Phalaris canariensis seeds hexane extract (Al-H) in high-fat diet- (HFD-) induced obese CD1 mice and in streptozotocin-induced mild diabetic (MD) and severely diabetic (SD) mice.AL-H was orally administered to MD and SD mice at a dose of 400 mg/kg once a day for 30 days, and a set of biochemical parameters were studied: glucose, cholesterol, triglycerides, lipid peroxidation, liver and muscle glycogen, ALP, SGOT, SGPT, glucose-6-phosphatase, glucokinase, hexokinase, SOD, CAT, GSH, GPX activities, and the effect on insulin level. HS-H significantly reduced the intake of food and water and body weight loss as well as levels of blood glucose, serum cholesterol, triglyceride, lipoprotein, oxidative stress, showed a protective hepatic effect, and increased HDL-cholesterol, serum insulin in diabetic mice. The mice fed on the high-fat diet and treated with AL-H showed inhibitory activity on the lipid metabolism decreasing body weight and weight of the liver and visceral adipose tissues and cholesterol and triglycerides in the liver. We conclude that AL-H can efficiently reduce serum glucose and inhibit insulin resistance, lipid abnormalities, and oxidative stress in MD and SD mice. Our results demonstrate an antiobesity effect reducing lipid droplet accumulation in the liver, indicating that its therapeutic properties may be due to the interaction plant components soluble in the hexane extract, with any of the multiple targets involved in obesity and diabetes pathogenesis.Obesity is one of the major factors to increase various disorders like diabetes. The present paper emphasizes study related to the antiobesity effect of  Obesity is a metabolic disease of pandemic proportions largely arising from positive energy balance, a consequence of sedentary life style, conditioned by environmental and genetic factors . Obesity  the past, canary seed was not seen as a viable cereal for human consumption due to the harmful effects associated with the siliceous hairs that cover the hull of the seed. These hairs are highly irritating when they come in contact with human skin or lungs and have been linked to esophageal cancer [Adipose tissue, besides its primary function of fat storage, also regulates appetite and metabolism by secretion of certain chemicals like an endocrine gland . Thus, pl cancer . In 1997l cancer . The varPhalaris canariensis L., is a member of a family of grasses (Graminaceae), and it is used in folk medicine in the form of tea as a coadjuvant in the treatment of hypertension, diabetes mellitus, and hypercholesterolemia [P. canariensis seed infusion in normotensive rats [Phalaris canariensis.The canary seed or alpiste, erolemia , with orerolemia ; howeverive rats ; howeverP. canariensis were collected in Morelos State, Mexico. A voucher specimen (number 8054) was deposited in the Herbarium of the National School of Biological Sciences, for further reference.Seeds of P. canariensis were dried and powdered in a mechanical grinder. The grinded material was extracted with 5 L of hexane, chloroform, and methanol consecutively using a soxhlet apparatus. These extracts were filtered and concentrated by a rotary vacuum evaporator and kept in a vacuum desiccator for complete solvent removal.A total of 1000 g of the seeds of The study was conducted in CD1 mice of both sexes, weighing about 25–30 g. Before and during the experiment, animals were fed a standard laboratory diet  with free access to water. Mice were procured from the bioterium of ENCB and were housed in microloan boxes in a controlled environment (temperature 25 ± 2°C). Animals were acclimatized for a period of three days in their new environment before the initiation of experiment. Litter in cages was renewed three times a week to ensure hygiene and maximum comfort for animals. The experiments reported in this study were carried following the guidelines stated in Principles of Laboratory Animal Care National Institute of Health publication (NIH) publication 85-23, revised 1985 and the Mexican Official Normativity (NOM-062-Z00-1999). All animals procedures were performed in accordance with the recommendations for the care and use of laboratory animals (756/lab/ENCB).P. canariensis extracts. Each extract at dose 400 mg/kg was prepared by dissolving in 1% of Tween 80. The supplementations were given daily via oral gavage for 10 weeks, at a volume of 0.1 mL. The NC and obese control (OC) groups were given distilled water as a placebo.Thirty-five male CD1 mice, 2 months old and weighing between 20 and 25 g, were acclimatized under room temperature (28 ± 2°C) with a regular light/dark cycle and free access to food and water for 1 week before use. Following acclimatization, the animals were randomly segregated into five groups of seven rats each. We formulated experimental high-fat diets consisting of 50% normal rat chow pellet, 24% corn oil, 20% full-cream milk powder, and 6% sugar, as suggested by Martinello et al. . AnotherP. canariensis were used in the experiment: 200 and 400 mg/kg. The extracts were prepared by dissolving in 1% of Tween 80. The supplementations were given daily via oral gavage for 10 weeks, at a volume of 0.1 mL. The NC and obese control (OC) groups were given distilled water as a placebo. Contrary from the induction period, all groups were given normal rat chow throughout the treatment period. Treatment with extracts began after 10 weeks of obesity induction and scored until week 10. Two different doses of Severe diabetes was induced in overnight fasting male mice by a single intraperitoneal injection of 50 mg/kg of streptozotocin in a volume 1 ml/kg body weight dissolved in cold citrate buffer (pH 4.5) . Hyperglb.w. STZ in 0.1 mol/L cold citrate buffer (pH 4.5), 15 min after the intraperitoneal administration of 120 mg/kg nicotinamide. The STZ treated animals were allowed to drink 5% glucose solution overnight to overcome drug induced hypoglycemia. After 10 days of development of diabetes, mice with moderate diabetes having persistent glycosuria and hyperglycaemia (blood glucose > 250 mg/dl) were used for further experimentation [Mild diabetes was induced in overnight fasting mice by administering a single intraperitoneal injection of 60 mg/kg entation .After the mice had been denied access to food/water overnight, they were randomly divided into eight groups (six mice per group) matched for body weight. Normal mice administered distilled water daily for 30 days. Diabetic control mice administered distilled water daily for 30 days. Diabetic mice administered extract of hexane (100 mg/kg), for 30 days. Diabetic mice administered extract of hexane (200 mg/kg), for 30 days. Diabetic mice administered extract of hexane (400 mg/kg), for 30 days. The other diabetic groups were orally administered 100, 200 and 400 mg/kg body weight (b.w.) of extracts of chloroform (AL-C) and methanol (AL-M) suspended in Tween 80, 1% via gavage). Diabetic treated mice received glibenclamide (GB) a dose of 5 mg/kg b.w as standard drug. Blood samples were collected from the tail vein at 0, 2, 4, 6, 8, and 12 h after the administration. The plasma glucose concentration was determined by an enzymatic colorimetric method using a commercial kit .n = 10) of diabetic mice were used to determine the chronic effect of AL-H extract. Each group was submitted to a specific treatment as follows. Normal control and severe and mild diabetic mice groups were fed with normal diet and drinking water ad libitum and were given saline by gastric gavage. Severe and mild diabetic mice that received Al-H extract by gastric gavage (400 mg per kg of body weight) every day [In a parallel study eleven groups  into a tail vein for 10 min [At the end of obesity and chronic diabetes experiments all mice were anesthetized with 1.0% pentobarbital sodium and blood was obtained from the retroorbital plexus of each animal following the injection of heparin . The blood samples were then centrifuged at 1600 ×g for 15 min at 4°C for the preparation of plasma. Concentrations of plasma glucose, total cholesterol (TC), triglycerides (TG), and HDL-cholesterol were measured with enzymatic assay kit (Genzyme Diagnostics), and LDL-cholesterol was calculated as the remaining difference of total cholesterol and HDL. Blood glucose levels were measured employing the glucose oxidase-peroxidase (GOD-POD) method [μM/g of liver and kidney tissue. Serum glutamate oxaloacetate transaminase (SGOT), glutamate pyruvate transaminase (SGPT), serum alkaline phosphatase (SALP), and total protein, using a commercial Diagnostic Kit Biocompare, BioVision, Biocompare and Thermo scientific, respectively. Malondialdehyde (MDA) as thiobarbituric acid reactive substances was measured at 532 nm spectrophotometrically [Blood samples were collected from tail vein of the mice into microcentrifuge tubes containing heparin (10 ) method . Lipid p) method  and exprtrically .P. canariensis aqueous extracts at doses of 400 mg/kg body weight on a daily basis for 30 days. At the end of the experiment, an oral glucose tolerance test (OGTT) was performed to assess the animals' sensitivity to a high glucose load. Overnight fasting mice were fed orally 2 g glucose/kg b.w. Blood samples were collected from the caudal vein from a small incision at the end of the tail at 0 min (immediately after glucose load), 30, 60, 90, and 120 min after glucose administration.Mice of each group were orally administered 2SO4 method [2SO4) and the absorbance was measured at 620 nm.Mice were  sacrificed by decapitation; livers and kidneys were extracted. Frozen livers were thawed, weighed, and homogenized in Tris-HCl  buffer containing 2 mmol/L EDTA. Homogenates were centrifuged at 1000 ×g for 15 min at 4°C. The supernatant was mixed with glucose-6-phosphate dehydrogenase and NADPH and the activity of hexokinase was determined at 37°C for 3 min at 30 s intervals at 340 nm  The hepa4 method . BrieflyThe hepatic G6Pase (glucose-6-phosphatase) activity was assayed by the method of Baginiski et al. . ShortlyGlucokinase (GK) activity was measured using a spectrophotometric method as previously described by Panserat et al. . Briefly) assay kit Bioxytech GR-340 for GR activity , glutathione peroxidase (GPx) assay kit GPx-340 for GPx , and lipid peroxidation using malondialdehyde level by commercial kit (TBARS assay kit). In the pancreas the protein concentration was determined by the Bradford method as described in the Bio-Rad protein assay kit.After 30 days of treatment, mice fasted overnight and euthanized by anesthesia. Antioxidant enzyme activities in the liver, pancreas, and kidney were assayed using commercial kits: superoxide dismutase (SOD) assay kit Bioxytech SOD-525 for SOD activity , catalase assay kit for catalase activity (CAT) , glutathione reductase  was homogenized with chloroform/methanol , and then chloroform (1.25 mL) and distilled water (1.25 mL) were added to the homogenate and mixed well. After centrifugation (1500 ×g for 10 min), the lower clear organic phase solution was transferred into a new glass tube and then lyophilized. The lyophilized powder was dissolved in chloroform/methanol (1 : 2) as the hepatic lipid extracts and stored at −20°C for less than 3 days . The hepμIU/mL.Diabetic mice serum and pancreatic insulin were measured by a Glazyme Insulin-EIA Test according to the manufacturer's instructions . Blood sP value < 0.05 was considered to be significant.Statistical analysis was carried out by using commercially available software SigmaStat 3.5. Values are expressed as mean ± SEM. For multiple comparisons, one-way ANOVA was used followed by Tuckey and Dunnett's test. Treatment with chloroform and methanol extracts did not significantly inhibit the rise in blood glucose levels in normal and diabetic mice and in oral glucose tolerance. However, mice fed the high-fat diet treated with hexane, chloroform, and methanol extracts showed a decrease similarly; for this reason it was decided to study only hexane extract due to the fact that it presents hypoglycemic and antiobesity activities. However, chloroform and methanol extracts did not.The body weights and food intake are shown in The mice fed on the high-fat diet for 10 weeks had a significantly higher body weight and significantly heavier visceral adipose tissues than the mice fed on the normal diet. In the mice fed on the high-fat diet, the body weight elevation that took place over the initial 6 weeks on the high-fat diet was significantly reduced  and the P. canariensis extracts (200 mg/kg per day) also showed significantly lower values of plasma TG 35%, 44%, and 32% for hexane, chloroform, and methanol extract, respectively, compared