Marianna Murdjeva

Experimental study on the role of 5-HT2 serotonin receptors in the mechanism of anti-inflammatory and antihyperalgesic action of antidepressant fluoxetine.

ABSTRACT INTRODUCTION: Fluoxetine is an antidepressant that has anti-inflammatory and antihyperalgesic effects in experimental models of pain and inflammation. The AIM of the present study was to determine the role of 5-HT2 receptors in the mechanism of anti-inflammatory and antihyperalgesic action of fluoxetine after single and repeated administration of the drug. MATERIALS AND METHODS: 40 male Wistar rats were randomly divided in five groups (n = 8) treated for 14 days with saline (control), diclofenac (positive control), fluoxetine, cyproheptadine (5-HT2 antagonist), and fluoxetine + cyproheptadine, respectively. We used the experimental model of inflammation induced by intraplantar injection of carrageenan and nociceptive test with mechanical pressure on the inflamed hind paw. RESULTS: Single and repeated administration of fluoxetine showed that it had significant anti-inflammatory and antihyperalgesic effects when compared with the control (p < 0.05). Cyproheptadine did not change significantly the anti-inflammatory effect of fluoxetine in the first 4 hours, after a single administration. At 24 hours the combination did not differ statistically when compared with the control. Cyproheptadin did not change significantly the anti-inflammatory effect of fluoxetine after repeated administration. After prolonged treatment the group that received fluoxetine + cyproheptadine showed a statistically significant increase in paw pressure to withdraw the hind paw compared with that treated with fluoxetine alone (p < 0.05). CONCLUSIONS: Fluoxetine has anti-inflammatory and antihyperalgesic effects in the carrageenan model of inflammation. 5-HT2 receptor mediated its anti-inflammatory effect in single dose treated animals. Spinal 5-HT2 receptors are involved in the antihyperalgesic effect of fluoxetine after repeated administration РЕЗЮМЕ ВВЕДЕНИЕ: Флуоксетин представляет собой антидепресант, обладающий противовоспалительным и антигиперальгезическим эффектами при экспериментальных моделях боли и воспаления. ЦЕЛ: Настоящее исследование ставит себе целью установить роль 5-НТ2 рецепторов в механизме противовоспалительного и антигиперальгезического действий флуоксетина при однократном и многократном применениях. МАТЕРИАЛ И МЕТОДЫ: На 40 мужских крыс породы „Вистар”, разделенных на 5 групп (n = 8), в течение 14 дней воздействовали физиологическим раствором (контрольная группа); диклофенаком (позитивная контрольная группа); флуоксетином, ципрохептадином (5-НТ2 антагонист) и флуоксетином + ципрохептадином. Использованы модель воспаления (интрапланетарноe введениe карагенина) и ноцицептивный тест (механическоe давлениe на воспаленную лапку). РЕЗУЛЬТАТЫ: При однократном и многократном применениях флуоксетина наблюдались сигнификантный противовоспалительныйи антигиперальгезический эффекты - сравнение с контрольной группой (р < 0.05). При однократном воздействии ципрохептадин достоверно не изменяет протививовоспалительный эффект флуоксетина в первые четыре часа, но на 24-ый час комбинация не показывает статистическую разницу при сравнении с контрольной группой. При многократном воздействии ципрохептадин достоверно не изменяет противовоспалительный эффект флуоксетина. Группа крыс, подвергнутая продолжительному воздействию флуоксетином и ципрохептадином, показала сигнификантное увеличение силы давления при отдергивании лапки по сравнению с группой, получившей только флуоксетин (р < 0.05). ВЫВОДЫ: Флуоксетин обладает противовоспалительным и антигиперальгезическим эффектами при карагениновой модели воспаления. 5-НТ2 рецепторы медиируют его противовоспалительный эффект при однократном воздействии. Спинальные 5- НТ2 рецепторы участвуют в реализации антигиперальгезического эффекта флуоксетина при его продолжительном применении

