Lactobacillus populations. Furthermore, antibiotic treatment resulted in a significant decrease in the tolerogenic CD11c+/CD11b+/CD8α- mesenteric lymph node DCs independent of tolerizing treatment. These results provide evidence that antibiotic treatment, potentially through its effects on tolerogenic DCs and intestinal microflora, may contribute to autoimmune and atopic disorders via a breakdown in tolerance and support prior epidemiologic studies correlating increased antibiotic usage with the development of these disorders.The mucosal immune system is constantly exposed to antigen, whether it be food antigen, commensal bacteria, or harmful antigen. It is essential that the mucosal immune system can distinguish between harmful and non-harmful antigens, and initiate an active immune response to clear the harmful antigens, while initiating a suppressive immune response (tolerance) to non-harmful antigens. Oral tolerance is an immunologic hyporesponsiveness to an orally administered antigen and is important in preventing unnecessary gastrointestinal tract inflammation, which can result in a number of autoimmune and hypersensitivity diseases. Probiotics , T regulatory cells, and dendritic cells (DCs) are all essential for generating tolerance. Antibiotics are commonly prescribed to fight infections and often necessary for maintaining health, but they can disrupt the normal intestinal probiotic populations. There is increasing epidemiologic evidence that suggests that antibiotic usage correlates with the development of atopic or irritable bowel disorders, which often result due to a breakdown in immune tolerance. This study investigated the effect of the antibiotic erythromycin on oral tolerance induction to ovalbumin. The results demonstrated that antibiotic treatment prior to exposure to fed antigen prevents tolerance to that antigen, which may be associated with a reduction in intestinal  The mucosal immune system is the first line of defense for the body to eliminate antigens. This system favors the induction of a suppressive immune response, one that is tolerant to antigen instead of mounting an active immune response against it, and promotes the induction of oral tolerance . The phe+/CD25+ T cells that express Foxp3 and suppress immune responses toward self-antigens, allergens, food antigens, and other non-harmful antigens  and IL-10  tract . These iacterium . Studiese models . L. parand IL-10 . Importaon VSL#3  and the ntiation . Furtherntiation .Disruption of the intestinal microflora, particularly depletion of probiotic species, can lead to a variety of consequences such as diarrhea or yeast infections that are common side-effects of antibiotic treatment . The effWhile there are increasing numbers of clinical and epidemiologic studies on the benefits of probiotics and their influence on mucosal integrity and immune processes, there is little research providing information about the effect of antibiotic treatment on probiotic populations and the subsequent consequence on specific immune responses. It is possible that the development of atopic disorders and irritable bowel disorders following antibiotic treatment results from a breakdown in tolerance, which may occur due to the depletion of probiotic species. The goal of this study was to examine the effect of antibiotic treatment on oral tolerance induction to ovalbumin (OVA). Our results demonstrate that a 7-day course of the broad-spectrum antibiotic erythromycin, immediately followed with a single tolerizing dose of OVA, is sufficient to impair tolerance induction to OVA. Concomitantly, Treg and tolerogenic DC populations in the MLNs were examined to identify alterations that may correlate with the hindrance in tolerance.Balb/c mice between the ages of 8 and 12 weeks, bred from mating pairs purchased from The Jackson Laboratory , were used for each study. Mice were housed individually in cages and separated into four treatment groups, with no fewer than three mice per group and with equal numbers of each sex between groups. Methods involving mice were approved by the Ball State University Animal Care and Use Committee.n = 3–4/group for each experiment): NT, OT, NTAb, and OTAb . On each of days 1–5, all mice were administered a probiotic solution to establish an intestinal probiotic flora. Mice were allowed to rest for 2 days, and then on day 8 fecal matter was collected from all mice, plated, and feces cultured to determine intestinal microbial numbers pre-antibiotic treatment. On days 8–13, the antibiotic erythromycin was consecutively administered to NTAb and OTAb mice with water given to NT and OT mice. On day 14, a high-dose of OVA was added to the water (OT) or antibiotic solution (OTAb) of the OT and OTAb mice to induce tolerance. On day 15, all treatments were discontinued and fecal matter was collected from all mice, plated, and feces cultured to determine intestinal microbial numbers post-antibiotic treatment. On day 19, five days after OVA treatment, all mice were immunized with OVA, and they were again immunized on day 26. On day 33, all mice were sacrificed and their serum and MLNs isolated.For each experiment, there were four treatment groups  plates, which select for Lactobacilli. Plates were incubated at 37To ensure an established probiotic population in the GI tract of all mice prior to the start of the experiment, the probiotic capsules Acidophilus with Pectin (The Vitamin Shoppe) were dissolved in 3 mL of 10% sugar water per capsule. About 300 μL of the probiotic solution was administered via syringe feeding (placing tip of a syringe with no needle into the mouth of the mouse and administering the solution drop-wise into the mouse’s mouth) to each mouse for five consecutive days. For all other treatments, mice were fed via intragastric gavage using ball-tipped feeding needles . Mice received 5 mg erythromycin (Sigma) in 200 μL water total volume (antibiotic-treated groups) or water alone (control) daily for seven consecutive days. On day 7, 20 mg of OVA (Sigma) was added to the 200 μL antibiotic or water solution and administered only to the treatment groups that were to be tolerized to OVA.Five days after ceasing control and experimental feeding treatments, all mice were immunized intraperitoneally with 0.1 mg of OVA (Sigma) prepared in phosphate buffered saline (PBS) with equal parts alum (Thermo Scientific) to stimulate an immune response. The final injection volume was 200 μL. One week after the initial immunization, all mice were again immunized with 0.1 mg of OVA.2 asphyxiation. Blood was collected by cardiac puncture and blood serum was isolated. Levels of OVA-specific IgG present in the serum were detected using an indirect enzyme-linked immunosorbent assay (ELISA). Plates were coated with 400 μg/100 μL/well OVA (Sigma) in coating buffer  for 2 h at room temperature (RT). Plates were washed three times in wash buffer (Bethyl Laboratories) between every step. Plates were then blocked for 30 min with blocking solution (Bethyl Laboratories). Serum from each mouse was diluted 1:100, 1:5000, 1:2500, and 1:12,500 for the IgG ELISA, and 1:10, 1:100, and 1:1000 for the IgE ELISA. Samples were then added to the plate, and incubated for 2 h at RT. Rat anti-mouse IgG-AP or rat anti-mouse IgE-AP, human adsorbed  was diluted 1:1000 and used for detection of the appropriate antibodies. Samples were analyzed in duplicate using a microplate reader .One week following the second immunization, all mice were sacrificed by CO-5 M 2-mercaptoethanol . The resulting cell suspensions were washed in RPMI-1640 complete medium, and then red blood cell lysis was performed for 5 min, followed by two washes in RPMI-1640. Cells were then immediately re-suspended in FACS buffer (1× PBS with 2% BSA and 0.1% NaN3) and immediately stained for flow cytometric analysis.Following cardiac puncture, MLNs were harvested from each mouse and homogenized in RPMI-1640 (Sigma) supplemented with 10% heat-inactivated FBS , penicillin–streptomycin, sodium pyruvate, non-essential amino acids, L-glutamine, HEPES, and 5 × 10°C. All staining was done for 10–15 min at 4°C followed by two washes with FACS buffer, and then fixed in PBS with 2% BSA, 0.1% NaN3 and 0.5% formaldehyde (fix buffer). Samples were incubated with antibodies (specific for the cell surface proteins described in each experiment) conjugated to FITC, PE, or CyChrome . To enumerate Tregs, cells isolated from MLNs were stained using a Treg identification kit (eBioscience) according to the manufacturer’s instructions. Cells were analyzed on an Accuri C6 flow cytometer using CFlow Plus software .Multi-color flow cytometric analysis was performed to identify populations of DCs and Tregs in the MLNs. To enumerate DC populations, cells isolated from MLNs were resuspended in FACS buffer. Cells were then blocked with 15 μg rat serum in 5 μL FACS buffer for 5 min at 4t-test was performed on pre- and post-treatment colony counts. To determine statistically significant differences in levels of OVA-specific antibodies, data from the ELISAs were analyzed by one-way ANOVA followed by Student–Newman–Keuls post hoc analysis. To determine statistically significant differences in percentages of DCs and Tregs present in MLNs, the data from flow cytometric analyses were analyzed by one-way ANOVA followed by Student–Newman–Keuls post hoc analysis or Kruskal–Wallis ANOVA on ranks. Differences between groups were considered statistically significant at p ≤ 0.05.All statistical analyses were performed using Sigma Plot . Results are represented as mean ± SEM. To determine significant decreases in intestinal bacteria following antibiotic treatment, a Student’s paired The goal of this study was to demonstrate the effect of antibiotic usage on oral tolerance induction and tolerogenic immune populations in the MLNs using OVA as the antigenic stimulus.Figure 1A). However, in the OTAb group OVA-specific IgG was markedly elevated from the tolerized control, and very similar to the IgG levels observed in the NT control group . OTAb levels of OVA-IgG that are comparable to the NT group demonstrate an inability to suppress an antibody response to OVA, suggesting that tolerance to OVA was not achieved in the antibiotic-treated group.Antibiotic treatment has been implicated in the development of various autoimmune diseases in humans such as allergy, asthma , and CroFigure 1B), demonstrating that an IgE response was not generated to OVA in our model system.It has previously been demonstrated that germ-free mice produce IgE, generating allergic reactions to orally fed antigens, but replacement of individual intestinal microbial species restores oral tolerance . FurtherLactobacillus is a model probiotic genera .One major complication of antibiotic treatment is its alteration of intestinal microbial communities . Alteredc genera  and can bacillus . Fecal s+/CD11b+/CD8α- DCs, CD11c+/CD103+ DCs, and CD11c+/MHC Class II+/CD103+ DCs , demonstrating that antibiotic treatment alone may be altering populations of tolerizing DCs in the MLNs.To explore the mechanisms behind this hindrance of tolerance induction, subsets of tolerogenic DCs were examined. While tolerogenic DC subsets differ between various blood and lymphoid tissues, the tolerogenic subsets found in the MLNs are CD11c103+ DCs . Examina+/CD103+ DCs and CD11c+/MHC Class II+/CD103+ DCs were examined. There were no significant differences between treatment groups for CD11c+/CD103+ DCs  or CD11c+/MHC Class II+/CD103+ DCs . To identify any alterations that may occur shortly after tolerance induction, all three MLN DC subset populations were enumerated 24 h after oral administration of OVA. No significant differences between treatment groups was observed in any of the DC populations at this time point (data not shown). Taken together, these data suggest that there is no direct association between the inability to induce tolerance after reduction in intestinal Lactobacilli and tolerogenic DC populations in the MLNs, although antibiotic treatment alone may be sufficient to decrease some tolerogenic DC populations.To further investigate the effect of tolerance and antibiotic treatment on other DC populations in the MLNs, populations of CD11c+/CD4+/CD25+/FoxP3+ Treg populations in the MLNs were enumerated. There were no differences observed in the MLN Treg populations between the different treatment groups 14 days  or 24 h (data not shown) after tolerance induction, suggesting there is no direct link between alterations in total Treg populations and the hindrance of tolerance following antibiotic treatment.Because DCs in the MLNs have a primary role in naïve T cell differentiation, specifically Treg differentiation, CD3+/CD11b+/CD8α- MLN DCs in both the tolerized and non-tolerized antibiotic-treated mice, indicating that antibiotic treatment alone may be sufficient to alter intestinal mucosal DC populations. These are novel studies that provide evidence of antibiotic effects that exceed “common” side-effects and can contribute to the breakdown of normal tolerant immune responses.Given the emerging evidence highlighting the importance of probiotics and the normal intestinal flora in health and disease, this study sought to examine alterations in immune mechanisms brought about by antibiotic treatment. The data presented in this study demonstrate that treatment with erythromycin prior to the oral introduction of antigen hinders the induction of oral tolerance, such that mice tolerized to antigen after antibiotic treatment have an immune response that is comparable to non-tolerized controls, and this may be associated with the alteration in normal intestinal flora due to antibiotic treatment, as intestinal populations of Lactobacilli were reduced following erythromycin treatment. Furthermore, this study examined populations of DCs and Tregs in the MLNs to identify changes in those cellular populations that may correlate with the inability to induce tolerance in the antibiotic-treated mice. Although no significant alterations in DC or Treg populations were identified following antibiotic and tolerance treatments, there was a decrease in tolerogenic CD11cThe effects of antibiotic treatment on oral tolerance have been examined previously. The role of probiotics in oral tolerance induction has been debated, and this study provides further information for this debate. Figure 5), indicating that there is no correlation between alterations in the Treg populace and the hindrance of tolerance in the antibiotic-treated group. However, there are two limitations to our study that may contribute to this finding. First, total Treg populations, and not OVA-specific populations, were examined. It is possible that alterations in the OVA-specific Treg population exist while total Treg populations remain unchanged. Additionally, it is possible that the method of oral tolerance induction in our study design contributed to a lack of observable change in Treg populations. Our study utilized the high-dose method for tolerance induction. While this was the best method of inducing tolerance in our model, it can be argued that this mechanism for initiating oral tolerance leads to the deletion of autoreactive cells and of Tregs. It has previously been demonstrated that the feeding dosage of antigen has a profound effect on the mechanism by which oral tolerance is achieved. While animals fed with a low-dose (1 mg) of antigen over several days induced tolerance via active suppression of immune cells and increased secretion of the TGF-β, animals fed with a one-time high-dose (20 mg) of antigen induced tolerance by anergy and an increased secretion of the cytokine IL-4  that correlates with a hindrance in oral tolerance induction. No changes in the MLN Treg populations were observed between treatment groups , suggesting that the antibiotic treatment itself, excluding the tolerizing antigen, has an effect on this tolerogenic DC subset. It is possible that this is due to alterations in the intestinal epithelium as a result of the antibiotic treatment. Disruption of the intestinal probiotic population can result in intestinal alterations and inflammation .Interestingly, antibiotic treatment in both the non-tolerized and oral-tolerized treatment groups resulted in a decrease in CD11cammation . It is pIn conclusion, this was a novel study that demonstrated a break in tolerance resulting from a 7-day course of erythromycin that was associated with a decrease in a subset of tolerogenic DCs potentially and may involve alterations in the normal intestinal microflora. Our findings, coupled with the finding that antibiotic treatment-induced alterations of intestinal microflora extend past antibiotic treatment and permanently alter the composition of the microflora , supportThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In oral implantology, there is no consensus on the most appropriate regimen for antibiotics prescribing, the decision to prescribe antibiotic is usually based on procedure, patient and clinician related factors. The aim of this study was to investigate the rationale of antibiotic prescribing among Jordanian clinicians who practice oral implantology.The target sample for the study was the 250 Jordan Dental Implant Group members. A five page questionnaire contained 41 questions, both closed and open questions were used to collect data. Statistical analysis was performed using SPSS Windows 16.0 . Descriptive statistics were generated.The response rate was (70.4%) 176/250. Mean age was 37.2 yrs, 49.4% always prescribe antibiotics mainly oral amoxicillin and amoxicillin with clavulinic acid. Antibiotics prescribing increased with flap raising, multiple implants and sinus or bone augmentation. Patient medical condition, periodontitis and oral hygiene were the most important clinical factors in antibiotic prescribing, non-clinical factors were; reading scientific materials, courses and lectures, knowledge gained during training, and the effectiveness and previous experience with the drug.Wide variations in antibiotics types, routes, dose and duration of administration were found. Recommendations on antibiotic prescribing are needed to prevent antibiotic overprescribing and misuse. The practice of oral implantology has been expanding widely over the last few decades, and more patients and dental practitioners are showing interest in this field ,2. High 20. The clinician decision to prescribe an antibiotic or not for a certain procedure is usually based on several factors, some factors are procedure related; the type, site, complications, sterility and duration of the procedure [Over prescribing antibiotics has a negative results on the general health and economy, therefore the proper selection of antibiotic regimen in clinical practice has a great value rocedure ,22, patirocedure ,22 and crocedure . Regulatrocedure -30, the rocedure ,32.In Jordan, oral implantology is practiced in the private sector, the two University Hospitals, the Royal Military Medical Services Hospitals and recently in some of the public hospitals. Jordan is a Middle Eastern country with a population of around 6 millions served by around 7000 dentists. Although it is not obligatory, most dentists who practice oral implantology in Jordan are members of the Jordan Dental Implant Group (JDIG). It had been shown that the Jordanian general dental practitioners (GDPs) inappropriately prescribed antibiotics and poorly adhered to recommended guidelines for optimum dosage and course duration . No prevThe aim of this study was to investigate the rational of antibiotic prescription among Jordanian clinicians who practice oral implantology, and to investigate the influence of the procedure, patient and clinician factors on the selection of the type, dose, duration and method of administration of the antibiotic.The study was an observational study based on information collected from dentists who are members in the Jordan Dental Implant Group, therefore no ethical approval was obtained and the participants were not consented for participation in the study. The target sample was the JDIG members, as most of the Jordanian dentists who practice oral implantology are JDIG members and JGDI represent all sectors that provide the oral implantology service in Jordan. The exact number of dentists who practice oral implantology is not known but it is not expected to exceed significantly the number of the JDIG members which is currently 250, 137 are general dental practitioners (GDP) and 113 are specialists; mainly oral surgeon, prosthodontists and periodontists. A list of the names addresses and telephone numbers of the JGDI members was obtained from the JDIG, members were informed about the study by a telephone call and if agreed, a specifically designed questionnaire was sent by hand and collected a week later. Members who did not complete the questionnaire after the first week were reminded to do so by a telephone call after two weeks and again after four weeks. In case of no positive response after the third follow up call the member was considered non-responsive.The 5 page questionnaire  affect his or her decision. The fourth section was composed of two parts, the first part included open questions where clinicians are asked to write the type, dose, method of administration and duration of the antibiotic they