The activities of this natural extract have mainly been recognized through clinical experience, but there have been relatively little scientific studies on its biological actions. Here we investigated whether EO extract is able to affect the phagocytic ability of human monocyte derived macrophages (MDMs) in vitro and of rat peripheral blood monocytes/granulocytes in vivo in absence or in presence of immuno-suppression induced by the chemotherapeutic agent 5-fluorouracil (5-FU).Besides few data concerning the antiseptic properties against a range of microbial agents and the anti-inflammatory potential both in vitro in EO treated and untreated MDMs, by confocal microscopy after fluorescent beads administration; ii) in vivo in monocytes/granulocytes from peripheral blood of immuno-competent or 5-FU immuno-suppressed rats, after EO oral administration, by flow cytometry using fluorescein-labelled E. coli. Cytokine release by MDMs was determined using the BD Cytometric Bead Array human Th1/Th2 cytokine kit.Morphological activation of human MDMs was analysed by scanning electron microscopy. Phagocytic activity was tested: i) EO is able to induce activation of MDMs, dramatically stimulating their phagocytic response. EO-stimulated internalization is coupled to low release of pro-inflammatory cytokines and requires integrity of the microtubule network, suggesting that EO may act by means of complement receptor-mediated phagocytosis. Implementation of innate cell-mediated immune response was also observed in vivo after EO administration, mainly involving the peripheral blood monocytes/granulocytes. The 5-FU/EO combined treatment inhibited the 5-FU induced myelotoxicity and raised the phagocytic activity of the granulocytic/monocytic system, significantly decreased by the chemotherapic.Eucalyptus oil extract is able to implement the innate cell-mediated immune response, provide scientific support for an additional use of this plant extract, besides those concerning its antiseptic and anti-inflammatory properties and stimulate further investigations also using single components of this essential oil. This might drive development of a possible new family of immuno-regulatory agents, useful as adjuvant in immuno-suppressive pathologies, in infectious disease and after tumour chemotherapy.Our data, demonstrating that  The monocytic/granulocytic system as well as differentiated macrophages constitute the primary cellular effectors of the immune response, playing a pivotal role in the detection and elimination of foreign bodies such as pathogenic microorganisms. Recognition of foreign microorganisms by these cells ultimately results in phagocytosis and in the eventual destruction of pathogens by lysosomal enzymes. Macrophages perform a variety of functions other than phagocytosis . The phaEucalyptus oil have been demonstrated to possess antiseptic properties against a range of bacteria [in vitro and in vivo. Monoterpenoid components of aromatic constituents of Eucalyptus oil are traditionally used as analgesic, anti-inflammatory, and antipyretic remedies and are commercially available for the treatment of the common cold and other symptoms of respiratory infections. Phytochemical analysis has shown that the profile of the monoterpenoids changes among the Eucalyptus species, with potential variations in therapeutic properties. In Eucalyptus globulus, the major monoterpenoid component is eucalyptol , constituting the 60–90% [Eucalyptus oil, although a direct relationship is still to be demonstrated.Natural oils are extensively used in cosmetics as well as in folk medicine for the treatment of a growing number of more or less specific pathologies. Recently the clinical use of essential oils has expanded worldwide also including therapy against various kinds of inflammatory diseases, such as allergy, rheumatism and arthritis. These activities have mainly been recognized through clinical experience, but there have been relatively little scientific studies on biological actions of these natural extracts. For instance, the tea tree oil as well as bacteria -8 and habacteria ,10 and lbacteria  are ablebacteria ,13 and cbacteria  in in vie 60–90% , that hae 60–90% ,16. An ae 60–90% . These fEucalyptus essential oil on the cellular components of the immune system, the only exception is for the effect on some cytokine production [Eucalyptus globulus (EO) is able to affect the phagocytic activity of human monocyte-derived macrophages (MDMs) in vitro and of peripheral blood monocytes/granulocytes from immuno-competent rats treated in vivo in absence or in presence of immuno-suppression induced by administration of the chemotherapeutic agent 5-fluorouracil (5-FU).To our knowledge, actually there are no available data concerning the influence of oduction ,16. The EO treatment, at both concentrations used in our experiments, did not affect the macrophage viability, as revealed by the Trypan blue exclusion method , increased so dramatically the phagocytic activity of MDMs, showing up to 64.8 phagocytosed beads/cell, that the majority of cells engulfed with indigestible beads possibly died, leading to a registration of only 10% of phagocytic cells. Moreover, pre-treatment with EO 24 h before the addition of LPS to cell culture was able to increase, without any cytotoxicity and in a dose-dependent manner, the phagocytic activity of human MDMs in comparison with the treatment with LPS alone  and the Tea Tree oil (TeaTreeO) in parallel with EO. The results obtained . Moreover, in accordance with the anti-inflammatory properties ascribed to EO, pre-treatment with EO 24h before the addition of LPS to cell culture was able to significantly decrease the LPS-induced cytokines production . No significant effect on the production of the other immune-modulating cytokines tested  was recorded for both EO or LPS treatments .In spite of the morphological features typical of activated macrophages, and of the increased phagocytic ability acquired by one Fig. . This efEO-induced internalization and cytokine production were associated with differences in the cytoskeletal elements that mediate phagocytosis. In particular, we verified if the phagocytic ability of LPS- or EO- stimulated MDMs required integrity of the microtubular network. To this purpose, control cells and macrophages, pre-treated with LPS or EO, were treated with the microtubule-destabilizing agent nocodazole and the phagocytic ability was tested after fluorescent beads administration. Confocal microscopic observation , in EO-stimulated cells bead uptake was almost completely inhibited . These results clearly suggest that LPS- and EO-induced phagocytosis occur possibly by means of different mechanisms, since only the EO-stimulated internalization requires integrity of the microtubule network.We also investigated whether the observed differences in LPS- or ion Fig.  showed tion Fig. , while cion Fig. d1, d2, ited Fig. d3. TheseEO stimulation is able to provoke a more dramatic reorganization of microtubular filaments respect to LPS, strongly suggesting that tubulin plays an important role in the EO-induced macrophage activation.Moreover, as shown in Fig. EO on peripheral blood mononuclear cells (PBMCs) after in vivo administration to immuno-competent animals, following the scheme of treatment reported in Fig. 15 revealed that EO treatment was able to significantly increase the percentage of circulating monocytes in peripheral blood of treated rats compared to the untreated controls, while no significant modification in the percentage of circulating granulocytes and lymphocytes was evidenced .We also examined the monocytic/granulocytic fraction for the expression of surface molecules that may be indicative of leucocytes activation. In particular we evaluated the expression of CD44, the receptor for the extracellular matrix component hyaluronan that mediates the leukocyte/endothelial interactions leading to extravasation , and of ion Fig. . No signEx vivo evaluation of the phagocytic activity of monocytes derived macrophages (MDMs) obtained from treated and untreated animals at T15, revealed that the stimulatory effect recorded in circulating monocytes was also retained in differentiated macrophages , a chemotherapeutic agent extensively used in the treatment of different types of cancer [15 revealed that EO treatment was able to induce a recovery of the percentage of circulating granulocytes that was significantly reduced by 5-FU treatment, while no modification was recorded in the percentage of circulating monocytes, that, on the other hand, was not influenced by the chemotherapeutic agent  extract such as its antiseptic properties against a range of microbial agents [in vitro [in vivo [Some biological effects of l agents ,7 and, rin vitro ,24 and i[in vivo ,17,25, mEO from Eucalyptus globulus is able to induce morphological and functional activation of human MDMs in vitro, dramatically stimulating the phagocytic response of these effectors of the innate immune defence. The acquisition of morphological features of EO activated macrophages and the increased phagocytic ability is coupled to a low release of IL-4, IL-6 and TNF-α pro-inflammatory cytokines, in contrast to that recorded under LPS stimulation.In this study we present findings indicating that EO-induced cytokine production were associated with differences in the cytoskeletal elements that mediate phagocytosis, since EO-stimulated internalization requires integrity of the microtubule network, while LPS-stimulation does not. This suggests, almost preliminary, that LPS and EO might act by means of different mechanisms, possibly involving different phagocytic receptors. It is actually known that internalization by macrophages occurs by a restricted number of phagocytic receptors present on their surface [EO might implement pathogens internalization stimulating the CR-mediated phagocytosis. EO-induced implementation of innate cell-mediated immune response, coupled to reduced production of pro-inflammatory cytokines and toxic oxygen intermediates, might have profound implications for the inflammatory response during pathogen infections.The differences in LPS- or  surface . Specifi surface , while C surface . On thisEO treated