Wnt signalling in gynaecological cancers: A future target for personalised medicine?

The three major gynaecological cancers, ovarian, uterine and cervical, contribute a significant burden to global cancer mortality, and affect women in both developed and developing countries. However, unlike other cancer types that have seen rapid advances and incorporation of targeted treatments in recent years, personalised medicine is not yet a reality in the treatment of gynaecological cancers. Advances in sequencing technology and international collaborations and initiatives such as The Cancer Genome Atlas are now revealing the molecular basis of these cancers, and highlighting key signalling pathways involved. One pathway which plays a role in all three cancer types, is the Wnt signalling pathway. This complex developmental pathway is altered in most human malignancies, and members of this pathway, particularly the recently linked ROR receptor tyrosine kinases may be attractive future therapeutic targets. This review provides an up-to-date summary of research into Wnt signalling and ovarian, uterine and cervical cancers, and discusses the potential of the Wnt pathway as a future target for personalised medicine in gynaecological cancers.

Monitoring of antibiotic consumption and development of resistance by enterobacteria in a tertiary care hospital.

WHAT IS KNOWN AND OBJECTIVE: Antibiotics are the most frequently used drugs in hospitalized patients, but studies have shown that the prescribed antibiotics may be inappropriate and may contribute to antibiotic resistance. We carried out a survey of antibiotic consumption and antibiotic resistance in our tertiary care university hospital, from 2005 to 2013. We focus on cephalosporins, one of the most prescribed groups of antibiotics in the tertiary health care. The objective was to identify any relationship between ceftriaxone consumption and resistance by enterobacteria. METHODS: Antibiotics consumption and antimicrobial resistance were monitored in the tertiary care university hospital from 2005 to 2013. Data on the use of antibiotics in surgical inpatients were obtained and expressed as defined daily doses per 100 bed days. Bacterial resistances were given as percentages of resistant isolates. RESULTS AND DISCUSSION: There was an increasing trend in cephalosporins consumption from 9·56 DBD (2005) to 23·32 DBD (2013), with ceftriaxone as the most frequently used cephalosporin, 3·6 DBD (2005) to 10·78 DBD (2013). E. coli and P. mirabilis resistance to ceftriaxone increased significantly from 22% in 2005 to 47% in 2013 and from 31% in 2005 to 60% in 2013, respectively. We found a significant correlation between ceftriaxone consumption and E. coli resistance (r = 0·895, P < 0·05). WHAT IS NEW AND CONCLUSION: Our study shows that cephalosporin consumption increased from 2005 to 2013, with ceftriaxone as the most prescribed antibiotic. E. coli and P. mirabilis resistance to ceftriaxone increased significantly over the study period. E. coli resistance increased with ceftriaxone consumption.

Population pharmacokinetics of tacrolimus in kidney transplant patients.

OBJECTIVE: The purpose of this study was to derive population pharmacokinetics (PPK) model of tacrolimus clearance, identify and describe factors that influence it in Serbian kidney transplant patients. METHODS: Population pharmacokinetics analysis was performed using nonlinear mixed-effects model (NONMEM) program from Serbian adult kidney transplant patients receiving triple immunosuppressive therapy, including oral tacrolimus. Details of drug dosage history, sampling time and tacrolimus concentration in 63 patients (44 males and 19 females), 27 - 57 years old (age mean 40.88 +/- 7.01 years) were collected retrospectively. Effects of several covariates on tacrolimus clearance were tested: total body weight, gender, age, posttransplantation days, hemoglobin count, CRP, alanine aminotransferase/aspartate aminotransferase, total daily dose of tacrolimus, co-medication with cotrimoxasole, omeprazole, mycophenolate mofetil and prednisone (> 25 mg). RESULTS: Typical mean value of tacrolimus clearance, estimated by the base model (without covariates), in our population was 1.03 l h-1. The final model showed that tacrolimus clearance increased with total daily dose and concomitant administration of high-dose prednisone (> 25 mg). The magnitude of prednisone effect was + 1.16 l h-1. Final model was validated in a group of 17 patients, showing good predictive performance. CONCLUSIONS: The derived model describes well tacrolimus clearance in terms of characteristics of Serbian kidney transplant patients, offering basis for rational individualization of tacrolimus dosing regimens.

Chronic stress differentially affects antioxidant enzymes and modifies the acute stress response in liver of Wistar rats.

