Awareness and acceptance of the pandemic influenza (H1N1v 2009) vaccination among antenatal patients in a district general hospital.

Pandemic H1N1 influenza virus was the cause of worldwide respiratory infection in 2009. The majority of these infections were self-limiting, however, high-risk groups, including pregnant women were at increased risk of mortality and morbidity from swine flu. Because of these risks, the World Health Organization recommended that pregnant women should receive the swine flu vaccine during pregnancy. The swine flu vaccine, like the seasonal flu vaccine, is safe to use in pregnancy. In view of the obvious benefits and safety of the pandemic flu vaccine, we decided to undertake a survey to assess the awareness and uptake of the vaccine among pregnant women in our local community. In our survey, lack of counselling from healthcare providers and fears of risks from the vaccine are the main reasons for refusal. For these reasons, healthcare professionals are provided with up-to-date information about the vaccine and are asked to provide this information to pregnant women at all stages of pregnancy in order to increase their awareness and acceptance of the vaccine.

Microarray coupled to quantitative RT-PCR analysis of androgen-regulated genes in human LNCaP prostate cancer cells.

The androgen receptor (AR) mediates the growth-stimulatory effects of androgens in prostate cancer cells. Identification of androgen-regulated genes in prostate cancer cells is therefore of considerable importance for defining the mechanisms of prostate-cancer development and progression. Although several studies have used microarrays to identify AR-regulated genes in prostate cancer cell lines and in prostate tumours, we present here the results of gene expression microarray profiling of the androgen-responsive LNCaP prostate-cancer cell line treated with R1881 for the identification of androgen-regulated genes. We show that the expression of 319 genes is stimulated by 24 h after R1881 addition, with a similar number (300) of genes being significantly repressed. Expression of the upregulated genes, as well as of 60 of the most robustly downregulated genes, was carried out using quantitative RT-PCR (Q-RT-PCR) over a time-course of R1881 treatment from 0 to 72 h. Q-RT-PCR was also carried out following treatment with other AR agonists (dihydrotestosterone, estradiol and medroxyprogesterone) and antagonists (cyproterone acetate, hydroxyflutamide and bicalutamide). This study provides a comprehensive analysis of androgen-regulated gene expression in the LNCaP prostate cancer cell line, and identifies a number of androgen-regulated genes, not described previously, as candidates for mediating androgen responses in prostate cancer cells.

Capsaicin-sensitive primary sensory neurons in the mouse express N-Acyl phosphatidylethanolamine phospholipase D.

Our previous finding, that the capsaicin- and KCl-induced Ca(2+)-dependent production of the intra- and intercellular signaling molecule N-arachidonoyl ethanolamine (anandamide) in cultured primary sensory neurons could be abolished and reduced by approximately 2/3 by capsaicin-induced degeneration of capsaicin-sensitive neurons, respectively suggests that a major sub-population of capsaicin-sensitive cells together with a group of non-capsaicin-sensitive cells should express enzymes involved in Ca(2+)-dependent anandamide synthesis. N-acyl phosphotidylethanolamine phospholipase D (NAPE-PLD) is known to be involved in Ca(2+)-dependent anandamide production. Hence, here, we used reverse transcriptase and quantitative real time polymerase chain reaction to study NAPE-PLD expression in dorsal root ganglia and to clarify the sub-population of cells expressing this enzyme. Cultures prepared from mouse dorsal root ganglia were grown either in the absence or presence of the neurotoxin, capsaicin (10 muM) overnight. We report, that NAPE-PLD is expressed both in dorsal root ganglia and cultures prepared from dorsal root ganglia and grown in the absence of capsaicin. Furthermore, we also report that capsaicin application downregulates the expression of NAPE-PLD as well as the capsaicin receptor, transient receptor potential vanilloid type 1 ion channel, by about 70% in the cultures prepared from dorsal root ganglia. These findings indicate that a major sub-population of capsaicin-sensitive primary sensory neurons expresses NAPE-PLD, and suggest that NAPE-PLD is expressed predominantly by capsaicin-sensitive neurons in dorsal root ganglia. These data also suggest that NAPE-PLD might be a target to control the activity and excitability of a major sub-population of nociceptive primary sensory neurons.