with the vehicle-treated counterparts . The HFD caused elevation of hepatic TC and TG. All P. canariensis extracts decreased the level of hepatic TC . The moderate doses of P. canariensis extracts significantly reduced the TG hepatic . On the one hand, there were no significant differences in lipid profile and hepatic lipid in all groups. The hepatic tryglicerides levels in the normal group, high-fat diet group, and high-fat + P. canariensis extracts  diet group were 86.2 ± 6.7, 84.5 ± 5.8, and 87.3 ± 2.0 mg/g liver, respectively. In addition, plasma tryglicerides levels tended to be reduced in the high-fat diet group because of a decrease in triacylglycerol secretion from the liver.terparts . The modMDA, which is the final product of lipid peroxidation, was determined spectrophotometrically. HFD increased the MDA level in plasma when compared with that of the normal group. HFD group treatment with AL extracts showed a significant reduction in MDA levels .in vivo, indicated with reduced ALP, SGOT, and SGPT levels. It was found that mice fed with high-fat diet alone developed a high degree of steatosis. The effect of increased liver enzymes levels and the formation of steatosis in the high-fat diet-fed group correlates with an increase of liver weight (As shown in r weight . The admP < 0.001) decreased when compared with normal control group. The extract at dose of 400 mg/kg b.w. significantly (P < 0.001) maintained the body weight and liver and kidney weights toward normal as compared with STZ control.During the study period of 4 weeks, body weight and food and water intake of each mouse were recorded daily but data is presented only at day 0 and the end of the experimentation period . The bodP < 0.001) elevated as compared with the normal control group. Treatment with  AL at the dose of 400 mg/kg b.w. significantly (P < 0.001) brought the SGOT, SGPT, SALP, and serum protein toward the normal values. The total protein was found to be significantly (P < 0.001) decreased in the STZ controls groups as compared with the normal control group indicating a lower capacity for protein synthesis in the livers of the diabetic animals; the administration of hexane extract in types SD and MD diabetic animals significantly (P < 0.001) prevented the loss of total protein content as compared with the STZ control group . Oral treatment with glibenclamide (4 mg/kg) caused a light reduction in blood glucose levels. Reduction in the blood glucose level caused by AL-H at all doses is higher than that of standard drug, glibenclamide.The oral administration of AL at doses of 100, 200 mg/kg, and 400 mg/kg produced a significant hypoglycaemic effect in normal fasting mice after 4 h. The most pronounced effect of AL was observed after 6 h . The dos  STZ-nicotinamide-induced MD diabetic mice is shown in P < 0.005) on streptozotocin-induced severe and mildly diabetic mice after 2 weeks of treatment. Treatment of diabetic mice with glibenclamide (4 mg/kg) produced a significant fall in blood glucose after 4 weeks (68%). Hexane extract (AL-H) at 400 mg/kg dose gradually decreased blood glucose level 4 weeks after administration . In diabetes mildly, continued glucose lowering effect at the end of the study when compared to diabetic control.The antihyperglycemic effect of AL-H on the fasting blood glucose levels in STZ-induced SD andP. canariensis produced a significant (P < 0.05) reduction of glucose in blood at 120 min, showing a 45% and 47% decrease of glucose in blood in severe and mild diabetes, respectively, compared with diabetic mice . Treatment with glibenclamide for 30 days restored insulin levels. After three weeks of administration of the P. canariensis hexane extract there was also a notorious elevation in serum insulin and pancreatic insulin levels. This is shown in In streptozotocin-induced diabetic mice insulin in serum and in pancreas decrease markedly, as low as 1.5 P. canariensis in obese animals, has shown that there was a significant difference in the body weights between the high-fat and normal diet groups; no significant difference in the daily food intake between groups was observed. The high-fat diet groups continuously consumed similar quantities of food, regardless of the higher calories content in the diet. As a result, the caloric intake was raised in the high-fat diet group compared to the normal group counterpart. The supplementation of obese rats with AL-H at 400 mg/kg conversely causes a remarkable reduction of body weights compared to the untreated obese group. The findings demonstrated that AL-H is capable of preventing body weight gain and showed a significant reduction in their body weight after 4 weeks of treatment. AL-H has shown that oral administration of P. canariensis modulates lipid homeostasis in the development of HFD-induced obesity. In this study, feeding a hyperlipidaemic diet containing 45% energy from fat for 10 weeks caused obesity, hyperlipidaemia, and hyperglycaemia in mice [P. canariensis supplementation not only suppressed excessive gains of weight in body, liver, kidney, visceral adipose tissue (VAT) and subcutaneous abdominal adipose tissue (SAT), by affecting adipose tissue growth through modulation of HFD-induced hypertrophy of adipocytes, but also reduced serum lipids, which is defined as the main risk for dyslipidaemia [Obesity induction via dietary means in animal models has been considered as the most popular reference among researchers due to its high similarity of mimicking the usual route of obesity occurrence in humans. It is generally known that high-fat diet is one of the major factors causing obesity and that the long-term intake of high-fat diet evokes a significant increase in abdominal fat weight in mammals . The pre in mice , Howeverpidaemia . As showpidaemia .in vivo. The groups treated with AL-H showed a significant elevation in their SOD, GPx, GSH, and CAT activities compared to the untreated obese rats. These results are aligned with the reduction of MDA levels, which may be related to the ability of AL-H to suppress lipid peroxidation due to having an increase in the activity of antioxidant enzymes, regardless of the availability of lipid substrates.The increased MDA level in the untreated obese group clearly demonstrated that high fat consumption was attributed to increased oxidative stress. AL-H supplementation was found to improve the endogenous antioxidant defense system by enhancing the antioxidant enzymes activities P. canariensis is a kind of traditional medicine which has long been used to effectively treat obesity and diabetesl by local people. In the present findings, we evaluated the antidiabetic effect of AL-H on SD and MD induced by STZ. Our date clearly showed hypoglycemic activity and glucose tolerance pattern was significantly altered comparable to that of glibenclamide. This activity improved glucose tolerance suggesting a decrease in insulin resistance and helping to maintain blood glucose levels steady which may indicate certain induction of peripheral utilization of glucose.Streptozotocin is known for its selective pancreatic islet beta cells cytotoxicity and has been widely used to induce diabetes mellitus. Besides alteration in the carbohydrate and lipid metabolism, rats treated with STZ also exhibited reduced total protein and liver glycogen levels, increased liver glucose transfer, and decline in liver glycogen content in STZ diabetic rats . From glFurthermore, the attenuating effect of this extract on experimental physiological symptoms of streptozotocin-induced severe and mild diabetes has been confirmed here by the study of glucose-6-phosphatase activity in liver, as well as the quantification of glycogen in liver and skeletal muscle, which are very important indicators of diabetes mellitus. In our study, the hexane extract of AL-H seeds had a beneficial effect in terms of peripheral glucose utilization.P. canariensis decreased G6Pase activity and increased GK activity in liver, which indicates that this can be an increase in hepatic glucose uptake and decrease in hepatic glucose release. Therefore, this study strongly suggests that P. canariensis enhances hypoglycemic activity probably by reducing HGO via decreasing G6Pase activity and increasing GK activity. One of the key enzymes in the catabolism of glucose is hexokinase, which phosphorylates glucose and converts it into glucose-6-phosphate. The increased activity of hexokinase can promote glycolysis and increase utilization of glucose for energy production [P. canariensis to the diabetic mice increased the activity of hepatic hexokinase causing an increase in glycolysis. The hepatic glycogen was found to be increased in both liver and skeletal muscle in diabetic rats and suggested also a reduction in glycogenolysis and an increase in glycogenesis.Control of hepatic glucose (HGO) may occur through regulation of gluconeogenesis or glycogenolysis. However, the common final pathway of glucose uptake and release involved the phosphorylation and dephosphorylation of glucose via GK and G6Pase, respectively . In thisoduction . The admP. canariensis improved insulin sensitivity.Insulin deficiency is associated with hypercholesterolaemia and hypertriglyceridaemia. STZ-induced diabetes showed increased plasma levels of cholesterol, triglyceride, free fatty acid, and phospholipids. Insulin deficiency or insulin resistance could be responsible for dyslipidaemia because insulin increases fatty acid as well as triglyceride synthesis in adipose tissue and liver. It inhibits lipolysis, partly via dephosphorylation (and hence inactivation) of lipases . Insulin  particular, the diabetes-associated free radical injury, accumulation of lipid peroxidation products, depletion of GSH, decrease in GSH/GSSG ratio, and downregulation of key antioxidant enzymes [2•−; GPx and CAT were shown to be responsible for the detoxification of significant amounts of H2O2. In addition, administration of AL-H extract showed increased activities of SOD, GPx, and CAT after 30 days of treatment in STZ rats indicating that the AL-H extract can reduce reactive oxygen free radicals and improve the activities of the antioxidant enzymes [We studied the antioxidant effect of the AL-H extract over tissue oxidative markers. Diabetic mice showed a significant reduction in SOD, CAT, GSH, and GPx in hepatic and renal tissues as a result of a persistent oxidative stress. One of the main consequences of chronic hyperglycemia is the enhanced oxidative stress resulting from the imbalance between production and neutralization of reactive oxygen species (ROS), in enzymes . Accordi enzymes . The enh enzymes . P. canariensis prevents oxidative stress, acts as a suppressor of liver cell damages and inhibit, the progression of liver dysfunction induced by chronic hyperglycemia. AL-H extract has a potent effect over antioxidant enzymes activities in pancreatic tissue, enhancing them compared to diabetic control.The effects of the AL-H on transaminase  activity was also studied in hyperlipemic mice. Transaminases are important enzymes for the study of liver toxicity. ALP is found predominately in the liver, with lesser quantities in the kidneys, heart, and skeletal muscles. As a result, ALP is a more specific indicator of liver inflammation than SGPT and SGOT and may also be elevated in diseases that affect other organs, such as the heart and muscles. Our results indicate that only treatment with the AL-H of hyperlipemic mice reduced the activity of these enzymes, suggesting that this extract effectively reduced the toxic effect on these enzymes. These results suggest that P. canariensis improves glucose metabolism by reducing insulin resistance and by protecting pancreatic β-cells from oxidative stress and exhibited excellent hypoglycemic activity, enhancing glucose uptake by adipose and muscle tissues, along with beneficial lipid regulation ability. A reduction in the activities of SGOT, SGPT, and ALP in plasma and an increase in glucokinase and HK and a decrease in G6Pase indicated the hepatoprotective role. We demonstrated that daily consumption of P. canariensis tended to suppress body weight in fatty mice with hypertriglyceridemia. Although the effect of P. canariensis is important, it is interesting that P. canariensis suppressed body weight without affecting food consumption. P. canariensis possesses lipid lowering effect in obesity-induced mice, as well as its weight-reducing ability. Due to the promising effects of P. canariensis in STZ-induced diabetes mice and diet-induced obesity, further studies are sought in order to determine the active principle from this plant.