routinely prescribe preoperatively, postoperatively or both in dental implant insertion for healthy individuals not allergic to any medications. In the second part the clinicians where asked if their choice of an antibiotic regimen was affected by the patient preference, reading scientific materials, knowledge gained during undergraduate or postgraduate training, attending courses or lectures, availability of the drug in the nearby pharmacy, advertisement, cost of the antibiotic, recommendation of other colleagues, previous experience with the drug and to specify if there were other factors.Statistical analysis was performed using SPSS for Windows release 16.0 . Descriptive statistics were generated.Of the 250 JDIG members to whom the structured questionnaires were distributed, 176 (70.4%) returned answered questionnaires. Four of these answered questionnaires were excluded because of missing data. The demographic and professional characteristics of the 172 respondents are shown in Table Amoxicillin plus Clavulanic acid or amoxicillin alone were the most common preoperative and postoperative antibiotics prescribed, the routes of administration, the dosages, frequencies and the length of the courses are shown in Table The sample was representative of all the sectors that provide the oral implantology service in Jordan, and it was clear from the results that young practitioners had more interest in oral implantology and slightly less than half of the service providers were specialists with postgraduate degrees, most of them work in the private sector and many were interested in continuous education. Similar findings related to the increased interest of young generations in oral implantology had been found in other studies in Hong Kong  and SwitAntibiotics prescribing was influenced by flap raising, number of implants inserted, the timing of implant insertion in the presence or absence of active infection, bone augmentation and sinus lifting procedures. Although no evidence could be found in the literature, the participants in this study had considered the flap type, number of implants and the timing of implant insertion as factors for antibiotic prescribing. An interesting finding in the study was that 13% of the participants prescribe antibiotics at time of gingival former insertion and more interesting was that around 8% prescribe antibiotics at time of impression taking and at time of crown delivery. On the other hand, regarding timing of antibiotic prescribing, although the pre-operative administration of 2g amoxicillin had been recommended to reduce chances of implant failure ,36, and The participants in this study were nearly equally divided on whether antibiotics should always be prescribed prior to implant insertion regardless of any possible related factors. This does reflect the conflicting results and opinions found in the literature regarding oral implantology and antibiotics -16. For Wide variations in the types, routes, dose and duration of administration of antibiotics were found in the study, both amoxicillin and amoxicillin with calvulinic acid were most frequently used antibiotics pre-operatively or postoperatively. The two antibiotics are widely used in oral implantology and their role in reducing implant failure was investigated in several clinical trials ,19,35,36Other antibiotic regimen used included clindamycin, lincomycin, metronidazole, penicillin, cephalosporins, and some participants followed antibiotic regimens which included a combination of two or three drugs. One familiar combination which had been widely used in dentistry, was amoxicillin and metronidazole -39, but Based on the result of this observational study, the main oral implantology service providers were young clinicians, many possess high level of education and had put effort to obtain knowledge mainly by reading scientific materials and attending courses. Despite this, wide variations in antibiotic prescribing practices were found, and some practices may not be justified as it might be considered as antibiotic overprescribing and more importantly might be harmful on the patient. In the authors opinion, recommendations on antibiotic prescribing are needed from international oral implantology regulating bodies based on the available evidence in the literature to help clinicians avoid antibiotic misuse, and meanwhile it might be sensible to suggest that for dental implant insertion, clinicians might give no antibiotics, a single preoperative dose or a short postoperative course.The authors declare that they have no competing interests.All authors had reviewed and accepted the final manuscript. AA contribution was in literature review, designing the study, data analysis, write up and editing; KAA contributed in designing the study; AK, NK, HH contributed in collecting and entering data; AH contribution was in write up and editing and FS contributed in data analysis.Questionnaire. 5 page questionnaire composed of four sections and contains 41 questions.Click here for file


Bombyx mori, participate in 20E signal transduction. Met is 20E responsive and its expression peaks during molting and pupation, when the 20E titer is high. As found with results from RNAi knockdown of EcR-USP (the ecdysone receptor genes), RNAi knockdown of Met at the early wandering stage disrupts the 20E-triggered transcriptional cascade, preventing tissue remodeling  and causing lethality during the larval-pupal transition. MET physically interacts with EcR-USP. Moreover, MET, EcR-USP and the 20E-response element (EcRE) form a protein-DNA complex, implying that MET might modulate 20E-induced gene transcription by interacting with EcR-USP. In conclusion, the 20E induction of MET is required for the maximal action of 20E during Bombyx metamorphosis.Little is known about how the putative juvenile hormone (JH) receptor, the bHLH-PAS transcription factor MET, is involved in 20-hydroxyecdysone  action. Here we report that two MET proteins found in the silkworm,  Drosophila melanogaster, Methoprene-tolerant (MET), a bHLH-PAS transcription factor in vitroMet and gce null single mutants are fully viable, Met gce double mutants die during the larval-pupal transition Tribolium castaneum, MET plays a similar key role in JH action during the larval-pupal metamorphosis Tribolium, suggesting strongly that MET is the actual JH receptor The molting hormone, 20-hydroxyecdysone (20E), and juvenile hormone (JH) coordinately control insect molting and metamorphosis. Overall, 20E orchestrates the molting process, whereas JH determines the nature of the molt. In the fruitfly, Br-C, E74, E75, and E93) and, subsequently, the 20E secondary-response genes Br-C, E74, E75, E93 and other 20E response genes positively impact 20E signaling. For example, E93 binds to many 20E response genes and cell death genes on polytene chromosomes. The expression of these genes is defective in E93 mutants, while E93 overexpression results in the upregulation of these genes A great deal more is known about the 20E signal transduction pathway. The 20E nuclear receptor complex is a heterodimer composed of ecdysone receptor (EcR) and ultraspiracle (USP) DrosophilaAedes aegyptiAedes, Drosophila, and Tribolium, suggesting a role in enhancing JH-20E crosstalk One major function of JH is to inhibit some of the actions of 20E EcR-USP RNAi) during the early wandering stage in the silkworm, Bombyx mori. EcR-USP RNAi was shown to disrupt the 20E-triggered transcriptional cascade, preventing tissue remodeling and resulting in lethality during metamorphosis Met (Met RNAi) during this stage resembles the data resulting from EcR-USP RNAi. MET physically interacts with EcR-USP, which forms a protein-DNA complex with the 20E-response element (EcRE) supporting the conclusion that MET is required for the maximal action of 20E during metamorphosis in Bombyx.Previously, we performed RNAi knockdown studies of the ecdysone receptor  . The developmental profiles show that Met mRNA levels reach a small peak during the 4th larval molt and are very high during the larval-pupal transition   [30]. Meansition , suggestr larvae  and S1B.ng stage . Furtherectively . In geneMet RNAi (10 µg dsRNA per larva) was performed at the initiation of the early wandering stage. Met RNAi resulted in lethality during the larval-pupal-adult metamorphosis, with a higher percentage of lethality occurring from Met2 RNAi (∼80%) compared to Met1 RNAi (∼50%)  . Althoug thinner , and the thinner . Some Meng stage  or durinng stage , while sng stage . OverallThrough the ecdysone receptor, the 20E-triggered transcriptional cascade is important in removing obsolete larval tissues via programmed cell death  and generating adult structures from progenitor cells during metamorphosis Met RNAi treated animals results in lethal phenotypes similar to those observed in EcR-USP RNAi treated animals, we investigated the effects of Met RNAi on larval tissue remodeling during Bombyx metamorphosis to determine the possible role of MET in PCD. Eighteen hr after treatment, Met RNAi significantly prevented apoptotic events in the fat body as estimated by TUNEL labeling and quantification of caspase 3 activity Met RNAi nearly abolished autophagy, as estimated by LysoTracker staining Met RNAi dramatically inhibited fat body cell dissociation  and they had shortened and distorted legs or unexpanded wings  were stronger than for Met1 RNAi (50–90%)  . Moreove(50–90%)  as well (50–90%)  revealedt dsRNAs  which ext dsRNAs . Since ttal data , we stilegfp RNAi treated fat body, while this upregulation was dramatically decreased when Met2 RNAi was performed  and USP strongly associate with one another. Weak associations were observed between MET1 and MET2, MET1 and MET1, and MET2 and MET2, while intermediate associations were formed between the two MET proteins and the ecdysone receptor  but EcR-USP did . Thus, it appears that the 20E induction of those 20E-response genes (including the Met genes) is required for the maximal action of 20E to induce gene expression in Bombyx. In Drosophila, mutation of Br-C, E74, E75, or E93 interrupts the 20E signaling, with more pronounced effects in E93 mutants. It has been well documented that the 20E induction of E93 determines a PCD response by positively impacting the 20E signaling Both Kr-h1 expression and to antagonize 20E-induced Br-C expression Met and gce mutants. It might be incomparable between the function of Met and gce in Drosophila and that of Met1 and Met2 in Bombyx, since the duplication events in these two insect species are evolutionary independent Tribolium, Met RNAi during the early quiescent stage also disrupts the 20E-triggered transcriptional