Lifestyle, body composition, and physical fitness changes in Hungarian school boys (1975-2005).

General socioeconomic conditions as well as the physical environment have undergone remarkable changes in Hungary during the past 30 years. Unfortunately, these positive processes have resulted in a reduction of habitual physical activity along with unfavorable changes in dietary habits. Therefore, the purpose of the present study was to compare some selected morphological and functional parameters of 7-14-year-old Hungarian schoolboys living in the middle of the 1970s and at the beginning of the new millennium. It was hypothesized that there would be significant differences in morphological and functional characteristics of the Hungarian schoolboy populations, because they were assessed 30 years apart. Means of height, body mass, body mass index (BMI), the sum of five skinfold tests, percentage of body fat, and two running performance times (400 m and 1,200 m) of the boys (N = 3,672) studied in 1975 were compared to those of the boys (N = 3,758) in 2005. Data were analyzed using two-tailed independent samples t tests (p < .05). We observed significant secular changes in body mass and height. In addition, boys in 2005 had significantly more subcutaneous fat compared to 1975. The running times for the two distances were significantly poorer at the time of the second investigation. The remarkable and unfavorable changes in body composition and cardiorespiratory performance were attributed to the continuously decreasing intensity of habitual physical exercise and a lifestyle that had become more sedentary (watching TV playing computer games, etc.). Radical interventions are necessary to reduce these risks associated with the high prevalence of cardiovascular disease in Hungary, and the challenge to resolve the problem requires combined efforts at the educational, societal, corporate, and governmental levels.

Effects of 3 hours a week of physical activity on body fat and cardio-respiratory parameters in obese boys.

The prevalence of overweight or fat children and adolescents has markedly increased in Hungary during the past three decades. Among the possible factors insufficient physical activity and a relative or absolute excess of calorie intake associated to it can be regarded as the most important ones. The aim of the study was to analyse the effects of a 20-week aerobic exercise on body composition and on the exercise tested cardio-respiratory functions in 10-year-old obese boys. Obesity was defined by a BMI greater than the cut-off value reported by Cole and co-workers (5) and a relative body fat content above 30% (13). Of the study group 21 volunteer children completed the program; the contrast group contained 28 obese boys. Mean calendar age was 10.03 +/- 0.26 in the study group (S) and 9.88 +/- 0.29 in the control group (C). The members of group S had two curricular physical education (PE) classes a week and three extracurricular aerobic physical activity sessions of 60 min net time in the afternoon, on Mondays (swimming and water games), Wednesdays (folk dance) and Fridays (soccer). Group C had only 2 PE classes a week. Anthropometric and spiroergometric data were collected in the middle of January and June of 2004. Relative body fat content and BMI did not increase during the observation period in contrast to the significant increase of both in the control group. Peak minute ventilation, aerobic power, oxygen pulse, and running distance (performed on a treadmill) increased in group S, and did not change in group C. The program was considered successful despite that the changes in the observed physiological and physical indicators appeared to be slight. However, the 5-month elevated level of physical activity brought about such development in the physical status of the obese subjects that might be an appropriate basis for regular training. Fortunately, the cardio-respiratory functions of the investigated boys were not affected yet by obesity, consequently the really dramatic change in their further lifestyle exclusively depends on their decision.

Overweight and obesity in 7 to 10-year-old Hungarian boys. Short communication.

Increasing prevalence of overweight and obesity is a serious social and health problem both in the economically developed and developing countries. Despite this fact the nation-wide growth studies completed in Hungary during the past 30 years had not categorised the children either by body fat content or nutritional status. The aim of the study was to estimate the prevalence of overweight and obese boys in the country at the beginning of the new millennium. Height, body mass and four skinfold thicknesses were measured in 7173 volunteer boys aged between 7 and 10 and living in various urban and rural settlements of Hungary between 2003 and 2005. Nutritional status was qualified by the BMI and relative body fat content. The significantly increasing prevalence with age of overweight and obesity ranged between 10.3 and 23.4%. The results showed the joint effects of a secular trend of growth and of a remarkably changed lifestyle. Of these the consequences of the lifestyle are the more important ones. The high and possibly further increasing prevalence of child-age overweight and obesity reminds one of the observations of Kopp and associates (5), namely that of the increased prevalence of chronic childhood diseases during the past 15 years. More intense habitual physical activity and dramatic changes in dietary habits still promise some solution. No one should reckon, however, with the efficiency of physical education at the schools with its very few classes.

