Eimeria (E.) tenella-challenged condition. Two thousand male Partridge Shank broiler chicks were obtained from a hatchery. After hatch, birds were weighed, color-marked and allocated equally into two anticoccidial treatments, namely medicated and vaccinated groups. Chicks were either fed, from 1 d of age, diets containing coccidiostat (narasin) or diets without the coccidiostat but were inoculated with an anticoccidial vaccine at 3 d of age. At 22 d of age, 1080 chicks among them were randomly allocated evenly into 6 groups under a 2 × 3 treatment with 2 anticoccidial programs and 3 dietary methionine (Met) levels. Chicks medicated or vaccinated against coccidia were fed diets containing 0.45%, 0.56% or 0.68% of Met from 22 to 42 d of age. All chicks were orally introduced with an amount of 5 × 104 sporulated oocysts of E. tenella at 24 d of age. The growth performance, serum anti-oxidative indexes, intestinal morphology, cecal lesion scores, fecal oocyst counts and immune parameters were measured.Coccidiosis is a prevalent problem in chicken production. Dietary addition of coccidiostats and vaccination are two approaches used to suppress coccidia in the practical production. Methionine (Met) is usually the first limiting amino acid that plays important roles in protein metabolism and immune functions in chickens. The present study is aimed to investigate whether increasing dietary Met levels will improve the anticoccidial effects in broilers medicated or vaccinated against coccidia under E. tenella infection, as indicated by improved intestinal morphology and immune functions as well as decreased cecal lesion and fecal oocyst counts.The results showed increasing dietary Met level from 0.45% to 0.56% and 0.68% improved weight gain and feed conversion of broilers medicated against coccidia. In contrast, higher dietary levels of Met did not improve growth performance of the vaccinated chickens. Higher Met levels helped the medicated chickens resist E. tenella-challenged condition.Anticoccidial vaccination is a better strategy for controlling coccidiosis than feeding narasin, due to not only greater growth performance, but also the lower Met supplementation. Furthermore, higher dietary Met levels improved growth performance of chickens medicated rather than vaccinated against coccidia under  Methionine (Met) is the first limiting amino acid in broiler chickens fed maize-soybean diets . SupplemEimeria, is prevalent in the poultry industry and continues to cause the most severe health and welfare problems. Malabsorption and diarrhea are typical symptoms of coccidiosis. Chickens with coccidiosis thus have depressed growth rate, impaired feed conversion rate, and, in worst cases, increased mortality [Coccidiosis, caused by several species of ortality . Dietaryortality . Their pEimeria strains, which conversely decreases anticoccidial effectiveness of drugs [Eimeria strains influence the nutrient absorption of chicks remains unclear. Coccidial vaccination is generally associated with secondary enteritis, or even sporadically, necrotic enteritis incidence [However, both strategies have their shortcomings. The use of coccidiostats over the past 50 years has caused some degree of resistance of of drugs . But to ncidence . In addincidence . It has ncidence . The avancidence . Maroufyncidence  found thncidence  discoverncidence .Eimeria (E.) tenella-challenged condition.However, the effects of dietary Met levels on the resistance to coccidia in broilers under different anticoccidial programs remain unclear. Therefore, this study is aimed to investigate whether increasing dietary Met levels will improve the anticoccidial effects and growth performance in broilers medicated or vaccinated against coccidia under 4 sporulated oocysts of E. tenella at 24 d of age.Two thousand male Partridge Shank broiler chicks were obtained from a hatchery in which eggs were set and hatched in a common incubator. After hatch, birds were weighed, color-marked and allocated equally into two anticoccidial treatments, namely medicated and vaccinated groups. In the medicated group, chicks were fed diets always containing 60 mg/kg narasin  in the starter diet and the grower diet, whereas chicks in the vaccinated group were inoculated with an anticoccidial live vaccine  at 3 d of age without dietary addition of anticoccidial drugs. All chicks received a complete starter diet Table . These dAverage daily gain (ADG), average daily feed intake (ADFI) and feed to gain ratio (F/G) were measured through d 22 to d 42. Mortality data were collected and recorded on a daily basis, and the mortality was low in all treatment groups. Excreta was observed daily after oocyst challenge and oocyst counts were determined in excreta samples taken from each pen on d 6 and d 7 after challenge. Oocysts population was enumerated by microscopy according to the procedure of Long and Joyner . The fin+ and CD8+ lymphocyte populations (about 5 ml each), which were taken from wing vein, were stored in EDTA anticoagulation tubes. After blood collection, birds were killed by cervical dislocation. The ileum and cecum were separated and excised for histological examination. Intestinal contents from the cecum were dissolved into 0.9% NaCl solution  and then centrifuged at 10000×g for 1 min. The supernatant was used to detect the concentration of secreted immunoglobulin A (sIgA) in the lumen. The cecal tonsils were separated and stored in liquid nitrogen until Reverse transcription-PCR (RT-PCR) analysis.At 42 d of age, 30 birds per treatment were randomly selected (6 pens per treatment and 5 birds from each pen) after feed deprivation for 12 h. Blood samples (about 5 mL each) were taken from the wing vein and centrifuged at 3000×g for 15 min at 4 °C to separate serum, which was frozen at − 20 °C for further analysis. Blood samples for testing CD4The lesion scores of the cecum were determined and recorded according to the method of Johnson et al. . The les2SO4 and the absorbance was determined at 492 nm in Multiskan ELISA plate reader .sIgA levels in cecal lumina were examined using the commercially available ELISA kit  following the manufacture protocols. Briefly, a 96 well coated with one specific antibody at the bottom was incubated at the room temperature. After washing, suitably diluted samples were added to designated wells with subsequent addition of secondary antibodies and ortho-phenylenediamine. Following an incubation period, the reaction was stopped with H-∆∆CT method. β-tubulin expression was used as an internal control to calculate the relative expression levels of targeted genes . Reverse transcription of total RNA was completed using Prime Script RT Reagent Kit (TaKaRa). Primers for RT-PCR were presented in Table es Table . The mRN+ and CD8+ lymphocyte populations in peripheral blood. The fluorescein isothiocyanate (FITC)-CD4+ and phycoerythrin (PE)-CD8+ mouse anti-chicken antibodies  and blood samples were added to each tube and mixed by gentle shaking at 4 °C for 30 min. Erythrocytes were then lysed with 2 mL of Lysing Solution (Becton Dickinson (BD), Franklin Lakes, NJ, USA) at room temperature for 20 min. The lysate was centrifuged at 1500×g for 5 min and the supernatant was removed. The pellet was resuspended in phosphate-buffered saline (PBS). The lysate was centrifuged again at 1500×g for 5 min, the supernatant was removed, and the pellet was resuspended in 500 mL of PBS for assay performed on a flow cytometer .Flow cytometry was used to assay CD42O2 and a peroxidase (metmyoglobin) results in the production of the blue-green radical cation ABTS+. Antioxidants in the serum suppress this color production proportionally to their concentration. The system was standardized using Trolox, a water-soluble vitamin E analogue. The results were expressed as mmol Trolox equivalent. In malondialdehyde (MDA) assay, 2 mL 0.6% thiobarbituric acid was added to 2 mL of the serum in a 10 mL tube. This tube was next kept in boiling water for 15 min and placed on ice to cool down, before the optical density was measured at 532 nm. The results were expressed as nmol MDA/mL.GHS peroxidase (GPx) in the serum was tested using detection kits , according to the manufacturer’s instructions. Total antioxidant capacity (T-AOC) of the serum was measured using the azino-diethyl-benzthiazoline sulfate (ABTS) method. Incubation of ABTS with HP < 0.05. Results are reported as mean ± standard deviation.Statistical analyses were performed using the general linear procedure of the SAS software . A two  by three (dietary Met levels) factorial arrangement of treatments in a randomized complete block design was employed in the analysis of variance. All data