cascade, showing that the single MET protein in Tribolium and the two MET proteins in Bombyx have similar functions in modulating 20E signaling during metamorphosis.Previously, we demonstrated that MET and GCE are functionally redundant in transducing JH signal to induce Bombyx. Our preliminary results suggest that MET might bind transcriptional co-activators, including CBP/p300 CBP/p300 RNAi attenuated, but did not abolish, 20E-induced luciferase activity driven by EcRE, suggesting that the receptor complex may consist of other histone-modifying transcriptional co-activators. A good candidate is p160/SRC/NCoA, which has been demonstrated in Aedes, Drosophila, and TriboliumWe have also tried to dissected out the molecular mechanism how MET is required for the maximal action of 20E in Bombyx MET2 might act as a JH receptor. In the presence of JH, MET2 associates with SRC, the p160/SRC/NCoA-like molecule in Bombyx, to interact the JHRE in inducing Kr-h1 expression Very recently, it has been documented that the Bombyx (P50) TriboliumBombyx DZNU-Bm-12 cells Met1 and Met2 cDNA sequences (GenBank accession numbers: EU249371 and EU249372) were cloned using RACE. Details of qPCR and Western blotting were previously described The full-length Bombyx  was more sensitive to RNAi treatments than the other tested strains Bombyx was used throughout this study. RNAi knockdown was performed at two developmental stages, including initiation of the early wandering stage and 6 hr after pupation. After the RNAi treatment , fat body from the abdominal segments was collected for bioassays. Three biological replicates were used, each of which consisted of 10 silkworms. The details of hormone treatment in vivo  were previously described dsRNAs were generated using the T7 RiboMAX™ Express RNAi System (Promega). Preliminary data showed that the P50 strain of RNAi knockdown in DZNU-Bm-12 cells was performed using the Effectene transfection reagent (Qiagen) for 48 hr at a final concentration of 2 µg/ml dsRNA. To determine the 20E primary-response genes, 1 µM 20E and 10 µg/ml cycloheximide (Sigma Aldrich) were used Bombyx MET1 and Br-C antibodies were produced by the Abmart Company (Shanghai). A cDNA fragment encoding amino acids 151M to 350Q of MET1 and the full-length Br-C Z4 cDNA were expressed in E. coli and their protein products were purified. Antigen-purified rabbit polyclonal antibodies against MET1 and Br-C were generated. The AB11 USP-specific monoclonal antibody was provided by Dr. K.F. Kafatos (Harvard University). The monoclonal antibodies against the V5 tag (Sigma Aldrich), cMyc tag (Santa Cruz), and Tubulin (Invitrogen) were also used.The th abdominal segment by immunohistochemistry with the above primary antibodies. The fluorescein-conjugated secondary antibodies (Jackson ImmunoResearch) were FITC-conjugated Affinipure Goat Anti-Mouse IgG for USP and Cy3-conjugated Affinipure Goat Anti-Rabbit IgG for Br-C. Fluorescence signals were detected using the Olympus Fluoview FV1000 confocal microscope.USP and Br-C were detected in explanted fat body from the 5cdc25 allele (cdc25H) in yeast when Sos is targeted to the plasma membrane through bait-prey interactions. This system has been well characterized for protein-protein interaction studies between transcription factors and their associated proteins (SR6). First, the full length MET1, MET2, EcR-B1 (EcR) and USP1 (USP) were amplified from silkworm genome, then these genes were cloned into bait or prey vector. MET1, MET2, EcR or USP was expressed as a fusion protein with human Sos as the bait protein. On the other hand, MET1, MET2, EcR and USP were expressed as prey proteins fused with a myristoylation (Myr) signal, targeting the proteins to the cell membrane. Expression of the prey is controlled by the GAL1 promoter, which is induced on galactose, and repressed on glucose medium. When bait and prey are co-transformed into the cdc25H strain, the only cells capable of growing at restrictive temperatures on galactose medium are those that have been rescued by the bait-prey interactions that recruit Sos to the cell membrane.Yeast two-hybrid assays were carried out using the CytoTrap system (Stratagene), which is based on the ability of human Sos to complement a temperature-sensitive V5-Met1 and cMyc-Met2 was pEGFP-N1 (Clontech) under the control of the ie1 promoter. After transfection, cells were treated with 1 µM 20E, followed by immunoprecipitation, qPCR, and luciferase assay.Transient transfection assay in DZNU-Bm-12 cells was carried out for 48 hr using Effectene according to the manufacturer's instructions. The final DNA concentration was 2 µg/ml, and the DNA∶Effectene ratio was 1∶25. The vector used to overexpress MET1, MET2, EcR-B1 (EcR) and USP were amplified from the silkworm genome, and the HA, FLAG, V5, cMyc tags were fused in their 5-ends, respectively and cloned into the pcDNA 3.1(+) vector (Invitrogen). After transfection, the cells were harvested for immunopreciptation, luciferase assay and EMSA.Transient transfection assays in HEK 293 cells were performed using Lipofectamine 2000 (Invitrogen). The full length After treatment, DZNU-Bm-12 cells and HEK 293 cells were harvested and lysed in ice-cold NP-40 lysis buffer (Beyotime). Lysates were incubated with FLAG, V5, or cMyc antibody or IgG for 4 hr, followed by incubation with protein G  overnight at 4°C. After extensive washing with cold NP-40 buffer, the samples were treated with RIPA lysis buffer (Beyotime) about 15min on the ice. Then immunoprecipitates were separated by SDS-PAGE and analyzed by Western blots after measured the protein concentration by the enhanced BCA protein assay kit (Beyotime).GACAAGGGTTCAATGCACTTGTC) and a hsp70 mini promoter was used for the luciferase reporter. And the reference pRL vector (Promega) carrying Renila-luciferase driven by actin3 promoter was co-transfected into the cell with the reporter vector. The dual luciferase double reporter assay system and substrates were purchased from Promega.Luciferase assays were carried out using the Dual Luciferase Assay System (Promega) and a Modulus Luminometer (Turner BioSystems). The reporter pGL3 vector (Promega) containing four repeated EcRE sequences . The minimal EcRE  was biotin-labeled as a probe using the Biotin 3′ End DNA Labeling Kit (Pierce). After binding, the nuclear extract (15 µg) containing the biotin-labeled EcRE and the protein-DNA complexes were separated on 5% nondenaturing PAGE gel. HA, FLAG, V5, and cMyc antibodies were added to the nuclear extract to detect the shift of EcRE. EMSA was performed using the LightShift Chemiluminescent EMSA Kit (Pierce).The Figure S1The diagram of the three sets of Met dsRNA and confirmation of the MET and Br-C antibodies. (A) The diagram illustrates the three sets of Met dsRNA. Red bar: #1 set of Met1 (491–916) and Met2 (491–916) dsRNA; green bar: #2 set of Met1 (141–586) and Met2 (1925–2336) dsRNA; yellow bar: #3 set of Met1 (948–1348) and Met2 (245–669) dsRNA. (B and C) Western blotting confirmation of the MET1 and Br-C antibody after Met1 and Br-C RNAi. The arrow points to the MET1 protein and the Br-C protein isoforms with ideal molecular weights. efgp RNAi was used as a control. Tubulin was used as a loading control.(PDF)Click here for additional data file.Figure S2Met RNAi prevents removal of obsolete larval tissues and generation of adult structures. dsRNA (10 µg per larva) was injected into larvae during initiation of the early wandering stage. More than 30 silkworms were used in each group. egfp dsRNA was used as a control. (A) Met RNAi larvae form thinner cocoons. Cocoon images were collected after the silkworms stopped spinning. (B) Met RNAi prevented silk gland lysis 24 hr after pupation. The inhibiting effects, particularly on the middle silk gland, by Met2 RNAi were stronger than Met1 RNAi. (C) Met1 RNAi affected adult structure formation. Many of the surviving Met1 RNAi treated adults exhibited shortened and distorted legs (left panel) or unexpanded wings (right panel).(PDF)Click here for additional data file.Figure S3Met RNAi disrupts the 20E-triggered transcriptional cascade during the early wandering stage and in DZNU-Bm-12 Cells. Three biological replicates were used and one was represented (A–C). In each biological replicate, more than 10 larvae were used (A and B). egfp dsRNA was used as a control. (A) The other two sets (#2 and #3) of Met dsRNA (30 µg per larva) also disrupt the 20E-triggered transcriptional cascade during initiation of the early wandering stage. See Met dsRNA. (B) 20E treatment fails to induce expression of 20E-response genes in fat body explanted from the Met2 RNAi silkworms during the early wandering stage. (C) Met1 RNAi disrupts the 20E-triggered transcriptional cascade, except Met2 whose expression level is extremely low, in Bombyx DZNU-Bm-12 cells. RNAi knockdown was performed using the Effectene transfection reagent (Qiagen) for 48 hr at a final concentration of 2 µg/ml dsRNA. The cells were treated with 20E for 6 hr at a final concentration of 1 µM.(PDF)Click here for additional data file.Figure S4Met is required for 20E action in Tribolium. RNAi knockdown of either EcR or Met (∼4 ng per larva) in Tribolium during the early quiescent stage resulted in lethality (A), significantly delayed the larval-pupal transition (A and B), and disrupted the 20E-triggered transcriptional cascade (C). egfp dsRNA was used as a control. (A) Larval, prepupal, and pupal numbers were counted 24 and 48 hr after RNAi treatment. Total lethality caused by egfp, Met and EcR dsRNAs was compared. (B) Phenotypic images were collected from the above experimental animals 24 (left) and 48 hr (right) after RNAi treatment. (C) Met, EcR, USP, E74, and Br-C mRNA levels, as determined by qPCR, were significantly down-regulated 24 hr after Met RNAi.(PDF)Click here for additional data file.Figure S5The negative controls for the IP and EMSA experiments. (A) The HA-EcR, FLAG-USP, and V5-Met1 constructs were co-transfected into human HEK 293 cells, the cells were treated by 20E for 6 hr at a final concentration of 1 µM. The negative control IgG was not able to pull down HA-EcR, FLAG-USP, and V5-Met1. IP, immunoprecipitate; Blot, Western blot. (B) The HA-EcR and FLAG-USP or V5-Met1 and cMyc-Met2 constructs were co-transfected into the human HEK 293 cells. After nuclear extracts were bound with biotin-labeled EcRE, the protein-DNA complexes were separated on a 5% native PAGE gel followed by EMSA. The shift was indicated by a black arrow in comparison with a gray arrow.(PDF)Click here for additional data file.Table S1A list of all primers used in this paper.(DOC)Click here for additional data file.