Melanocyte-Directed enzyme prodrug therapy (MDEPT): development of second generation prodrugs for targeted treatment of malignant melanoma.

Evaluation of second generation prodrugs for MDEPT, by oximetry, has highlighted structural properties that are advantageous and disadvantageous for efficient oxidation using mushroom tyrosinase. In particular, a sterically undemanding prodrug bis-(2-chloroethyl)amino-4-hydroxyphenylaminomethanone 28 was synthesised and found to be oxidised by mushroom tyrosinase at a superior rate to tyrosine methyl ester, the carboxylic acid of which is the natural substrate for tyrosinase. The more sterically demanding phenyl mustard prodrugs 9 and 10 were oxidised by mushroom tyrosinase at a similar rate to tyrosine methyl ester. In contrast, tyramine chain elongation via heteroatom insertion was detrimental and the rate of mushroom tyrosinase oxidation of phenyl mustard prodrugs 21 and 22 decreased by 10 nanomol/min.

Absence of the t(14;18) chromosomal translocation in primary cutaneous B-cell lymphoma.

BACKGROUND: The t(14;18)(q32;q21) chromosomal translocation is found in the majority of nodal follicular lymphomas and in a lower percentage of systemic high-grade diffuse large B-cell lymphomas. The translocation results in the juxtaposition of the bcl-2 gene on chromosome 18 with the immunoglobulin heavy chain joining region on chromosome 14. Bcl-2 protein prevents apoptosis and the translocation leads to overexpression of a functionally normal Bcl-2 protein that prevents apoptosis of neoplastic cells. OBJECTIVES: The purpose of our study was to analyse cases of primary cutaneous B-cell lymphoma (PCBCL) for the presence of the t(14;18) translocation and to correlate the results with Bcl-2 expression and histological subtype. METHODS: Forty-four cutaneous B-cell lymphoid proliferations (36 PCBCL, four follicular B-cell lymphomas with cutaneous presentation and four reactive B-cell infiltrates) were analysed by polymerase chain reaction amplification and polyacrylamide gel electrophoresis using consensus primers for the joining region on the immunoglobulin heavy chain gene in combination with either a primer for the major breakpoint region (MBR) or the minor cluster region (mcr) on chromosome 18. RESULTS: None of 36 PCBCL analysed demonstrated a t(14;18) translocation; however, three of four systemic follicular B-cell lymphomas presenting in the skin were found to have a translocation in the MBR, which was confirmed by sequence analysis. Correlation with Bcl-2 immunostaining showed that of seven patients with high-grade cutaneous diffuse large B-cell lymphoma, four were Bcl-2 positive but had no evidence of a t(14;18) translocation. In the five cases classified as primary cutaneous follicle centre cell lymphoma, the neoplastic cells within the germinal centres failed to express Bcl-2. However, Bcl-2-positive neoplastic cells were present in all four cases of systemic follicular lymphoma, including the case that did not show a t(14;18) translocation. In all cases of marginal zone lymphoma the marginal zone lymphocytes were Bcl-2 positive. CONCLUSIONS: These findings indicate that the t(14;18) translocation does not occur in PCBCL, which suggests the involvement of different pathogenetic mechanisms compared with their nodal counterparts. Furthermore, the detection of a t(14;18) translocation in cutaneous B-cell lymphoma should suggest the presence of systemic disease, which underlies the need for exhaustive staging procedures.

In vitro cytotoxicity of norviburtinal and isopinnatal from Kigelia pinnata against cancer cell lines.