cultures of numerous phagocyting polarised cells, exhibiting elongated lamellopodia and filopodia, indicates that stimulation of pseudopodial activity and cell motility might contribute to the augmented phagocytic capability. The in vitro stimulatory effect was achieved using concentrations of the essential oil ranging from 0.008% and 0.016% (v/v), doses that resulted absolutely non toxic and up to 100 fold lower than those used in vitro by other authors [The presence in the  authors ,29.in vitro experiments but also in treated BDIX rats after 15 days EO administration. The stimulatory effect in treated animals, mainly involving the peripheral blood monocytes/granulocytes, led to a raise in the percentage of circulating monocytes as well as to an increment in the phagocytic activity of granulocytes and mostly of monocytic cells. These latter cells also exhibited an enhanced potential of extravasation ability, as suggested by increased expression of CD44 on their cellular membrane [+ cells in circulating monocytes, compared to the untreated control rats.Implementation of innate cell-mediated immune response has been observed not only in membrane , as wellmembrane ,20, as iex vivo experiments. The EO dose used in the in vivo experiments (12 mg/Kg/die) did not produce evident toxic effect on treated rats, as suggested by the lack of body weight reduction and of animals mortality. This dose was comparable to doses recommended for oral therapy in humans and much more lower than dose that have been described to exhibit sub-acute toxicity in rats (about 300 mg/kg/die) [The stimulatory effect on phagocytic ability recorded in circulating monocytes was also retained in monocyte-derived macrophages from treated animals, as showed by the EO on the cell-mediated immune response in immuno-competent rats, this essential oil is also able to induce a dramatic recovery of granulocytes/monocytes activity after bone marrow suppression produced by 5-FU administration. In fact, combined treatment 5-FU/EO not only inhibited myelotoxicity, as resulted by the restoration of granulocytes number to normal values, but also raised the phagocytic activity of granulocytes and monocytes as well as of the ex vivo analyzed monocyte-derived macrophages, significantly decreased in animals treated with the chemotherapic alone. Since myelosuppression continues to be a major dose-limiting side effect for most chemotherapeutic agent such as 5-FU [EO with 5-FU might be taken into account for the prevention of immunotoxicity and myelotoxicity caused by 5-FU administration, even if additional studies should be carried out to demonstrate that the antitumor activity of 5-FU is not influenced by this combined therapeutic strategy.Besides the effect of  as 5-FU , our resin vitro and in vivo, could be due to the major monoterpenoid component eucalyptol , constituting the 60–90% of Eucalyptus oil [Moreover, further experiments are in progress to determine whether the immuno-stimulatory effect, observed both ptus oil , or is rMost knowledge of the therapeutic use of plant essential oils is acquired through folklore, and only some activities of these natural extracts are actually supported by scientific studies. But plants are an important source for drug discovery, and investigations on biological actions of plant medicinal extracts, as well as the understanding of the mechanisms underlying these actions drive the search for novel drugs.Eucalyptus essential oil from Eucalyptus globulus is able to implement the innate cell-mediated immune response, provide scientific support for an additional use of this plant extract, besides those concerning its known antiseptic and anti-inflammatory properties. Thus, the present study stimulates further investigations also using single components of essential oil extracts from various species of Eucalyptus for development of a possible new class of immuno-regulatory agents useful as adjuvant in immuno-suppressive pathologies, in infectious disease as well as in tumour chemotherapy.Overall, our data, demonstrating that 2 flasks and maintained at 37°C in 5% CO2, to generate adhering MDMs. After 1 h of culture, non-adhering cells were removed and the residual adhering MDMs were maintained in culture for 7 days to obtain partially differentiated macrophages. At this time, cells were detached using cold PBS, seeded at a density of 2.5 × 104 cells/cm2 in 35 mm culture plates or on cover-slips (∅ 10 mm) and allowed to adhere for 4–5 days before treatments.Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats of healthy donors by density gradient centrifugation using Lympholyte-H . The lymphocytic/monocytic fraction was then re-suspended in RPMI 1640 medium  supplemented with 10% (v/v) heat-inactivated fetal calf serum (FCS) (Hyclone), L-glutamine (2 mM), penicillin (100 IU/ml) and streptomycin (100 mg/ml) and cells were seeded on 175 cmEucalyptus globulus was purchased from Cruciani Prodotti Crual® Srl, . To exclude that the oil preparation used contained any endotoxins, we tested the EO extract using the Limulus Amebocyte Lysate (LAL) test  The LAL test is a qualitative test for Gram-negative bacterial endotoxin utilizing a lysate prepared from the circulating amebocytes of the horseshoe crab (Limulus polyphemus) standardized to detect the labelled concentration (EU/ml) of the FDA Reference Standard Endotoxin. Limulus Amebocyte Lysate was mixed in equal parts with the solution being tested and incubated 1 h at 37°C. In the presence of endotoxin, a positive reaction is characterized by the formation of a firm gel; in the absence of endotoxin, gelation does not occur. The assay was done as a yes/no test. The EO samples and controls were diluted in sterile water, performed in triplicate and run in parallel. Positive controls consisted in 2 ng/ml (1 EU/ml) E. coli Control Standard Endotoxin or 0.1 μg/ml LPS; negative control consisted in sterile water. The LAL test excluded that the EO extract contained any endotoxins.  for 24 h. Concentrations were selected on the basis of the lowest, non toxic, effective doses found in a preliminary dose-response experiment  for 6 h were used as positive control of macrophage activation. The effect of EO 24 h pre-treatment before stimulation with LPS was also analysed. Essential oils from Lavender oil and the Tea Tree oil , were used as controls to exclude a non specific effect on macrophages phagocytic activity caused by the oil preparation. For any microscopic analysis cells were grown on cover-slips.Human MDMs were treated with 0.008% v/v in RPMI 1640 medium (corresponding to about 50 μg/ml) or 0.016% v/v (about 100 μg/ml) of 4 in the same buffer for 1 h at 4°C and dehydrated using increasing acetone concentrations. The specimens were critical-point dried using liquid CO2 and sputter-coated with gold before examination on a Stereoscan 240 scanning electron microscope .Analysis of morphological feature of activated MDMs, was carried out by phase-contrast microscopy after Wright Giemsa staining and by scanning electron microscopy (SEM). For SEM observation, MDMs were fixed with 2.5% glutharaldehyde in 0.1 M Millonig's phosphate buffer (MPB) at 4°C for 1 h. After washing in MPB, cells were post-fixed with 1% OsO7 beads/ml of yellow-green fluorescent polystyrene beads  with excitation/emission wavelengths of approximately 495 nm/515 nm . After 30 min, cells were fixed with paraformaldehyde, counter-stained with 1 μg/ml propidium iodide (PI – Sigma-Aldrich) and observed by the confocal microscope LEICA TCS SP5 . The excitation/emission wavelengths employed for PI staining were 568 nm/590 nm. A minimum of 500 cells per sample were observed, the number of phagocytic MDMs (reported as percent of phagocytic cells), as well as the number of beads per cell, were counted. MDMs subjected to beads administration and maintained at 0°C to block internalization, was used as negative control of uptake. Quantitative assessment was done in a blinded fashion. The experiment was repeated three times and the mean values were plotted. We analyzed at least 20 buffy coats from different healthy donors.The phagocytic activity of treated and untreated MDMs was tested by adding to cultures 2 × 10in vitro EO treatment were determined by using the BD Cytometric Bead Array human Th1/Th2 cytokine kit (BD Pharmingen) according to the manufacturer's protocol [EO pre-treatment on the pro-inflammatory and immune modulating cytokines production by macrophages stimulated with LPS was also evaluated.The concentration of IL-2, IL-4, IL-6, IL-10, TNF-α, and IFN-γ secreted into the culture media by human macrophages after protocol . Flow cyEO, were treated with 2 μg/ml nocodazole for 30 min. For phagocytic activity testing, after nocodazole treatment, fluorescent polystyrene beads were added to the cell culture, samples were processed as described above and analysed by confocal microscopy. The effect of nocodazole on microtubular network was analysed after immuno-staining, using an anti-human tubulin mouse monoclonal antibody (Molecular Probes) revealed with the secondary Alexa Fluor 488-conjugated anti-mouse IgG (Molecular Probes), by confocal microscopy. Cells were counter-stained with PI (Sigma-Aldrich).To depolymerize microtubules, control cells and macrophages, pre-treated with LPS or EO was administrated for 15 days per os, at a dose of 12 mg/Kg/day, by adding, every evening, the essential oil extract directly to drinking water . Two sets of in vivo experiments were carried out. In the first one, in which we tested the effect of EO on peripheral blood mononuclear cells of immuno-competent rats, animals were divided in two groups , an untreated control group and a group treated for 15 days with EO  were intra-peritonally injected with a single dose of 100 mg/Kg of the chemotherapeutic agent 5-fluorouracil (5-FU) on day 7 from the beginning of EO treatment , on day 7 (T7), on day 15 (T15) and on day 20 (T20). For evaluation of haematological parameters, after erythrocytes lysis by FACS Lysing Solution (Becton Dickinson), mononuclear cell fractions in RPMI 1640 medium were analyzed for forward (FSC) and sideward (SSC) scatter patterns in a fluorescence-activated cell sorter . Gates were defined to identify populations