The covalent attachment of nanoparticles to therapeutic proteins enhances their bioavail- ability, prolongs half-lives and reduces immunogenicity . Ten products conjugated to nanoparticles have been FDA-approved, and four are blockbuster drugs : conjugated IFN- α2a, IFN-α2b, G-CSF and epoietin. Conjugated IFN-α2a and IFN-α2b are more effective and less toxic than their plain forms, and are the treatment of choice for hepatitis B and C [47,48]. Conjugating GM-CSFto nanoparticles is feasible, and enhances DCs activity . Mouse re- combinant GM-CSF in subcutaneous administration had a distribution half life of 0.92±0.04 minutes and an elimination half-life of 11.75±3.89 minutes, much shorter than PEGylated GM-CSF with distribution half-life of 15.9±1.5 minutes and an elimination half-life of 5.3 hours ±13 minutes . We deployed a different approach and conjugated mGM-CSFto Qdot (625) to increase its size and consequently increase its retention in the alveolar space. When we treated IAV-infected C57Bl/6 wild type mice with either Qdot-mGM-CSF, PBS, or plain mGM-CSF, all three groups displayed comparable morbidity, as evidenced by similar weight loss patterns, indicating similar infection rate. However, whereas all of the mice received Qdot- mGM-CSF survived, 40% of PBS-treated group (2 out of 5), 20% of Qdot only treated group and 29% of plain mGM-CSF-treated group (2 out of 7) succumbed (Fig 5C). This demonstrat- ed that despite the reduced in vitro biological activity (Fig 5), the Qdot-mGM-CSF displayed greater protective efficacy in treating influenza infected mice compared to the plain mGM-CSF. Taken together, these results somewhat confirms our hypothesis that retaining mGM-CSF in the alveolar space is a pragmatic strategy to address IAV-induced lung injury and emphasizes on needs for additional work to optimize this strategy. This will open new avenues for thera- peutic use ofGM-CSF in IAV and other acute lung infections.
As shown above, influenza DNA vaccination can repre- sent a safe alternative to current commercial vaccines. De- spite all the referred advantages there are still some draw- backs of this therapy that also need to be overcome (Table 2). Although the good immunogenicity of pDNA bio- molecules, they are especially susceptible to degradation by nucleases, thus affecting the expression of the transgenes. To make up for this limitation the plasmid construction can be altered either by targeting the expressed antigen to pro- fessional APCs and thereby ensuring the efficient MHC-I and MHC-II compartment activation, by including adju- vants or by using specific delivery methods. Even though it has not been proved, the presence of prokaryotic protein subunits in pDNA vaccines backbone can trigger an aller- gic reaction or cause the mutation of the elements in the plasmid. So, minicircle DNA without unnecessary bacterial material has emerged as an alternative to conventional pDNA vaccination, because of its easiness to manipulate, produce and transfect [64, 65]. This strategy only encodes an antigen expression cassette (promoter, antigen and polyA region) and has shown to be immunogenic, inducing both a cellular and humoral responses which means that it can be used as a potential strategy to combat influenza in- fections. In this fight, as previously referred, current vac- cines need to be updated every year in order to be able toprotectagainst newly emerging seasonal or pandemic flu strains. To fulfil this objective, the development of univer- sal vaccines with the most conserved portions ofinfluenza virus such as M2 , NA  or NP  are been de- signed. These antigens, with low drift, should be able to recognize and neutralize several strains without the usual selection of the strains to be inserted in the annual vaccine. However, antibodies against these proteins are not gener- ally protective without adjuvants or other components that
Particle mediated epidermal delivery (PMED) of DNA vaccines involves the use of a needle-free device to deliver plasmid vaccines directly into cells of the epidermis of the skin including both non- professional (i.e. keratinocytes) and professional antigen presenting cells (i.e. Langerhans cells) [6,7,8]. PMED influenza DNA vaccines have been extensively studied in relevant animal models including mice, ferrets, and swine and have been shown to induce protective antibody and cytotoxic T cell responses that provided complete protection against both homologous and drifted strains ofinfluenza A viruses [9,10,11,12,13,14,15,16]. Importantly, PMED-based DNA vaccination may prove to be particularly advantageous over other DNA vaccine delivery methods due to its apparent