Age-Specific Features of the Levels of Reference Proteins Actin, Tubulin, and GAPDH in Different Organs of Male Wistar Rats.

Western blot analysis is used for evaluation of the level of proteins production in organs and tissues, and housekeeping proteins GAPDH, actin, and tubulin are usually used as the reference proteins. The signal of the target protein is normalized to the corresponding signal of the reference protein. The data on the intensity of actin, tubulin, and GAPDH synthesis are fragmentary: their expression differs in different organs and can vary depending on age, which is often not taken into account in experimental studies. We studied the features of the production of reference proteins in the liver, heart, brain, and lungs of newborn, prepubertal, and adult male Wistar rats. Age-related differences in the expression of ß-actin, ß-tubulin, and GAPDH in the myocardium and dorsal prefrontal cortex were revealed. GAPDH expression in the dorsal prefrontal cortex in adult rats was significantly higher than in prepubertal rats; GAPDH expression in the myocardium of adult rats was significantly higher than in newborns. The level of actin in the dorsal prefrontal cortex in newborn rats was significantly higher than in prepubertal and adult rats. In the liver and lungs, the expression of actin, tubulin, and GAPDH did not differ in newborn, prepubertal, and adult rats. When choosing the reference protein for Western blotting, animals age and the studied organ should be taken into account.

[Revealing the genetic determinants of Pks-pathogenicity island in clinical strains of enterobacteria].

AIM: Detection by PCR the frequency of clbB, clbN, clbA H clbQ genes of Pks-pathogenicity island in clinical strains ofenterobacteria. MATERIALS AND METHODS: 112 strains various genera and species of enterobacteria, including 16 museum and 96 clinical are investigated. Isolated strains represents Escherichia species (n = 68), Klebsiella (n = 16), Enterobacter (n = 9), Serratia (n = 7) and others minor species of Enterobacteriaceae family (n = 12). Fifty nine strains isolated from urine of urinary tract infection, 26 isolates from intestines of patients with dysbiosis and 11--from children with complications after a liver transplantation. A total bacterial isolates were screened by multiplex PCRforthe presence ofclbB, clbN, clbA and clbQ genes. RESULTS: Among 41 uropathogenic E.coli it is revealed 15 (36,6%) Pks-positive strains carring all of clbB, clbN, clbA ? clbQ genes, that composed 27,1% from total number of the enterobacteria, isolates from urine. Among 44 clinical isolates of various species of enterobacteria only one Pks-positive strain K. pneumoniae was revealed. Strains enterobacteria, isolated at pyoinflammatory complications after liver transplantation (n = 11) and isolates from intestinal tract in dysbiosis (n = 26), were Pks-negative. CONCLUSION: The clbB, clbN, clbA ? clbQ genes of the Pks-island which have been detected in 36,6 % E. coli urological strains are markers of pathogenicity of clinical isolates of extraintestinal origin and advisable of their detection by PCR.

[Bacterial mixed infection in women with chronic recurrent cystitis].

AIM: To study microbial repertoire of urine in healthy women and patients with chronic recurrent cystitis (CRC) including facultative anaerobic (FA) and non-clostridial anaerobic (NCA) bacteria. MATERIALS AND METHODS. Triple bacteriological study of urine was performed in three groups of women: group I--22 healthy virgin women aged 18- 25 years, group II--24 women aged 18 - 25 years with regular sexual contacts, group III--72 women aged 20 - 60 years with CRC, before antibacterial therapy. Bacteriological method was used to study qualitative and quantitative composition of urine microflora. RESULTS: In all subjects from groups I and II aerobic-anaerobic associations with predomination of coagulase-negative staphylococci (CNS), corynebacteria, peptococci, and peptostreptococci were isolated from urine. Quantity of isolated NCA bacteria was significantly higher than that of FA. In etiologic structure of CRC, NCA bacteria, enterobacteria, and CNS predominated. Spectrum of NCA bacteria isolated from patients with CRC was wider and level of bacteriuria--higher (p < 0.05) compared to groups I and II. Bacteria were identified in aerobic-anaerobic associations. In 85.7% of cases following NCA were identified in biopsy samples: Propionibacterium sp. (41.8%), Peptococcus sp. (35.7%), Eubacterium sp. (28.6%), Peptostreptococcus sp. (14.3%), and Bacteroides sp. (14.3%). Aerobic-anaerobic associations were observed in 7.1% of samples. CONCLUSION: Urine of healthy women is not sterile. Aerobic-anaerobic mixed infections were detected in patients with CRC that should take into account during diagnostics and treatment of this disease.

[Etiological structure and antibiotic sensitivity of uropathogens in chronic recurrent infection of the lower urinary tract].

While acute infections of the lower urinary tract (UT) have been studied in detail and antibiotic therapy of such infections is well known, etiology and choice of antibacterial treatment in recurrent chronic UT infection are not so clear. In our trial we aimed at elucidation of etiological structure of chronic cystitis recurrences by means of microbiological investigation of urine samples from 72 women on extended spectrum of nutrient media. In all the cases urine was infected with both aerobic and non-clostridial anaerobic bacteria. We determined prevalent pathogens and their antibiotic sensitivity and compared them with standard pathogens and their sensitivity in acute lower UT infection. We came to the conclusion that it is necessary to develop new recommendations on antibiotic treatment of recurrent chronic cystitis in women.