of cells with different FSC/SSC characteristics, corresponding to granulocytes, lymphocytes or monocytes populations and results were expressed as percent of total cells. In addition, rat peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using Lympholyte-H . The lymphocytic/monocytic fraction was then re-suspended in RPMI 1640 medium and rat adhering macrophages were obtained as described previously for human MDMs and used for the ex vivo experiments.Inbred male BDIX rats , 8 weeks old and weighing 220–250 g, were held for 7 days, housed in a pathogen-free animal facility with free access to water and standard food and kept in accordance with European Community guidelines. Experiments were approved by the local committee on animal experimentation, and were performed under strict governmental and international guidelines. in vivo EO administration, in absence or in presence of bone marrow suppression by 5-FU administration, was carried out by cytofluorimetric analysis using the phagotest kit , following the manual's protocol. This test allows the quantitative determination of leukocyte phagocytosis in heparinized whole blood. It contains fluorescein (FITC)-labelled opsonized bacteria (E. coli-FITC) and necessary reagents, and measures the overall percentage of phagocytic monocytes and granulocytes (that have ingested one or more bacteria per cell) and the individual cellular phagocytic activity (number of bacteria per cell) recorded as Mean Fluorescence Intensity (MFI).The evaluation of phagocytic activity of monocytes/granulocytes from peripheral blood of BDIX rats, after ex vivo by confocal microscopy after fluorescent beads administration, as previously described for the in vitro experiments on human MDMs. To test the phagocytic ability towards microbial pathogens, MDMs from treated and untreated immuno-competent animals were subjected to an in vitro infection with a suspension of Staphylococcus aureus (1 × 105bacteria/cell), and observed after 6 h of culture by confocal microscopy.The phagocytic activity of rat MDMs, obtained from treated and untreated animals and grown on cover-slips, was evaluated + cells in circulating monocytes from treated and untreated rats were evaluated by cytofluorimetric analysis. Living cells were incubated with a mouse anti-rat CD44H , detected using a PE-conjugated polyclonal anti-mouse IgG (BD Pharmingen) or with a FITC-conjugated mouse anti-rat CD25 , using a FACScan flow cytometer (Becton Dickinson).The expression of CD44 and the percentage of CD25t test was used. For the in vitro evaluations, at least three independent experiments have been carried out and data are given as the mean ± SD. For the in vivo and ex vivo experiments, results are reported as mean of 12 animals/group ± SD. For all analyses, significance was calculated with a P value < 0.01.For statistical analysis the two-tailed Student's EO: Eucalyptus oil; 5-FU: 5-fluorouracil; LavO: Lavender oil; LPS: bacterial lipopolysaccharide; MDMs: monocyte-derived macrophages; MPB: Millonig's phosphate buffer; PBMCs: peripheral blood mononuclear cells; SEM: scanning electron microscopy; TeaTreeO: Tea Tree oil.in vivo study. MZ carried out the in vivo study, and the preparation of samples for cytofluorimetric analysis, participated in the macrophage cultures maintenance and treatment. LM participated in the in vivo study, and in the macrophage cultures maintenance and treatment. MF participated in the design of the study and has been involved in revising the manuscript critically. GR has been involved in drafting the manuscript and revising it critically. EG has been involved in revising the manuscript critically. PP participated in the design of the experiments, carried out all cytofluorimetric analysis, and has been involved in drafting the manuscript and revising it critically.AS conceived of the study and designed the experiments, carried out all microscopic analyses, collected and interpreted data, drafted the manuscript. PSV has been involved in drafting the manuscript and revising it critically. FA performed the macrophage cultures and treatment, carried out preparation of samples for confocal and electron microscopy observations, participated in the Eucalyptus oil used in the in vitro studies. The data provided report the cell survival and the phagocytic activity of MDMs after 24 h treatment with increasing concentrations of EODose-response experiment performed to select the lowest, non toxic, effective doses of Click here for fileIn vitro effect of Lavender oil (LavO) or Tea Tree oil (TeaTreeO) treatments on phagocytic activity of human MDMs. The data provided show the phagocytic activity of MDMs treated for 24 h with 0.008% and 0.016% LavO or TeaTreeO, compared to EO used at the same concentrations.Click here for fileIn vitro effect of LavO or TeaTreeO treatments on viability of human MDMs. The data provided show the viability of MDMs treated for 24 h with 0.008% and 0.016% LavO or TeaTreeO, compared to EO used at the same concentrations.Click here for fileEndotoxin detection by Limulus Amebocyte Lysate (LAL) test. The data, provided in a Table, show the results of LAL test performed on EO extract used in this study and in negative (sterile water) or positive (E. coli Control Standard Endotoxin or LPS) controls.Click here for file


Traditionally, interleukin-2 and interferon alfa have been administered in this setting, with high toxicity and limited improvement in cancer-specific survival. However, newer agents such as sunitinib, sorafenib, bevacizumab, and temsirolimus have demonstrated great potential and provide a new frontier in the management of high-risk rcc.Non-localized renal cell carcinoma (ovid databases for English articles from 1950 to December 2008 using the keywords “renal cell carcinoma,” “high risk” and “renal cell carcinoma,” and “neoadjuvant.” Articles from these searches and the reference lists of relevant articles were obtained. Articles published between 1996 and 2008 were included in the present review.We queried PubMed and the Medline iii trials. Sporadic case reports have demonstrated promising results with neoadjuvant use of these agents, and a pilot study of neoadjuvant temsirolimus is currently underway at our centre.Risk stratification is imperative for optimal patient selection in adjuvant, neoadjuvant, and research settings. Utilization of interferon alfa and interleukin-2 has not demonstrated improved disease-free survival in the adjuvant setting. A number of adjuvant vaccines have also failed to demonstrate improved survival. The adjuvant role of targeted small-molecule inhibitors such as sorafenib, sunitinib, and temsirolimus is currently under investigation in phase rcc remains to be delineated. Ideally, clinicians will be able to identify high-risk patients and offer treatment to those who would benefit most from adjuvant and neoadjuvant therapy, while minimizing toxicity in low-risk patients.The role, efficacy, and toxicity of adjuvant and neoadjuvant targeted small-molecule inhibitors in high-risk  The analysis revealed that the 1997 TNM staging system, tumour size greater than 5 cm, nuclear grade, and histologic necrosis are predictive of cancer-specific mortality ssign scores of 0–2, 3–4, 5–6, and 7–9 have 5-year cancer-specific survival rates of 100%, 91%, 64%, and 47% respectively; all patients scoring 10 or more die of their disease within 2 years Recently devised integrated stratification systems attribute a value to various clinical and histologic features, and these combinations permit risk assessment within a defined patient population. The two most extensively studied integrated stratification systems for uiss system uses a combination of 1997 TNM stage, Fuhrman grade, and Eastern Cooperative Oncology Group performance status (ecog-ps) that was identified by Zisman et al., through an analysis of 661 patients at ucla, as significantly predictive of cancer-specific survival uiss categories i, ii, iii, iv, and v being 94%, 67%, 39%, 23%, and 0% respectively rcc not unlike the system used in prostate cancer ecog-ps. In addition, analysis of freedom from recurrence in nonmetastatic patients revealed that 91% of low-risk, 64% of intermediate-risk, and 37% of high-risk patients are free from any recurrence at 5 years. Given these statistically significant differences in recurrence, and a tendency for low-risk patients to recur in the chest (high-risk patients recur in the abdomen), various postoperative surveillance regimens have been defined The uiss with at least 8249 patients, and the ssign with 2656 patients ssign score appears to be slightly superior to the uiss in nonmetastatic patients, having shown a predictive accuracy of 0.830 as compared with 0.760 in one study of 388 patients. However, the authors of the latter study did not report whether the difference was statistically significant, and clinical significance has yet to be established uiss increases its predictive accuracy to 0.903 uiss system is currently limited by the complexity and technical demands of obtaining molecular markers from all patients. A potential limitation of the ssign system is its reliance on histologic tumour necrosis, which does not have a standardized definition, consensus for reporting, or availability at many centres ssign is applicable only to patients with ccrcc  and with interleukin-2 (il-2) therapy in the context of metastatic rcc, but these immune modulators do not currently have a defined role in the adjuvant setting. In randomized trials, adjuvant ifnα and the widely available recombinant ifnα2b have been shown not to contribute to survival or relapse-free survival iii trial investigating adjuvant il-2 in high-dose bolus form was closed early because an interim analysis revealed that disease-free survival was not affected il-2 in low-dose subcutaneous form was also ineffective with respect to disease-free survival Immunotherapy has been another area of active investigation as an adjuvant strategy in rcc has come in the form of tumour vaccines. Galligioni et al. investigated the use of autologous irradiated tumour cells mixed with bacillus Calmette–Guèrin as an adjuvant strategy and found no statistically significant improvement in overall or disease-free