To quantitatively evaluate the safety and related-toxicities of intensity modulated radiotherapy (IMRT) dose–volume histograms (DVHs), as compared to the conventional three-dimensional conformal radiotherapy (3D-CRT), in gynecologic malignancy patients by systematic review of the related publications and meta-analysis.Relevant articles were retrieved from the PubMed, Embase, and Cochrane Library databases up to August 2011. Two independent reviewers assessed the included studies and extracted data. Pooled average percent irradiated volumes of adjacent non-cancerous tissues were calculated and compared between IMRT and 3D-CRT for a range of common radiation doses (5-45Gy).p = 0.004), 27.00% , 37.30% , and 39.50% . Reduction in irradiated small bowel was also observed for IMRT-delivered 40 Gy and 45 Gy (by 17.80% (p = 0.043) and 17.30% (p = 0.012), respectively), as compared with 3D-CRT. However, there were no significant differences in the IMRT and 3D-CRT pooled average percent volumes of irradiated small bowel or rectum from lower doses, or in the bladder or bone marrow from any of the doses. IMRT-treated patients did not experience more severe acute or chronic toxicities than 3D-CRT-treated patients.In total, 13 articles comprised of 222 IMRT-treated and 233 3D-CRT-treated patients were included. For rectum receiving doses ≥30 Gy, the IMRT pooled average irradiated volumes were less than those from 3D-CRT by 26.40% (30 Gy, IMRT-delivered high radiation dose produced significantly less average percent volumes of irradiated rectum and small bowel than 3D-CRT, but did not differentially affect the average percent volumes in the bladder and bone marrow. Radiotherapy (RT) plays an important role in the adjuvant treatment of gynecologic malignancies, particularly in cervical and endometrial cancer. While RT has greatly improved local regional control of primary tumors -3, it haIMRT has also proven an efficacious and safe method of treating head, neck, lung, central nervous system, breast, and prostate cancers -23. WhilNonetheless, the previous successes of IMRT in other cancer patients have promoted significant research interest to evaluate its promise for treating gynecologic malignancy patients . In addiThe PubMed, Embase, and Cochrane Library databases were searched for relevant publications by using the following keywords: “radiotherapy, intensity modulated”, “IMRT”, “cervical cancer”, “cervix cancer”, “cervical carcinoma”, “cervix carcinoma”, “endometrial cancer”, “endometrial carcinoma”, and “gynecologic malignancies”. The upper publication date was August 2011 and no lower date was set. These terms were then combined with the search terms for the following study designs: “practice guideline”, “systematic review”, “meta-analysis”, and “review”. In addition, the reference lists of all pertinent articles found in PubMed were manually searched.The Physician Data Query (PDQ) clinical trials database and the proceedings of the 1980–2010 annual meetings of the American Society of Clinical Oncology (ASCO) and the American Society of Radiation Therapist (ASTRO) were also searched for reports of new or on-going trials.A study was selected for inclusion if it provided information on DVHs of different irradiated organs that had been treated with IMRT or 3D-CRT. Those studies were then selected for the following criteria: 1) prescription dose of Gy or 50.4 Gy, for either IMRT or 3D-CRT, with all patients having received radiation doses of 1.8 Gy/day; 2) irradiated normal organs at risk being small bowel, bladder, rectum, and bone marrow; 3) data from the DVHs in irradiated organs at risk being relative number and not the actual measured value; and 4) studies being independent and not replicates of a single population. If studies were found to overlap, the largest dataset was selected for inclusion. A flowchart of the strategy used for this systematic review of the literature is presented in Figure The data from each included study were extracted by two reviewers (Lin Zhu and Baojuan Yang), who worked independently and used a standardized form for data collection. Any subsequent discrepancies were discussed and revised until a consensus was achieved. Information extracted from each article included the first author, country of origin, number of patients, normal organs irradiated, prescribed dose of IMRT and 3D-CRT, and average percent irradiated volumes of the organs at risk (OARs) at various radiation doses  in DVHs.In regards to the DVHs information, if the authors did not list the average percent irradiated volumes of OARs, we measured it based on the figures in the article. If the prescribed dose in the study was 50.4 Gy , we only extracted the data for radiated doses <45 Gy.I2 was less than 50%; otherwise, a random effect model was used. The pooled average percent irradiated volumes for IMRT and 3D-CRT treatment plans were compared at each radiation dose in OARs . Differences were considered to be statistically significant if the p-value was less than 0.05. Publication bias was evaluated by funnel plot, followed by a quantitative analysis using a linear regression approach and rank correlation method [To assess heterogeneity of the studies, a fixed effect model based on the Mantel-Haenszel method was used to calculate the pooled average percent irradiated volumes, if n method ,35. For In total, 13 articles, which compared the extents of DVHs in IMRT and 3D-CRT for endometrial and cervical cancer patients, were included. Ten of these reported on the irradiated volumes of small bowel, nine on rectum, eight on bladder, and six on bone marrow. The 13 articles comprised a total of 222 patients treated with IMRT and 233 patients treated with 3D-CRT. The prescribed dose was either 45 Gy or 50.4 Gy, and all patients received treatment at 1.8 Gy/y. The characteristics of the included articles are summarized in Table p = 0.004), 27.00% (p = 0.040), 37.30% (p = 0.006), and 39.50% (p = 0.002), respectively. Furthermore, a statistically significant dose–response relationship was observed between increasing Gy of irradiation doses and decreasing pooled average percent volumes (P = 0.003).The pooled average percent irradiated volumes of IMRT and 3D-CRT were calculated for different OARs and compared for each irradiated level Table . For recp = 0.043). Similarly, at a dose of 45 Gy, the pooled average percent volumes were 17.30% lower in IMRT (IMRT: 18.60% (95% CI: 12.60-26.70) and 3D-CRT: 35.90% (95% CI: 21.90-52.80); p = 0.012). At low doses (<20 Gy), the pooled average percent volumes of small bowel irradiated with IMRT were similar to those for patients who received 3D-CRT treatment (p > 0.05). Likewise, the doses between 25 Gy and 35 Gy did not produce significantly different effects (p > 0.05), but irradiation with IMRT did yield > 10% less percent pooled average percent volumes than 3D-CRT.In small bowel, the pooled average percent volumes were significantly lower (by 17.80%) for IMRT than for 3D-CRT at a radiation dose of 40 Gy (IMRT: 24.70% (95% CI: 17.30-34.10) and 3D-CRT: 42.50% (95% CI: 24.00-63.40); The results of bladder and bone marrow from our meta-analysis revealed that the pooled average irradiated volumes in IMRT were lower than those in 3D-CRT. Although the differences were more obvious for the higher doses of irradiation, none reached statistical significance Table .The graphical funnel plots of pooled average percentage volumes of small bowel irradiated at 45 Gy and rectum irradiated at 30 Gy by IMRT and 3D-CRT are shown in Figure The acute and chronic toxicity effects of IMRT and 3D-CRT were evaluated by investigating the reported side effects of gastrointestinal (GI), genitourinary (GU), and hematologic toxicity for each. Five studies that reported on the toxic effects of IMRT and 3D-CRT were identified, including four on acute toxicity ,36,42,43et al.[et al.[et al.[et al.[In the studies by Mundt et al. and Chenl.[et al., none ofl.[et al. and Hassl.[et al., severe et al.[et al.[For chronic toxicities, most of the patients receiving IMRT had no or mild side effects of GI and GU. The incidence of severe GI side effects with IMRT was 2.20% (4/180), and with 3D-CRT, the rate reached up to 4.60% (3/65). Although Hasselle et al. concludel.[et al., one patBecause IMRT can deliver treatment to target organs while reducing the volumes of proximal normal structures that are irradiated, it appears to offer several advantages over conventional techniques for the treatment of malignancies  . Howeveet al.[et al.[et al.[et al.[p < 0.05). However, the study by Mell et al.[P = 0.003). Since some publication bias existed in our meta-analysis, we adjusted the reduction of volumes irradiated by using the trim and fill method, and we found that the differences retained statistical significance. This result illustrated that, the higher the radiation dose prescribed, the better IMRT was at reducing the average percent irradiated volumes for the rectum, as compared to 3D-CRT.The studies by Heron et al., Igdem el.[et al., and Roel.[et al. reportedl.[et al. reportedll et al. found noet al.[et al.[p = 0.043) and 17.30% (p = 0.012), respectively. However, at 35 Gy and below, no statistically significant reduction was found between IMRT and 3D-CRT in the pooled average percent of irradiated volumes. In this meta-analysis, there was no publication bias detected for data related to IMRT-delivered 20 Gy, 30 Gy, or 35 Gy or for 3D-CRT-delivered 5 Gy, 20 Gy, or 25 Gy. Although publication bias was observed for data related to both IMRT- and 3D-CRT-delivered 40 Gy and 45 Gy radiation doses, the reduction of pooled average percent irradiated volumes remained significant after adjusting with the trim and fill method.Some studies have reported that IMRT treatment of gynecologic malignancies more effectively protects the small bowel compared to the 3D-CRT technique, especially when radiation doses <20 Gy are used . In the et al. and Roesl.[et al., it was l.[et al.,44 . OurThis meta-analysis found no statistically significant evidence to support the theory that IMRT was an effective approach to reduce the irradiated volumes of the bladder.et al.[p < 0.001). In the studies by Lujun et al.[et al.[et al.[p > 0.05). Publication bias was observed only for the IMRT-delivered dose of 15 Gy and 3D-CRT-delivered doses of 15 Gy, 20 Gy, and 30 Gy. After adjusting the reduction of irradiated volumes by using the trim and fill method, there was still no statistically significant reduction found between IMRT and 3D-CRT.Considering the effects of IMRT and 3D-CRT on bone marrow, Brixey et al. showed tun et al. and Ahmel.[et al., the avel.[et al.demonstraHigh heterogeneity was found for the data of bladder and rectum irradiation from high radiation doses delivered by IMRT and for the data of small bowel and bladder irradiation from 40 Gy and 45 Gy doses delivered by 3D-CRT. Potential explanations exist to explain these instances of heterogeneity. First, the OARs in the abdominal cavity are not static and are in continual motion, and the volumes of irradiated organs are known to be impacted by different postures. Second, the RT physicians defined the extent of OARs that were reported in each study, and they may not have abided by a unified standard. Third, the data from each study was generated independently and may have been influenced by the particular study design.et al.[et al.[et al.[et al.[et al.[In our meta-analysis, we determined that toxicity occurred with significantly lower frequency in the IMRT treated patients than in the 3D-CRT patients ,39,46. Iet al. and Chenl.[et al., adjuvanl.[et al. and Hassl.[et al. sufferedl.[et al.. Likewisl.[et al. providedFinally, the collected IMRT dosimetric data from gynecologic patients used in our meta-analysis suggested that IMRT is safe for use as a treatment of gynecologic cancers. However, all of the research studies with which our meta-analysis was carried out were observational. It is generally believed that findings from observational studies are not as accurate as those from randomized controlled trials, since they can easily overestimate the magnitude of effects. Another limitation in our study was the small sample size and uneven quality of the samples. Thus, our conclusions need to be validated by larger samples and more studies to confirm the benefits of IMRT in patients with gynecologic malignancy and to further study the different acute and chronic toxicities produced by IMRT and 3D-CRT.This study suggested that IMRT significantly reduced the average percent irradiated volume of the rectum resulting from >30 Gy doses and of the small bowel from 45 Gy. Furthermore, in the bladder and bone marrow, the advantages of IMRT over 3D-CRT were not significant for any of the radiation doses examined.IMRT: Intensity modulated radiotherapy; DVHs: Dose–volume histograms; 3D-CRT: Three-dimensional conformal radiotherapy; RT: Radiotherapy; WPRT: Whole pelvic radiotherapy; NCCN: National comprehensive cancer network; PDQ: Physician data query; ASCO: The American society of clinical oncology; ASTRO: American society of radiation therapist; OAR: Organ at risk.The author(s) declare that they have no competing interests.BY carried out the molecular genetic studies, participated in the sequence alignment and drafted the manuscript. LZ carried out the immunoassays. HC participated in the sequence alignment. QL participated in the design of the study and performed the statistical analysis. YZ and YZ conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.