were analyzed using anticoccidial programs, dietary Met levels, and their interactions as main effects. Differences among treatment means were classified by Duncan’s multiple range test and significant difference was defined at E. tenella-challenged conditions, anticoccidial programs and dietary Met levels had significant effects on final weight, ADG and ADFI . There were interactions between anticoccidial programs and dietary Met levels for final weight, ADG and F/G ratio (P < 0.01). With regard to the anticoccidial programs, the vaccinated chicks displayed better or identical performance than the medicated chicks except for the high diet Met level (0.68%). In regard to dietary Met levels, feeding 0.45% Met to medicated chicks and 0.68% Met to vaccinated chicks resulted in the lowest final weight, and medicated chickens showed increased ADG and decreased F/G ratio with increasing dietary Met concentrations from 0.45 through to 0.68% (P < 0.05). For medicated chickens, high supplemental levels of Met had no effects on ADFI (P > 0.05). In contrast, feeding 0.68% Met to vaccinated chickens decreased final weight, ADG and ADFI compared to 0.45% and 0.56% Met (P < 0.05). Increasing dietary levels of Met had no beneficial effects on F/G ratio in vaccinated chickens (P > 0.05).Under P < 0.05 or 0.01; Table P < 0.05). For all these indexes, anticoccidial programs and dietary Met levels had an interaction (P < 0.05 or 0.01). Medicated chickens showed increased VH and V/C after feeding 0.56% and 0.68% Met compared to 0.45% Met (P < 0.05). The CD was decreased after medicated chickens were fed 0.56% and 0.68% Met (P < 0.05). However, vaccinated chickens had decreased VH after increasing Met levels (P < 0.05). Feeding high levels of Met had no effect on CD of vaccinated chickens (P > 0.05). Feeding 0.68% Met decreased V/C in vaccinated chickens (P < 0.05).The VH, V/C, cecal lesion scores and excreted oocyst numbers were influenced by anticoccidial programs (P < 0.05). But, vaccinated chickens fed 0.68% Met exhibited increased cecal lesion scores, compared with those fed 0.45% and 0.56% Met (P < 0.05). The number of excreted coccidial oocysts was decreased when medicated chickens were fed 0.56% and 0.68% Met (P < 0.05). High supplemental levels of Met in the diet did not affect the number of excreted coccidial oocysts in vaccinated chicks.Vaccinated chickens showed lower cecal lesion scores and excreted coccidial oocysts than medicated chickens, irrespective of Met levels in diet Table . Medicat+ cells in the blood and the mRNA expression of IFN-γ in the cecal tonsil tissue (P > 0.05), but influenced CD8+ cell percentages, CD4+/CD8+, cecal luminal sIgA levels and the mRNA expression of TNF-α and IL-2 . The data showed that increasing dietary Met concentrations to 0.56% and 0.68% elevated the percentages of blood CD4+ cells in medicated chicks (P < 0.05). However, the increase in dietary Met concentrations decreased the CD4+ cell percentages in vaccinated chickens (P < 0.05). CD8+ cell percentages in medicated chickens were notably increased as well after feeding 0.56% and 0.68% Met (P < 0.05). In vaccinated chickens, CD8+ cell percentages were reduced after feeding 0.68% Met (P < 0.05). The CD4+/CD8+ was not influenced by Met concentrations in the diet in medicated chickens (P > 0.05), but decreased by high diet Met concentrations in vaccinated chickens (P < 0.05).Anticoccidial programs had no effects on the percentages of CD4P < 0.05), but had no effect on sIgA level in vaccinated chickens (P > 0.05). When Met concentrations in the diet for medicated chickens were increased to 0.56 and 0.68%, TNF-α and IL-2 mRNA expression levels were dramatically increased (P < 0.05). mRNA expression of TNF-α and IL-2 in vaccinated chickens was not affected by dietary Met concentrations. IFN-γ mRNA expression was increased in medicated chickens, but decreased in vaccinated chickens, with Met level increased to 0.56% and 0.68% (P < 0.05). Feeding 0.45% Met to vaccinated chickens had the highest IFN-γ mRNA expression among all treatments.Feeding 0.56% and 0.68% Met was associated with elevated sIgA level in medicated chickens (P > 0.05), but impacted on GPx activity and T-AOC (P < 0.05). Dietary Met levels affected all these indexes (P < 0.05). There were interactions between anticoccidial programs and dietary Met levels for GPx activity and T-AOC (P < 0.05). Serum GPx activity in medicated chickens was increased by feeding 0.56% Met (P < 0.05), compared to 0.45% Met. But the beneficial effect was not evident by feeding 0.68% Met (P > 0.05). High Met levels in diets did not affect serum GPx activity in vaccinated chickens (P > 0.05).As indicated in Table P < 0.05) and 10.84 nmol/mL (P < 0.01) from 6.93 nmol/mL in medicated chickens, and to 8.66 (P < 0.05) and 9.40 nmol/mL (P < 0.05) from 7.74 nmol/mL in vaccinated chickens. Serum T-AOC in medicated chickens was enhanced by feeding 0.56% Met (P < 0.05), but not by feeding 0.68% Met (P > 0.05). High Met levels in diets did not affect T-AOC in serum in vaccinated chickens (P > 0.05).Compared to 0.45% Met, feeding 0.56 and 0.68% Met increased MDA in serum to 8.62  to medicated chicks improved the growth performance compared to 0.45% Met. Previous reports showed that feeding increased Met either had no effects or decreased the growth performance of chicks that were not challenged by coccidia [E. tenella -challenged conditions.Coccidiosis is a major parasitic disease of poultry, associated with malabsorption, inefficient feed utilization, impaired growth rate and increased mortality in broilers. chickens . Previouchickens . Anticoccoccidia , 19. In coccidia . Increascoccidia , 22. Narcoccidia . Our datEimeria species. Narasin is an ionophore coccidiostat, processing a potent, broad-spectrum anticoccidial activity against Eimeria species. The control mechanism of ionophore coccidiostats is altering the ion transport and disrupting osmotic balance, which can damage the coccidial sporozoites during their release into the lumen [E. tenella from Dutch broilers in 1996 was sensitive to narasin, however, two isolated E. tenella in 1999 showed resistance to narasin. Another study found that replacing a proportion of nicarbazin with narasin results in reduced sensitivity to most coccidial species [E. tenella probably still survive after the exposure to narasin and cause malabsorption and inefficient feed utilization of chickens. This may partly explain the increased Met requirement of the medicated broiler chickens under E. tenella-challenged conditions. In addition, we found that feeding increased Met to medicated chickens improved small intestinal morphology, as indicated by the improved VH, CD and V/C as well as reduced cecal lesion and fecal oocyst output. Immunological assays further revealed that immune functions of chickens fed anticoccidial drug were enhanced with the increase of Met levels in diet. These data suggested that increasing Met levels in diet helped medicated chickens antagonize the challenge of E. tenella through the stimulation of immune functions. Met is known to play an important role in the immune functions by regulating antibody production and cell-mediated immune responses. We hypothesized that anticoccidial drug narasin may be incapable of most effectively controlling the number of E. tenella probably due to the drug resistance under a heavy load of E. tenella infection. Many survived E. tenella induced the host immune response that is conducive to eliminating these pathogenic micro-organisms. Increased Met in diet just meets the increased requirement for the stimulated immune functions. Following exposure to Eimeria species either naturally or via vaccination, chicks gradually develop acquired immunity and become resistant to reinfection of coccidia [Eimeria infection can induce a significant increase of CD8+ cells in the intraepithelial leucocytes, tonsil lymphocytes and peripheral blood [+ cells were also increased in the intraepithelial leucocytes, but decreased in the peripheral blood and spleen [Eimeria, but not the secondary infection [+ cells reduced IFN-γ mRNA expression in the cecal tonsil and intraepithelial lymphocytes and increased production of oocysts of E. tenella in chicks [+ lymphocytes in controlling primary infection with E. tenella. The selective elimination of CD8+ cells resulted in the exacerbation of disease as evidenced by increased oocyst shedding after secondary infection with E. tenella or E. acervulina [. It is known that CD4+ cells play an important helper function in protective immune responses, and CD8+ cells act as effector cells in these processes [Eimeria species compared with non-infected