Crude dichloromethane extracts of Kigelia pinnata stem bark and fruit showed cytotoxic activity in vitro against cultured melanoma and other cancer cell lines using the Sulphorhodamine B assay, which was used for bioassay-guided fractionation. Thin layer chromatography (TLC) examination of the most active fractions of both stem bark and fruits showed the presence of the same major components which were found to be norviburtinal and beta-sitosterol. Norviburtinal was found to be the most active compound but had little selectivity for melanoma cell lines whilst isopinnatal also showed some cytotoxic activity. beta-Sitosterol was found to be comparatively inactive. HPLC analysis of the crude extract showed that the amount of norviburtinal present in the plant material did not account for all of the activity of the total extracts.

Melanocyte-directed enzyme prodrug therapy (MDEPT): development of a targeted treatment for malignant melanoma.

A novel prodrug rationally designed to function as a tyrosinase substrate has been synthesised to allow targeted treatment of malignant melanoma. This agent has been evaluated for tyrosinase-mediated drug release, and has been shown to act in the desired manner. Furthermore, differential cytotoxicity has been demonstrated in cell lines which express tyrosinase and those which do not.

In vitro synergy of paclitaxel (Taxol) and vinorelbine (navelbine) against human melanoma cell lines.

Paclitaxel (PTXL) (Taxol), a taxane, and vinorelbine (VRB), a semisynthetic vinca alkaloid drug, have tubulin as their common intracellular target, but inhibit growth by binding to different sites. We evaluated in vitro the antiproliferative activity of these two drugs as single agents and in combination, against two human melanoma cell lines, G361 and StM111a. The SRB (sulphorhodamine B) assay was used to determine growth inhibition. Possible drug-drug interaction at the cellular level was assessed by constructing Isoboles (Isobologram analysis) and applying the concept of an 'envelope of additivity'. Both agents were active in the nanomolar range at clinically achievable concentrations. The mean IC50 for G361 was 46.6 nM (PTXL) and 19.9 nM (VRB) after a 1 h drug exposure. Mean IC50 (1 h) for StM111a was 9.7 nM (PTXL) and 26.9 nM (VRB). Isobole analysis at the isoeffect levels of 25%, 50% and 75% indicated that drug interaction was predominantly synergistic (supra-additive) when paclitaxel and VRB were added concurrently for 1 h to cultures of StM11 1a or G361. In some experiments, this synergy was observed with particularly low concentrations of paclitaxel (3 nM) and VRB (0.01 nM). A new points were located within the envelope of additivity or in the subadditive (antagonism) region of the isobole. An overall synergy was also found if the data were analysed by the median effect analysis. The effect of these agents on the cytoskeleton and ultrastructure were studied with immunofluorescence and electron microscopy, respectively. These results confirm the in vitro inhibitory activity of paclitaxel and VRB against malignant melanoma, but more importantly the two drugs appear to act synergistically at relatively low concentrations.

The combination of paclitaxel with cisplatin exhibits antagonism in vitro against human melanoma.

The in vitro cytotoxicity of paclitaxel and cisplatin, alone and in combination, was evaluated against the established human melanoma cell line, G361, with either 1 or 24 h asynchronous paclitaxel exposure using the sulforhodamine B assay. As single agent, the mean cisplatin concentration which inhibited G361 cell growth by 50% (IC50) was 10,000 nM for 1 h exposure. IC50 values obtained with 1 and 24 h paclitaxel exposure were, respectively, 63 and 3.8 nM, concentrations clinically achievable. The combination of paclitaxel with cisplatin was found to be antagonistic by the classical isobologram method, independent of drug sequence and of paclitaxel exposure time. The antagonism was significantly more pronounced for the sequence of paclitaxel followed by cisplatin compared with the reverse sequence for both 1 and 24 h paclitaxel exposure time (p < 0.05). Future clinical protocols employing paclitaxel and cisplatin, both active single agents for the treatment of metastatic malignant melanoma, should take into consideration that the combination of the two drugs may result in significant antagonism, irrespective of drug sequence, if administered within a short interval of each other.

Antibody-directed enzyme prodrug therapy (ADEPT).