High-risk human papilloma virus (HR-HPV) infection is associated with the development of cervical cancer. HPV vaccination reduces the risk of developing malignant lesions and is expected to change the dynamics of HPV transmission. Data from non-vaccinated women may provide an important benchmark to allow the impact of HPV vaccination programs to be assessed.This study was designed to prospectively determine the changing dynamics of HR-HPV infection and associated genital diseases in young women, most of whom were non-vaccinated.Data from a population-based cohort study, comprising women of two predefined birth cohorts (women born in 1983/84 or 1988/89), were analyzed between 19 October 2009 and 31 December 2010 to determine risk factors for high-risk HPV infection and the association between specific HR-HPV types and atypical Pap smear test results. HPV status was determined by Hybrid Capture 2 (HC2) assay and genotyping.The prevalence of HR-HPV was 22.8% in the 1983/84 cohort (150/659) and 23.7% in the 1988/99 cohort (142/599). Only the number of sexual partners was a significant risk factor for HPV infection  in multivariate analysis. HPV16 positive-women were significantly more likely to have abnormal Pap smears of any degree than HPV16-negative women . CIN3 was diagnosed in six women 84 cohort,/84 cohort and two in the 1988/89 cohort. All women with CIN3 tested positive for HC2-HR and all six CIN3 cases 84 cohort,/84 cohort tested positive for HPV16. In the 1988/89 cohort, the rate of HPV16 infection was significantly lower in vaccinated than non-vaccinated women .HR-HPV infection was highly prevalent in both cohorts and associated with an increased risk of abnormal Pap smears and biopsy proven CIN2+. HPV16 infection was associated with a high risk of clinically relevant lesions. HPV vaccination significantly decreased the risk of HPV16 infection. Thirteen types of human papilloma virus (HPV) are associated with the development of cervical cancer and are designated as high-risk (HR) . Among tVaccination programs are expected to change the dynamics of HPV transmission  and, theThe Wolfsburg HPV Epidemiological Study (WOLVES) is a prospective, population-based surveillance study on the prevalence and incidence of HPV infections and associated diseases in women of three predefined birth cohorts. All women born in 1983/84, 1988/89, and 1993/94 with a first residency in Wolfsburg, Germany , will be invited to participate. This analysis includes baseline data for the 1983/84 and 1988/89 cohorts.The residents’ registration office provided a list of women with a first residency in Wolfsburg born in either 1983/84 or 1988/89 and all 2850 women were invited by letter to attend cervical cancer screening. The invitation included information on the study objectives and made it clear that participation was voluntary. To be recruited, women had to attend one of 20 gynecologists in private practices in the city of Wolfsburg for routine Pap smear screening, according to the standard screening concept in Germany. All participants gave written consent and the study was approved by the ethics committee of the physicians’ association of Lower Saxony in Hannover, Germany.Participants completed a short, standardized questionnaire in the private practice. The questionnaire included questions on education, birth country, marital status, pregnancies, parity, contraception, smoking, number of sexual partners, age at sexual debut, prior screening for cervical cancer, history of abnormal Pap smears, sexually transmitted infections and genital warts. Furthermore, the referring gynecologist collected information on HPV vaccination status by checking the certificate of vaccination.Participants underwent a pelvic examination with visualization of the uterine cervix. Pap smears were taken using spatula and endocervical brush. A second sample was then obtained with a Qiagen Cervical Sampler , and suspended in 1 ml of specimen transport medium  for HPV DNA testing.All primary HPV testing was undertaken using the Hybrid Capture 2 (HC2) assay . All samples were analyzed for the presence of at least 13 HR-HPV types  following the manufacturer’s instructions. A positive HR-HPV result in this study refers to a subject positive for the HR-HPV probe mix. In this analysis, HR HPV subtypes were counted individually. In patients with multiple infections, HR HPV subtypes were reported separately. LR-HPV types  are reported in a separate publication .All samples that tested positive for HR-HPV with HC2 and 10% (every tenth) of all HC2-negative samples underwent HPV genotyping. HPV genotyping was performed as described previously using SPF-10-PCR, followed by Reverse Line Probe Assay LiPA Extra (SPF-10-PCR) . BrieflyA standardized, risk-adapted follow-up protocol was used for the management of participants according to the results of conventional Pap smear testing and HPV status, as described previously .Patients were transferred for colposcopy if they had abnormal Pap smears conspicuous of high-grade lesions or had Pap smears classified as borderline/low-grade and tested positive for HR-HPV. Colposcopists classified the type of transformation zones according to the Barcelona nomenclature of the International Federation for Cervical Pathology and Colposcopy (IFCPC) . ColposcThis paper presents a one-time cross-sectional analysis of data for the 1983/84 cohort and baseline data for the 1988/89 cohort. As this is an observational study, no formal hypothesis was tested and the statistical analysis was descriptive for all evaluable variables. All statistical analyses were undertaken by an independent statistician who did not participate in the collection of data. The association between HR-HPV infection and exploratory variables was analyzed in univariate analysis with a level of significance defined as p < 0.05 (two-sided testing). Multivariate analyses include data on exploratory variables for all HPV subtypes. All statistical analyses were performed with the validated program Testimate Version 6.5 from IDV Gauting .Between 19 October 2009 and 31 December 2010, 659 (43.8%) of 1504 registered women born in 1983/84 and 599 (44.5%) of 1346 women born in 1988/89 were recruited. The characteristics and risk factors of interest for the two cohorts are shown in Table HC2 test results for HC2 HR-positive and LR-/HR-positive are shown in Figure The type-specific HR-HPV prevalence is shown in Figure Univariate analysis showed that the number of sex partners (strong), early age at first intercourse (strong), and smoking (weak) were significant risk factors for HR-HPV infection in the 1983/84 cohort, whereas there were weaker associations in the 1988/89 cohort  versus HPV-negative (reference). In the 1983/84 cohort, the association between HPV16 infection and both the number of partners  and early age at first sex (p = 0.0065) was stronger than for HR-types overall. No association was evident in the 1988/89 cohort.The three cofactors associated with HR-HPV infection were analyzed by specific HR-HPV type using the Mann Witney In multivariate analysis, only the number of sexual partners was a significant risk factor for HPV infection. The odds ratios (ORs) were 22.687 (95% CI 8.032–64.086) and 6.124 (95% CI 3.195–11.738) for more than five partners versus one partner in the 1983/84 and 1988/89 cohorts, respectively.The association between atypical Pap smear test results and specific HR-HPV types is detailed in Table In HPV18-positive women in the 1983/84 cohort, the corresponding risks were significantly increased for atypical Pap smears classified borderline or more (p < 0.0034), LSIL or more (p < 0.0088) and CIN1+ (p < 0.04). No significant differences were observed in the 1988/89 cohort.Type-specific univariate analyses of data from the 1983/84 cohort showed significant interactions between other specific HPV types and atypical Pap smear test results. There was a weak association between HPV39 and Pap LSIL or more (p < 0.0239). HPV51 showed a weak association with Pap ASC-US or more (p < 0.0318), but a stronger association with Pap smears LSIL or more (p < 0.0047) and histology of CIN1+ (p < 0.0066). HPV53 showed a significant association with Pap ASC-US or more (p < 0.0093) and LSIL or more (p < 0.0107), but not with CIN1+. HPV66 showed significant associations with Pap ASC-US + (p < 0.0005), LSIL (p < 0.0005) and CIN1+ (p < 0.0134). HPV types 31, 33, 35, 45 and 52 showed no associations at all.For the 1988/89 cohort, type-specific univariate analysis found no association between HPV types 31, 33, 35, 39, 45, 51, 53 and 66 and atypical smear tests; however, there was a significant association between HPV52 (n = 27) and Pap smears LSIL or more (p < 0.0284)Overall eight women were diagnosed with CIN3, six in the 1983/84 cohort and two in the 1988/89 cohort. All women with CIN3 tested positive for HC2-HR. Genotyping showed that all six CIN3 cases in the 1983/84 cohort tested positive for HPV16 . For HPV16-infected women in the 1983/84 cohort the corresponding risk for CIN3 was 6/50 (12%). In the 1988/89 cohort, genotyping showed one case of co-infection with HPV16 and HPV52, but failed in the other case.Overall 11 women were diagnosed with CIN2, six in the 1983/84 cohort and five in the 1988/89 cohort. All tested positive for HC2-HR. Genotyping showed that three cases of CIN2 in the 1983/84 cohort were associated with HPV16  while the remaining cases were associated with HPV18 (n = 1), HPV18 and 31 (n = 1) and HPV53 (n = 1). In the 1988/89 cohort, genotyping showed four cases of CIN2 were associated with HPV16  and one case was associated with HPV51.Five women in the 1983/84 cohort had a history of conization because of CIN3, whereas there were no cases with a history of conization in the 1988/89 cohort.In total, 46 women were referred to colposcopy, all of whom had transformation zones either type 1 (n = 28) or type 2 (n = 18). Colposcopy was considered to be satisfactory in all of these cases. Colposcopy findings were classified as major changes in 11 cases, minor changes in 25 cases and as normal in 10 cases.All eight CIN3 lesions showed major changes with fast acetic acid reaction, dense acetowhite to opaque flat epithelium (n = 4), coarse mosaic (n = 1), coarse punctuation (n = 2) or coral-like with internal borders (n = 1). All CIN3 lesions were associated with HPV16.Only three CIN2 lesions showed major changes with coarse punctuation (n = 2) or flat dense acetowhite (n = 1), and all were associated with HPV16 or HPV18, while the remaining eight CIN2 lesions showed minor changes, mainly regular punctuation and flat acetowhite epithelium. Among cases with normal histology or CIN1 and minor changes (n = 27), no association was demonstrated between specific HPV types and defined morphological changes on colposcopy.Analysis of HPV vaccination was not useful in the 1983/84 cohort because of the low number of vaccinated participants (42/659). However, 126 of 599 women in the 1988/89 cohort had received a full course of three doses of HPV vaccines, which was more than expected at the start of the study. Only two of 126 were diagnosed with HPV16 infection (1.59%) compared with 42 of 476 non-vaccinated women (8.82%); the difference was statistically significant (p = 0.003). There was no CIN associated with HPV 16 or 18 in the vaccinated group.WOLVES has provided the first real-life data in Germany on the changing dynamics of HR-HPV and associated diseases in women aged 20–30 years who have not been routinely vaccinated. Essentially, the study represents