chicks, and higher levels of IFN-γ coincide with a protective immune response to the infection [E. tenella [E. tenella, as reviewed by Lillehoj et al. [+ and CD8+ cells in the blood and suppressed IFN-γ mRNA expression in cecal tonsils, suggesting an inhibited immune function against E. tenella. Maybe the reason is that coccidial vaccination confers the protection of the host from coccidial infection, so 0.45% Met is sufficient for chickens vaccinated. Excess Met could be detrimental to the anti-oxidative system and immune function. It was reported that feeding Met-sufficient diets (containing 0.50 and 0.70% Met) decreased the growth, feed intake and efficiency of feed utilization of chickens that were injected with some components of microbial pathogens, like Escherichia coli lipopolysaccharide or heat-killed Staphylococcus aureus [E. tenella infection. TNF-α leads to the development of a protective immunity on the one hand, but, on the other hand, it participates in the pathogenesis of coccidiosis, because treatment of chickens with antibodies against TNF-α results in a partial abrogation of E. tenella-induced body weight loss in chickens [TNF-α mRNA expression in cecal tonsils of the vaccinated chickens was not affected by increased diet Met levels, it was unclear whether the influence on TNF-α occurs in other immune organs, such as the spleen and intraepithelial lymphocytes. Further studies are warranted to investigate TNF-α alterations in these locations.There is increasing concern about rising levels of drug resistance of he lumen . Howeverhe lumen  found th species . Due to coccidia . Eimeriaal blood –30. CD4+d spleen , 31. Thenfection , 31. To n chicks , 32, whirocesses . Both CDrocesses . IFN-γ pnfection . Previou tenella , 35. Apas aureus . This stEimeria infection increases the activity of NADPH oxidase, an important enzyme for production of oxidative species in infected mucosa [d mucosa . It is kd mucosa . Broilerd mucosa . Wen et d mucosa  found thd mucosa . In the E. tenella-challenged condition, but has no beneficial effects on that of vaccinated chickens.In the present study, vaccinated chickens that were given 0.45% Met showed greater growth performance than medicated chickens that were provided basal (0.45%) or even increased levels of Met. Therefore, inoculation with anticoccidial vaccine is a better strategy for controlling coccidiosis than feeding narasin, not only due to greater growth performance obtained by the vaccination, but also due to the lower Met supplementation to achieve optimized performance. Furthermore, the present study for the first time revealed that high dietary Met levels benefit the growth performance of mediated chickens under


In vitro studies suggest a role for Met and its ligand HGF. To study this suggested role, MET and HGF gene copy numbers were determined by droplet digital PCR in tumours from 205 pre-menopausal and 184 post-menopausal patients, both cohorts randomised to receive either chemo- or radiotherapy. MET amplification was found in 8% of the patients in both cohorts and HGF amplification in 7% and 6% of the patients in the pre- and post-menopausal cohort, respectively. Met, phosphorylated Met (pMet), and HGF protein expression was determined by immunohistochemistry in the pre-menopausal cohort. Met, pMet, and HGF was expressed in 33%, 53%, and 49% of the tumours, respectively. MET amplification was associated with increased risk of distant recurrence for patients receiving chemotherapy. For the pre-menopausal patients, expression of cytoplasmic pMet and HGF significantly predicted benefit from radiotherapy in terms of loco-regional recurrence. Similar trends were seen for MET and HGF copy gain. In the post-menopausal cohort, no significant association of benefit from radiotherapy with neither genes nor proteins was found. The present results do not support that inhibition of Met prior to radiotherapy would be favourable for pre-menopausal breast cancer, as previously suggested.Few biomarkers exist to predict radiotherapy response in breast cancer.  In vitro studies suggest a role for Met in radiotherapy response [Post-operative radiotherapy is an important treatment to target any remaining cancer cells in the operated area and reduce the risk of loco-regional recurrence. Although radiotherapy has a high success rate, not all breast cancer patients benefit from the treatment . Accordiresponse .The Met oncoprotein is a transmembrane tyrosine kinase receptor, with major activation sites being Tyr1234/1235 in the kinase domain and Tyr1349 in the multi-docking site. The receptor is activated by extracellular binding of its ligand hepatocyte growth factor (HGF), leading to downstream signalling of pathways like the phosphoinositide 3-kinase (PI3K)/Akt pathway, stimulating tumour growth, cell survival, proliferation, migration, and invasion . The MetMET led to increased radiosensitivity [MET and HGF gene copy number, Met/HGF expression, and Met phosphorylation in breast tumours of patients randomised to receive either chemo- or radiotherapy, in order to study correlations with clinicopathological parameters and response to radiotherapy.Previous studies have shown that Met protein expression and ligand-independent activation rose after ionising radiation, and silencing of sitivity , 2, 14. MET and HGF in the tumours, copy number variation assays of MET and HGF were performed with droplet digital PCR (ddPCR). MET amplification, defined as four or more copies, was found in 8% of the patients in both cohorts . Copy gain, defined as three or more copies, was found in 33% (66/205) and 27% (50/184) in cohort 1 and 2, respectively. HGF amplification was detected in 6% (11/205) and 7% (12/184) of the tumours, and copy gain in 21% (41/205) and 27% (50/184), in cohort 1 and 2, respectively. MET gain and HGF gain were significantly correlated with each other in both cohorts .To establish the gene copy numbers of MET and HGF changed copy numbers, the genes were analysed in relation to clinicopathological parameters. For cohort 1, these are shown in Table MET amplification was correlated with high cell proliferation or high tumour grade. Increased MET copy number, either amplification or copy gain, was inversely correlated with the luminal A subtype, and thereby oestrogen receptor (ER) status, in cohort 1. In cohort 2, the same was true for MET amplification. In addition, MET amplification was more frequent in triple-negative breast cancer (TNBC) in this cohort. HGF copy gain was significantly correlated with a negative pAkt status in cohort 1  than tumours with fewer HGF copies.To explore the impact of cohort 1 . In cohoProtein expression levels of Met, pMet, HGF, and pAkt, a key protein in Met signalling, were studied in cohort 1 by use of immunohistochemical staining. High expression of Met in the membrane was found in 20% (45/228) of the tumours, and high cytoplasmic staining in 33% (73/228) of the cases. High pMet expression was found in 25% (55/228) and 53% (116/228) of the tumours in the membrane and the cytoplasm, respectively. High stromal HGF was found in 51% (110/228) of the tumours and high cytoplasmic staining in 49% (105/228). pAkt was highly expressed in 46% (105/228) of the tumours. Interrelationships between the proteins can be found in Albeit neither Met nor stromal HGF expression correlated to any of the clinicopathological parameters, membranous pMet showed a correlation with human epidermal growth factor receptor-2 (HER2) status, and cytoplasmic HGF and pMet were positively correlated with pAkt status. Both high cytoplasmic and membranous pMet were predominant in luminal B1 tumours. However, whilst membranous pMet was more often low in luminal A tumours, cytoplasmic pMet tended to be abundant in this subtype. Associations between proteins and clinicopathological parameters can be found in Table MET amplification tended to result in a shorter DRFS  = 2.52; 95% confidence interval (CI): 1.04-6.1, p=0.04). This could only be seen on gene level, and not on protein level. Although HGF copy number was not prognostic in patients with triple-negative tumours in either of the cohorts, high stromal HGF, but not cytoplasmic HGF, was correlated with a shorter DRFS in these patients in cohort 1 .Distant recurrence-free survival (DRFS) was used as endpoint to explore the prognostic importance of the proteins and genes of interest. In both cohorts, S Figure . In cohon Figure . A similMET amplification was further explored for patients who received chemotherapy with cyclophosphamide, methotrexate, and 5-fluorouracil (CMF). In multivariable Cox regression analysis, including both cohorts adjusting for lymph node involvement, tumour