Clinical reports suggest close interactions between stressors, particularly those of long duration, and liver diseases, such as hepatic inflammation, that is proposed to occur via reactive oxygen species. In the present study we have used 21-day social isolation of male Wistar rats as a model of chronic stress to investigate protein expression/activity of liver antioxidant enzymes: superoxide dismutases (SODs), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GLR), and protein expression of their upstream regulators: glucocorticoid receptor (GR) and nuclear factor kappa B (NFkB). We have also characterized these parameters in either naive or chronically stressed animals that were challenged by 30-min acute immobilization. We found that chronic isolation caused decrease in serum corticosterone (CORT) and blood glucose (GLU), increase in NFkB signaling, and disproportion between CuZnSOD, peroxidases (CAT, GPx) and GLR, thus promoting H2O2 accumulation and prooxidative state in liver. The overall results suggested that chronic stress exaggerated responsiveness to subsequent stressor at the level of CORT and GLU, and potentiated GLR response, but compromised the restoration of oxido-reductive balance due to irreversible alterations in MnSOD and GPx.

Long-term effects of chromium on morphological and immunological parameters of Wistar rats.

Hexavalent chromium raises high concern because of its wide industrial applications and reported toxicity. Long-term (135 days) oral exposure of Wistar rats to chromium in the form of K2Cr2O7 (exposed group~20 mg/kg/day) led to a decrease in thymus mass and thymocytes' number and caused structural and functional changes in the lymph nodes and spleen, namely lymphoreticular hyperplasia and plasmocytic macrophage transformation. Programmed cell death was increased in both thymocytes and splenocytes and decreased in lymphocytes in the T-zones of spleen and lymph nodes. Moreover, Cr (VI) administration decreased myeloid cells' and neutrophils' number, while it increased lymphoid and erythroid cells' number in bone marrow. Cr (VI) immune system effects seem to be related to oxidative stress induction, as depicted by the increased levels of diene conjugates and malondialdehyde in the spleen and liver and by the decreased activity of catalase and superoxide dismutase in rats' erythrocytes. In addition, exposure to Cr (VI) decreased copper, nickel and iron concentrations in blood and liver, while Cr levels in blood, spleen and liver were increased, as expected. The observed changes in the series of immunological parameters studied contribute to the development of new approaches for the prevention of low level Cr exposure toxicity.

Cardioprotective effects of fullerenol C(60)(Oh)(24) on a single dose doxorubicin-induced cardiotoxicity in rats with malignant neoplasm.

The therapeutic utility of the anthracycline antibiotic doxorubicin is limited due to its cardiotoxicity. Our aim was to investigate the efficacy of fullerenol C(60)(OH)(24) in preventing single, high-dose doxorubicin-induced cardiotoxicity in rats with malignant neoplasm. Experiment was performed on adult female Sprague Dawley rats with chemically induced mammary carcinomas. The animals were sacrificed two days after the application of doxorubicin and/or fullerenol, and the serum activities of CK, LDH and alpha-HBDH, as well as the levels of MDA, GSH, GSSG, GSH-Px, SOD, CAT, GR, and TAS in the heart, were determined. The results obtained from the enzymatic activity in the serum show that the administration of a single dose of 8 mg/kg in all treated groups induces statistically significant damage. There are significant changes in the enzymes of LDH and CK (p < 0.05), after an i.p. administration of doxorubicin/fullerenol and fullerenol. Comparing all groups with untreated control group, point to the conclusion that in the case of a lower alpha-HBDH/LDH ratio, results in more serious the liver parenchymal damage. The results revealed that doxorubicin induced oxidative damage and that the fullerenol antioxidative influence caused significant changes in MDA, GSH, GSSG, GSH-Px, SOD, CAT, GR, and TAS level in the heart (p < 0.05). Therefore, it is suggested that fullerenol might be a potential cardioprotector in doxorubicin-treated individuals.

Analysis of the tennis racket vibrations during forehand drives: Selection of the mother wavelet.

The time-frequency analysis of the tennis racket and hand vibrations is of great interest for discomfort and pathology prevention. This study aimed to (i) to assess the stationarity of the vibratory signal of the racket and hand and (ii) to identify the best mother wavelet to perform future time-frequency analysis, (iii) to determine if the stroke spin, racket characteristics and impact zone can influence the selection of the best mother wavelet. A total of 2364 topspin and flat forehand drives were performed by fourteen male competitive tennis players with six different rackets. One tri-axial and one mono-axial accelerometer were taped on the racket throat and dominant hand respectively. The signal stationarity was tested through the wavelet spectrum test. Eighty-nine mother wavelet were tested to select the best mother wavelet based on continuous and discrete transforms. On average only 25±17%, 2±5%, 5±7% and 27±27% of the signal tested respected the hypothesis of stationarity for the three axes of the racket and the hand respectively. Regarding the two methods for the detection of the best mother wavelet, the Daubechy 45 wavelet presented the highest average ranking. No effect of the stroke spin, racket characteristics and impact zone was observed for the selection of the best mother wavelet. It was concluded that alternative approach to Fast Fourier Transform should be used to interpret tennis vibration signals. In the case where wavelet transform is chosen, the Daubechy 45 mother wavelet appeared to be the most suitable.