Rabies was undetected in terrestrial wildlife of northern Arizona until 2001, when rabies was diagnosed in 19 rabid skunks in Flagstaff. Laboratory analyses showed causative rabies viruses associated with bats, which indicated cross-species transmission of unprecedented magnitude. Public health infrastructure must be maintained to address emerging zoonotic diseases. Mephitis mephitis) and gray foxes (Urocyon cinereoargenteus) maintain independent rabies enzootic cycles, and in indigenous bats, rabies has been diagnosed in 14 of 28 species . Although skunks live throughout Arizona, until 2001, rabid skunks had been found only in the southeastern quadrant of the state.In North America, >90% of cases of rabies in animals occur in wildlife  and late winter (when they are mating).The Flagstaff epizootic peak coincided with nationwide seasonal trends of rabid skunks (Eptesicus fuscus) and Myotis bats in the western United States  bats shared >95% identity with Flagstaff skunk RABVV. An additional 44 samples, representing 11 bat species, differed by >8% from Flagstaff skunk RABVV.Viruses isolated from the rabid skunks exhibited monoclonal antibody patterns similar to RABVV associated with big brown  collected in 1999 and 1997 than to the 2 bat RABVV collected locally during the outbreak. In clade B, subclade 1 RABVV were collected from January through early April from the northeastern region of the outbreak, whereas subclade 2 RABVV were collected from early March through July from the southeastern and western regions of the outbreak  even in situations lacking human or pet exposures. Comprehensive animal disease surveillance provides direct benefits to public health and animal health by promoting early recognition of risk and opportunities for disease control and prevention interventions.–Unpredictable health threats related to emerging zoonoses, especially those involving wildlife reservoirs, pose notable surveillance and control challenges (


We conducted a bibliometric evaluation to get insight into research activities and tendencies.The search criteria in PubMed were: (1) “human T-lymphotropic virus 1” as medical subject headings (MeSH) major topic; and (2) publication date from January 2001-December 2010. We retrieved publication year, author names, journal, country of first author, language, free availability, and MeSH terms.The search retrieved 1189 publications by 3969 authors. Twenty-eight authors published >=15 articles. The number of publications did not increase: 600 in 2001-2005 and 589 in 2006-2010. The journals with the highest number of publications are: Journal of virology (126), Retrovirology (58), Blood (47), The Journal of biological chemistry (45) and Virology (41). In 349 publications (30%), the first author was from the USA; Japan contributed 329 publications (28%); Brazil 115 (10%); France 91 (8%); and the UK 54 (5%). The proportion of publications with a first author from Latin America or the Caribbean increased over time ; the proportion with a North American first author decreased . Ninety-six percent (1137) of the publications are in English. The full text of 582 publications (49%) is freely available. The most frequent MeSH terms relate to virology. Clinical/epidemiological MeSH terms are more frequent in publications from Latin America and the Caribbean than elsewhere.The constantly modest number of publications characterizes HTLV-1 as a neglected health problem. The MeSH terms suggest that clinical/epidemiological aspects are investigated much less frequently than virological aspects.


Acinetobacter baumannii (MRA) from October 2009 to May 2010. A multifaceted strategy involving segregation, enhanced infection control procedures, and microbiological surveillance was implemented. We evaluated its impact on antibiotic stewardship.A 12-bed ICU experienced an outbreak of multiresistant ® was conducted: 90 consecutive patients before the outbreak (January to June 2008) and 91 thereafter (October 2010 to May 2011). Data included patient profiles, admission criteria, ICU survival, antimicrobials used, antibiotic days, number of patients on antibiotics, prescribing cost and the demographic of microbes isolated.A retrospective review of patient notes and results using AcuBaseStaphylococcus and Klebsiella and an increase in Enterobacter. The use of cefuroxime (3.2 vs. 2.3 antibiotic days/patient) and quinolones (6 vs. 2) decreased. There was a reduction in average antibiotic days per patient episode (5.1 vs. 4.2) (P = 0.0291) and the prescribing cost savings were £13,558 (47%). See Figure Following the outbreak, enhanced infection control measures were implemented alongside the Matching Michigan protocols. Daily operational critical care and elective planning meetings and a staff education programme were undertaken. ICU mortality (31 (14%) vs. 43 (16%)) was unchanged. Microbiological isolates were overall similar, with a reduction in coagulase-negative The need for a complex strategy to manage and eradicate an MRA outbreak in the ICU led to a clinically significant decrease in antibiotic use and prescribing cost saving following eradication of MRA. Improved antibiotic stewardship is achievable through better infection control strategies.


Antibiotic surveillance is mandatory to optimise antibiotic therapy. Our objectives were to evaluate antibiotic use in our pediatric intensive care unit (PICU) and to implement a simple achievable intervention aimed at improving antibiotic therapy.Prospective, 3 months surveillance of antibiotic use on PICU (phase I) and evaluation according to the CDC 12-step campaign with development of an attainable intervention. 3 months surveillance (phase II) after implementation of intervention with comparison of antibiotic use.Appropriate antibiotic use for culture-negative infection-like symptoms and targeted therapy for proven infections were the main areas for potential improvement. The intervention was a mandatory checklist requiring indication and recording likelihood of infection at start of antibiotic therapy and a review of the continuing need for therapy at 48 h and 5 days, reasons for continuation and possible target pathogen. The percentage of appropriate empiric antibiotic therapy courses for culture-negative infection-like symptoms increased from 18% (10/53) to 74% , duration of therapy <3 days increased from 18% (10/53) to 35%  and correct targeting of pathogen increased from 58% (7/12) to 83% .Antibiotic surveillance using the CDC 12-step campaign can help to evaluate institutional antibiotic therapy. Development of an attainable intervention using a checklist can show improved antibiotic use with minimal expense. Antibiotic surveillance is mandatory to optimise antibiotic therapy and to prevent antimicrobial resistance-3. GuideThe Center for Disease Control and Prevention (CDC) 12-step program to prevent antimicrobial resistance was launched in 2000 to educate clinicians about antimicrobial resistance and provide strategies to improve clinical practice. Patel aIn a recently published concise review, Newland and Hersh concluded that prospective audit with feedback was the most favourable and efficient strategy for antimicrobial stewardship programs in paediatrics. They asWe performed a prospective, single-centre interventional audit of antibiotic use on our PICU. Antibiotic therapy was assessed looking for adherence best practice recommendations of the CDC 12-step campaign. Basic data collection was carried out for 3 months (phase I). Interventions were then planned based on areas for potential improvement, that were simple and attainable, required minimal personnel resources and were low in cost (no funding available). Data collection was repeated after the intervention based on the power calculation (phase II). The audit was supervised by the departments of Paediatrics, Pharmacy and Microbiology at the Royal Brompton and Harefield NHS Foundation Trust. The results of phases I and II were reviewed as part of the clinical governance program. This audit with an implemented intervention that is part of standard care on our PICU without a change in policy didn't required ethical approval.st April 2010 and 31st June 2010 (91 days) were included in phase I, and then between 10th November 2010 and 28th February 2011 (110 days) in phase II.This audit was conducted on the PICU at the Royal Brompton and Harefield NHS Foundation Trust in London, UK. Our unit is a specialised, tertiary unit for paediatric cardio-respiratory care with approximately 700 admissions per year. Newborns and children up to 16 years of age with a broad spectrum of cardiac malformations, cardiac diseases, rhythm disturbances, postoperative cardiorespiratory care and respiratory problems are treated in our facility. All admissions between 1Staff of the unit includes physicians  working as consultants, fellows and senior house officers; nursing staff working usually in 1:1 care (1 nurse / 1 patient), partially 1:2 if patients were stable and not invasively ventilated; pharmacists and allied health professionals. The group of physicians prescribing antibiotics remained unchanged in regard of number and level of expertise during both study periods: 5 consultant intensivists, 9 fellow intensivists and 9 senior house officers (Paediatrics). Infection control is part of our standard practises on our unit and two infection-focused care bundles are actively practised with prospective data collection: i) care bundle for prophylaxis of catheter-related infections, ii) care bundle for prophylaxis of ventilator-associated pneumonia. Both care bundles remained unchanged and there were no other stewardship interventions in place during the study phase.Data collection in both phases was carried out using the electronic prescribing system for medication (ICIP), the electronic microbiology data system (EPR), the electronic system for nursing reports and all available medical reports. With the electronic medication and microbiology data system we were able to assess all doses of antibiotic therapy administered to patients on PICU, and results of all microbiologically tested samples . Indications for antibiotic therapy were obtained from medical and nursing reports in phase I, and additionally from the study intervention form during phase II.The design of our study was an antibiotic surveillance audit, based on the CDC 12-step recommendations focused at the 2 strategies “Diagnose and treat infection effectively” (steps 3–5) and “Use antimicrobials wisely” (steps 6–10). The unit of antibiotic analysis was an antibiotic course, defined as one or more antibiotics given concurrently for one or more days. The possible reasons for therapy were categorised as 1) prophylaxis (post-surgery), 2) empiric therapy  or 3) therapy for proven infection . Appropriateness of antibiotic use was compared using institutional antimicrobial guidelines for prophylaxis and empiric therapy for suspected infections. Appropriate duration of empiric therapy for culture negative infection-like symptoms was defined as less than 3 days unless there were clear and reasonable documented reasons for continuing antibiotic therapy.After defining 2 main areas for potential improvement in phase I, we created a checklist as a simple achievable intervention Figure . The cheA power calculation was done based on the aim of our primary intervention, which was to reduce inappropriate duration of empiric antibiotic therapy with culture negative results. In phase I, 18% (10/53) of patients on empiric therapy received it for an appropriate duration. We hypothesised, that it was possible to increase this to 36% (duplication) with the implemented intervention. With the definition of alpha=0.05 and a power of 80%, 42 courses of empiric therapy would be required to demonstrate a significant result. Calculating a possible compliance of 80% to the intervention our aim was to observe 52 courses of empiric antibiotic therapy during phase II. A Chi-squared test was calculated if all frequencies were equal to or greater than 5, otherwise a Fisher’s Exact two-tailed test was used.Assessing antibiotic use according to the CDC 12-step campaign revealed 2 main areas in phase I for potential improvement: i) Step 10: Empiric antibiotic therapy for < 3 days for unproven infections (culture negative); ii) Step 4: targeting of pathogens as therapy for proven infections (culture positive). 