size, ER and HER2 status, and stratified for cohort, MET amplification was associated with a significantly increased rate of distant recurrence . Likewise, for patients with TNBC, treated with CMF, MET amplification showed prognostic significance with adjustments made for nodal status and tumours size .The increased rate of distant recurrence seen in relation to MET and HGF in terms of loco-regional recurrence. Patients having tumours with HGF gain relapsed at a higher rate than those having tumours with one or two copies of the gene in cohort 1. In cohort 2 this was merely a trend, likewise with MET gain in both cohorts . The role of Met in HER2 positive breast cancer is debated. In cohort 2, no correlation was found between MET gain and the HER2 positive subtype, which is in agreement with the meta-analysis conducted by Yan et al. [MET gain and the HER2 positive subtype. Another study, not included in the meta-analysis, revealed Met to be co-expressed with HER2 in breast cancer [MET and HGF amplification was interrelated, which in part might be explained by the fact that both genes are located in close proximity to one another, on chromosome 7.In agreement with other studies, increased R status , 20. It  studies –27, highn et al. . Cohort t cancer . In addiIn vitro, Met and HGF have been demonstrated to negatively influence response towards ionising radiation, and it has been suggested that Met inhibition might overcome radioresistance [MET or HGF gain, or high cytoplasmic pMet or HGF expression, had more benefit from radiotherapy versus chemotherapy, as compared with those with no copy gain or low expression. This effect was not seen in post-menopausal patients, nor in relation to non-phosphorylated Met or stromal HGF in cohort 1. One confounding factor in the post-menopausal cohort might be that 50% of the patients were randomised to receive tamoxifen, known to effectively reduce loco-regional recurrence rates. It can, however, not be excluded that the HGF/Met axis differently influences pre- and post-menopausal patients and their response to radiotherapy. In cohort 2 it was previously shown that low pAkt predicted a favourable response to radiotherapy [sistance , 29, 30.sistance . Whereassistance ; up to notherapy . In contin vitro studies suggest that Met inhibition might overcome radioresistance, the novel finding presented here suggests that it might not be prudent to inhibit the HGF/Met axis prior to radiotherapy, as previously suggested after in vitro studies. However, more research is needed to elucidate the role of phosphorylated Met and HGF in radiotherapy response.In summary, we show that Met and HGF have a multi-factorial relationship to the biology and outcome of breast cancer, influenced by gene copy number and protein expression, activation status, stromal environment, and cellular localisation. Even though several 2, orally once a day, methotrexate 40 mg/m2, intravenous on days 1 and 8, 5-fluorouracil 600 mg/m2 intravenous on days 1 and 8) [From 1976 to 1990, 547 pre-menopausal and 679 post-menopausal women with breast cancer in the Stockholm region were randomised to adjuvant chemotherapy or post-operative radiotherapy . The pos1 and 8) . Modifie1 and 8) . Tables 1 and 8) . HER2 ov1 and 8) . Phospho1 and 8) . Notting1 and 8) .TM Fluorometer (Promega).Genomic DNA was previously extracted from fresh frozen tumour tissues of patients in cohort 2 . GenomicMET and HGF  in both cohorts, using AP3B1 (Bio-Rad) as reference gene  and The Cancer Genome Atlas (TCGA: http://cancergenome.nih.gov), which is why it is considered a suitable reference gene. It has previously been reported that absolute gene copy numbers can be successfully determined in DNA derived from FFPE tissues, by using ddPCR [Copy number variations were evaluated with ddPCR for nce gene . Copy nung ddPCR . The recng ddPCR . In shorFFPE tissues were available for the pre-menopausal cohort. Representative tissue blocks were selected as donor blocks for the TMAs. Sections were cut from each donor block and stained with Haematoxylin and Eosin. From these slides, three morphologically representative regions were chosen in all tumour samples. Three cylindrical tissue cores with a diameter of 0.8 mm were taken from these areas and mounted in recipient blocks. The TMAs were constructed using a manual arrayer . Five μm sections were cut from the TMA blocks and transferred to microscope slides for IHC analysis.2O2) solution, and Mayer's Haematoxylin for counterstaining . The slides were dehydrated using serial dilutions of ethanol. Images were obtained with an AxioCam ICc5 camera attached to an Axiolab A1 microscope, using Zen 2012 (blue edition) software .TMA sections were stained overnight with monoclonal antibodies against Met , pMet , HGF  and pAkt-S473 . The same lot of each antibody have been used throughout this study. Sections were deparaffinised, rehydrated, and antigen retrieved using a DAKO PT module  with DAKO PT Low  or High  pH Buffer . Serum-free protein block  was used for 60 minutes on all sections and PBS/0.1% bovine serum albumin was used for washing. After overnight incubation at 4°C with the primary antibodies, the sections were incubated at room temperature for 30 minutes with an appropriate secondary antibody . Colour was developed by incubating with 3′-diaminobenzidine tetrahydrochloride , without knowledge of clinical data. The samples were categorised into different groups. For Met and pMet, the tissues were scored following Herceptest Guidelines for membrane staining (Dako). The scoring for both the cytoplasm and membrane were divided into four groups based on intensity: negative (−), weak (+), moderate (++) and strong (+++). Met/pMet protein expression was considered high if staining was weak to strong, otherwise it was considered low. HGF was visualised in the stroma and the cytoplasm of the tumour cells. Stromal staining was either considered negative  or positive . Cytoplasmic staining was divided into negative (−), weak (+) or strong (++), based on intensity and was considered high if staining was strong. Both cell membranous and cytoplasmic staining for Met/pMet, and cytoplasmic and stromal staining for HGF were used in statistical analysis. Phospho-Akt was visualised in the cytoplasm and nucleus. Intensity was scored in the cytoplasm as negative (−), weak (+), moderate (++) or strong (+++), nuclear staining was divided in negative (−), intermediate (+), and strong (++). In this study, only cytoplasmic pAkt was used for analysis and dichotomised into low (negative to weak) and high (moderate to strong). Representative images of Met, pMet and HGF staining are shown in Figure et al. .To verify the specificity of the Met phospho-antibody, a lambda protein phosphatase test was carried out. After pre-treatment using DAKO PT module, the slides were either treated with 4000U Lambda Protein Phosphatase  or with washing buffer; after two hours of incubation at 30°C, the slides were treated as described above, though with an antibody dilution of 1:50. Representative images can be seen in Figure MET or HGF copy numbers, pAkt, pMet, Met or HGF protein expression. Multivariate Cox models were also used in the analysis of DRFS in relation to MET amplification and known prognostic factors, stratified for cohort.Patient survival in multiple groups was computed with Kaplan-Meier and the log-rank test was carried out to estimate statistical differences. Survival time for the given end point was defined as the period of time elapsed between the diagnosis of the primary tumour and the distant recurrence (DRFS) or loco-regional recurrence . The Hazard Ratio was calculated using the Cox proportional hazard regression model and Pearson's chi-squared test was executed to assess the relationships between the different variables. A multivariate Cox model was used to test the interaction between benefit from post-operative radiotherapy versus chemotherapy and Breast cancers were categorised into five main subtypes: Luminal A  I or II or low S-phase (≤10%)); Luminal B1 ); Luminal B2 ; HER2 positive (ER- and HER2+) and triple-negative breast cancer (TNBC) (ER- and HER2-). Progesterone receptor data was not available for the majority of the patients; hence it was not used for the categorisation of subtypes. To analyse correlations between these subtypes and either protein expression or copy number the Pearson's chi-squared test was performed for each subgroup separately.Statistical analyses were performed with Statistica software version 12.0 . The criterion for statistical significance was P≤0.05 and reported in bold in the tables. Hazard-ratios were reported with 95% confidence interval.