survival iii randomized controlled trial of adjuvant autologous tumour-cell vaccine conducted in Germany show a statistically significant disease-free survival benefit A more passive method of immunomodulation and its application to adjuvant therapy in rcc. Following encouraging results in a phase ii trial, a 728-patient multicentre open-label randomized phase iii trial compared adjuvant HSPPC-96 with observation following nephrectomy and found no difference in recurrence-free survival after a median follow-up of 1.9 years Another vaccine strategy has focused on the use of heat shock proteins. The heat shock protein peptide complex HSPPC-96 (vitespen) was developed from autologous tumours in rcc. Recently, an immunoglobulin G1 antibody known as cG250 (WX-G250) was found to bind carbonic anhydrase ix, which is a rcc-specific tumour antigen. A phase iii trial investigating the use of cG250 as compared with placebo following nephrectomy [the ariser  study] is currently in progress, with results expected in 2013 The use of monoclonal antibodies has also been considered in the adjuvant treatment of rcc has focused on the use of small-molecule kinase inhibitors and anti-vascular agents. Although no substantial studies have yet justified the use of these agents in the adjuvant setting, sorafenib rcc. These agents are thought to work through a mechanism influencing the involvement of the tumor-suppressor VHL gene in the pathogenesis of rcc. Spontaneous inactivation of VHL in sporadic cases of rcc results in the overexpression of vegfs, platelet-derived growth factors (pdgfs) and hypoxia-inducible factors (hifs) vegf receptors 1, 2, and 3; pdgf beta receptors; and fms-like tyrosine kinase 3 (Flt3), among others hifa, which drives the downstream regulation of a number of pro-angiogenic factors A new frontier in the adjuvant treatment of rcc, several trials are now underway to evaluate their use as adjuvant therapies in high-risk surgically resectable rcc Given the favourable effect of these agents in the treatment of metastatic able rcc ii. The asorce trial is a multicentre double-blind randomized study with an estimated enrolment of 1656 patients with resected primary rcc at high- or intermediate-risk of relapse (defined by investigators as a Leibovich score of 3–11). Patients are to be randomized to receive either sorafenib for 1 year, sorafenib for 3 years, or placebo. The primary outcome measure is disease-free survival; secondary outcome measures are metastasis-free survival, overall survival, cost effectiveness, and toxicity. The sorce trial opened recruitment in June 2007 and is estimated to be completed by August 2012. Another ongoing adjuvant trial called the s-trac addresses the use of adjuvant sunitinib. With a multicentre double-blind randomized design, this study will compare 1 year of oral sunitinib with placebo in 290 patients at high risk of recurrence (based on uiss criteria) following nephrectomy. The primary endpoint of the study is disease-free survival, and secondary endpoints to be assessed will be overall survival, safety, and patient-recorded outcomes. The projected timeline for this investigation is July 2007 to March 2011 www.clinicaltrials.gov/ct2/search)A third trial known as 2.3rcc. Until recently, cytokine-based immunotherapy had been the mainstay of systemic therapy in rcc, but its significant toxicity and poor primary tumour response are believed to have limited any investigations in the neoadjuvant setting rccA review of the current literature failed to reveal any prospective studies in the field of neoadjuvant therapy for high-risk 3.rcc to small-molecule inhibitors in vivo. This, along with the exciting potential of the application of these agents in the neoadjuvant setting of high-risk rcc patients, has compelled our centre to undertake a nonrandomized open-label outpatient pilot study investigating the use of temsirolimus before nephrectomy in patients with high-risk and metastatic rcc. Patients receive 25 mg temsirolimus by intravenous infusion on a weekly basis for 12 weeks before nephrectomy. Patients with metastatic disease continue to receive the same dose postoperatively for a maximum of 24 months, or until disease progression. A renal-mass biopsy and biomarker analysis is performed upon entry into the study. Efficacy will be evaluated using the Response Evaluation Criteria in Solid Tumors, and safety will be evaluated using the National Cancer Institute Common Toxicity Criteria for Adverse Events. The approximate duration of this study is 5 years, with an estimated enrolment of 20 subjects.Currently, there is a paucity of knowledge on the biologic response of ned (no evidence of disease) at 6 months post-nephrectomy. Patient 2  is ned at 3 months post-nephrectomy. Patient 3  has recently undergone uneventful surgery, and follow-up computed tomography is pending. Patients 4 and 5 are receiving weekly temsirolimus, and surgery is planned.To date, 5 patients have been enrolled in the trial. Three patients have undergone uneventful laparoscopic radical nephrectomy. Patient 1  is rcc may induce disease regression post-surgery, and may even lead to disease resolution in patients with low-volume disease. Longer term follow-up is necessary to assess overall progression-free survival and overall survival.These preliminary and early results suggest that neoadjuvant temsirolimus before radical nephrectomy for advanced 4.rcc remain to be delineated. Ideally, clinicians will be able to identify high-risk patients and to offer treatment to those who would benefit most from adjuvant and neoadjuvant therapy, while minimizing toxicity in low-risk patients.In summary, the role, efficacy, and toxicity of adjuvant and neoadjuvant targeted small-molecule inhibitors in high-risk


Although cigarette smoking is a well-established risk factor for vascular disease, the genetic mechanisms that link cigarette smoking to an increased incidence of stroke are not well understood. Genetic variations within the genes of the inflammatory pathways are thought to partially mediate this risk. Here we evaluate the association of several inflammatory gene single nucleotide polymorphisms (SNPs) with ischemic stroke risk among young women, further stratified by current cigarette smoking status.A population-based case-control study of stroke among women aged 15–49 identified 224 cases of first ischemic stroke (47.3% African-American) and 211 age-comparable control subjects (43.1% African-American). Several inflammatory candidate gene SNPs chosen through literature review were genotyped in the study population and assessed for association with stroke and interaction with smoking status.IL6 SNP rs2069832  was found to be significantly associated with stroke using an additive model, and this was only among African-Americans . When stratified by smoking status, two SNPs demonstrated statistically significant gene-environment interactions. First, the T allele (frequency = 5%) of IL6 SNP rs2069830 was found to be protective among non-smokers , but not among smokers ; genotype by smoking interaction (p = 0.036). Second, the C allele (frequency = 39%) of CD14 SNP rs2569190 was found to increase risk among smokers , but not among non-smokers ; genotype by smoking interaction (p = 0.039).Of the 8 SNPs (across 6 genes) analyzed, only IL6) and that cigarette smoking may modulate stroke risk through a gene-environment interaction (IL6 and CD14). Our finding replicates a prior study showing an interaction with smoking and the C allele of CD14 SNP rs2569190.This study demonstrates that inflammatory gene SNPs are associated with early-onset ischemic stroke among African-American women ( IL1A; OMIM 147760), interleukin-1 receptor – type I precursor , interleukin-1B , interleukin-6 , stromelysin-1 , and monocyte endotoxin receptor . In this study, we analyze the relationships between various polymorphisms of these genes, ischemic stroke risk, and smoking status.Cigarette smoking is a well-established risk factor for vascular disease and stroke. Various mechanisms that link cigarette smoking to these risks have been described, including: vasomotor dysfunction -3 modifiThe Stroke Prevention in Young Women Study 2 (SPYW2) is a population-based case-control study that was designed to examine genetic risk factors for ischemic stroke in young women. The term "population-based" indicates that the cases and the comparison control group were identified from the same population including all of Maryland (except the far Western panhandle), Washington DC, and the southern portions of both Pennsylvania and Delaware. Two hundred thirty nine female cases age 15 to 49 years of age with a first cerebral infarction were identified by discharge surveillance at 51 regional hospitals and through direct referral by regional neurologists. The methods for discharge surveillance, chart abstraction, and case adjudication have been described previously -13. We dCases and controls were grouped into the following race-ethnic categories: Caucasian (non-Hispanic) (95 cases and 99 controls), African-American (105 cases and 91 controls), and other  (24 cases and 21 controls). Because of the small size and heterogeneity of the latter group, it was not analyzed separately, but was included within the combined total study group (224 cases and 211 controls). Strokes were further classified by subtype: the atherosclerotic group included 27 cases with either probable or possible atherosclerotic mechanism, the cardiac group included 14 cases with a probable cardiac source of embolism, the probable dissection group included 13 cases confirmed by neuroimaging, the lacunar group included 45 cases of symptomatic small deep lesions on neuroimaging studies or classic lacunar syndromes regardless of other potential causes, and the probable hematologic group included 9 cases. These categories were not mutually exclusive. There were 125 non-lacunar stroke cases of undetermined etiology. were evaluated. After weighing these considerations, the final list of genes and SNPs analyzed in this study included: CD14 (rs2569190), IL1A (rs17561), IL1R1 (rs3917318), IL1B (rs3917365), IL6 , and MMP3 (rs679620).We identified genes and SNPs that influence inflammation through a