the final opportunity to monitor a population of young women at full risk from HPV and provides benchmark longitudinal data that will allow the impact of HPV vaccination to be measured. The overall prevalence of HR-HPV in this study was 23%. HPV16 was the most common type in 1983/84 cohort and the second most common type after HPV51 in the 1988/89 cohort. The risk for HR-HPV infection was increased by the number of sexual partners, age at sexual debut and smoking (weak association). Multivariate analysis demonstrated that only the number of sexual partners was a significant risk factor for HPV infection. This finding supports the concept that the number of sexual partners is the single most important risk factor for HPV infection, although other factors may be more important for HPV persistency [Other national studies in Europe, using heterogeneous sources and methodologies, have provided HPV prevalence data in women aged <30 years prior to widespread HPV vaccination. In a German cross-sectional study , 377/169In other European studies of non-vaccinated women aged 18 to <30 years, the prevalence of HR-HPV varied considerably from <10% to >50% -32. It iWith its well-defined target population, the ongoing WOLVES study has a number of advantages compared with one-time, cross-sectional studies in mixed screening populations. The 1988/89 cohort will be followed by annual examination for 5 years. In 2014/15, women born in 1993/94 will be invited for a one-time examination. WOLVES will finally comprise three different age cohorts  allowing a comparison of changes over time. At the end of the trial in 2014/15, the 1993/94 cohort will be as old as the 1988/89 cohort was at the beginning of the trial in 2009, while the 1988/89 cohort will be as old as the 1983/84 cohort was in 2009.A particular strength of the WOLVES study design is careful monitoring of defined patient pathways in participants with abnormal screening results or clinical findings. As a result, compliance was high and almost 90% of referred participants underwent a colposcopy examination. A strong association (Mann Whitney 0.3388) was found between HPV16 infection and ASC-US (>PapII), LISL, and CIN + in 1983/84 cohort, but only for ASC-US (>PapII) in 1988/89 cohort. This age-related pattern is probably explained by a high persistency rate and increased risk of oncogenic transformation over time, consistent with observations reported by Kjaer and colleagues showing that the increased risk of CIN3 or cancer associated with HR-HPV infection, especially HPV16, is a continuous trend over several decades . Along wAn association between HPV52 and LSIL or more was observed in the 1988/89 cohort, but not in 1983/84 cohort. LSIL can result from harmless transient HPV infections and the difference between cohorts is most likely to be a chance finding. An imbalance in conization history between the 1983/84 and 1988/89 cohorts can be explained by the conservative policy adopted in the colposcopy clinic in Wolfsburg, which has been the referral center for most cases of abnormal smears in the city for more than 8 years. The corresponding figures in other German regions may be significantly higher.Correlative data for HPV infection and associated pathological abnormalities will help to further refine the clinical management of young women at risk for malignancies associated with HR-HPV infection, especially HPV16. The data from the WOLVES study support recommendations for a later start of cervical screening, at the age of 25 years or later, no routine HPV testing in women younger than 25 years and conservative management of individuals presenting with abnormal Pap smears. In addition, preliminary data from the WOLVES study showed that HPV vaccination significantly reduces the rate of HPV16 infection, corroborating recent data from a national study in Australia .A limitation of this first analysis of the WOLVES study is that it provides only prevalence data and as yet does not show the dynamics of HPV infection and changes in pathology findings over time. Nevertheless, the WOLVES data are in accordance with published data from similar European trials of HPV types commonly associated with atypical Pap smears/cervical neoplasia ,29,37,38Only a high number of sexual partners (>5) was an independent risk factor for HPV infection. The effect of cofactors for the risk of CIN3 and cancer, such as smoking and use of contraceptives, may not yet be apparent because these factors probably trigger HPV persistency rather than acquisition. There was a high prevalence of HR-HPV infection in women aged 20 and 25 years in 2009, which was associated with an increased risk of abnormal Pap smears and biopsy proven CIN2+. Women with HPV16 infection have a high risk of clinically relevant lesions that seems to increase over time (as the comparison of the two age cohorts shows) and there was a significant impact of HPV vaccination on HPV16 infections, although the majority of these women must have been sexually active already at the time of vaccination and, therefore, the efficiency must be considered to be suboptimal.ASC-US: Atypical squamous cells of uncertain significance; CIN: Cervical intraepithelial neoplasia; HC2: Hybrid capture 2; HR-HPV: High-risk human papilloma virus; LR-HPV: Low-risk human papilloma virus; LSIL: Low-grade squamous intraepithelial lesion; Pap: Papanicolaou test; PCR: Polymerase chain reaction; WOLVES: Wolfsburg HPV epidemiological studyWOLVES is financed mainly by an unrestricted grant from Sanofi Pasteur MSD and supported by free samplers distributed by Qiagen and Hologic. KUP received speaker´s honorarium from Beckton Dickinson, Qiagen and Roche Diagnostics. RS-R is employee of Sanofi Pasteur MSD. TI has received institutional grants from GSK, Gen-Probe, Sanofi-Pasteur MSD and Hologic. The remaining authors declare they have no competing interests.KUP was responsible for the overall study design, data analysis, interpretation, writing of the manuscript and data collection. AL was responsible for data collection and interpretation. AJ was responsible for statistical analyses and interpretation. AI was responsible for HC2-testing, HPV genotyping and sequencing, data collection and interpretation. SS was responsible for trial coordination, quality control and data collection. AR-L was responsible for histology. RSR was responsible for trial coordination and interpretation. EG was responsible for recruitment, data collection and interpretation. TI was responsible for the study design, HPV testing, data collection and interpretation. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/13/135/prepub


Indigofera suffruticosa leaves obtained by infusion was used to evaluate the oviposition, its effect on development of eggs and larvae, and morphological changes in larvae of Aedes aegypti. The bioassays were carried out with aqueous extract in different concentrations on eggs, larvae, and female mosquitoes, and the morphological changes were observed in midgut of larvae. The extract showed repellent activity on A. aegypti mosquitoes, reducing significantly the egg laying by females with control substrate (343 (185–406)) compared with the treated substrate (88 (13–210)). No eclosion of A. aegypti eggs at different concentrations studied was observed. The controleclodedin 35%. At concentration of 250 μg/mL, 93.3% of larvae remained in the second instar of development and at concentrations of 500, 750, and 1000 μg/mL the inhibitory effect was lower with percentages of 20%, 53.3%, and 46.6%, respectively. Morphological changes like disruption on the peritrophic envelope (PE), discontinued underlying epithelium, increased gut lumen, and segments with hypertrophic aspects were observed in anterior region of medium midgut of larvae of A. aegypti. The results showed repellent activity, specific embryotoxicity, and general growth retardation in A. aegypti by medium containing aqueous extract of I. suffruticosa leaves.Aqueous extract of  Aedes aegypti Linnaeus is a vector and promotes the spreading of four serotypes of dengue virus. However, a decrease in the effective vector control has been described due to larval tolerance to chemical insecticides [A. aegypti continues to pose serious public health problems [The mosquito cticides . The inccticides . Despiteproblems . A denguproblems . A. aegypti larvae treated with an aqueous extract of Derris urucu showed histological alterations in the midgut, and larval mortality was associated with peritrophic matrix damage [A. aegypti, and larvicidal bioassays have been conducted using third (L3) and fourth (L4) instars or comparing the effect of plant extracts on larval development of L1–L4 [It has been demonstrated that insect gut is the target of many insecticidal compounds. Transmission electron microscopy of x damage . The perx damage . Plants of L1–L4 . Variousof L1–L4 .Indigofera suffruticosa Mill (Fabaceae) is a plant found in tropical and subtropical areas and well adapted to growth in semiarid regions and soil of low fertility [ertility . This plertility , 10. In ertility .I. suffruticosa) in Natural Products Alert (NAPRALERT) [A chemical investigation of this species (PRALERT)  and ChemI. suffruticosa [Recently, antitumoral and antimicrobial activities and mice embryotoxic effects have been tested with extract of leaves of ruticosa , 12–14.A. aegypti, and morphological changes in larvae treated with aqueous extract from leaves of I. suffruticosa. In the present study, we have investigated the process of oviposition, early development on eggs and larvae of I. suffruticosa were collected in October 2005 in Igarassu, State of Pernambuco, Brazil, and authenticated by the Biologist Marlene Barbosa from the Botanic Department, Universidade Federal de Pernambuco (UFPE). A voucher specimen number 32859 has been deposited at the Herbarium of the above-cited department.The leaves of A. aegypti were originally obtained from Centro de Vigilância Ambiental da Prefeitura Municipal do Recife, Pernambuco, Brazil, and female mosquitoes from the ecology laboratory of Chemistry Department of Universidade Federal de Pernambuco/UFPE. Adult mosquitoes (F0 generation) were fed with 10% glucose and with chicken blood and were reared in a room maintained at 27°C in humidified cages. Eggs of these mosquitoes were counted using a stereoscopic microscope. The larvae generated were fed with commercial cat food. Eggs and the 1st instars larvae were used in the experiments.Eggs and larvae of μL of distilled water in microcentrifuge tubes, then diluting in water to the appropriate concentration 250, 500, 750, and 1000 μg/mL to evaluate the embryotoxicity on eggs and larvae.Leaves (75 g) were weighed and chopped. The plant material was successively extracted in infusion with solvents of increasing polarity . The solvents were removed by rotary evaporation. The percentage yields were hexane (0.67%), ethyl acetate (0.39%) methanol (3.9%), and (w/w) in terms of newly collected plant material. After the extraction processes with the aforementioned solvents, the same plant material was extracted with distilled water, resulting in the aqueous extract. To the egg-laying evaluation, 25 mL of aqueous extract was used with female mosquitoes. The other part of extract was lyophilized, and the dried powder plant material (4.2%) was stored at 20°C. This dry residue aqueous extract was homogenized using 100 A. aegypti were stored in polypropylene cages (30 × 30 × 30 cm)  with sacarose solution 10% at 25°C. Females were exposed to 18 substrates (paper