Glioblastoma (GBM) is a lethal brain tumor with dismal prognosis. Current therapeutic options, consisting of surgery, chemotherapy and radiation, have only served to marginally increase patient survival. Receptor tyrosine kinases (RTKs) are dysregulated in approximately 90% of GBM; attributed to this, research has focused on inhibiting RTKs as a novel and effective therapy for GBM. Overexpression of RTK mesenchymal epithelial transition (MET), and its ligand, hepatocyte growth factor (HGF), in GBM highlights a promising new therapeutic target. This review will discuss the role of MET in cell cycle regulation, cell proliferation, evasion of apoptosis, cell migration and invasion, angiogenesis and therapeutic resistance in GBM. It will also discuss the modes of deregulation of HGF/MET and their regulation by microRNAs. As the HGF/MET pathway is a vital regulator of multiple pro-survival pathways, efforts and strategies for its exploitation for GBM therapy are also described. Glioblastoma (GBM) is the most common and lethal primary malignant brain tumor with an incidence rate of approximately 3.19/100,000 per year . The CanGiven the dismal prognosis of GBM, research has focused on identifying novel targets for therapy. A particular area of interest are RTKs that regulate many essential cellular processes within normal cells, such as cell proliferation, differentiation and survival ,9. DeregThe RTK MET is coded for by the MET proto-oncogene located on chromosome 7q21–31 ,11. MET The MET receptor is a dimeric, 190 kD tyrosine receptor kinase expressed on the surface of epithelial and endothelial cells and at low levels in the brain. The dimer features a 50 kD extracellular α-chain and a 140 kD transmembrane β-chain, linked together by disulfide bridges. The only known ligand for MET is hepatocyte growth factor (HGF) , which is a multifunctional two-chain cytokine secreted by mesenchymal cells  10,19].,19.10,19The oncogenic effect of MET is mediated through the recruitment of multiple effectors including GRB2, GAB1, PI3K, Shc, Src and SHP-2, ultimately leading to the activation of several downstream signal transduction pathways  8,14,16,1614,16.MicroRNAs (miRNAs) provide a novel layer of signaling regulation outside of the traditional central dogma. miRNAs are non-protein-coding RNAs that bind their mRNA targets through imperfect base pairing. Once bound, miRNAs function by either inhibiting the translation of their mRNA targets or promoting their degradation . A subsemiR-410 , miR-144Two other important miRNAs are miR-34a  and miR-Conversely, miRNAs also participate in a feedback loop within the MET and other RTK pathways. RTKs repress the transcription of these miRNAs, which usually repress the RTK signaling, thus forming a positive feedback loop; an example is miR-134 . VariousThe prevalence and complexity of MET/HGF signaling in high-grade GBM such as MET amplification, elevated HGF and pathway orchestration through intricate miRNA networks emphasize the importance of novel therapeutic strategies for targeting MET/HGF signaling for optimal therapy. With this in mind, we will discuss below the different therapeutic approaches being developed and discuss past and ongoing clinical trials targeting MET/HGF for therapy.Under normal conditions, HGF-induced MET activation is regulated by paracrine ligand delivery, ligand activation and ligand activated receptor internalization and degradation. Given the many molecular effectors that are known to enter into or result from MET recruitment, there are many mechanisms that lead to aberrant MET signaling including: overexpression of MET, elevated paracrine or autocrine ligand production, constitutive MET activation in the absence of MET gene amplification, and MET mutations . ComplicA well-known mechanism of transformation is the ability of the cell to by-pass the critical cell cycle check point machinery and to advance into pro-growth processes; in particular, the main obstacle to clear is the G1 phase checkpoint, monitored by p53, where DNA integrity is assessed before replication. The p53 pathway is commonly dysregulated in GBM, where mutations or loss of p53 are common in secondary GBM and are present less frequently in primary GBM. Since p53 responds to DNA damage by inducing cell cycle arrest and apoptosis, loss or loss of function mutations of this tumor suppressor leads to deregulation of the cell cycle and enhanced tumor cell growth . The HGFHGF induced activation of MET or mutations in the receptor that lead to constitutive activation stimulate the initiation of many downstream signaling pathways that promote tumor cell growth. The PI3K/Akt pathway, frequently altered in GBM, is deregulated through two mechanisms: aberrant activation of an RTK, such as MET, or deletion of the tumor suppressor PTEN. PTEN negatively regulates the PI3K/Akt pathway but is frequently lost or mutated in GBM. Up-regulation of the PI3K/Akt pathway in GBM leads to uncontrolled tumor cell growth and survival through aiding nuclear translocation of nuclear factor Κb (NFκB) which activates many cell survival and anti-apoptotic genes . MoreoveUpon HGF stimulation of MET, brain tumor vascular cells express matrix metalloproteinases and urokinase, promoting matrix degradation and invasion . Tumor cHGF regulates angiogenesis through activation of MET on vascular endothelial cells, promoting vascular endothelial cell proliferation, migration, survival, and extracellular matrix degradation . PreviouInterestingly, MET plays an important role in drug-acquired resistance. For example, in Bevacizumab sensitive GBM tumors, MET and vascular endothelial growth factor receptor 2 (VEGFR2) form complexes that suppress HGF mediated growth and invasion. However, in GBM tumors resistant to Bevacizumab, a VEGF-A neutralizing antibody, MET is further upregulated preventing this regulation and advancing invasion ,42.The cancer stem cell (CSC) model proposes an alternative view on tumorigenesis and cancer treatment. Drawing analogy from the normal stem cell biology, the CSC model states that the development and maintenance of a tumor is driven by a small population of CSCs within the tumor . AccordiMET is a key signaling pathway in GBM stem cells (GSCs). Primary GBM cells with high MET expression contain higher number of GSCs and exhibit higher tumorigenicity . EctopicIn addition to maintaining the GSC population, MET also plays a crucial role in conferring resistance to conventional therapies in GSCs. First, it has been known for many years that inhibiting MET enhances GBM responses to radiation ,55,56. RMET/HGF mutations and overexpression are present in a high percentage of GBM tumors and previous studies have demonstrated that inhibition of MET in tumor cells leads to growth inhibition, tumor regression and decreased metastatic potential. MET-amplification and overexpression has been reported in 5% and 13% of GBMs respectively, whereas MET gains are present in 47% of primary GBMs and 44% of secondary GBMs . This fiCompetitors of MET vie with the HGF ligand for binding to the receptor to prevent activation and subsequent initiation of downstream signalling pathways. NK2 and NK4, HGF antagonists, are intra-molecular fragments of HGF that compete for binding to the receptor to inhibit activity ,69,70. ADue to the ubiquitous and prolific involvement of receptor tyrosine kinases in cancer pathology, many classes of small molecule inhibitors have been developed to combat RTKs through multiple modes of inhibition. Inhibitor types range from Types I–VI and are categorized by their binding patterns, kinetics and conformations, in addition to inhibiting multiple RTKs across different families . InhibitAdvances in genotyping and centralized tissue bank systems  have allowed for the cataloguing and categorization of genetic alterations such as gene amplification, mutation, deletions and other modifications implicated in multiple cancer types; many of these alterations happen, unsurprisingly, in RTKs. Therefore, when designing small-molecule inhibitors for RTKs, it is imperative to determine the rates of amplification, mutation and deletions within a given cancer and to avoid potential modes for resistance. Berthou and colleagues show one mode of this strategy, where they demonstrated the compound SU11274 targets wild type MET and two mutants, M1268T and H1112Y in stably expressed NIH 3T3 cells . This muAlongside of type I and II ATP-competitive inhibitors, other inhibitors can maintain inhibition modes outside of ATP competition, such as allosteric inhibitors and covalent inhibitors (types III through types VI) .One common type Ia member is the MET/ALK inhibitor crizotinib/PF-02341066, a small molecule inhibitor developed by Pfizer. Crizotinib has been shown to effectively inhibit MET-dependent growth, invasion and survival  and is cOther small molecule inhibitors currently being investigated in other forms of cancer, excluding GBM, include: Foretinib/GSK1363089 (Exelixis), an ATP-competitive inhibitor of MET and VEGFR2, has been investigated clinically in phase I and II trials in many cancer types including head and neck, NSCLC, breast, liver and metastatic gastric cancer with varying results; Amuvatinib/MP470 (SuperGen), an inhibitor of MET, c-Kit, PDGFRα and Flt-3, has completed phase I and II trials in multiple tumor types including small cell lung cancer, solid tumors and lymphoma ; MK-2461Monoclonal antibodies act to obstruct binding of HGF to the MET receptor preventing activation of downstream pathways leading to growth inhibition, activation of apoptosis and inhibition of metastasis. One major drawback to using monoclonal antibodies for cancer therapies is the decreased penetration of the blood-brain barrier observed compared to small molecule inhibitors. This issue is addressed with the use of novel delivery techniques ,86.AMG102/Rilotumumab , a human IgG2 monoclonal antibody, binds to MET and inhibits the binding of HGF to MET. AMG102 has completed phase I and II clinical trials in GBM patients with reports of partial response and stable disease  87]. On. On87]. Inhibition of MET has demonstrated clinical benefit for patients with multiple types of cancer. In addition to showing that MET inhibitors are generally well-tolerated, previous clinical trials  have alsFurthermore, therapeutic regulation of miRNAs in GBM tumors will likely lead to increased survival for patients. In the past, lack of an efficient delivery system for miRNAs, especially into the central nervous system, was the main hurdle. In recent years, various delivery mechanisms have been investigated, for example, miRNAs can be conjugated to modified gold nanoparticles, which are able to carry miRNAs across the blood-brain barrier, as in the case of miR-182 . Gold naHGF/MET inhibitors, specifically small molecule inhibitors and monoclonal antibodies, have displayed mixed results in clinical trials in GBM patients that emphasize the importance of patient selection. Combining HGF/MET inhibitors with currently used chemotherapeutics or inhibitors for other growth pathways would prove to be a more effective treatment options for many cancer patients.