Fullerenes in biomedicine.

Studies on the biological properties of fullerene C(60) and its derivatives started a decade ago as curiosity-driven studies and are now flourishing as an area of transdisciplinary research. This paper summarizes the results of studies on the biological activity and applications of selected functionalized fullerenes that were published in the last few years. Apart from literature data, we present most of our results of in vitro and in vivo studies with fullerenol C(60)(OH)(24) anti-oxidative and free radical scavenger activities in chemical and biological systems; cytotoxicity against human tumor cell lines; protective effects against various cytotoxic drugs and irradiation; effects on cell cycle and apoptosis, and in vivo radioprotective and cardioprotective effects. The fullerene family of carbon molecules has been a central focus in the emerging fields of nanotechnology and nanomedicine. Fullerenes take an important place in the development of nanobiotechnology and nanomedicine-related products.

Migration and invasion is inhibited by silencing ROR1 and ROR2 in chemoresistant ovarian cancer.

Ovarian cancer survival remains poor despite recent advances in our understanding of genetic profiles. Unfortunately, the majority of ovarian cancer patients have recurrent disease after chemotherapy and lack other treatment options. Wnt signalling has been extensively implicated in cancer progression and chemoresistance. Therefore, we investigated the previously described Wnt receptors ROR1 and ROR2 as regulators of epithelial-to-mesenchymal transition (EMT) in a clinically relevant cell line model. The parental A2780- and cisplatin-resistant A2780-cis cell lines were used as a model of ovarian cancer chemoresistance. Proliferation, adhesion, migration and invasion were measured after transient overexpression of ROR1 and ROR2 in the parental A2780 cell line, and silencing of ROR1 and ROR2 in the A2780-cis cell line. Here we show that ROR1 and ROR2 expression is increased in A2780-cis cells, alongside ß-catenin-independent Wnt targets. Knockdown of ROR1 and ROR2 significantly inhibited cell migration and invasion and simultaneous knockdown of ROR1 and ROR2 significantly sensitised cells to cisplatin, whilereas ROR overexpression in the parental cell line increased cell invasion. Therefore, ROR1 and ROR2 have the potential as novel drug targets in metastatic and recurrent ovarian cancer patients.

Factors related to the process of seeking and completing treatment for drug abuse (qualitative methods in drug abuse research).

Numerous studies have demonstrated the effectiveness of drug abuse treatment. Yet many drug abusers do not enter treatment, many who do enter leave prematurely, and relapse following treatment is common. Understanding motivation for change and treatment readiness is key to understanding how to induct and engage drug users in treatment. To the extent that treatment programs focus initially on reducing drug use, rather than psychosocial problems that motivate individuals to seek treatment, treatment programs may fail to meet the primary needs of users and thus fail to attract or engage them. Outcomes of substance abuse treatment programs historically have been measured by successful program completion, reduced drug use and illegal activity, and improved social functioning (employment, education etc). There is minimal reference to client expectations of treatment and factors that influenced treatment-seeking behavior. Studies that have assessed client dropout from substance abuse treatment have generally focused upon quantitative measures that attempt to determine what types of clients drop out or stay, or what types of characteristics best predict client dropout. Qualitative methods are the most appropriate to fill these gaps in substance abuse treatment research.

Protein kinase A regulates Lewis lung carcinoma adherence to extracellular matrix components and spontaneous metastasis.

Tumor cell adhesion to and migration through the extracellular matrix (ECM) can influence their capacity to disseminate. Since prior studies with Lewis lung carcinoma (LLC) tumors had shown metastatic clones to have more protein kinase A (PKA) activity than nonmetastatic clones, the present study assessed if PKA regulates the interaction between tumor and the ECM, and how this may be associated with the metastatic capacity of the tumor cells. This was accomplished with the use of metastatic (LLC-LN7) and nonmetastatic (LLC-C8) variants that had been stably transfected to overexpress the PKA Calpha subunit or to have blocked PKA activity. Cells with increased PKA activity were less adherent to vitronectin, laminin, and collagen I, and could more readily migrate through these ECM components than could transfectants with reduced PKA activity. PKA did not regulate adhesion to or migration through fibronectin, and did not appear to be associated with changes in expression of surface integrins. In addition to modulating tumor adhesion and migration in vitro, PKA activation caused an increased formation of metastases from s.c. tumors, but did not regulate formation of experimental metastases by i.v. injected tumor cells. These results suggest that PKA signaling is important for modulating the tumor-ECM interaction and can facilitate tumor transit from the primary tumor site.