literature review seeking an association of the genes and SNPs with vascular disease . If the evidence suggested that smoking might modify the relationship between gene or SNP and stroke through a gene-environment interaction, a higher priority for inclusion in our analyses was assigned to that gene or SNP. Because we sought to evaluate common SNPs, only those SNPs with a minor allele frequency of 0.05 or greater among African-Americans or Caucasians as per the NCBI SNP website  and submitted to Autoprimer.com . For each SNP, three primers were designed, two for PCR amplification and an internal primer with a 5' DNA sequence tag. Pairs of primers were used to initiate PCR amplification. The free primers were removed by enzymatic digestion using Exonuclease I and Shrimp Alkaline Phosphotase . The internal primers were used to initiate a sequencing reaction that adds one labeled base for the alternative nucleotides of each SNP to have distinct labels. The labeled products are separated on a SNP-IT plate consisting of 384 mini-arrays with 16-spots each . For each individual DNA sample, 16 spots hybridized to the two homozygotes, the heterozygote, a negative control and the 12-labeled primers associated with the 12-SNPs. Thus, every PCR and labeling reaction had internal controls to confirm the success of the reactions and the appropriateness of the fluorescent outputs for each DNA sample. SNPs genotyped using this method include: IL1R1 (rs3917318); IL1B (rs3917365); IL6 .Genotyping was conducted on DNA isolated from whole blood using the QIAamp DNA Blood Maxi Kit . SNP genotyping was performed by one of two methods. The first method, developed for a SNP stream Ultra-High Throughput machine  is capable of genotyping up to 12-SNPs simultaneously. Sequences surrounding the SNPs were obtained from GenBank CD14 (rs2569190); IL1A (rs17561); IL6 (rs2069830); MMP3 (rs679620).The second genotyping method was Taqman (Applied Biosystems). This method is based on four primers, two flanking the SNP that are used to amplify the DNA surrounding the SNP and two, one for each alternative allele, that were labeled with different fluorescent dyes. The original form of the labeled primer has a quencher in close proximity to the dye. However, when the exonuclease activity of DNA polymerase disrupts the primer hybridized to the single strand DNA during the PCR, then quencher and the dye are released and the fluorescence can be measured. The reaction itself follows manufacturer's instructions included with each individual primer set. SNPs genotyped using this method include: ®, Version 9.1 . We compared means by two-sided t-tests and proportions by χ2 tests. All SNPs were verified to be in Hardy-Weinberg equilibrium. Adjusted odds ratios from logistic regression were used to determine whether the presence of the risk allele was associated with an increased risk for ischemic stroke after controlling for potential confounders.All statistical analyses were performed using SASTwo additive models were used to determine the relation between SNP and outcome. The first model  was adjusted for age and race  or only age when the analysis was stratified by race (i.e. African-Americans and Caucasians). The second model, (adjusted model) added history of hypertension, diabetes, smoking, oral contraceptive (OCP) use and angina pectoris or myocardial infarction (angina-MI), to age (or age and race) to the independent variables used in the first model. The results of the analyses were expressed as odds ratios which quantify the increased risk of ischemic stroke associated with each additional risk allele. Age, race, current cigarette smoking status, and OCP use were determined by subject reports . Hypertension and diabetes mellitus were determined by asking study participants (or a proxy) if a physician had ever told them they had the condition. Current smoking was defined as having one or more cigarettes in the month (31 days) preceding their stroke, or for controls, in the month prior to their interview. Current oral contraceptive use was also defined as use in the prior month.In order to evaluate each SNP for a genotype by smoking interaction , study participants were stratified by smoking status and evaluated in an age-, race-adjusted additive model. For those SNPs that demonstrated a potential genotype by smoking interaction the significance of the difference between the point estimates (i.e. the log odds ratios) in smokers and non-smokers was tested using a z-test. Lastly, SNPs with significant z-test results were additionally evaluated for potential race-specific smoking interactions in an age-adjusted additive model, with the SNP stratified by smoking status further stratified by race (Caucasian and African-American).In secondary analyses, significantly associated SNPs from the primary analyses  underwent further analyses to evaluate groups stratified by other standard risk factors  and ischemic stroke subtype ) with the later groups compared to all controls. Additionally, analyses consisting of dominant and recessive genetic models that included similar groups and covariates to the primary analyses were also performed. We did not adjust for multiple comparisons because our study was considered to be hypothesis generating.Demographic and risk factor characteristics by case-control status are described in Table IL6 SNP rs2069832  was found to be significantly associated with stroke, and this was only among African-Americans . Dominant and recessive models did not strengthen the association (data not shown). Stratifying participants by other risk factors and evaluating stroke risk in relation to SNP rs2069832 genotype revealed no associations with stroke (data not shown). Stratifying participants by stroke subtype as compared to all controls and evaluating stroke risk in relation to SNP rs2069832 genotype revealed no significant associations (data not shown).Table CD-14 SNP rs2569190  was found to be associated with increased risk of stroke among smokers , but not non-smokers . The two odds ratios were significantly different (p = 0.039). Secondly, IL6 SNP rs2069830  was found to be protective among non-smokers , but not among smokers . The two odds ratios were significantly different (p = 0.036).Table Results of the race-stratified smoking-genotype interaction analyses demonstrated no significant interactions. However, the race-stratified point estimates (ORs) remained similar to the combined population point estimates as detailed in the preceding paragraph. For CD-14 SNP rs2569190, similar ORs were seen among both smokers of both races  and non-smokers of both races . For IL-6 SNP rs2069830, among African-Americans, similar ORs as found in the combined population were seen among smokers  and non-smokers , consistent with a protective effect for the T allele among non-smokers. For IL-6 SNP rs2069830, among both Caucasian non-smokers and smokers, the logistic regression models did not converge due to the low frequency (2%) of the T allele.IL6 (rs2069832) was found to be associated with stroke, and only among African-Americans. However, it did identify two polymorphisms that appear to influence stroke risk via a gene-environment interaction with cigarette smoking, IL6 rs2069830 and CD-14 rs2569190.Our study did not demonstrate strong relationships between the inflammatory gene polymorphisms and ischemic stroke risk. Only IL-6 plays a key role in the acute inflammatory response and in regulation of the production of acute phase proteins such as C-reactive protein ,16. It cCD14 SNP rs2569190 is a C > T substitution in the proximal CD14 promoter GC box at position -260 from the translation start site and results in a HaeIII restriction site [CD14 SNP rs2569190 [CD14 is a surface protein preferentially expressed on monocytes and macrophages that acts to bind lipopolysaccharide-binding protein . Endotoxin is a potent mediator of inflammation, and smokers have elevated plasma levels of endotoxin. Hence, it is believed that the risk of atherosclerosis from endotoxemia is increased in smokers . The CD1ion site  and two ion site . Contrarion site , and wasion site ,9, speciion site . Still oion site ,32. Our s2569190 .Our study has several limitations. Most notably, we only evaluated a limited number of SNPs, which were not sufficient to comprehensively evaluate stroke risk or smoking interaction for any of the genes studied. Secondly, our study did not provide any mechanistic information regarding stroke risk or protection. Specifically, we did not determine if our SNPs were associated with increased or decreased protein activity. Furthermore, our study population is relatively small and is underpowered to detect modest effects, particularly in the stroke subtype analyses. Although we have attempted to minimize phenotypic heterogeneity through identifying ischemic stroke subtypes during adjudication, we recognize that residual phenotypic heterogeneity may exist. Lastly, though we performed numerous analyses, no correction was made for multiple comparisons. This allows for the possibility that our results could be attained through chance alone, although confirming CD-14 rs2569190 as a mediator of vascular risk, and specifically among smokers of both races, makes such a false positive association less likely.IL6) and that cigarette smoking may modulate stroke risk through a gene-environment interaction (IL6 and CD14).This study demonstrates that inflammatory gene SNPs may be associated with early-onset ischemic stroke among African-American women (The authors declare that they have no competing interests.All authors certify that they participated in the conceptual design of this work, the analysis of the data, and the writing of the manuscript to take public responsibility for it. All authors reviewed the final version of the manuscript and approve it for publication. JWC, OCS, and SJK participated in the writing of the initial draft. OCS participated in the genotyping. JWC OCS, MAW, BJS, MJS, MD, LS, NZ, and SJK participated in DNA and data collection. JWC, DWB, WHG, JRO, BDM, JDS, and LJR participated in the data analysis. All authors provided critiques of the final manuscript.The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention.