Lactobacillus (L.) casei Zhang as a model.The extensive use of antibiotics in medicine has raised serious concerns about biosafety. However, the effect of antibiotic application on the adaptive evolution of microorganisms, especially to probiotic bacteria, has not been well characterized. Thus, the objective of the current work was to investigate how antibiotic selection forces might drive genome adaptation using L. casei Zhang. We then monitored the mutations in the bacterial genome and changes in the minimum inhibitory concentrations (MICs) of these two antibiotics along a 2000-generation-cultivation lasted over 10 months.Two antibiotics, amoxicillin and gentamicin, were consistently applied to the laboratory culture of L. casei Zhang, i.e. 3.5 × 10-9 per base pair per generation under either amoxicillin or gentamicin stress, when compared with the parallel controls grown without application of any antibiotics. The increase in mutation frequency is significantly lower than that previously reported in Escherichia (E.) coli. The rate of de novo mutations, i.e. 20 per genome, remained low and stable throughout the long-term cultivation. Moreover, the accumulation of new mutations stopped shortly after the maximum bacterial fitness (i.e. the antibiotic MICs) was reached.We found an approximately 4-fold increase in the genome mutation frequency of L. casei Zhang, has high genome stability even in the presence of long-term antibiotic stresses. However, whether this is a species-specific or universal characteristic for all probiotic bacteria remains to be explored.Our study has shown that the probiotic species, The online version of this article (doi:10.1186/s12864-017-3710-x) contains supplementary material, which is available to authorized users. Escherichia (E.) coli  and cell wall/membrane/envelope biogenesis [M] Fig. . ComparePrcK (LCAZH_2350), and a DNA-directed RNA polymerase subunit beta (LCAZH_2481). Among them, the regulators of OmpR family is directly associated with antibiotic, extreme pH, temperature, and oxidative stresses ,capacity .L. casei Zhang in the later generations even with continuous antibiotic exposure. Instead, the mutation rate returned to the baseline level similar to that of the control, suggesting that both the bacterial genome and fitness level to antibiotics were stabilized. Our findings show that L. casei Zhang is a stable strain and presents limited biosafety risks even subject to long-term antibiotic exposure. However, the scope of the current study is limited to the analysis of mutation accumulation under in vitro cultivation conditions. Another important mechanism that drives evolution is horizontal gene transfer, which has not been addressed here. Particularly, when the probiotic bacteria arrive in the host gastrointestinal tract, in addition to antibiotic exposure, they will also be exposed to an enormous and complex microbial community within the colon. Horizontal gene transfer may then become a significant player in the genome stability and evolution of these bacteria in in vivo situations.Some pathogens have been shown to adapt rapidly upon antibiotic exposure by continuous accumulation of point mutations and thus raise serious health and safety concerns to the public . HoweverL. casei Zhang in a long-term evolution experiment with and without antibiotic (amoxicillin or gentamicin) selection pressure. We found that L. casei Zhang has high genome stability even subject to long-term antibiotic stresses. However, whether this is a species-specific or universal characteristic for all probiotic bacteria remains to be explored.In the present study, we monitored the genotypic and phenotypic changes of


Background: Antibiotic resistance is a global threat. Scarce knowledge about safe and appropriate antibiotic use is coupled with frequent self-administration, e.g., in China. This repeated self-medication poses potential risk in terms of antibiotic resistance. Low-resource countries are facing an elevated burden of antibiotic self-medication as compared to developed ones. Thus, this study focused on evaluating the pervasiveness of antibiotic self-medication in 3 universities of Southern Punjab, Pakistan. Methods: We conducted a descriptive cross-sectional survey in three government sector universities of Southern Punjab, Pakistan. The study was carried out with self-administered paper-based questionnaires. Data was analyzed using SPSS version 18.0 . Results: Seven hundred twenty-seven students out of 750 (response rate 97%) with a mean age ± SD of 23.0 ± 3.4 years agreed to participate in the study. The proportion of females was slightly greater (52%) compared with males (48%), and almost one-third of the respondents (36%) were in their 2nd year of university. Out of the total, 58.3% practiced self-medication in the preceding six months, and 326 (45%) confirmed the use of antibiotics. Metronidazole was the most frequently self-medicated antibiotic (48%). Out of the total, 72% demonstrated awareness regarding the side effects of antibiotics. Diarrhea was the well-known adverse effect (38%). Forty-three percent affirmed having antibiotic resistance knowledge, and 30% knew that the irregular use of antibiotics would lead to increased antibiotic resistance. Conclusion: Despite having ample awareness of the adverse antibiotic reactions, self-medication among the university students was high and antibiotic resistance was a fairly unknown term. In our daily lives, the practice of self-medication (SM) is frequently employed for self-care . The mosWHO affirmed that appropriate and responsible SM can be of assistance to treat and prevent disease more economically and without a doctor’s consultation , whereasA drastic increase in antimicrobial resistance (AMR), the severity of disease, the duration of the disease, complication risk, mortality rate, and health care cost was observed due to unorthodox use of antibiotics. Among all, AMR is of immense concern . A numbeSeveral studies have been performed around the globe to evaluate the prevalence of SM among university students ,13. PrevA cross-sectional descriptive study design was adopted to estimate the prevalence of SM with antibiotics among non-medical students in three different universities of Punjab, Pakistan. This study was conducted between July 2016 and September 2016. All of the selected universities were public sector universities with a diverse range of offered programs. In addition to pharmacy, pharmacology, and other medical departments, these universities provide higher education in fields of business administration, agriculture, fine art, economics, and humanities.Undergraduate and postgraduate students of any age and gender, familiar with the English language and enrolled in non-medical departments in any of the target universities, were included in the study. Students in paramedical and pharmacy departments and those who were refused to participate in the study were not included.The use of any kind of medication in the previous6 months of an individual’s own volition without a doctor’s consultation was the definition of SM used in this study. A period of 6 months was selected to minimize bias in recall. This period was considered adequate for one to recall if he had used the antibiotics.To attain the appropriate sample size, the convenient sampling method was adopted. The study objectives were clarified to authorities and prior consent from respective departments was obtained. The data collectors were instructed to approach the students in the target area for interviews. Students taking part in the study gave both written and verbal consent. A self-administered questionnaire was circulated among the study participants, and they were asked to complete it on the spot. The filled questionnaire was then returned to the researchers.A 3 section, 8-itempaper-based study instrument, which was validated and used for data collection in previous studies, was adopted . Each seAlthough this questionnaire was validated and pretested in other parts of Pakistan , consistData screening and entry was performed by researchers and further cross-checked by colleagues. Data were carefully entered into EpiData 3.1  and were analyzed using SPSS . Descriptive statistics was used to calculate the pervasiveness of SM; similarly, frequency of possible reasons for SM, frequency of the SM of different types of antibiotics, knowledge of the most common side effects, frequency of knowledge with respect to antibiotic resistance, and the effect of antibiotic use on resistance was evaluated by generating frequency tables in SPSS.Permission was taken from three universities prior to commencement of the study. Written and verbal consent was sought from each and every individual. Request for the name or any other private info was not made purposefully. This study was approved by the Ethics Committee for Medical Research of Xi’an Jiaotong University Shaanxi, China (ANT-17-21).A total of 750 students were approached, and among them 727 agreed to take part in the study with are sponse rate of 97%. The mean age ± SD of respondents was 23.0 ± 3.4 years with a minimum age of 18 to a maximum of 40. The proportion of females (52%) was slightly higher than that of males (48%), and the majority of respondents (36%) were in their second year of university. Of the total, 245 (34%) had a monthly household income below 15,000 PKR, and the majority were single (91%). The demographic characteristics were elaborated in Out of the total respondents, 326 (45%) stated that they treated themselves with antibiotics without a doctor’s consultation in the previous six months. Gender and year of education showed statistical significance with frequency of antibiotic SM, whereas marital status and monthly household income showed no significance.Metronidazole was the most frequently self-medicated antibiotic amongst the university students, and 156 individuals reported using it. It was followed by ciprofloxacin, amoxicillin and co-trimoxazole. Ampicillin and erythromycin was used by 7% and 6% of the same respondents, respectively, while ampicillin/cloxacillin was amongst the least commonly used antibiotic. The results are shown in Ninety-eight (13%) respondents mistakenly considered other drugs as antibiotics; for example, 78 (11%) participants reported that Panadol was an antibiotic. This was determined by the “other antibiotics” question in Section 2. Here, respondents were asked to report any antibiotic that they used in the previous six months and were not given in the list of antibiotics