Development of a new serum-free cell culture system, McCoy-Plovdiv

Dear Editor: McCoy are synovial cell lines of either human (McCoy A) or murine (McCoy B) origin. These ceils are cultured in Earles minimal essential medium (EMEM), Liverpool-Waymouth medium, or RPMI-1640, supplemented with 5 to 10% of fetal calf serum (Karayiannis et al., 1981; Bose et al., 1986; Meysick et al., 1990; Perotti et al., 1991; Kluytmans et al., 1993; Varanda et al., 1997). McCoy cells are widely used as a model for in vitro cytotoxicity experiments (Vento et al., 1990; Shrivastava et al., 1992; Andersen et al., 1993; Machmoud et al., 1995; Pascual et al., 1997; Varanda et al., 1997; Docheva et al., 1998; Garsia et al., 1998). The presence of serum, however, can lead to interference of serum proteins with the test substances (Stark et al., 1986) and mask the cytotoxic effects. Here we describe the development, stabilization, and the conditions of a continuous culture and preservation of McCoy-Plovdiv clone as a new serum-free cell culture system. McCoy A cell line was obtained from the National Bank of Industrial Microorganisms and Cell Cuhures (NBIMCC), Sofia, Bulgaria, and cultured in MEM+ 10% FCS. In the process of cell adaptation of the new clone to the medium conditions and total absence of serum the effects of three defined growth media were compared: Hams F-12, DMEM, and RPMI-1640. Media were mixed at different ratios as follows: Hams F-12:DMEM (3:1), Hams F-12: DMEM (1:1), Hams F-12:RPMI 1640 (3:1), and Hams F-12:RPM1 1640 (1:1). Cells were evaluated microscopically on their ability for adhesion, spreading, shape, division, monolayer formation, monolayer stability, subconfluent and postconfluent state of the monolayer. These observations determined Hams F-12:DMEM 1:1 (HD) medium as the most suitable for stably maintaining cells in culture. In this growth medium supplemented with DMSO, cells were successfully preserved in liquid nitrogen. We found that 2, 5, 7, or 10% of the cryoprotector in the medium provided 81.7, 83.9, 95.8 and 91.5% cell survival, respectively. Thus for routine storage we use HD + 7% DMSO. Shown in Fig. 1 are microphotographs of McCoy-Plovdiv cells, cultured on cover slips. Inoculation density of 1 • 10 s cell/ml permitted the cells to rapidly adhere to the glass as well as to plastic cell culture flasks. Sixty minutes after inoculation ceils were firmly attached (Fig. 1A). Short and clearly distinct cytoplasmic extensions with various shapes could be observed. Ceils with bipolar fibroblast-like shape were prevailing in the culture 24 h after inoculation (not shown). A confluent culture could be obtained 96 h after inoculation (Fig. 1B). In older cultures cells with epithelial-like shape were predominant. The culture retained its viability as a monolayer 15-17 d after inoculation without change of the medium. Cell death in the culture under these conditions was only moderate. We found that trypsin had to be removed by centrifugation following trypsinization. If this step was omitted, the culture did not develop normally. Cells adhered but did not spread, forming spherical grape-shaped clusters (Fig. 1C).

Guidelines for cultivation and preservation of the serum-free cell line McCoy-Plovdiv

In this paper, we offer detailed information concerning manipulations with the novel serum-free cell line McCoy-Plovdiv. Guidelines for procedures as trypsinization of the monolayer, subculturing, as well as freezing and thawing conditions are proposed. Our results give us grounds to assume that this is a cell line entirely serum-independent at any step of the process of culturing and preservation. The serum-free cell line McCoy-Plovdiv enriches the collection of mammalian serum-free cell lines. The easier cultivation, the lower expenses and the higher sensitivity in comparison with the serum-supplemented McCoy cells are discussed as possibilities for broad applications of McCoy-Plovdiv cells in different types of laboratory investigations.

Study on anti-inflammatory and immunomodulatory effects of fluoxetine in rat models of inflammation

The aim of the present study was to evaluate the anti-inflammatory effect of fluoxetine in carrageenan- and lipoplysaccharide-induced models of inflammation by investigating the changes in serum levels of pro-inflammatory cytokine TNF-α and anti-inflammatory cytokines IL-10 and TGF-β after single and repeated administration of the drug. To study the effect of a single and repeated dose fluoxetine on carrageenan-induced paw edema male Wistar rats were divided into five groups (n = 8): control group; positive control group; and three experimental groups treated with 5, 10, and 20 mg/kg bodyweight (bw) fluoxetine, respectively. To study the effect of a single and repeated dose of fluoxetine on serum cytokine levels, the animals were divided in four groups (n = 8): two control groups treated with saline and two experimental groups treated with fluoxetine 20 mg/kg bw. Carrageenan and LPS were injected immediately after fluoxetine or saline injection. Serum cytokine concentrations were tested by enzyme immunoassay. In single administration only the highest dose used inhibited carrageenan-induced inflammation. Edema inhibition was seen with 10 and 20 mg/kg bw fluoxetine after repeated administration. At 24 h a statistically significant effect on inhibition of carrageenan edema was found only in rats treated with 20 mg/kg bw fluoxetine In carrageenan-induced inflammation, fluoxetine significantly increased Il-10 and decreased TNF-α after repeated administration. Surprisingly, in single-dose treated animals an increase in TNF-α values upon fluoxetine administration was observed in this model of inflammation. In LPS-induced inflammation, fluoxetine significantly decreased TNF-α after single and repeated treatment. Fluoxetine has anti-inflammatory and immunomodulatory effect in the carrageenan-induced model of exudative inflammation. In LPS-induced inflammation it showed an immunomodulatory effect manifested with a decrease in pro-inflammatory cytokine TNF-α.