Investigating severe maternal morbidity (near-miss) is a newly recognised tool that identifies women at highest risk of maternal death and helps allocate resources especially in low income countries. This study aims to i. document the frequency and nature of maternal near-miss at hospital level in Damascus, Capital of Syria, ii. evaluate the level of care at maternal life-saving emergency services by comparatively analysing near-misses and maternal mortalities.Retrospective facility-based review of cases of near-miss and maternal mortality that took place in the years 2006-2007 at Damascus Maternity University Hospital, Syria. Near-miss cases were defined based on disease-specific criteria (Filippi 2005) including: haemorrhage, hypertensive disorders in pregnancy, dystocia, infection and anaemia. Main outcomes included maternal mortality ratio (MMR), maternal near miss ratio (MNMR), mortality indices and proportion of near-miss cases and mortality cases to hospital admissions.There were 28 025 deliveries, 15 maternal deaths and 901 near-miss cases. The study showed a MNMR of 32.9/1000 live births, a MMR of 54.8/100 000 live births and a relatively low mortality index of 1.7%. Hypertensive disorders (52%) and haemorrhage (34%) were the top causes of near-misses. Late pregnancy haemorrhage was the leading cause of maternal mortality (60%) while sepsis had the highest mortality index (7.4%). Most cases (93%) were referred in critical conditions from other facilities; namely traditional birth attendants homes (67%), primary (5%) and secondary (10%) healthcare unites and private practices (11%). 26% of near-miss cases were admitted to Intensive Care Unit (ICU).Near-miss analyses provide valuable information on obstetric care. The study highlights the need to improve antenatal care which would help early identification of high risk pregnancies. It also emphasises the importance of both: developing protocols to prevent/manage post-partum haemorrhage and training health care professionals to manage infrequent but fatal conditions like sepsis. An urgent review of the referral system and the emergency obstetric care in Syria is highly recommended. Since the declaration of the Millennium Development Goals (MDGs) in 2000, reproductive health, the 5th goal of the MDGs, has been recognised as fundamental for human development. National governments and the international community have increasingly embraced language which supports reproductive health. However, re-orienting policies and programmes has been more challenging, particularly in the Middle East and North Africa region (MENA) . DecisioEach year approximately 529 000 women die of complications during pregnancy and delivery; almost all of which (99%) occur in low-resource countries . HoweverNear-miss is a serious adverse event that leads to harm and morbidity in the mother, but from which she survives . Since 'Essentially, three different methods have been used for identifying near-miss cases. These different approaches depend on either a set of clinical criteria defining common diagnostic categories; a set of management-based criteria related to specific interventions or on organ(s)/system(s) failure .Although a considerable body of obstetric near-miss analyses have been recently conducted in developing countries -11, suchIn this retrospective study, we aim to document the frequency and nature of maternal near-misses at a hospital level in Damascus, the Capital of Syria. We also aim to evaluate the level of care at the maternal emergency services in Syria by comparatively analysing maternal near-misses and mortalities. This review is expected to serve as a guiding tool for policy-makers by highlighting the most common and the infrequent but serious maternal morbidities to help direct the expenditure of the modest national health budget towards health care priorities.We conducted a facility-based retrospective study between February and July 2008 for all admissions to Damascus Maternity University Hospital (DMUH), the women teaching hospital for Damascus University, in the years 2006 and 2007. DMUH is one of two maternity referral hospitals in Damascus which serve a region populated by over 3 million inhabitants. This 250-bed hospital has approximately 21,000 admissions yearly, 60% of which are accepted in labour and pregnancy units. This study was approved by Damascus Faculty of Medicine (21 Jan 2007).A near-miss event is 'a woman who nearly died but survived a complication that occurred during pregnancy, childbirth or within 42 days of termination of pregnancy' .et al [The disease-specific criteria set by Filippi et al  were empIn 2009, the World Health Organisation (WHO) published a consensus on maternal near-miss definition and set a criteria for cases identification . These cAll maternal near-misses which occurred before or after arrival at the hospital were included in this study. These were further classified into direct (resulting from complications occurring during or after pregnancy) or indirect (resulting from previously existing disease aggravated by pregnancy). Causes of maternal mortality, as defined by the International Classification of Diseases (ICD-10), were alMaternal deaths and near-miss cases were retrospectively identified by searching the Medical Records Register at DMUH. Each patient's discharge summary includes 'primary' and 'secondary' diagnoses sections. It also has a 'complication' section, where all complications which happened as a result of either the primary or the secondary diagnoses are listed. Discharge summaries also include 'investigations'  and 'interventions'  sections.Using the data documented in the admission/discharge sheets of the hospital, we identified cases where any of Filippi's main disease-specific categories was mentioned . The full notes of these cases were then scrutinised and carefully checked against Filippi's criteria for final inclusion. The data collection was done by 3 investigators using a pre-coded form specially developed for this study. All data was then inserted into a Microsoft Excel spreadsheet. Causes of maternal deaths were generally based on clinical or surgical findings. Data on deliveries and live births were derived from statistics reported by the DMUH's Statistical Department.For each case, we collected data on demographic characteristics including patient's age, parity, previous deliveries, and gestational age at delivery. We also collected data on the nature of the obstetric complication(s) responsible and where it developed , type of delivery, foetal outcomes, any ICU admissions including length of stay, and any special procedure carried out during the care of the woman. Special procedures were defined as propaedeutic or therapeutic interventions not normally used during prenatal, intra-partum, or postnatal care e.g. echocardiography, hysterectomy, transfusion ≥ 4 units, electric cardioversion and iliac arteries ligation.SPSS 13.0 software  was used for descriptive statistical analysis. Results are presented as frequencies, percentages and descriptive statistics. MMRs and MNMRs were calculated using live births as the denominator. In order to evaluate the standard of care provided for each disease category, we calculated the mortality index for each obstetric condition. This was defined as the number of maternal deaths due to a particular obstetric condition divided by the sum of near-miss morbidities and maternal deaths which resulted from this condition, expressed as a percentage .During the 2-year period reviewed, there were 28 025 hospital deliveries, 27 350 live births, 901 near-miss cases and 15 maternal deaths. This resulted in a total MMR of 54.8/100 000 live births. The total MNMR was 32.9/1000 live births and the total mortality index for near-miss cases was 1.67% . Most of the cases  were referred to the hospital with the complication while only 7% developed the complication within the hospital.During the study period, 54% of women with near-miss delivered by caesarean section . Foetal outcomes were 82% liveborn; 11% abortions and 6% stillbirths. Other demographic characteristics of women who sustained near-miss complications and those who died are presented in table 14 (93.3%) of the 15 maternal deaths were due to causes directly related to pregnancy, with severe late pregnancy haemorrhage leading the causes (n = 9), followed by hypertensive disorders  and sepsis (n = 2). One additional death followed uterine rupture and one death was due to an exacerbation of a pre-existing cardiac problem. The mean 'stay in hospital to death' period was 2.6 days (SD = 4.2), table The disease profile for near-miss morbidity differed from that of maternal mortality table . The mosThe majority of the near-miss cases (93%) were referred in critical condition from other facilities namely traditional birth attendant homes (67%), primary (5%) and secondary (10%) healthcare units and private practices (11%) within Damascus city and countryside. This figure varied according to categories . All severe haemorrhage in early pregnancy cases (n = 66) were referrals while 13% (n = 32) of late pregnancy haemorrhages developed in the hospital.Two hundreds and forty five women with near-miss (27%) were admitted to ICU, with a mean stay of 3.5 days  while sepsis had the higher mortality index (7.4%). Most of the cases (93%) had near-miss upon arrival at the hospital. Almost quarter of near-miss cases were admitted to ICU.The MNMR which this study describes (32.9 per 1000 live births) is located within the wide range of ratios reported in studies from other developing countries which used similar criteria for near-miss definition (12.3 - 82.3 per 1000 deliveries) . On the Studying cases of women who nearly died but survived a complication during pregnancy, childbirth or postpartum  is increasingly recognised as a useful means to examine the quality of obstetric care. Nevertheless, routine implementation and wider application of this concept in reviewing clinical care has been limited due to the lack of a standard definition and uniform case-identification criteria.The WHO initiated a process aiming to develop