provided by the researcher.One hundred seventy-eight (55%) stated that they chose antibiotics for themselves, 34 (10%) followed the instructions of friends, 71 (22%) followed parental instruction, and 46 (14%) followed a pharmacist’s instructions for SM. Out of the total, 571 (78%) knew what antibiotics were, and 522 (72%) of the respondents believed that antibiotics can cause side effects.Among the 326 who self-medicated themselves with antibiotics, 154 (47%) used them to relieve gastro intestinal problems, 136 (42%) to relieve pain, 118 (36%) to relieve respiratory symptoms, 84 (26%) to treat fever, and 26 (8%) were used to treat urinary problems. The students were asked about the most common adverse effects that they think can be caused by antibiotics in Section 3. According to the results, the most common side effect reported was by was diarrhea (38%), followed by sleep disturbances (27%), allergic reactions (24%), headache (21%), fever (20%), nausea (17%), tiredness (15%), teeth discoloration (13%), and heart problems (13%). A complete list of awareness of side effects is given in n = 311) of the 727 respondents reported that they were aware of this expression and 33% were ignorant of this term. However, it is shown in However, 22% of the respondents experienced side effects of antibiotic use. Of 68 students who specified a particular side effect, 18 reported of having gastrointestinal problem after antibiotic use (26%), 16 reported allergic reactions (23%), 13 experienced allergic reactions (19%), 11 experienced sleep problems (16%), and 9 reported headache (13%). Furthermore, the students were then asked if they knew about the term “antibiotic resistance”; 43% , that the antibiotic resolved a problem experienced previously , and that it bypasses the hassle of seeing a doctor . These results are predictable, as it is known that, most of the time, health care costs are exorbitant and that primary, secondary, and tertiary health care facilities are overloaded and botched, unable to fulfill the healthcare needs of individuals. To clarify this situation, further investigative actions regarding patient satisfaction regarding the health care system should be taken. These results coincide with the study in Karachi, where the most common reason for self-medication was that “it saved time” . AnotherMost frequent antibiotic used amongst the list given to the students was metronidazole and ciprofloxacin. The results in previous studies in Karachi were in line with our results in that the most frequently used antibiotics was metronidazole , while rA small proportion of respondents (24.5%) chose antibiotics for themselves. The target population was a non-medical group, so lack of knowledge about safe and appropriate use rendered them to use antibiotics. A study from Peru showed that the largest portion of the students who self-medicated themselves received recommendations from a pharmacist and that one-fourth chose of their own volition . FurtherMore than two-thirds of our respondents, however, were aware of the expression “antibiotic resistance,” and less than two-thirds knew that the unorthodox use of antibiotics will lead to increased antibiotic resistance, while the rest were deprived of the knowledge about the arbitrary use of antibiotics and antibiotic resistance. These results can be explained by the fact that our study population mainly consisted of a non-medical individuals and the term “antibiotic resistance” is generally used in the medical system. The same results were found from the previous study in Karachi .Ultimately, it is the responsibility of government to ensure that any population administers SM responsibly. The government should implement policies to regulate the sale of “prescription-only drugs” upon the presence of prescription. It should be known by the community pharmacist, dispenser, or medical storekeeper that the sale of antibiotics or of any prescription drugs, without prescription, is unethical and is a criminal act. Government, media, and health care authorities should impart their role in educating and motivating users about the safe use of antibiotics. Properly planned educational seminars, lectures, and workshops should be instigated in all universities on anational level. The education should not only focus on the safe use of antibiotics but also potential hazards, the extent of utilization, and accurate dosage.The outcome of our study will be supportive in designing programs that will be of assistance in creating awareness of the side effects of antibiotic SM. The pervasiveness of antibiotic SM amongst respondents was high. A large number of study respondents were aware of the possible side effects of the self-use of antibiotics. Nevertheless, the practice of self-utilization of antibiotics was observed.It is recommended that necessary steps be taken to improve awareness via educational programs, highlighting the risks of unconventional antibiotic use. Physicians should discourage the unnecessary use of antibiotics and prescribe them as a last resort. The health care provider’s role should be to eradicate this practice, especially the community pharmacist who can discourage the purchase of antibiotics without prescription and impart knowledge regarding their safe use. Great negligence on account of the government and health care department was also demonstrated here. The government thus must ensure that the sale of antibiotics is on a prescription basis only.Our study had few limitations. Firstly, the data is based on university students and thus represent only an educated portion of the general population; however, this sample should be assessed because the educated portion of the community is bound to have the greatest influence on the community. Secondly, we approached students by convenience sampling rather than random sampling. We attempted to restrain the effects by selecting large samples and multiple universities.


Antibiotics are commonly administered to hospitalized patients with infiltrates for possible bacterial pneumonia, often leading to unnecessary treatment and increasing the risk for resistance emergence. Therefore, we performed a study to determine if an enhanced antibiotic de-escalation practice could improve antibiotic utilization in mechanically ventilated patients with suspected pneumonia cared for in an academic closed intensive care unit (ICU).This was a prospective cross-over trial comparing routine antibiotic management (RAM) and enhanced antimicrobial de-escalation (EAD) performed within two medical ICUs  at Barnes-Jewish Hospital, an academic referral center. Patients in the EAD group had their antibiotic orders and microbiology results reviewed daily by a dedicated team comprised of a second-year critical care fellow, an ICU attending physician and an ICU pharmacist. Antibiotic de-escalation recommendations were made when appropriate based on microbiologic test results and clinical response to therapy.P = 0.438). In the remaining patients, antimicrobial de-escalation occurred in 70 (66.0%) and 70 (67.3%), respectively (P = 0.845). There was no difference between groups in total antibiotic days ((median (interquartile range)) 7.0 days  versus 7.0 days  (P = 0.616)); hospital mortality (25.2% versus 35.4% (P = 0.061)); or hospital duration  versus 11.0 days  (P = 0.918).There were 283 patients evaluable, with suspected pneumonia requiring mechanical ventilation: 139 (49.1%) patients in the RAM group and 144 (50.9%) in the EAD group. Early treatment failure based on clinical deterioration occurred in 33 (23.7%) and 40 (27.8%) patients, respectively  contains supplementary material, which is available to authorized users. Escalating rates of antibiotic resistance add substantially to the morbidity, mortality, and costs related to infection in hospitalized patients, especially those in the intensive care unit (ICU) setting –5. The rBecause not all serious infections are due to multidrug-resistant (MDR) organisms, clinicians must have a strategy for determining which patients should receive empiric broad-spectrum antibiotics . The praGiven the importance of balancing IAAT with the avoidance of unnecessary antibiotic exposure, we performed a clinical trial with the goal of determining whether an enhanced antimicrobial de-escalation (EAD) practice could improve antibiotic utilization and outcomes in mechanically ventilated patients with suspected pneumonia. Our hypothesis was that introduction of an EAD practice would increase rates of antimicrobial de-escalation in the ICU population. We also wanted to determine whether a practice of EAD impacted other outcomes including mortality and lengths of stay.The study was conducted within the two medical ICUs . at Barnes-Jewish Hospital, an academic referral center of 1250 beds. The medical ICUs are geographically co-located closed units with shared physician, nursing, pharmacist, and respiratory therapist staff. The medical ICUs are staffed 24 hours per day and 7 days per week by these teams, including intensivists board-certified in critical care. This investigation was approved by the Washington University School of Medicine Human Studies Committee and the need for informed consent was waived # 201509075; ClinicalTrials.gov Identifier: NCT02685930). All mechanically ventilated patients with suspected pneumonia, from 1 January 2016 through 31 December 2016 were eligible for inclusion. Patients were excluded if they had a concomitant non-pulmonary infection requiring antibiotic therapy, bronchiectasis, or significant immune suppression. Data were prospectively collected from the electronic health record and from patients’ ICU teams.The primary objective of this study was to determine whether and how frequently antibiotic de-escalation occurred following the initial prescription of antibiotics for suspected pneumonia. The secondary outcomes assessed included antibiotic duration, failure of initial antibiotic therapy, hospital mortality, ICU and hospital duration, and subsequent infection with antibiotic-resistant strains of bacteria.The two medical ICUs were initially randomly assigned so that subjects admitted to each ICU would receive either routine antibiotic management (RAM) for suspected pneumonia or EAD. Patients in the RAM group had all antibiotic decisions determined by their ICU team composed of a critical care board-certified attending physician, a critical care fellow, resident physicians, and a critical care specialty pharmacist. Antibiotic decision-making was similar in patients in the EAD group except that all