A conjugate of the bacterial enzyme carboxypeptidase G2 and the F(ab)(2) fragment of the anti-CEA monoclonal antibody A5B7 was directed in vitro at the human colon tumour cell line LS174T and the human non-small cell lung line COR-L23. Indirect immunofluorescence microscopy was used to show that the conjugate bound to LS174T cells but not to COR-L23. The cytotoxicity generated by addition of a phenol mustard prodrug to each cell line after pre-incubation with conjugate was found to be significantly greater for LS174T cells (IC50=0.24 mu M) than COR-L23 cells (IC50=108 mu M). However, for a 1:1 mixture of these cells an IC50, of 3.4 mu M was obtained. These data show that phenol mustard released by a localised conjugate can exert a bystander effect on neighbouring cells to which the conjugate does not bind.

Activity of platinum drugs against melanoma cell lines: is it modulated in vitro in the presence of tamoxifen?

Cisplatin and carboplatin have been used against human malignant melanoma as single agents and in combination. Tamoxifen is used in the treatment of breast cancer, but has no significant activity against human malignant melanoma. Tamoxifen, however, has been promoted as a modulator in some drug regimens. The addition of tamoxifen to cisplatin or carboplatin has been reported to enhance their activity against the human melanoma cell line T-289. We investigated whether tamoxifen potentiates, in vitro, the activity of cisplatin and carboplatin against C32, G361 and StMl11a melanoma cell lines. Tamoxifen alone at clinically achievable concentrations of 0.1 and 1.0 microM (168 hrs exposure) had no significant effect on growth. No chemopotentiation of the activity of cisplatin or carboplatin was observed with the addition of tamoxifen (0.1 and 1.0 microM). The platinum drugs were added for 1 hr (serially diluted from 100.0 microM). Against the G361 line there was a trend towards chemopotentiation of cisplatin by 0.1 microM of tamoxifen. However, this did not reach statistical significance. Tamoxifen (5.0 and 10.0 microM) produced some inhibitory activity, and a trend towards synergy with cisplatin was observed. However, these concentrations are not clinically feasible. Previous reports detecting synergistic interaction between tamoxifen (0.1 and 1.0 microM), and the platinum compounds against the T-289 melanoma cell line cannot be supported in our in vitro system.

Activity of extracts of Kigelia pinnata against melanoma and renal carcinoma cell lines.

Serial dilutions of standardised water, ethanol, and dichloromethane extracts of the stembark and fruits of Kigelia pinnata were tested for their growth inhibitory effects against four melanoma cell lines and a renal cell carcinoma line (Caki-2) using two different (MTT and SRB) assays. Lapachol, a possible constituent of these extracts, together with known therapeutic antineoplastic agents, was also tested in the same way. The IC50 of each extract was measured after extracts were diluted to 100 micrograms/ml in 1% ethanol or water. Significant inhibitory activity was shown by the dichloromethane extract of the stembark and lapachol (continuous exposure). Moreover, activity was dose-dependent, the extract being less active after 1 h exposure. Chemosensitivity of the melanoma cell lines to the stembark was greater than that seen for the renal adenocarcinoma line. In marked contrast, sensitivity to lapachol was similar amongst the five cell lines. Lapachol was not detected in the stembark extract.

Growth inhibitory activity of S12363, a novel vinca alkaloid derivative on human melanoma cell lines.