a uniform set of criteria for identifying maternal near-miss cases. According to the new WHO criteria, published in 2009, near-miss cases are identified by dysfunctional system  based on a set of clinical criteria, laboratory markers, or management-based proxies which were strictly defined . It is hIn 2008, when the data collection of our study commenced, the WHO consensus on maternal near-miss definition was not yet published. The clinical criteria method, which we chose to define our near-miss cases, has many advantages including its easy interpretation, retrospective nature, and ability to assess both complication rates and quality of care of a particular disease .Although case identification using admission to ICU is easier; it would have heavily underestimated severe morbidity, as generally <10% of near-miss cases in low-resource settings receive intensive care ,24  which implies effective management of these complications upon arrival at hospital.Of the various types of life-threatening obstetric haemorrhage, postpartum haemorrhage (PPH) constituted the greatest danger to affected women. While accounting for almost tenth of the maternal morbidities, PPH is responsible of 40% of maternal deaths. This suggests that either women had no access to care or poor care was provided by health professional .Another area that needs improvement is management of sepsis which has the highest mortality index (12:1). This, indeed, constitutes a significant threat to the survival of affected patients although it is the least frequent cause of life-threatening obstetric conditions (0.9/1000 live births).One critical defect this study helped expose is the delay in referrals to higher levels of care. Surprisingly, the majority of women with near-miss morbidity (93%) arrived at the hospital in critical condition having been referred from other hospitals, private practices or homes. This fact underscores the significance of pre-hospital barriers even in this setting with its availability of free maternal health care .One possible barrier is related to transporting patients to hospital. A recent study on maternal mortality in Syria showed that in 31% of maternal deaths where deliveries took place at home, women died on the way to the hospital. Besides, in 18% of hospital deliveries, women died in another hospital as they were referred from hospitals which refused to admit them due to incapability of dealing with such cases. Small proportion of women were transported by ambulance (6%), while almost two thirds of them were brought by private cars or taxis .Another obstacle to timely arrival are to be found in 'the first delay' , which iHowever, even if women arrive late, much can be done to save them and their babies, and a review into the care received in the hospital may lead to positive changes in the procedures and resources available for managing these complications . This faIt is important to mention that our study has limitations. Its retrospective nature carries the possibility of underestimating near-miss cases due to poor documentation . Other lNear miss cases share many characteristics with maternal deaths and can directly inform about obstacles that had to be overcome after the onset of an acute complication, hence providing valuable information on obstetric care. This allows for corrective action to be taken on identified problems to reduce related mortality and long-term morbidity. Based on our findings, we recommend a number of actions to avert future maternal deaths: i. improving antenatal care to help early identification of high risk pregnancies including pre-eclampsia, ii. developing protocols to prevent/manage post-partum haemorrhage including raising the awareness about using active management during third stage, iii. training obstetric health professionals on managing infrequent but fatal conditions like sepsis, iv. urgently reviewing the referral system and emergency obstetric care in Syria.The authors declare that they have no competing interests.YA was responsible for the study design, together with AA, MQA and HEM. YA, AA and YS collected the data. MQA and HEM did the analysis and wrote the first draft in consultation with YA. AA revised the manuscript and all authors agreed on the final draft.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2393/10/65/prepub


Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is currently being investigated as a therapeutic agent for a variety of malignancies, as it triggers apoptosis specifically in transformed cells. However, TRAIL use as a stand alone therapeutic is hampered by the fact that many primary tumor cells are resistant to TRAIL-mediated apoptosis. Here, we investigated the extent to which pretreatment of TRAIL-resistant primary B-cell chronic lymphocytic leukemia (B-CLL) cells with histone deacetylase inhibitors (HDACis) could render them susceptible to killing by TRAIL. We found that HDAC inhibition in B-CLL cells led to increased TRAIL receptor expression, increased caspase activation, decreased expression of antiapoptotic regulators such as Bcl-2, and ultimately, enhanced TRAIL-induced apoptosis. Importantly, untransformed peripheral blood mononuclear cells remained largely resistant to TRAIL, even in the presence of HDACis. These results suggest that combination therapies using HDAC inhibition and TRAIL could prove beneficial for the treatment of B-CLL. Inducing apoptosis in tumor cells is a promising therapeutic approach for the treatment of both solid and lymphatic tumors. The TNF family member tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an apoptotic mediator that has received much attention, due to its ability to bring about cell death specifically in tumor cells without affecting untransformed cells or tissues –3. In huG0/G1 phase of the cell cycle [B-cell chronic lymphocytic leukemia (B-CLL) is the most frequent type of leukemia found in western countries . It is cll cycle . Not surll cycle . The reaHDACi are a promising group of anticancer agents that have been used successfully alone or in combination to induce tumor cell death in vitro and in vivo , 21. HDAL and Bcl-2. Importantly, the combination therapies showed limited toxicity against normal PBMC. Thus, our study illustrates the feasibility of using HDACi and TRAIL in combination to mediate apoptosis of B-CLL cells.Based on previous studies by our group and others, in which the combination of HDACi and TRAIL was able to bring about apoptosis of tumor cells that were resistant to single therapy with either agent alone –30, we sThe HDACi Trichostatin A (resuspended in EtOH) was purchased from Sigma ; Oxamflatin (resuspended in DMSO) was purchased from Calbiochem . All other HDACis are as previously described . AntibodPeripheral blood samples were obtained from patients at the University of Iowa Holden Comprehensive Cancer Center who were diagnosed with B-CLL, or from healthy control donors. Experiments were repeated 3–5 times, using cells from different donors. Lymphocytes were enriched by density gradient centrifugation using Ficoll-Hypaque. Cells were cultured in RPMI 1640 supplemented with 10% FCS, 1% nonessential amino acids, 1 mM sodium pyruvate, and 1% penicillin/streptomycin solution. This study was conducted according to protocols approved by the institutional review board at the University of Iowa, and informed consent was obtained from all patients before blood sample collection.4 cells/well in 96-well plates. Cells were either cultured in medium alone or in the presence of individual HDACi at the indicated doses, for 16 hours. After this pretreatment, recombinant human TRAIL was added at 100 ng/mL, and cells were cultured for an additional 4 hours. Cell death was determined by costaining for Annexin V and propidium iodide, to permit identification of apoptotic versus necrotic cells.Cells were cultured in complete medium at 2 × 10Briefly, cells were cultured in complete medium, in the presence or absence of individual HDACi for 16 hours, followed by a 4 hour treatment with TRAIL as above. Caspase activity was quantitated as described previously . BrieflyTotal RNA was isolated from cultured cells with Trizol reagent  according to the manufacturer's protocol. Reverse transcription of total RNA was done by using Superscript II. The real-time qRT-PCR primer and probe sets were purchased from Applied Biosystems . cDNA (250 ng) was used as a template for TaqMan Assays and the internal control of rRNA. The TaqMan reaction was conducted as previously described .To evaluate the potential therapeutic efficacy of combining HDACi with recombinant TRAIL for the treatment of B-CLL, we began by examining the ability of a panel of HDACi to upregulate TRAIL receptor expression on B-CLL. Human B-CLL were cultured for 16 hours in the presence or absence of individual HDACi. As shown in Because we have shown previously that combination therapy with TRAIL and HDACi can lead to TRAIL sensitivity in untransformed cells , it was To assess whether the upregulated surface expression of TRAIL-R1 and TRAIL-R2 on B-CLL cells was due to changes in the quantity of mRNA present, we cultured cells for 16 hours in the absence or presence of varying doses of Oxam or TSA. Both Oxam and TSA were found to increase mRNA levels of TRAIL- R2 , suggestμM Oxam or 500 ng/mL TSA for 16 hours prior to the addition of recombinant TRAIL at 100 ng/ml. Cultures were harvested 4 hours later, and the percentage of TRAIL-induced apoptosis measured by Annexin V staining. As controls, normal PBMCs were cultured simultaneously. In agreement with previous reports [Once we had determined that Oxam and TSA upregulated TRAIL receptor expression on B-CLL cells, we then determined whether this translated into an increase in susceptibility to TRAIL-mediated apoptosis. B-CLL cells were again cultured alone (untreated) or in the presence of 2.5  reports , 33, 34,We next wanted to determine the mechanism by which Oxam and TSA sensitized B-CLL cells to TRAIL-mediated killing. TRAIL induces apoptosis via activation of downstream caspases, including caspase 8, and this signal can be augmented by the loss of mitochondrial