18% (10/53) of empiric antibiotic courses had a duration of < 3 days and there was no clear and reasonable documentation for courses of longer durations. In 58% (7/12) a cultured pathogen was targeted with a change of antibiotics unless antibiotics were already susceptible. The other CDC steps showed good results, i.e. 95% of all prophylactic courses complied with institutional guidelines and vancomycin, carbapenems and third generation cephalosporins were rarely used and did not appear in the top 5 most commonly used antibiotics (results not shown).Baseline comparisons of patient demographics, overview of antibiotic courses and outcomes are shown in Table Regarding possible adverse outcomes due to early discontinuation of antibiotic therapy there was no statistically significant difference in overall mortality, infection-related mortality and infection relapse rate (Tab 1). There was no case of death on our unit during the phase I of the study, whereas 3 children died during phase II, none of them infection related: i) cardiac failure in a newborn baby with critical aortic stenosis, ii) cardiac failure in a baby with unbalanced atrioventricular septum defect and complete AV-block III , iii) ischemic necrotizing enterocolitis after cardiopulmonary bypass surgery (antibiotic therapy continued after surgery). Secondary courses of antibiotic therapy during the same hospitalisation after a first course of suspected or proven infection (possible relapses of infection) were even less frequent in phase II. The significantly higher rate of proven infections in phase II was due to an increased rate of pulmonary infections in the winter months November – February compared to phase I in April – June.As with Patel et al. who evaluated antibiotic use according to the CDC 12-step program on a NICU, we used the CDC recommendations as the evaluation tool for our audit,10. ProlThe first step in building an intervention for antibiotic surveillance/stewardship is to select the most appropriate strategy that leads to a sustained improvement in a defined area. In the guidelines for developing an institutional program to enhance antimicrobial stewardship there are two core strategies described: 1) prospective audit with feedback and 2) formulary restriction and pre-authorisation. InitiatThe importance of a high compliance rate  for a successful intervention is emphasised with the statistically not significant increase of antibiotic courses of empiric therapy < 3 days. In contrast to the aviation industry, there is still scepticism by some physicians about the use of checklists for the safe routine care and emergency situations in medicine, illustrated with a citation from A. Gawande in his recently published book “The Checklist Manifesto”: “It runs counter to deeply held beliefs about how the truly great among us – those we aspire to be – handle situations of high stakes and complexity”. A mandaThe limitations of our audit were the short surveillance time, the low compliance to the intervention (<80%) and the low numbers of proven infection with positive cultures (no conclusions regarding susceptibility patterns possible). The main strength of our audit was that it was a simple and achievable intervention which was low in cost  and resources. The construction of an easy attainable strategy is very important due to the fact that lack of resources and time are major barriers to implementing an antimicrobial stewardship program in paediatrics.The 12 steps of the CDC campaign are a possible tool to evaluate institutional antibiotic therapy. A mandatory checklist as an audit-feedback-mechanism can be a simple achievable intervention with possible improvement in antibiotic use at minimal expense. Further studies with mandatory, computerised checklists within the prescribing system need to be carried out to show compliance and sustainable results over a longer period of time.All authors declared that they have not received support  from any companies for the submitted work; no authors have any relationships with companies that may have an interest in the submitted work; their spouses, partners, or children.MS was responsible for the design of the study, data acquisition and analysis and the final manuscript; FE participated in the study and was responsible for data acquisition; BW was responsible for statistical data analysis and interpretation; JC participated in the study and made substantial contributions to the manuscript; DM helped in the design of the study and made substantial contributions to the manuscript; AF helped in the design of the study, participated in data acquisition and analysis and made substantial contributions to the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2431/12/196/prepub


An increased interest is given to the impact of high fat diet on health worldwide. Abnormalities in lipid metabolism induced by high cholesterol diet (HCD) were reported to exacerbate renal diseases via oxidative stress pathways. Rutin and ascorbic acid showed a protective role against oxidative stress-mediated diseases. Furthermore, both lipid metabolism and tissue response to oxidative stress damage was found to vary according to animal gender. Thus, the objective of this work was to examine possible gender-related differences and the possible protective effects of rutin and ascorbic acid supplementation on high cholesterol diet induced nephrotoxicity.96 young male and female Wistar albino rats were used. HCD supplemented animals were treated with rutin alone or in combination with ascorbic acid for 6 weeks. Creatinine plasma level was estimated. Furthermore, kidney levels of nucleic acids, total protein, malondialdehyde (MDA), reduced glutathione (GSH), total cholesterol, and triglycerides were determined. Finally, kidney tissues were used for histopathological examination.HCD supplementation decreased kidney level of nucleic acids, which was more prominent in female animals. Both vitamin combination significantly attenuated HCD induced decrease in nucleic acids. Moreover, kidney level of MDA was significantly altered by HCD in both genders, which was inhibited by rutin and ascorbic acid alone or in combination in male groups and by both vitamins in female groups. There was a reduction in kidney level of GSH by HCD, especially in male groups, which was attenuated by rutin and ascorbic acid combination. Kidney levels of total cholesterol and triglycerides were significantly increased by HCD supplementation in both genders. Coadministration with rutin and/or ascorbic acid protected from such increase, which was more obvious in both vitamins combination. Histopathological investigation supported vitamins protective effect, which was more prominent in male vitamins combination group.HCD-induced renal injury in female was higher than in male animals, suggesting a better anti-oxidative stress defense response in male's kidney. Moreover, the antioxidant and reno-protective effects of rutin and ascorbic acid were augmented following their combination. It is well known that lifestyle and diet play a role in the development of kidney disease. Several studies indicated that abnormalities in lipid metabolism can often accompany and exacerbate renal disease ,2. HyperRutin (RT), a quercetin-3-rutinosid or vitamin-P, is considered as one of flavonoid glycosides, which is found in onions, apples, tea and red wine . Rutin iGender difference currently plays an important role in the etiology of hyperlipidaemic-induced disorder including cardiovascular diseases (CVD). For instance, men are more susceptible to coronary heart disease than age-matched women. However, postmenopausal women have an equal chance of CVD with men ,19. The The current study was designed with major two goals; (1) to investigate gender-related differences in response to high cholesterol diet induced nephrotoxicity using Wistar albino rats as an animal model; (2) to evaluate the potential beneficial effects of rutin and/or ascorbic acid supplementation on high cholesterol diet induced nephrotoxicity.Young male and female Wistar albino rats  were provided by the Experimental Animal Care Center . Animals were roughly the same age, weighing 80-100 grams. Animal environment was maintained under controlled conditions of temperature (22 ± 1°C), humidity (50-55%), and light (12 h light/dark cycles). All methods including euthanasia procedure were conducted in accordance with the Guide for the Care and Use of Laboratory Animals, Institute for Laboratory Animal Research, National Institute of Health  and approved by the Ethical Guidelines of the Experimental Animal Care Center .All animals' diets were prepared in pellet form by adding test materials in rat chow powder following shade dry method. Table All animals were randomly divided in to eight groups  as follow; group-1: Control, group-2: RT, group-3: AA, group-4: RT + AA, group-5: HCD, group-6: RT + HCD, group-7: AA + HCD, group-8: RT + AA + HCD. The experimental diets were supplemented for 6 consecutive weeks. At end of the 6th week rats were sacrificed by decapitation and the trunk blood was collected in heparinized tubes. Both kidneys were dissected and weighed. Kidney tissues were immediately dipped in liquid nitrogen for 1 min and then preserved at -75°C  till analysis. The plasma was collected after centrifugation at 4,000 rpm for 15 min and stored in freezer at -20°C till analysis.Creatinine concentrations were estimated in plasma samples by using commercially available diagnostic kits .Kidney levels of nucleic acids (DNA and RNA) were estimated by the method described by Bregman, 1983 . Kidney Kidney levels of MDA were estimated as described by Ohkawa et al., 1979 . BrieflyThe method prescribed by Sediak and Lindsay, 1968  was usedLipids contents of kidney tissues were extracted using the method described by Folch et al., 1957 . In brieOne half of kidney from control, HCD, RT + HCD, AA + HCD and RT + AA + HCD male and female groups was removed for histopathological examination. Kidney tissues were fixed in 10% neutral buffered formalin, embedded in paraffin wax, sectioned at 3 μm, stained with Hematoxylin and Eosin (H & E) stain and placed in slides for light microscopic examination. To avoid any type of bias, the slides were coded and examined by a histopathologist who was blinded to the treatment groups. Grading of kidney injury was preformed according to Table P < 0.05. GraphPad prism program (version 5) was used as analyzing software.All data were expressed as mean ± Standard Deviation (SD). Data were statistically analyzed using one-way ANOVA followed by Student-Newman-Keuls multiple comparisons test. The differences were considered statistically significant at Kidney weights did not show significant difference between control group and other treated groups in both male and female animals Table . HCD vitCreatinine plasma level in both male and female rats was not significantly altered at any group as compared to control groups Table . FurtherIn male groups, kidney levels of both nucleic acids and total protein were not significantly altered by HCD administration either alone or with vitamins as compared to control group Table . DNA kidHCD administration to Wistar albino rats for 6 weeks significantly elevated kidney level of MDA in HCD male and female groups as well as in HCD vitamins treated female groups  as compared to control groups  normal cortical and medullary pattern with well-formed glomeruli in control group, (2) hypercellular glomeruli with mesangeal cells proliferation till occlusion of glomeruli and few shrink as well as a swelling in tubular epithelial cells in HCD group, (3) proliferated hypercellular glomeruli with mesangial cells proliferation along with some swollen epithelial