Oxidative stress in diabetic pregnancy: SOD, CAT and GSH-Px activity and lipid peroxidation products.

Diabetic pregnancy is often complicated by a number of pathological conditions among which is increased oxidative stress. This study was conducted to investigate the parameters of oxidative stress in 90 patients divided into the three groups: pregnant women with Type 1 diabetes mellitus, healthy pregnant women and non-pregnant women. In pregnancy groups all parameters were followed in 1st, 2nd and 3rd trimester. Diabetic control was monitored by fasting blood glucose and glycosylated hemoglobin (HbA(1c)) and these values, as well as measured biochemical parameters (urea, creatinine, total cholesterol and uric acid), were appropriate throughout the study. The concentration of TBARS, as a measure of lipid peroxidation, and activity of antioxidant enzymes superoxide dismutase (Cu, Zn-SOD), glutathione peroxidase (GSH-Px) and catalase (CAT) were investigated in hemolysate of erythrocytes. TBARS concentration increased significantly in pregnant women when compared with control group (non-pregnant women), as well as in pregnant diabetics compared with healthy pregnant women. The SOD activity was gradually increased in the group of normal pregnant women vs. non-pregnant group, but decreased significantly in the group of diabetic pregnant women. Catalase activity was significantly increased only in 3rd trimester diabetic pregnant women. Increased lipid peroxidation and reduced antioxidant status, despite good diabetic control, show that pregnant women are exposed to oxidative stress to a greater degree than controls.

Applicational possibilities of linear and non-linear (polynomial) regressions and analysis of variance: kinetics of chloramphenicol.

The dependance of the content and microbiological activity of Chloramphenicol (active substance) at dissolution on time and on pH, as well as that of the content and microbiological activity of Chloramphenicol (250 mg capsules) at release, on time, in in-vitro conditions, was determined using linear and non-linear (polynomial and generalized dilution) regressions. Based on the square error value, the dependance of the content and microbiological activity of Chloramphenicol (active substance) at dissolution on time and different pH values, as well as the dependance of the content and microbiological activity of Chloramphenicol (capsules) at release on time were best described by polynomial function. The comparison of the content and microbiological activity of Chloramphenicol (active substance) at dissolution at different pH values, as well as of Chloramphenicol (capsules) at release showed the significant correlation between these parameters (r = 0.999, P << 0.001). The comparision of the content, on one hand, and microbiological activity of Chloramphenicol (active substance), on the other, at dissolution at different pH values, as a function of time, was done using a modified method of one-way analysis of variance for linear regression comparisons. Based on the value of Fischer's coefficient (F), there is a statistically very significant difference between the contents and between the microbiological activities of chloramphenicol (active substance) at dissolution and different pH as a function of time (P << 0.005).

[Non-specific sacroiliitis]. / Nespecificni sakroileitis.

We present retrospectively five cases of non-specific sacroileitis which were established and treated in Special Orthopaedic Hospital Banjica Belgrade in the period of 12 years, from 1982 to 1994. Patients have been from 17 to 27 years of age. All of them had been treated initially in other institutions under different diagnosis: specific sacroileitis, specific coxitis, specific spondylitis, abscess of gluteal region and discarthrosis and lumboishialgia. All of the patients have been treated by antibiotics, one by tuberculostatics, one surgically by drainage and one by coxofemoral spica cast. In spite of short period of follow up between 1.5 month and 2 years, we have had excellent clinical and laboratory recovery.

Characteristics of agoraphobia in women and men with panic disorder with agoraphobia.

We compared female and male patients with panic disorder with agoraphobia (PDA) in terms of characteristics of agoraphobia (AG). Ninety-five patients (73 women and 22 men) with the SCID-based diagnosis of PDA were administered the National Institute of Mental Health Panic Questionnaire (NIMH PQ), and women and men were compared on the items of the NIMH PQ that pertain to AG and symptoms of panic attacks. Male and female patients did not differ significantly with respect to demographic characteristics, age of onset of panic disorder and AG, duration of PDA, and severity and frequency of symptoms experienced during panic attacks. Women avoided more situations than did men, but this difference was not statistically significant. Women avoided buses and being in unfamiliar places alone significantly more often. The only situation that was avoided more often by men, although not significantly, was staying at home alone. Women were significantly more likely to stay at home to avoid agoraphobic situations and significantly less likely to go outside of home alone. When going outside, women required a companion significantly more often. There were significantly more married women than married men who required a spouse as a companion, and significantly more women with children than men with children who required a child as a companion. Women thought that AG had affected the overall quality of their lives significantly more adversely. Whereas the overall "profile" of agoraphobic situations does not seem to distinguish between female and male patients with AG, females may be more impaired and appear more dependent than men in terms of requiring companions to move outside of the home. Cultural and psychological factors may be most likely to account for these findings.