filter) with distilled water (9 substrates) and 25 mL at 30% of aqueous extract of I. suffruticosa (9 substrates). The quantification of the eggs was assessed by observation under a stereomicroscope (1.2x). The oviposition bioassay was assayed as recommended by the World Health Organization [During 4 consecutive days, 90 female mosquitoes of nization .Aedes aegypti L., whose common name is dengue mosquito, belongs to the Arthropoda Phylum, Hexapoda Class, Diptera Order, and Culicidae Family. The effect of aqueous extract of I. suffruticosa leaves on egg outbreak and larval development of A. aegypti was assayed as recommended by the World Health Organization [A. aegypti were exposed to the extract in concentrations of 250, 500, 750, and 1000 μg/mL. Preliminary bioassay was performed using 40 eggs that were hatched in mineral water (200 mL) at 26°C–28°C. The test using larvae  were carried out in duplicate for each concentration. Larvae were placed into 200 mL disposable plastic cups containing 25 mL of the test solution and incubated at 27°C. The developmental stages of larvae was determined at the start of the experiment (0 h) and 24, 48, and 72 h thereafter, and developmental stages were assessed by observation under a stereomicroscope (1.2x).nization . Eggs anA. aegypti) larvae from control and treated groups were fixed with formaldehyde (2.5%) for morphologic evaluation and were photographed using a digital video camera (Leica) connected to an inverted microscope (magnification of 200x.).Mosquito (P < 0.001) using the SigmaStat (3.5 version) between the control and tested groups. The oviposition results were expressed in media (min-max).We used Mann-Whitney  the eggs were quantified (3.634 eggs) after 4 days using 30% of aqueous extract of I. suffruticosa. The substrate containing aqueous extract reduced significantly the posture of eggs (88 (13–210)), compared with the control treated with distilled water (343 (185–406)) ) .A. aegypti eggs in the different concentrations studied was observed. The same number of eggs (n = 40) was used as a control that ecloded in 35% (No eclosion of d in 35% .Aedes aegypti was observed from 0 to 72 h using concentrations from 250 to 1000 μg/mL of aqueous extract of I. suffruticosa leaves. I. suffruticosa at different concentrations.The embryonic development of larvae of first instar (L1) of μg/mL of extract stopped at second instar (L2) similarly to other concentrations , in which the inhibitory effect was lower with percentages of 20%, 53.3%, and 46.6%, respectively.Approximately 93.3% of live larvae treated with 250 I. suffruticosa ( Aedes aegypti in early development treated with aqueous extract of I. suffruticosa showed disruption on the peritrophic envelope (PE) structure consequently resulting in a discontinued underlying epithelium, increased gut lumen, and segments with hypertrophic aspects in comparison with control larvae. The developmental delay is directly dependent of morphological changes that occur when the larvae are growing in contact with different substances of the extract.Control Live L2 on distilled water  and trearuticosa  after 72Indigofera suffruticosa on oviposition and embryonic development of Aedes aegypti. The purpose of this study was to determine the repellent and toxic effects of A. aegypti by medium containing aqueous extract of I. suffruticosa leaves. The results showed significant repellent effect on egg posture and specific embryotoxicity and general growth retardation on Indigofera species were not found in literature, but many plants from the family Lamiaceae are toxic for insects including Ocimum basilicum, O. gratissimum, O. americanum, Cymbopogom nardus, Alpinia galanga, Syzyaium aromaticum e Thymus vulgaris, Mentha, Eucalyptus maculata citriodon, and Tagetus e Lantana camara, and they have been studied as natural alternative repellents [Studies reporting repellent effect with pellents .A. aegypti eggs did not outbreak and larvae in early development showed an increase of abnormalities, mainly in the peritrophic envelops at different concentrations. At 250 μg/mL concentration the extract could affect one of the phases of the life cycle of A. aegypti. Higher incidences of specific embryotoxicity were found at concentrations that also caused general growth retardation [in vivo: amaranth [in vitro, whereas penicillin [I. suffruticosa. Aqueous extract of I. suffruticosa leaves was studied for adverse effects in preimplantation mouse embryos. Two-cell mouse embryos were cultured for 94 h in human tubal fluid medium (HTF), and the extract at a concentration of 5 mg/mL showed a development from morula to blastocyst stages similar to the controls, and at a higher concentration (10 mg/mL), all embryos persisted at the two-cell stage [ardation . The in ardation . Four coamaranth  and isonamaranth  had onlynicillin  and saccnicillin  did not ll stage .In vertebrates, mucus is the primary secreted layer, lining and protecting the intestinal epithelium, while assisting the digestion process . HoweverA. aegypti. Furthermore, the inhibition of PE formation severely affected the early development of larvae. In controlling second instar larvae of A. aegypti, the anterior region of medium midgut was recovered by a continued PE. However, morphological observation of larvae submitted to aqueous extract of I. suffruticosa leaves showed disruption on the PE structure. Clearly, we are far away from completely elucidating the mechanisms of I. suffruticosa to induce growth retardation in animal models. However, studies from our group also demonstrated that this plant is an extremely powerful inducer of cancer cell death and possibly the bioactive compound from I. suffruticosa could act binding many molecular targets inside the cell activating alternative apoptotic pathways or inducing mitotic catastrophe which indicates a form of cell death that is caused by aberrant mitosis by caspase 3 activation and oligonucleosomal DNA degradation [I. suffruticosa can induce cell death via different molecular pathways and with different executing mechanisms, that is classical apoptosis, but also mitotic catastrophe. These activities and the main recognized molecular targets of I. suffruticosa are depicted in I. suffruticosa can impinge upon different conditions  from


Hypertension and dyslipidemia are often insufficiently controlled in persons with type 2 diabetes (T2D) in Germany. In the current study we evaluated individual characteristics that are assumed to influence the adequate treatment and control of hypertension and dyslipidemia and aimed to identify the patient group with the most urgent need for improved health care.The analysis was based on the DIAB-CORE project in which cross-sectional data from five regional population-based studies and one nationwide German study, conducted between 1997 and 2006, were pooled. We compared the frequencies of socio-economic and lifestyle factors along with comorbidities in hypertensive participants with or without the blood pressure target of < 140/90 mmHg. Similar studies were also performed in participants with dyslipidemia with and without the target of total cholesterol/HDL cholesterol ratio < 5. Furthermore, we compared participants who received antihypertensive/lipid lowering treatment with those who were untreated. Univariable and multivariable logistic regression models were used to assess the odds of potentially influential factors.We included 1287 participants with T2D of whom n = 1048 had hypertension and n = 636 had dyslipidemia. Uncontrolled blood pressure was associated with male sex, low body mass index (BMI), no history of myocardial infarction (MI) and study site. Uncontrolled blood lipid levels were associated with male sex, no history of MI and study site. The odds of receiving no pharmacotherapy for hypertension were significantly greater in men, younger participants, those with BMI < 30 kg/m2 and those without previous MI or stroke. Participants with dyslipidemia received lipid lowering medication less frequently if they were male and had not previously had an MI. The more recent studies HNR and CARLA had the greatest numbers of well controlled and treated participants.In the DIAB-CORE study, the patient group with the greatest odds of uncontrolled co-morbidities and no pharmacotherapy was more likely comprised of younger men with low BMI and no history of cardiovascular disease. Hypertension and dyslipidemia constitute major public health problems as they increase the risk of cardiovascular diseases (CVDs), especially in patients with concomitant type 2 diabetes (T2D)-4.Nevertheless, several epidemiological studies indicate disappointing deficiencies in the detection and adequate treatment of hypertension and dyslipidemia in Germany with minimal improvement over the last decades-13. AbouTo our knowledge, no population-based study in Germany has so far dealt with characteristics of patients with T2D and hypertension and/or dyslipidemia who are not sufficiently treated for these serious conditions. In our study, we describe demographic, socio-economic and lifestyle factors in DIAB-CORE participants with diabetes and concomitant hypertension or dyslipidemia and analyse the factors associated with insufficient disease control and absent pharmacotherapy. We therefore aim to identify the group of patients with the most urgent need for intensified care.The DIAB-CORE Consortium consists of the following population-based studies (from north to south): the Study of Health in Pomerania , the Dortmund Health Study , the Cardiovascular Disease, Living and Ageing ) Study, the Heinz Nixdorf-Recall  Study, the Cooperative Health Research in the Region of Augsburg  Study, and the nationwide German National Health Interview and Examination Survey 1998 (GNHIES98), see Table All studies were conducted between 1997 and 2006 and used similar instruments, questionnaires and medical measurements to assess data. Detailed descriptions of study designs, samples and procedures are available elsewhere-20. EthiParticipants were classified in five age groups using five year intervals.T2D was defined based on self-report or self-reported intake of oral anti-diabetic agents, insulin or a combination of both. Some studies lacked information on diabetes type. Thus, in order to exclude participants who probably had Type 1 diabetes, self-reported age at diagnosis of diabetes was used, and only those patients with an age at diagnosis of > 30 years were included in the T2D group.Hypertension was defined using the mean of the second and third blood pressure measurements (the first and second measurements in DHS) conducted at the study centres with systolic blood pressure ≥140 and/or diastolic blood pressure ≥90 mmHg, or intake of anti-hypertensive medication in participants with physician’s diagnosis of hypertension (“awareness”). Participants with hypertension were categorized into one of the following four subgroups: (1) aware (with physician’ diagnosis) and controlled treated to target levels of < 140/90 mmHg, (2) aware and treated, but not reaching target blood pressure values of < 140/90 mmHg, i.e. uncontrolled treated, (3) aware, but not treated, (4) unaware of hypertension. Thus, “awareness” of hypertension applied to participants in categories 1, 2 and 3, “treatment” applied to those in categories 1 and 2 and “control” to those in category 1.Total cholesterol, high-density lipoprotein (HDL) cholesterol and low-density lipoprotein (LDL) cholesterol levels were measured from random blood samples. Dyslipidemia was defined