cells and no interstitial inflammatory cells in RT + HCD group, (4) hypercellular glomeruli with mesangeal cell proliferation and occasional some shrinked glomeruli with no tubulointerstitial inflammatory cells in AA + HCD group, (5) a preserved cortex and medulla with preserved normal looking glomeruli surrounded by benign tubules with slight swollen epithelail cells of some tubules in RT + AA + HCD group  induced-nephrotoxicity in male and female Wistar albino rats were investigated. HCD administration for six consecutive weeks induced renal injury and nephrotoxicity, which was more in female than in male animals. Moreover, rutin and ascorbic acid were found to protect from HCD induced-nephrotoxicity. In addition, these nephro-protective effects were improved in our study by administration of both vitamins in combination to the HCD fed rats which was supported by results from histopathological investigation.HCD is well known to cause nephrotoxicity and renal injury in different animal models. In the present study, signs of increased renal oxidative damage induced by HCD supplementation for six weeks were studied. Mean kidney weights were not significantly changed after HCD feeding and these results are in agreement with earlier reports ,30. ThisThe present study also showed that HCD can induce more significant renal impairment and toxicity in female than male animals. This suggests that male antioxidant defense response to HCD-induced neohrotoxicity was higher than female response. It is well known that sexual steroid hormones can control many physiological and pathophysiological processes . Sexual One possible explanation of the degree of renal oxidative damage observed in female rats is that females are able to oxidize poly unsaturated fatty acids more than males. High fat diet was reported to reduce testosterone and to increase estrogen serum levels, which was associated with increased intra-abdominal fats in male than in female . That isIn the present study, HCD induced renal impairment was significantly attenuated by administration of rutin and ascorbic acid. Our results revealed that the combination of both vitamins could have an additive, or sometimes synergistic, antioxidant effects. These speculations are supported by the histopathological evaluation that showed rutin and ascorbic acid combination to prevent HCD-induced nephrotoxicity especially in male animals. Flavonoids such as rutin are now widely accepted as physiologic antioxidants which are known to have effective free radical scavenging activity -52. PrevIn addition, both vitamins combination significantly decreased MDA and elevate GSH kidney levels as compared to their altered levels in HCD group suggesting that the antioxidant properties of rutin may be attributed to its protective effects on lipid peroxidation. Rutin can augment cellular antioxidant defense mechanisms leading to the protection against oxidative tissue injury . Rutin mTaken together, we suggest that HCD can induce nephrotoxicity with higher extent in female than in male animals, suggesting a more antioxidant defense mechanism in kidneys of male than female animals. Moreover, we concluded that combining rutin and ascorbic acid can augmented their ability to attenuate ROS-induced lipid peroxidation which prevented HCD induce renal injury in Wistar albion rats.The authors declare that they have no competing interests.SS, HM and OA performed experimental work including animal treatments, biochemical analysis, statistical analysis, interpretation and discussion of the results related to their part of the work. SS and AM conceived, design and planning of the study, wrote the paper, drafting and revision of the manuscript. All authors read and approved the final manuscript.Table S1. Effect of rutin (RT) and/or ascorbic acid (AA) on kidney histopathological evaluation in high-cholesterol diet (HCD) fed rats following 6 weeks of supplementation.Click here for file


Use of information campaigns and educational interventions directed to citizens and supported by physicians, aimed at promoting the appropriate use of medicines, have been evaluated by several studies with conflicting results. These interventions are potentially relevant, favouring the reduction of unnecessary use of medicines and related risks. Several studies have specifically evaluated the promotion of the appropriate use of antibiotics in adults and children, with variable results. A controlled study is proposed to evaluate the feasibility and effectiveness of a multifaceted intervention aimed at reducing antibiotic prescription by increasing awareness on risks of their unnecessary use.Information will be provided to citizens through several media . Brochures with information on expected benefits and risks of antibiotics will be also available, either with direct access in waiting rooms and pharmacies or handed out and mediated by doctors. Physicians and pharmacists will get specific data on local antibiotic resistance. A small group of representative doctors have also actively participated in defining the campaign key messages. A sample of general practitioners and paediatricians will be trained in patient counselling strategies.The information campaign will be implemented in two Provinces of Emilia-Romagna during the fall-winter season (November 2011-February 2012). Change in the overall prescribing rate of antibiotics (expressed as DDD per 1000 inhabitants/day) in the intervention area will be compared versus other areas in the same Region. Knowledge and attitudes of the general population will be evaluated through a phone and internet survey on a representative sample.While the campaign messages will be mainly directed to the general population, doctors' prescribing will be assessed. The main rationale for this apparent discrepancy lies in the influence patients may have on physicians' prescribing behaviour (directly or indirectly) and in physicians' endorsement of the campaign goals, considering their participation in its design.This study could observe a reduction lower than 5% in the prescribing rate of antibiotics. Such a reduction would be of public health relevance and would determine average savings of almost twice as much as the campaign costs. Use of information campaigns and educational interventions directed to citizens and supported by physicians, aimed at promoting the appropriate use of medicines, have been evaluated by several studies with conflicting results. Evaluating the feasibility and effectiveness of these interventions is relevant, considering the influence citizens may have on physicians' prescribing habits, both directly (when patients request prescription filling) and indirectly (when physicians think their patients expect getting a prescription) . PhysiciAs for population targets: citizens are exposed to several information sources about the effects of medicines, either through traditional media  or through the internet with its constant evolution to more interactive tools (blogs and the many features of web 2.0). These information are often promoted by drug producers: in Europe, direct-to-consumer drug advertising is currently not allowed   but citiSeveral studies have shown the potential of mass media on the use of health interventions . MedicinAside from the specific topic focused on, many other variables may influence the effectiveness of multifaceted educational campaigns. Defining one or more relevant target audiences and key messages are essential elements. A thorough evaluation of local contexts and a wise mix of information tools is necessary as well, taking lessons from those who do commercial information and advertising. ''Social marketing'' techniques may help the promotion of evidence-based knowledge. This term refers to the application of marketing techniques to the solution of social and health problems . A sociaAs for doctor's role: information campaigns could be more effective if physicians facilitate the transmission of information to their patients . In ordePracticing physicians can also provide substantial help in developing campaign messages, since they are in a relevant position to evaluate comprehensibility and potential effects of specific information in the context of patient expectations in primary care. Such participation can also increase their sense of ownership of the campaign goals and facilitate their endorsement , which cSeveral studies have evaluated the effects of educational interventions focused on the appropriate use of antibiotics in adults and in children. A Cochrane systematic review, reporting the results of 39 among RCTs and observational studies . SeveralWithin a wider project called ProBA project (progetto Bambini Antibiotici), in 2003 the Health Agency of the Emilia-Romagna Region carried out a survey on 633 paediatricians  and 1388 parents, aimed at evaluating the main determinants of antibiotic prescriptions for children [Starting from these considerations and from local and international experiences on educational campaigns, we propose a study to evaluate the feasibility and effectiveness of a multifaceted intervention on antibiotic prescription in the general population, aimed at reducing it by increasing awareness on risk of unnecessary use, thus reducing patients' requests for unnecessary prescription. The specific objectives are:• primarily, to reduce the overall volume of antibiotic prescription;• secondarily, to reduce total costs associated to antibiotic prescriptions; to evaluate knowledge, attitudes and reported behaviours of a sample of citizens on benefits and risks of antibiotic use; and to evaluate trends in antibiotic resistance (in a three year period after the intervention) from sputum, throat swab, urine and blood samplesWe consider such intervention of high public health relevance, considering the limited development of new antibiotics and the public health threat due to antibiotic resistance, which is in part accounted for antibiotic use in humans. The proposed intervention will involve citizens, general practitioners and paediatricians in two Provinces of Emilia-Romagna. In addition to promoting the appropriate use of antibiotics, this project will help to evaluate the feasibility of carrying out information campaigns on benefits and risks of medicines promoted by Local Health Authorities, with the collaboration of local physicians.This study will be a community level, controlled, non randomized trial. An information campaign on benefits and risks of antibiotic use, in general and specifically in upper respiratory infections, will be implemented in the provinces of Modena and Parma, in the Emilia-Romagna region. Regional areas where no campaign will be implemented and no information provided to physicians either will be the control group; the presence of local policies on antibiotics that would possibly alter the comparison with intervention areas will be evaluated immediately before implementing the campaign, so that provinces which may supply misleading data would not be included in the control group. Anyway, prescription of antibiotics in the intervention and control areas shall be similar at baseline .Considering that the proposed educational intervention will be done at the community level and will be multifaceted using several media, and considering its implementation in only two provinces, we think a randomised design would not add strength to the study validity: in fact, it would be impossible to restrict exposure to half the population and there would be too few units of randomisation. Therefore, we choose a non randomised design which is more feasible given those circumstances.The proposed multifaceted intervention will target adult citizens and physicians. Specifically, citizens will be directly targeted by general information through brochures, posters and mass media. General practitioners and paediatricians will receive ad hoc information on antibiotic resistance and will have an active role in facilitating the campaign messages getting through, so that they may be also sensitized to the issue of