Do Human Endogenous Retroviruses Contribute to Multiple Sclerosis, and if So, How?

The gammaretroviral human endogenous retrovirus (HERV) families MRSV/HERV-W and HERV-H (including the closely related HERV-Fc1) are associated with an increased risk of multiple sclerosis (MS). Complete HERV sequences betray their endogenous retroviral origin, with open reading frames in gag, pro, pol and env being flanked by two long terminal repeats containing promoter and enhancer sequences with the capacity to regulate HERV transactivation and the activity of host genes in spite of endogenous epigenetic repression mechanisms. HERV virions, RNA, cDNA, Gag and Env, and antibodies to HERV transcriptional products, have variously been found in the blood and/or brain and/or cerebrospinal fluid of MS patients, with the HERV expression level being associated with disease status. Furthermore, some HERV-associated single nucleotide polymorphisms (SNPs), such as rs662139 T/C in a 3-kb region of Xq22.3 containing a HERV-W env locus, and rs391745, upstream of the HERV-Fc1 locus on the X chromosome, are associated with MS susceptibility, while a negative association has been reported with SNPs in the tripartite motif-containing (TRIM) protein-encoding genes TRIM5 and TRIM22. Factors affecting HERV transcription include immune activation and inflammation, since HERV promoter regions possess binding sites for related transcription factors; oxidative stress, with oxidation of guanine to 8-oxoguanine and conversion of cytosine to 5-hydroxymethylcytosine preventing binding of methyl groups transferred by DNA methyltransferases; oxidative stress also inhibits the activity of deacetylases, thereby favouring the acetylation of histone lysine residues favouring gene expression; interferon beta; natalizumab treatment; impaired epigenetic regulation; and the sex of patients.

Evaluation of antimicrobial resistance among Salmonella and Shigella isolates in the University Hospital “St. George,” Plovdiv, Bulgaria

The aim of this work is to study the epidemiology and antimicrobial resistance to the most commonly used antibiotics for the treatment of acute gastroenteritis caused by Salmonella and Shigella at the largest Bulgarian hospital—University Hospital “St. George,” Plovdiv—for the period 2009–2013. Two hundred ninety strains were in vitro tested for resistance to 15 antimicrobial agents. The presence of extended-spectrum beta-lactamases (ESBLs) was demonstrated by a variety of specialized tests. For comparison, a collection of 28 strains submitted by the National Reference Laboratory (NRL) “Enteric Infections” at the National Center of Infectious and Parasitic Diseases (NCIPD), Sofia, was also tested for the production of ESBLs. In isolates, phenotypically demonstrated as ESBL producers, polymerase chain reaction (PCR) detection of the genes bla-CTX-M, bla-SHV, and bla-TEM was performed. Among the 290 tested isolates, only two—Salmonella serotype Livingstone and Shigella flexneri—were phenotypically proven to be ESBL producers. Only 4 strains from the collection of 28, submitted from the NRL “Intestinal Infections” in NCIPD, Sofia, were phenotypically confirmed as ESBL producers. The presence of the bla-CTX-M gene was detected in all of the tested strains (4 from NRL, NCIPD, Sofia, and 2 from the University Hospital St. George, Plovdiv), the bla-SHV gene only in strain S. Livingstone from Plovdiv, and the bla-TEM gene in two from Sofia and one (again S. Livingstone) from Plovdiv. In conclusion, Salmonella and Shigella isolates from patients hospitalized at the University Hospital St. George, Plovdiv, with acute gastroenteritis demonstrate good susceptibility to the most commonly used antibiotic agents, including azithromycin.

Analysis of clinical and microbiological indicators of vulvovaginal candidiasis

ABSTRACT Vulvovaginal candidiasis (VVC) is the most common vaginal infection. It is considered to be the reason for 15%–30% of all vulvovaginal symptoms. A diagnosis based only on clinical signs and/or on a syndrome-based approach, recommended by some authors and health institutions, is in many cases incorrect. There is no indisputable diagnostic method that guarantees high specificity, sensitivity and predictive value. There is no pathognomonic clinical feature of VVC. The aim of the present study was to assess the role of selected clinical and mycological indicators in the diagnosis of VVC using the standard complex scoring system. The research is retrospective and was conducted in several stages to give a more precise and detailed assessment of the examined clinical and microbiological indicators. Clinical, microbiological and statistical methods were used. The results showed the lowest level of compliance between clinical and laboratory diagnosis in VVC: 67.65%. We did not observe a statistically significant relation between the density of the vaginal smear, inflammation, odour test and VVC (p > 0.05). The results showed a moderate association between the positive yellow swab test and VVC (p < 0.05). A significant positive association was established between VVC and presence of microscopic signs of invasiveness (p < 0.001). The most common isolate was Candida albicans: 72.81% of all vaginal Candida isolates.