analogously to hypertension using information on lipid-lowering medication intake, self-reported information on physician's diagnosis and a total cholesterol to HDL cholesterol ratio (TC/HDL) of > = 5,22.Self-reported data on myocardial infarction and stroke  was assessed identically in all studies.All study participants were asked to bring original packaging of their medications used during the last seven days to the examination. The variable “anti-hypertensive medication” included any prescription of medication belonging to the ATC subgroups C02 (antihypertensives), C03 (diuretics), C04 , C07 (beta blocking agents), C08  and C09 (agents reacting on the renin-angiotensin system).Medications of the ATC subgroup C10 (lipid modifying agents) were included in the variable “lipid-lowering medication”.The BMI (kg/m2) was calculated using standardized weight and height measurements.Two categories (current vs. ex- and never smoker) were defined to differentiate risk types. A current smoker reported smoking at least one cigarette per day. Persons who had reported that they had smoked at least one cigarette per day in the past but who quit smoking at least one year ago were defined as ex-smokers. Never-smokers were defined as those persons who had never smoked or smoked only occasionally (< 1 cigarette day).In all studies, physical activity was assessed by self-report only. A threshold of less than 1 h of physical activity per week was determined for a high risk lifestyle. Assessment of activity was restricted to all kinds of exercise training but did not comprise low level exercise such as stepping stairs or walking, as this type of exercise was not assessed in all studies.In all studies, the participants were asked for their highest level of school qualification obtained. We classified educational level as a dichotomous variable contrasting low with medium or high level. According to the German school system, low educational level includes participants with up to 9 years of schooling. Medium educational level is equivalent to 10 years of schooling and high educational level to 12 or 13 years of schooling, which is required to enter a university.Information on monthly net household income as well as on household size was obtained from interviews. As the ages of household members were not available consistently across all studies, it was not possible to calculate the equivalent income according to the OECD equivalence scale. That is why the equivalent income was calculated according to the Luxembourg Income Study (income/household size). PoolingThe pooled data set included 1,287 participants with T2D aged 45 to 74 years. In the nationwide survey GNHIES98, 3% non-German citizens were included, KORA, SHIP and CARLA focused on participants of German nationality, and the other studies collected information on birthplace only. Two-stage cluster sampling or stratified random sampling were used. Overall response ranged between 56 and 69%.The hypertension sub-analysis included 1,048 hypertensive participants. N = 17 participants were excluded due to missing information on: study blood pressure measurement (n = 3), physician’s diagnosis of hypertension (n = 3), and/or medication intake (n = 14).The dyslipidemia sub-analysis included 636 participants with dyslipidemia according to the definition specified above. N = 126 participants had to be excluded due to missing information on laboratory measurements (n = 32), physician’s diagnosis of dyslipidemia , and/or medication intake (n = 14). N = 16 participants were not included in either one of the two sub-analyses.T2D participants with hypertension, with or without the blood pressure target of < 140/90 mmHg, and T2D participants with dyslipidemia with or without the target of total cholesterol/HDL cholesterol < 5, as well as T2D participants with or without treatment were compared with respect to their lifestyle factors, cardiovascular burden, clinical measurements and medications. Continuous variables were characterized by means and standard deviations (SD), categorical variables were described as percentages. Differences between groups were calculated using t-tests and Wilcoxon tests (continuous variables) or chi-square tests and univariable logistic regression models . At first, univariable logistic regression models were programmed to identify factors associated with blood pressure, lipid ratio or medication intake. In a second step, variables found to be significantly associated with the respective outcome were included in multivariable models to examine adjusted effects.The goodness-of-fit of adjusted models was assessed by the c-value, and the Hosmer-Lemeshow test. The c-value describes the area under the ROC-curve, ranging from 0.5 (random correlations) to 1.0 (perfect fit). In epidemiologic studies, values between 0.6 and 0.8 are usually regarded as satisfactory. The Hosmer-Lemeshow test compares observed and predicted values with each other. If there is no significant difference (at the significance level of 0.05) the model is characterized as being appropriate.A two-sided alpha level of 0.05 was chosen as criterion for statistical significance. All analyses were carried out using SAS, version 9.2 .Of 1,287 T2D participants aged 45-74 years, n = 1,048 (82.5%) had hypertension and n = 636 (54.8%) had dyslipidemia. N = 530 (41.2%) had both conditions and were included in both sub-analyses.The frequency of hypertension in persons with T2D differed slightly between studies, ranging from 78.5% in HNR to 89.6% in CARLA. Likewise, the number of participants with dyslipidemia ranged from 48.6% in CARLA to 59.6% in GNHIES98. The mean age was 63.1 years (Std. 7.2 years), and was very similar across all studies  had controlled blood pressure of < 140/90 mmHg, and n = 808 (77.1%) were uncontrolled. Dyslipidemia was treated to TC/HDL ratio < 5 in 143 (22.5%) of 636 T2D participants and uncontrolled dyslipidemia was observed in 493 (77.5%) T2D participants  increased with age in participants with T2D, while diastolic blood pressure (DBP) decreased. Both measurements were consistently lower in women of all age groups than in their male counterparts (mean SBP in women: 149.8 mmHg (SD 20.8) vs. 154.4 mmHg (20.6) in men, mean DBP 83.2 (11.1) and 86.6 (11.2) in women and men, respectively)  vs. 216.4 mg/dl (48.0) and 53.9 (16.6) vs. 46.3 (14.0)) – albeit, 48.7% of women and 35.1% of men had less than 50 or less than 40 mg/dl HDL respectively. However, the TC/HDL ratio was lower in women (4.7 (1.7) vs. 5.0 (1.7)).More women than men were not affected with dyslipidemia as defined by a TC/HDL ratio < 5 (47.5% vs. 43.4%), 14.9% were treated and well-controlled (vs. 10.2% in men), 7.9% were treated but did not reach goal values (compared to 6.7% in men), 16.3% were not treated despite of a physician’s diagnosis (compared to 17.8% in men) and 13.4% had unknown dyslipidemia (compared to 21.9% in men) , BMI < 30 , and study. Age in five groups was not significantly associated but displayed a steady tendency towards having the greatest odds in the second oldest group (65-69 years) compared to the youngest group (45-54 years). With GNHIES98 as the reference study, HNR had the smallest odds of uncontrolled hypertension , followed by CARLA  and KORA . Multivariable analysis with these significant variables  showed consistent results as well as the associated factor ‘no previous MI’ which reached statistical significance . A model including only sex, BMI and study had a c-value of 0.68 and a non-significant Hosmer-Lemeshow test (p = 0.78).Uncontrolled dyslipidemia was significantly associated with sex , no previous MI  and study . Again, age was not significantly associated, but showed some tendency, albeit in the other direction, with the youngest age group displaying the greatest odds of uncontrolled dyslipidemia (70-74 years as reference). Multivariable analysis with sex, MI, and study generated consistent odds. A model including only these variables had a c-value of 0.69 and a non-significant Hosmer-Lemeshow test result (p = 0.96). Smoking, physical activity, educational level and income were not significantly associated with either control of hypertension or dyslipidemia , males , current smokers  and participants with BMI < 30 . Moreover, no previous MI , no previous stroke  and study  were significantly associated with untreated hypertension. In multivariable models adjusted for age, sex, BMI, MI, stroke, and study, the odds remained significant, except for smoking. A model including only these significant variables was characterized by a c-value of 0.71 and a non-significant Hosmer-Lemeshow test result (p = 0.46).Non-use of lipid lowering medication in participants with dyslipidemia was univariably associated with male sex  and no MI . Multivariable analysis adjusted for these two variables yielded additionally significant odds for no concomitant hypertension , physical inactivity  and the CARLA study . A model including only sex and MI had a c-value of 0.64 and a non-significant Hosmer-Lemeshow test (p = 0.62). Neither education nor income were associated with treatment of hypertension or dyslipidemia (Table Uncontrolled blood pressure in T2D participants with (diagnosed or unrecognized) hypertension was associated with male sex, BMI < 30 kg/m2, no previous MI, and study site. Similarly, uncontrolled blood lipid levels in T2D participants with dyslipidemia (diagnosed or unrecognized) were more frequent in men, those who had not suffered MI and attendees of the older DIAB-CORE studies.Lack of treatment for hypertension was related to younger age, male sex, smoking, BMI < 30, no history of MI or stroke, and study site. Male T2D participants without concomitant hypertension and who had not suffered MI had significantly greater odds of untreated dyslipidemia than other participants in DIAB-CORE.Socioeconomic features such as educational level and income were not significantly associated with either disease control or pharmacotherapy; lifestyle factors were only associated in some models.On the one hand, the results of our analyses indicate that the vast majority of patients with T2D in Germany are not adequately treated for hypertension and/or dyslipidemia, even though these conditions have been found to considerably increase the risk of co-morbidities and complications such as MI, stroke, nephropathy and retinopathy.On the other hand, we confirmed the presumption that patients with additional risk factors, such as advanced age, previous MI, previous stroke or obesity are treated more often for hypertension and dyslipidemia and (apart from those with older age) more frequently achieve adequate blood pressure and lipid target levels. The difference between female and male participants was pronounced and consistent over all sub-analyses.The essential strength of our study is the large population-based sample drawn from the general German population, aged 45 to 74 years, and the fact that both, laboratory measurements and information on medication intake were available.Differences in the frequencies of hypertension and dyslipidemia between studies were probably due to the relatively small numbers of affected participants within the individual studies and analyses. Due to the pooling process, only similarly collected and coded data of all six studies could be used and the least common denominator had to be found. Therefore, the definition of diabetes was based on self-report of physician’s diagnosis and treatment with anti-diabetic agents rather than on clinical diagnosis and medical