appropriate antibiotic prescribing Table .The campaign will be generally focused on the appropriate use of antibiotics in upper respiratory tract infections. Problems arising from antibiotic overuse, in particular antibiotic resistance, will be particularly pointed at. On the other side, it will be highlighted that antibiotics are necessary in specific circumstances and should be used when doctors say it. The main instruments of the proposed multifaceted intervention are discussed as follows.1. Written information material (brochures) targeted at the general population. Specifically, two different types of brochure will be available: type-A brochure will provide general information on benefits and risks of antibiotic therapies; type-B brochures will provide more specific information on three possible clinical situations patients (and doctors) may deal with: antibiotic is necessary and is prescribed; antibiotic is not necessary and is not prescribed; antibiotic may or may not be necessary, so that a "wait and see" period is warranted.In developing the specific messages, particular attention has been given to the evaluation of key determinants of potentially inappropriate antibiotic use. The occurrence of antimicrobial resistance both at population and individual level, and of side effects, will be particularly targeted. In this regard, the relevance of antibiotic prescribing on individual resistance has been further highlighted by a recent systematic review and meta-analysis . A smallThe brochures will be available in waiting rooms of general practices and local health services (type-A) and will be distributed to patients by general practitioners and paediatricians (type-B). Electronic versions will be available on the web. Translated versions in Albanian, Arab, Chinese, English, French, Polish, Rumanian, Russian will be also posted on the web, so that doctors could print them out to share information with their foreign patients (mostly immigrants).2. Posters developed through the same methodology as described above (see point 1). Given the nature of this media, specific attention has been given to focusing on few clear messages. Posters will be mainly available in general practices, pharmacies and local health services.3. Short videos to be transmitted through video terminals in highly accessed places , local TVs and websites of Local Health Authorities.4. Radio-spots to be transmitted in local radio stations5. Advertisements and concise articles in local newspapers, written in a scientifically sound but accessible way to the average reader.6. Web materials/banners in web sites of local newspapers.7. A newsletter on antibiotic resistance in Emilia-Romagna, available to physicians and pharmacists in the intervention areas.8. Training courses in counselling strategies, directed to general practitioners and paediatricians to facilitate information transfer on the campaign contents. More specifically, all general practitioners and paediatricians in the intervention areas will get a half-day presentation about the campaign and its instruments, discussing the importance of counselling strategies through examples relevant to the antibiotics campaign. A six-module course will be pilot-tested on about 120-150 physicians on a voluntary basis during the campaign implementation, to improve their ability to convey information and facilitate their patients' behavioural change.The proposed interventions will be implemented during a 4 month period (November 2011 - February 2012).Cumulative five-month (November to March) changes in prescribing rates of specific classes of antibiotics in general practice, expressed as DDD per 1000 inhabitants/day.Data on antibiotic prescriptions  will be retrieved from regional prescribing databases. They will include NHS prescriptions only from general practitioners  and will not assess the actual use of antibiotics. In Emilia-Romagna, prescriptions from paediatricians are expressed in pieces and not in DDD, and will be evaluated separately.- changes in antibiotic prescribing trends in eighteen months- changes in antibiotic expenditure per 1000 inhabitants/day (comparing to the same November to March period from the previous year)- knowledge of and attitudes about the campaign messages, through a telephone and internet survey on a sample of the target population.As for trends in antibiotic resistance, the regional antibiotic surveillance system regularly monitors and will keep monitoring antibiotic resistance through the analysis of blood, throat swab and sputum samples. Considering this circumstance, data about antibiotic resistance will be evaluated in a three year period after the intervention.In the first three months of 2011, antibiotic prescribing rate in the provinces chosen as possible intervention areas was 21,1 DDD per 1000 inhabitants/day. Ratio of prescribing rates in intervention vs other Provinces in Emilia-Romagna was 1. Standard deviation of the distribution of antibiotic prescribed by general practitioners was about 3,0 in a 2007 dataset of antibiotic prescriptions in the province of Modena. Assuming that ratio of prescribing rates has not changed, that standard deviation of antibiotic prescription is 6,0 (a very conservative doubled figure compared to the 2007 sample) and that is similar in exposed and not exposed areas, it will be possible to observe tiny reductions of antibiotic prescription, and surely meaningful reductions of at least 3-5%, with alfa = 5% and beta = 10%. Potential average economic savings of such reductions would more than offset the fund received to finance the campaign and would be more than three times the out-of-pocket expenses to implement the campaign.T-test (for continuing variables), chi-square test (for discrete variables), time series analysis and multiple linear regression will be used, the latter to investigate in secondary analyses how specific variables  can affect outcomes. Analysis of covariance (ANCOVA) will be used for evaluating primary outcomes in case relevant baseline imbalances will make statistical adjustment necessary.The protocol has been approved by the provincial ethics committees of Modena and ParmaThis study will explore the impact of a local multifaceted information campaign on antibiotics. While the campaign messages will be mainly directed to the general population, volume of antibiotics prescribed (therefore doctors' prescribing behaviour) will be the main outcome of interest. Three points make the rationale for this apparent discrepancy and for the relevant role that informing patients may play: prescribers may be influenced by actual or perceived patient pressure to get antibiotics; they may be influenced by the awareness that patients may be more informed about (and more sensitive to) the antibiotic resistance issue; they have been asked to actively participate in developing the campaign messages and facilitating their transmission to patients, so that their endorsement of the campaign goals may be more likely. Thus, the "complex" nature of the proposed intervention lies not only in the use of multiple instruments, but especially in such articulate interplay between targets of information and targets of behavioural change, mirroring some of the possible influences on drug prescription in clinical practice.The potential impact of such a low-cost multifaceted intervention implemented within Local Health Authorities and using local media is of general interest, as well as its feasibility. One further reason of interest is that, in Italy, data are lacking about the impact of information campaigns addressing health topics, especially if "hard" outcomes (such as drug prescribing) are considered. The likely presence of a parallel national campaign implemented in the same period  could theoretically weaken the potential impact of this intervention; on the other side, we would be more confident if any effect had to be shown and the added value of a local initiative, with the use of local media and especially the participation of physicians, would be further elucidated.As for ethical aspects, the information campaign will provide general, clear and carefully balanced messages explaining when antibiotics are necessary and risks of their unnecessary use. This information is expected to increase appropriate use of antibiotics and, ultimately, health of the exposed population. Since doctors  will have a fundamental role in mediating this information, the risk of its incorrect interpretation will be very limited.The author declares that they have no competing interests.This is a collaborative research, with a few research centres and professional groups involved (see the paragraph below). Each of the listed people within these centres have a relevant role in study implementation. Within the writing committee (see next paragraph) GF had the original idea and wrote the protocol while all other authors provided important intellectual contribution to study design and implementation. All the authors read and approved the final version.The study and the information campaign are coordinated by the proponent institution, CeVEAS , which has a key role in developing the information material, carrying out the training courses and evaluating prescribing and attitudinal data.CeVEAS is a Department of the Local Health Authority of Modena, collaborating with several stakeholders such as Local Health Authorities and Regional Health Agencies, the Italian National Health Institute and the Italian Drug Evaluation Agency, and is a WHO Collaborating Centre for Evidence-Based Research Synthesis and Guideline Development in Reproductive Health. CeVEAS was founded in 1999 to evaluate health interventions and health policies  mainly through the analysis of scientific literature, and to facilitate the access to the best evidence on health care through the transfer of relevant knowledge to physicians, policy makers and citizens. CeVEASThe Emilia Romagna Regional Health Agency (Infectious Diseases Risk Unit) has a key role in analysing the determinants of antibiotic use, in evaluating topics and key messages of the information material as well as reviewing it, in evaluating antibiotic resistance data and in developing a newsletter on this topic.The specific role of this Unit is to work on prevention and control of infectious diseases, with a special focus on healthcare associated infections, antimicrobial resistance, tuberculosis, and other emerging and re-emerging diseases .The Unit activities consist of identifying and assessing interventions to implement within the regional health system: developing and implementing surveillance system for the diseases described above, monitoring of control programmes and implementation of organizational changes.The Local Health Authorities of Modena and Parma, which are in charge of planning, organizing and providing health care services in the Province of Parma, will have a key role in organizing contacts with local media  and in organizing training courses for physicians. Representatives from these Units participate in developing a strategic plan for implementing the campaign.General practitioners and paediatricians in the Provinces of Modena and Parma will have a key role in promoting the campaign messages, that they contributed to develop and evaluatePharmacies in the Provinces of Modena and Parma will have a key role in the dissemination of the information material and in providing information on the correct use of antibioticsThe communication agency CHANGE will have a key role in carrying out training courses in counselling strategies.This agency, whose founders co-founded the Italian Society for Counselling in Medicine (SICIM), is a not for profit organization developing and disseminating throughout Italy a method of systemic counselling that can be applied to different professional contexts and is used, in particular, by health-care workers and professionals in helping relations with family systems in situations of difficulty, illness or distress.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/11/816/prepub