membrane potential (MMP). B-CLL cells were again cultured in the presence or absence of either Oxam or TSA for approximately 16 hours, followed by culture with recombinant TRAIL. Preculture of cells with Oxam or TSA led to striking increases in the activity of caspases 8, 9, and 3 after TRAIL administration . As expeBoth capsases 8 and 3 are activated during the extrinsic apoptosis pathway, whereas caspase 9 is associated with the intrinsic apoptosis pathway . TriggerL (The preceding experiments indicated that HDACi pretreatment sensitized B-CLL cells to subsequent apoptosis with TRAIL. Mechanistically, this appeared to result from a variety of factors, including enhanced TRAIL receptor expression, increased caspase activation, and loss of MMP. Additional mechanisms of sensitization could also include activation of pro-apoptotic regulators and inhibition of antiapoptotic molecules. To determine if these factors were also contributing to elevated TRAIL-mediated apoptosis following HDACi treatment, we examined changes in apoptotic regulators at the mRNA level. As before, B-CLL cells were treated with either Oxam or TSA for 16 hours, then examined for changes in mRNA levels of several key apoptotic regulators. Both Oxam and TSA led to modest increases in mRNA for the pro-apoptotic molecule Bax, but substantial increases for the pro-apoptotic molecule Mcl-1 short, resulting in a 13-fold increase in mRNA levels with TSA treatment . At the L . Therefo+ B lymphocytes in the bone marrow and blood [B-CLL is the most prevalent form of adult leukemia, and is characterized by an accumulation of mature, often CD52nd blood , 18. At nd blood . ClearlyTRAIL is a promising anticancer agent, given that it can specifically induce apoptosis in transformed cells, without affecting normal host cells or tissues –3. A majWe and others have previously explored the use of TRAIL in conjunction with HDACi administration –30. HDAC+ during the 4 hours TRAIL incubation period. This is in comparison to B-CLL cells treated with TRAIL alone, which produced apoptosis in fewer than 10% of cells. This reversal in susceptibility to TRAIL-mediated apoptosis indicates the efficacy of B-CLL pretreatment with HDACi. Importantly, normal PBMCs were much more resistant to the combination of HDACi + TRAIL, suggesting that this type of therapy may have applicability in B-CLL patients. The combination of Oxam + TRAIL produced less apoptosis in normal PBMC than did TSA + TRAIL, which indicates that it may be possible to modify the timecourse or dose of agents in vivo to further limit killing of untransformed cells. In this study, we found that pretreatment of primary B-CLL cells with either of two HDACis, Oxam, or TSA, led to upregulated TRAIL receptor expression, increased caspase 8 activation, triggered a loss of MMP, and decreased expression of antiapoptotic regulators such as Bcl-2. The net result was a significant increase in TRAIL-mediated killing of B-CLL cells, such that over 30% of B-CLL cells became Annexin-VOur observations extend work by Garofalo et al. in which they examined the role of PED, an antiapoptotic molecule, as a mechanism for B-CLL resistance to TRAIL-mediated killing . In thatWe show here that pretreatment of TRAIL resistant B-CLL cells with either of two HDACis, Oxam, or TSA, renders cells susceptible to subsequent killing by recombinant TRAIL protein. Both Oxam and TSA led to increased surface expression of TRAIL-R1 and TRAIL-R2, increased caspase 8 and caspase 3 activation, a loss of MMP, and changes in mRNA levels for key pro- and antiapoptotic regulators, all of which combined to sensitize B-CLL cells to TRAIL-induced apoptosis. Importantly, combination therapies preferentially sensitized transformed cells to apoptosis, as the same level of killing was not observed in normal PBMC. Thus, combination therapies using specific HDACi and recombinant TRAIL may prove to be efficacious for the treatment of B-CLL in humans.


The intersecting HIV and Tuberculosis epidemics in countries with a high disease burden of both infections pose many challenges and opportunities. For patients infected with HIV in high TB burden countries, the diagnosis of TB, ARV drug choices in treating HIV-TB coinfected patients, when to initiate ARV treatment in relation to TB treatment, managing immune reconstitution, minimising risk of getting infected with TB and/or managing recurrent TB, minimizing airborne transmission, and infection control are key issues. In addition, given the disproportionate burden of HIV in women in these settings, sexual reproductive health issues and particular high mortality rates associated with TB during pregnancy are important. The scaleup and resource allocation to access antiretroviral treatment in these high HIV and TB settings provide a unique opportunity to strengthen both services and impact positively in meeting Millennium Development Goal 6. Mycobacterium tuberculosis (MTB) remains one of the leading causes of morbidity and mortality globally [Infection with globally . The groglobally . Globallglobally . Althougglobally , 3. TB co-infection in HIV-infected pregnant women is emerging as a major non-obstetric cause of maternal deaths in sub-Saharan Africa . AccordiThe intersection of the TB and HIV epidemics and the challenges and opportunities they provide for addressing both epidemics is the focus of this paper. P  =  .007), with sensitivity significantly higher in HIV-infected compared to uninfected patients (P  =  .001), and in HIV-infected participants with a CD4 < 200 cells/mm3, compared to those with CD4 counts above 200 (P  =  .003). In this particular study, urine-LAM remained highly specific for TB both in HIV-uninfected and in HIV-infected patients irrespective of CD4 counts. Twenty-five percent of smear-negative but culture-positive HIV-infected patients with a CD4 < 200 cells/mm3 were positive for urine-LAM [TB is the most common presenting opportunistic infection among HIV-infected patients, who remain at high risk for TB throughout the course of their disease. HIV-infected patients on HAART have a TB incidence rate of 4.6 cases/100 patient-years, approximately 10-fold higher than TB incidence rates in HIV-negative patients in the same community  and are rine-LAM . It is iTo overcome diagnostic infrastructure and human resource constraints, a TB symptom screening questionnaire is increasingly being utilised to more effectively and efficiently screen for TB . Data frThough intertwined, HIV and TB are treated in separate programs and facilities. TB clinics have been slow to implement HIV care and the introduction of antiretroviral therapy (ART), which has resulted in the establishment of separate HIV clinics, that often do not focus on TB co-infection , 32. In TB IRIS results from either deterioration of TB while on anti-TB treatment, known as paradoxical TB IRIS, or a new presentation of previously subclinical TB referred to as unmasking TB IRIS. Mycobacteria account for approximately 40% of all cases of infective IRIS in patients initiated on ART –41. The μL [Data from the Development of AntiRetroviral Therapy in Africa (DART) trial have demonstrated that cotrimoxazole prophylaxis reduced mortality in patients with HIV infection on ART for up to 72 weeks regardless of CD4 count status, with mortality similarly reduced in patients with a current CD4 cell counts above and below 200 cells/μL . NotwithμL –55 throuμL . Concurrent administration of ART and IPT has demonstrated a TB risk reduction of 76–89% in observational studies from both South Africa and Brazil , 55. GivThe protection offered by IPT to those infected with HIV may depend on a number of factors including degree of immune suppression of the individual, duration of IPT, adherence to and potency of the regimen, as well as the general risk of reinfection in that setting , 59. DatWhile toxicity from coadministration of IPT and HAART is valid, the likelihood of stopping therapy as a result of adverse events has to date been low. The risk of adverse events was higher with rifampicin and pyrazinamide-containing regimens than INH monotherapy . The fea3 or at WHO Stage 4 HIV disease [Recurrent TB accounts for the majority of TB cases in countries with a high TB incidence rate  with HIV disease . HIV-inf disease . The “ri disease , 74. One disease . Of note disease , 76, whi disease . With in disease .Given the high rates of undiagnosed TB in AIDS patients and the excess mortality associated with HIV-TB co-infection, HIV clinics should pay particular attention to infection control including good ventilation to minimise further TB risk acquisition in AIDS patients. The Tugela Ferry XDR-TB outbreak in rural South Africa brought to public attention the consequences of nosocomial TB transmission in high HIV burden settings and has also highlighted the risk of acquiring multi-drug-resistant TB in health care workers and in the general public. A 3-tier approach for TB-infection control has been developed by the WHO for application in all settings and includes administrative and environmental controls and personal protection. The proposed interventions are simple and feasible and can be customised further based on available resources . AdminisMuch has been learnt about HIV-related TB and much remains to be learnt. Research efforts need to focus on protective TB vaccines for immuno-compromised populations, new first- and second-line TB drugs, point of care diagnostics for TB and TB IRIS, including strategies to optimise clinical outcomes in patients co-infected with HIV, and drug-resistant TB. The intersection of the HIV and TB epidemics provides a unique opportunity to strengthen TB and HIV services with substantial morbidity and mortality gains at the individual and population level. This level of public health benefit in the current fiscal climate and growing disease burden is critically important. The integration of HIV and TB services could serve as an important role model for other service delivery. Significantly, reducing and better managing TB in HIV-infected persons could make a substantial contribution to countries reaching their MDG health goals.