records. Blood pressure was calculated using the mean of the second and third measurements in all studies except for DHS, where only two measurements were performed and used to calculate the mean.Moreover, measurements of blood pressure and lipids based on a single testing opportunity provide evidence for the respective condition, but are not equal to a clinical diagnosis with repeated measurements. We cannot exclude cases of ‘white coat hypertension’, i.e. elevated blood pressure caused by the excitement of the unfamiliar situation.Due to compliance issues, women might have reported their medication intake more reliably than men, thus pharmacotherapy in men could have been underestimated and consequently, sex-specific differences overestimated in these analyses.Patient-centered studies in Germany and other countries have so far examined awareness, treatment, control and factors associated with insufficient control of co-morbidities in patients with and without diabetes. A systematic review by McLean et al. from 2008, which included 26 studies from different countries with 66,833 diabetes patients with co-morbid hypertension, concluded that 83% (range 32-100%) of patients were treated, yet only about 29% (range 5-59%) had their blood pressure controlled to < 140/90 mmHg. The proportions of treatment to control were similar and thus equally insufficient between studies and countries. Unfortunately, the authors did not report person-related factors associated with disease control.There are very few studies that have reported factors associated with cardiovascular disease control in patients with diabetes, as opposed to adults in general, irrespective of diabetes status. However, a number of recent studies that focused on gender differences-30 foundFindings on the influence of age on hypertension control are controversial e.g.,37). An . An 37])In our study obese participants (BMI ≥ 30 kg/m2) received medication more frequently and were more often well treated for hypertension, although they were more often affected with hypertension than leaner individuals (data not shown). A Swedish population-based study in hypertensive 60-year-old persons with and without diabetes by Carlsson et al. found an inverse, direct association of high waist circumference with uncontrolled hypertension. The samWe expected to find a significant negative association of high socio-economic status with uncontrolled co-morbidities, however no significant associations were observed with any socio-economic characteristics and the outcome variables. This might, in the case of school education, be due to the unequal frequencies of low (about 80%) vs. high and middle status, which owns to the high mean age of the study population (63 years) and the fact that older people mostly attended junior high school only. In the previously mentioned Swedish study, lack of health care due to low income was independently associated with uncontrolled hypertension in men  but not in women. In contrast, living in an apartment instead of a house (as an indicator of lower socio-economic status) remained a significantly protective factor in an adjusted model in women . The authors stated that the finding was puzzling and offered no explanation.Carlsson et al. confirmed that previous cardiovascular disease or coronary heart disease has a protective effect on uncontrolled hypertension, probably because of more intense treatment, better medication adherence and/or symptom relief of angina pectoris.There are a number of possible influences on uncontrolled hypertension and dyslipidemia that we could not consider. For example, the Swedish study by Carlsson et al. included data on nutrition and found that daily intake of fruit had an independent, protective effect in men but not in women.Steckelings, 2004 et al. used datAccording to a review by Düsing et al. 2006, more coFinally, the sex of the attending physician has been shown to play a role in the quality of risk factor control in patients with hypertension and dyslipidemia. In a cross-sectional study by Journath et al., diabetic men and women achieved goals for blood pressure control, and men achieved goals for cholesterol control more often if they were treated by female physicians. LikewisInterestingly, there appears to be an intriguing difference in hypertension prevalence and control, irrespective of diabetes status, between Europe and the United States/Canada that has not been appreciated and examined sufficiently. Wolf-Maier et al.,49 compaIn order to improve awareness, creative new approaches have been successfully implemented in the United States, e.g. by addressing the issue and counseling black men during a visit at a barbershop. Such inIn the DIAB-CORE study, participants with diabetes who were at the greatest odds of uncontrolled co-morbidities and no pharmacotherapy were male, younger, had lower BMI and no history of cardiovascular disease. Although the general risk profile appears relatively low, preventative efforts should not overlook, but specifically consider this group of patients.ATC: Anatomical Therapeutic Chemical Classification System; BMI: Body mass index; BP: Blood pressure; CARLA: Cardiovascular Disease Living and Ageing Study; CVD: Cardiovascular disease; DHS: Dortmund Health Study; DMP: Disease management program; GNHIES98: German National Health Interview and Examination Survey; HDL: High-density lipoprotein; KORA: Cooperative Health Research in the Region of Augsburg Study; LDL: Low-density lipoprotein; OAD: Oral anti-diabetic medication; HNR: Heinz Nixdorf-Recall Risk Factors Evaluation of Coronary Calcification and Lifestyle Study; SD: Standard deviation; SHIP: Study of Health in Pomerania; T2D: Type 2 diabetes; TC: Total cholesterol.The authors declare that they have no competing interests.I-MR performed the statistical analyses and wrote the manuscript. CM and WM worked on pooling of the data, contributed to the discussion and reviewed/edited the manuscript. AM contributed to the discussion and reviewed/edited the manuscript. TT and WR worked on pooling of the data and reviewed/edited the manuscript. SS, HV, AK, K-HG, KB, GM, UE, HN, SM and SA contributed data and reviewed/edited the manuscript. All authors read and approved the final manuscript.


Introduction. X-linked agammaglobulinemia (XLA) is a primary immunodeficiency characterized by agammaglobulinemia requiring replacement treatment with immunoglobulin. The association of XLA and membranoproliferative glomerulonephritis (MPGN) is unexpected and, to our knowledge, only one case was previously published. Case Report. The authors report the case of a 10-year-old boy with family history and prenatal diagnosis of XLA, treated from birth with intravenous immunoglobulin replacement therapy. He presented with pneumonia, macroscopic hematuria, nephrotic proteinuria, hypoalbuminemia, and hypercholesterolemia with normal renal function and serum complement levels. Renal histology showed immune complex mediated MPGN. He was started on high dose prednisolone and ramipril and switched to weekly subcutaneous immunoglobulin. After a 4-month treatment, hematuria and proteinuria significantly improved and prednisolone was gradually tapered without relapse. Conclusion. The pathogenic process underlying MPGN development in this patient is unknown but residual humoral immunity might play an important role. Thus, this case highlights the risk of autoimmune disorders among patients with XLA. Membranoproliferative glomerulonephritis (MPGN) is an uncommon cause of chronic nephritis that occurs primarily in children and young adults. In most cases, complement activation is prominent and immune complex formation and deposition in the glomeruli play an important role –3. On thThe authors report the case of a patient with XLA who developed MPGN during treatment with IVIG. The association of XLA and MPGN is unusual and, to our knowledge, only one case was previously reported , althougμmol/L (reference range 46.9–69.8) and estimated glomerular function rate of 2.62 mL/s/m2. Urinalysis revealed proteinuria (urinary protein over creatinine ratio of 632.8 mg/mmol) with the following urinary sediment: erythrocytes 50 per high power field (HPF), leucocytes 5–10/HPF, and red blood cells and hialines casts 1-2/HPF. Normal serum complement levels were observed, with C3 level of 1.51 g/L (reference range 0.81–1.67) and C4 of 0.32 g/L (reference range 0.11–0.41). The etiological diagnosis of pneumonia could not be established.The patient in this report has a family history of XLA with two affected siblings, with their mother being a carrier of the mutation. He was screened prenatally and the genetic analysis revealed a BTK mutation (R288Q). A masculine gender newborn was born after a full-term pregnancy with a normal delivery, weighing 3250 grams, with an otherwise unremarkable neonatal history. He was started on IVIG shortly after birth. He showed low but detectable levels of serum IgG , IgA , and IgM  with CD19+ 1.4% and CD 20+ 1.6% at the age of 8 months. Although the patient had a poor compliance with medical follow-up, including IVIG treatment, there was no record of major infections during childhood. At the age of 10 years, he was admitted with right lower lobe pneumonia and macroscopic hematuria. Physical examination revealed normal blood pressure and no peripheral edema. Initial laboratory evaluation showed hypoalbuminemia , hypercholesterolemia  and normal renal function with serum creatinine of 32.71 Further investigation disclosed normal autoantibodies serum levels and excluded HIVs 1 and 2, HBV, and HCV infection, with negative viral load and normal liver enzymes. A renal biopsy was performed and histology presented glomeruli with membranoproliferative patterns, without significant interstitial inflammation or tubular atrophy . ImmunofIVIG was switched to SCIG replacement therapy in a weekly regimen and prednisolone was also started at an initial dose of 2 mg/kg/day, which was gradually tapered. Ramipril was also started at a dose of 2,5 mg/day. After 18 months the patient maintained a normal blood pressure and normal renal function. The urinary sediment is near normal  and no proteinuria was detected . There were no major infections and he maintained an adequate growth (weight and stature z-scores of 0.81 and 0.31 at age of 12 years).The classic XLA phenotype is characterized by a reduced peripheral B-lymphocyte count and near complete absence of endogenous immunoglobulin. More than 500 different BTK mutations have been reported in patients with XLA but a consistent correlation between genotype and phenotype remains to be established . Given tImmune complex-mediated MPGN is most commonly secondary to viral infections (hepatites C and B) and less frequently to a chronic bacterial disease . Although the patient had a poor compliance with immunoglobulin treatment, no important infections were noted until the age of 10 years. Autoimmune diseases like systemic lupus erythematosus (particularly in the chronic phase of lupus nephritis) and, less often, Sjögren's syndrome or rheumatoid arthritis are also associated with MPGN. The patient had neither clinical manifestations nor autoantibodies compatible with these multisystemic disorders. Monoclonal gammopathy is another cause of secondary MPGN but is an extremely rare diagnosis in childhood and no laboratory findings support it. So, like reported in another XLA patient with MPGN, the most frequent secondary causes of MPGN were excluded, but a viral etiology is still to be suspected . The pre