antibiotic orders and microbiology results were reviewed on a daily basis by the study team  to specifically determine if further antibiotic de-escalation could occur. The study team performed these reviews from 7 a.m. to 6 p.m. on weekdays excluding weekends and holidays. After 6 months the two medical ICUs were crossed over in terms of the type of antibiotic management strategy delivered to patients.9cells/L), leukopenia (≤4 × 109cells/L), or purulent secretions. The presence of a new or progressive radiographic infiltrate was based on the interpretation of the chest radiograph by board-certified radiologists blinded to the study. Patients admitted from the community without prior radiographs were assumed to have a new infiltrate when present. Pneumonia was classified microbiologically according to the pathogen(s) identified as pathogen-negative, viral, antibiotic-susceptible, or antibiotic-resistant. For purposes of this investigation, antibiotic-susceptible was determined according to ceftriaxone susceptibility, as ceftriaxone represents the antimicrobial agent most frequently recommended for hospitalized patients with pneumonia coming from the community setting [Adult patients age >18 years) were identified prospectively with suspected pneumonia (community-onset pneumonia (COP), hospital-acquired pneumonia (HAP), or ventilator-associated pneumonia (VAP)) in accordance with the American Thoracic Society position statement on pneumonia . The dia setting . Additio8 years wEarly failure of initial antibiotic therapy was defined as progression to shock, mortality, or progression of the radiographic infiltrates without resolution of clinical parameters  within 48 hours of starting antibiotic therapy. Septic shock was defined as the need for vasopressors . Only the first episode of suspected pneumonia was recorded. Antimicrobial treatment was classified as IAAT if the initial regimen had in vitro activity demonstrated against the isolated pathogens. Immune suppression was defined as acquired immune deficiency syndrome, solid organ or bone marrow transplant, hematologic malignancies, solid cell cancers treated with chemotherapy or radiation, long-term corticosteroids (>10 mg/day), and other immunosuppressive drugs . Multidrug-resistant (MDR) pathogens had to demonstrate in vitro resistance to at least one agent from three distinct classes of antimicrobials that would normally have activity against that bacterium .Staphylococcus aureus), and (3) changing the antibiotic regimen to a more narrow-spectrum regimen. For purposes of this investigation, antibiotics were ranked according to their activity spectrum against pneumonia pathogens, emphasizing Gram-negative bacteria  and Gram-positive bacteria   a reduction in the number of antibiotics prescribed, (2) elimination of antibiotic coverage for a class of pathogens  had a clinical pharmacist who reviewed all antibiotic orders to ensure that dosing and interval of antibiotic administration was adequate for individual patients based on body size, renal function, and the resuscitation status of the patient. The duration of antibiotic therapy was at the discretion of the ICU treating team based on the patient’s microbiology results and response to therapy.The initial antibiotic dosages employed for the treatment of bacterial infections at Barnes-Jewish Hospital were as follows: cefepime, 1 to 2 g every 8 hours; piperacillin–tazobactam, 4.5 g every 6 hours; imipenem, 0.5 g every 6 hours; meropenem, 1 to 2 g every 8 hours; ceftolozone/tazobactam, 1.5 or 3 g every 8 hours; ceftazidime/avibactam, 2.5 g every 8 hours; ciprofloxacin, 400 mg every 8 hours; levofloxacin, 750 mg once daily; vancomycin, 15 mg/kg every 12 hours; linezolid, 600 mg every 12 hours; and ceftaroline, 600 mg every 8 hours. Subsequent antibiotic dose and frequency were adjusted, where appropriate.The microbiology laboratory performed antimicrobial susceptibility of the bacterial isolates using the disk diffusion method according to guidelines and breakpoints established by the Clinical Laboratory and Standards Institute and published during the inclusive years of the study . All clat test was used to analyze normally distributed continuous variables, whereas the Mann–Whitney U test was used to analyze non-normally distributed continuous variables. Categorical data were reported as frequency distributions and analyzed using the chi-square test. P values less than 0.05 were considered statistically significant, and all tests were two-tailed. All analyses were done using SPSS Statistics 21 .The sample size was determined by a convenience sample of all mechanically ventilated patients with suspected pneumonia identified in the medical ICUs during the study period. Continuous variables were expressed as mean and standard deviation (SD) or median and interquartile range (IQR) when appropriate. The n = 237; 64.9%), chronic obstructive pulmonary disease , congestive heart failure , renal failure , and altered mental status . HAP occurred in 99 patients (27.1%) and VAP in 29 patients (7.9%). Of the 365 screened patients, 82 (22.5%) were excluded due to the presence of immune suppression, bronchiectasis, or the presence of another non-pulmonary infection requiring antibiotic therapy  was the most common pathogen associated with pneumonia followed by rhinovirus (12.4%), Pseudomonas aeruginosa (9.8%), and influenza A (6.2%).The final study population comprised 283 patients of whom 144 (50.9%) were in the EAD group and 139 (49.1%) in the RAM group. The majority were white men admitted from home or transferred from an outside hospital, with approximately 10% of the patients residing in a nursing home or rehabilitation facility before admission (Table P = 0.001). There was no statistical difference in antibiotic de-escalation according to double empiric coverage for Gram-negative bacteria (n = 10) versus single-agent coverage (n = 273) . Antibiotic escalation took place in 16 patients (5.6%) due to the identification of a pathogen resistant to the initial antibiotic regimen, and no change in antibiotic therapy occurred in 127 (44.9%) patients. Hospital mortality was greatest among patients undergoing antibiotic escalation (50.0%) followed by patients having no change in antibiotic therapy (43.3%), and was lowest for patients receiving antibiotic de-escalation (16.4%) (P < 0.001).Antibiotic de-escalation occurred in 140 patients (49.5%) including a reduction in the number of antibiotics prescribed or elimination of antibiotic coverage for a class of pathogens in 39 patients (27.9%) and changing the antibiotic regimen to a narrower-spectrum regimen in 101 patients (72.1%)  and was similar between study groups Table . Among t5.8% and We found that the addition of an EAD program to a high-intensity ICU staffing model already practicing antimicrobial de-escalation was not associated with greater antibiotic de-escalation or a reduction in the overall duration of antibiotic therapy in patients with suspected pneumonia during mechanical ventilation. We also observed no significant effect on hospital mortality, lengths of stay, or subsequent occurrence of antibiotic-resistant infections.We selected antibiotic de-escalation as the primary outcome for our investigation of an EAD program. However, as emphasized in several recent systematic reviews, antibiotic de-escalation is dependent on multiple factors, such as risk for infection with resistant pathogens, severity of illness, clinical response to initial therapy, site of infection, microorganisms associated with infection, adequacy of source control, and institutional practice patterns , 15. MorOne major area of continuing uncertainty for ICUs implementing antimicrobial stewardship programs is the concept of antimicrobial de-escalation. Further research must be done to determine what defines antimicrobial de-escalation, when it is appropriate, how to integrate infectious disease consultation into the ICU, when to “re-escalate” therapy, and how to transition antimicrobial decision-making when patients move from the ICU to the hospital ward , 17. At Several limitations of our study should be recognized. First, the study design did not allow the research team to supplant the ICU teams in terms of overall antibiotic decision-making. The study team could only make recommendations to the ICU teams on de-escalation. Furthermore, the EAD study team was only available during daytime hours and not during weekends or holidays. This may have had an impact on the study team’s ability to influence overall antibiotic management in patients with pneumonia during the time periods the study team was not available. Second, the EAD team and the ICU teams comprised similar clinicians including intensivists and pharmacists with similar levels of training and experience. This may have contributed to our inability to detect any impact of the EAD program on antibiotic management or clinical outcomes. Based on prior experience, inclusion of an infectious disease specialist or microbiologist might have improved our ability to improve antibiotic utilization with the EAD program , 20. ThiAnother limitation of our study is that the data were derived from a single center, and this necessarily limited the generalizability of our findings. As such, our results may not reflect what one might see at other institutions. For example, all of the ICUs at Barnes-Jewish Hospital are closed units with high-intensity multidisciplinary staffing providing continuous patient care. There is also a long history of antimicrobial stewardship being practiced within these ICUs –23. The In conclusion, we found that the addition of an EAD program to a high-intensity staffing model in medical ICUs was not associated with greater antibiotic de-escalation or a reduction in the overall duration of antibiotic therapy in mechanically ventilated patients with suspected pneumonia. Future studies are needed to determine whether EAD programs can impact antibiotic management in other types of ICUs which may have a lesser focus on de-escalation practices and ICUs with different staffing models. Additionally, identifying the optimal methods for carrying out antimicrobial de-escalation in the ICU setting, to include the integration of infectious disease experts and rapid diagnostics, deserves further investigation. Until such studies are performed, critical care clinicians should incorporate antimicrobial review strategies into their daily practice in order to balance the need for administering IAAT and the avoidance of unnecessary antibiotic utilization.