The growth inhibitory activity of S12363, a new antineoplastic agent which belongs to the vinca alkaloid group incorporating an amino-phosphonate (bioester of valine), was studied on six human melanoma cell lines with different phenotypic characteristics and in vitro growth rates. S12363 was compared with vinblastine (VBL), vincristine (VCR) and vindesine (VDS) using the MTT assay. Inhibition was time- and dose-dependent. Overall, IC50 values ranged from 24-6770nM and 4.6-11.6 nM for the reference drugs and for S12363 respectively, after exposure for 1 hr. All the vinca alkaloids were more active when cells were exposed continuously for 72 hrs, inhibition by S12363 was greater than the reference drugs (p < 0.05 in 15/18 comparisons). The activities of VDS and S12363 were also compared using the clonogenic assay. IC50 values ranged from 45-500 nM and 17-75 nM respectively. On a molar basis, S12363 was significantly more active than VDS (ANOVA p < 0.0001). The shape of the cell survival curve obtained with S12363 was exponential, whereas that of VDS was of the exponential-plateau type. Furthermore, survival with higher concentrations of S12363 was inversely related to cells seeded. Cell cycle analysis showed these compounds to block cells in G2+M after exposure to their respective IC50 concentrations for 1 hr. This effect was obtained using a lower S12363 concentration. In summary, S12363 proved to be 18-83 times more active than the reference drugs in the MTT and 3-11 times more active than VDS in the clonogenic assay. Its high potency and dissimilar cell survival profile indicate that this compound possesses different biological properties, and therefore merits further in vivo evaluation.

Antiproliferative activity of vinorelbine (Navelbine) against six human melanoma cell lines.

We compared in vitro the cytotoxic activity of vinorelbine (VRB) (Navelbine, 5'-nor-anhydro-vinblastine) a novel Vinca alkaloid, with that of vinblastine (VBL) vincristine (VCR) and vindesine (VDS). Five continuous human melanoma cell lines (C32, G361, StMl11a, StMl12d and StMl14a) and a new line WHMel 1, were used in this study. In growth-inhibition assays, VDS and VRB exhibited comparable cytotoxicity against the C32 and G361 melanoma lines; the effect being dose- and time-dependent. VRB appeared less inhibitory compared to VDS in the lowest concentrations (0.1-1 nM). The same activity was observed with 0.1-1 microM. These drugs exhibited comparable growth inhibition at clinically achievable doses. The MTT assay was used to compare VBL, VCR, VDS, and VRB. Overall IC50 values (concentration required to reduce viability by 50%) ranged from 1 pM to 10 nM. The reduction in cell viability with VRB was similar to that observed with the reference drugs in four out of five lines tested by this method. However a trend was observed for IC50 values to be lower with VBL and VDS. In clonogenic assays (StMl11a, StMl12d and StMl14a lines, 1 h exposure) VRB and VDS produced the same reduction in survival. Survival curves were exponential followed by a terminal plateau. IC50 values ranged from 60 nM to 70 nM. Our results indicate that VRB has in vitro activity against six melanoma lines with differing phenotypic characteristics.

Modulating the oxygen tension in tumours by hypothermia and hyperbaric oxygen.

Hypothermia is associated with reduced metabolism of tissues and especially reduced oxygen consumption by tumours. If the blood supply to a hypothermic tumour can be maintained then the hypoxic fraction of cells should be reduced and the radiation response increased. This hypothesis has been tested with radiation under hyperbaric oxygen and increased tumour response has been demonstrated.

Effect of hypothermia on radiosensitization.

Hypothermia reduces metabolism and oxygen utilization by tissues. If the blood supply to a solid tumour can be maintained at a sufficient level, the hypoxic fraction of tumour cells may be reduced and radiosensitivity increased. This may be achieved if hyperbaric oxygen is used in combination with the hypothermia. The blood supply and oxygen tension have been measured in C3H mouse mammary tumours under hypothermia and hyperbaric oxygen, and the enhancement of radiosensitivity by hyperbaric oxygen has been estimated in mice irradiated at different temperatures with and without anaesthesia. Measurement of xenon-133 clearance showed that the blood supply of a tumour tended to increase when anaesthetized mice became hypothermic. Oxygen cathode data showed that the oxygen tension tended to be relatively higher in tumours and lower in subcutaneous tissue when mice exposed to hyperbaric oxygen became hypothermic under anaesthesia. Hyperbaric oxygen enhanced the radiation response of the tumour in terms of an increase in regrowth delay by a factor of 1.7 when the mice had been anaesthetized, whether or not they became hypothermic. A lower factor of 1.4 was obtained without anaesthesia although induced hypothermia increased the response to a small extent. We conclude that anaesthesia and hypothermia affect oxygen metabolism in tumours by different mechanisms.