[Immunologic characteristics of peritoneal fluid in endometriosis]. / Imunoloske karakteristike peritonealne tecnosti kod endometrioze.

Increased volume of peritoneal fluid is found more frequently in patients with endometriosis (51%) than in infertile patients without endometriosis (13%). Immunologic analysis of the peritoneal fluid shows that in patients with endometriosis the level of immunoglobulin G (IgG) increases. We analyzed 34 samples of peritoneal fluid from patients with and 13 from patients without endometriosis. The mean value of IgG in the group of patients with endometriosis was 7.73g/L and 3.94g/L in the control group. This difference is statistically significant, but there is no statistically significant difference in regard to immunoglobulin A (IgA), while it has been significant for immunoglobulin M (IgM) only in the third stage of the disease. In certain stages of illness there are no statistically significant differences in values of all three immunoglobulin types. A golden standard of immunoglobulin G in peritoneal fluid is 5g/L and in regard to this level we calculated the following: sensitivity, specificity, prognostic value and accuracy of the test. Sensitivity and positive prognostic value reached 85.3%. On the basis of these findings it can be concluded that if no endometriosis can be seen during laparascopy while the volume of peritoneal fluid is increased, immunologic analysis should be performed. If IgG values are 5g/L or higher, the patient should be treated as a patient with possible "precursor endometriosis".

Activation of the protein kinase a signal transduction pathway by granulocyte-macrophage colony-stimulating factor or by genetic manipulation reduces cytoskeletal organization in Lewis lung carcinoma variants.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) that is produced by metastatic Lewis lung carcinoma (LLC-LN7) cells functions as an autocrine stimulator of tumor-cell motility through protein kinase A (PKA) signal transduction. This GM-CSF-mediated enhancement of LLC-LN7 cell motility coincides with a reduction in the level of polymerized F-actin. In contrast, non-metastatic LLC-C8 tumor cells, which have a diminished level of PKA signaling, do not produce GM-CSF and do not respond to exogenous GM-CSF, since they remain non-motile and retain a high content of filamentous actin. The capacity of PKA to regulate the cytoskeletal organization of tumor cells was further studied with the use of LLC variants that had been stably transfected to over-express the C alpha subunit of PKA (CEV cells) or to express a mutant cAMP-resistant PKA RI alpha subunit resulting in a defective PKA (REV cells). When compared with wild-type metastatic LLC-LN7 cells, in which the F-actin staining was too diffuse to be clearly visualized microscopically, the PKA-defective REV-LN7 transfectants had an increased level of F-actin. In comparison with the wild-type non-metastatic LLC-C8 cells, which had a high content of F-actin, the CEV-C8 transfectants that over-expressed PKA activity had a reduced level of F-actin. The reduced polymerization of actin in these CEV-C8 transfectants was accompanied by reduced levels of the intermediate filament protein vimentin and a shift in the distribution both of F-actin and of vimentin to the periphery of the cells. These results show reduced cytoskeletal organization in metastatic LLC-LN7 cells as compared with that of non-metastatic LLC-C8 cells, and indicate that elevation of PKA activity, either by autologous GM-CSF or by genetic manipulation, diminishes cytoskeletal organization.

Prostaglandin E2-protein kinase A signaling and protein phosphatases-1 and -2A regulate human head and neck squamous cell carcinoma motility, adherence, and cytoskeletal organization.

Human head and neck squamous cell carcinoma (HNSCC) cultures were established from cancers of two patients. These cells were used to study if phosphorylation reactions by protein kinase A (PKA) and dephosphorylation reactions by protein phosphatases-1 and -2A (PP-1/2A) regulate tumor motility and adhesion to extracellular matrix components, and if this might be associated with cytoskeletal reorganization. Both cultures were motile and adherent to collagen I, fibronectin, vitronectin and laminin. Motility and adhesiveness was dependent on production of prostaglandin E2 PGE2 and on PKA activation. Blocking PP-1/2A activity with okadaic acid resulted in a PKA-dependent increase in m otility and, in some instances, adhesiveness by the HNSCC cells. The okadaic acid-induced increase in motility and adhesiveness coincided with a reduction in filamentous actin. These data suggest PKA and PP-1/2A have opposing effects in regulating the motility, adherence, and actin polymerization.