ability to induce mucosal immune responses. Strong mucosal immune responses induced in both the gut and lung by PMED DNA vaccines have contributed significantly to enhanced and complete protection against mucosal challenges with both AIDS viruses and influenza [13,14,17,18,19]. The ability of PMED DNA vaccination to elicit mucosal immunity is explained by the finding that the epidermis of the skin can serve as a potent inductive site for mucosal responses in distal mucosal sites such as lung and gut [20,21,22,23]. PMED influenza DNA vaccines have shown considerable promise in the clinic. In contrast to DNA vaccines employing intramuscular needle injection where milligram doses of plasmids induced poor responses in humans [24,25,26,27], PMED deliveryof 1–4 m g doses of an influenza DNA vaccine stimulated protective levels of antibody in 100% of the vaccinated subjects, and responses exceeded the minimum criteria for licensure established by the Committee for Human Medicinal Products . In a subsequent phase 1 clinical study where subjects were vaccinated with a trivalent PMED DNA influenza vaccine and then challenged with a controlled dose of an H3 influenza virus, the vaccine was well tolerated, induced anti-hemagglutinin (HA) antibody responses and provided protection against challenge with a vaccine efficacy of 53% against upper respiratory tract infection .
Foot-and-mouth disease (FMD) is one of the most feared diseases of livestock worldwide. Vaccination has been a very effective weapon in controlling the disease, however a number of concerns with the current vaccine including the inability of approved diagnostic tests to reliably distinguish vaccinated from infected animals and the need for high containment facilities for vaccine production, have limited its use during outbreaks in countries previously free of the disease. A number of FMD vaccine candidates have been tested and a replication-defective human adenovirus type 5 (Ad5) vector containing the FMDV capsid (P1-2A) and 3C protease coding regions has been shown to completely protect pigs against challenge with the homologous virus (FMDV A12 and A24). An Ad5-P1-2A+3C vaccine for FMDV O1 Campos (Ad5-O1C), however, only induced a low FMDV-specific neutralizing antibody response in swine potency tests. Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been successfully used to stimulate the immune response in vaccine formulations against a number of diseases, including HIV, hepatitis C and B. To attempt to improve the FMDV-specific immune response induced by Ad5-O1C, we inoculated swine with Ad5- O1C and an Ad5 vector containing the gene for porcine GM-CSF (pGM-CSF). However, in the conditions used in this trial, pGM-CSF did not improve the immune response to Ad5-O1C and adversely affected the level of protection of swine challenged with homologous FMDV.
Em conclusão, os achados tomográficos mais frequentes foram opacidades em vidro fosco, consolidações do espaço aéreo ou a associação desses padrões. Os achados foram predominantemente bilaterais, sem predileção quanto à distribuição axial ou craniocaudal na maior parte dos casos. Quando houve predomínio zonal, esse foi mais frequente nos terços inferiores e nas regiões periféricas dos pulmões. O derrame pleural, quando observado, foi, em geral, de pequena monta. Não foram observadas linfonodomegalias. Apesar de inespecíficos, é importante reconhecer os principais aspectos tomográficos da infecção pulmonar pelo vírus influenza A (H1N1) a fim de incluir essa possibilidade no diagnóstico diferencial de sintomas respiratórios.
Productivity losses were estimated based on the friction method to include losses due to absenteeism and losses due to premature death . Work loss was assumed to occur amongst employed Ontarians experiencing symptomatic influenza, an influenza-associated hospitaliza- tion, or an influenza-related death (limited to 90 days post-death). The cost to a firm due to the absence of an employee depends on several factors. If the firm must replace the employee with an overtime/temporary worker, then the cost to the firm is equal to the overtime/temporary worker wages. If the employee can’t be replaced through an overtime/temporary worker, a firm might be able to sustain some degree of production while the employee is off sick. In the case that full production is sustained while the employee is off sick; then there is no cost to the firm as a result of the illness. In contrast, if the firm loses all the production associated with that employee not being at work, then the cost to the firm is the wages it pays the employee while they are at home, plus the opportunity cost, i.e. the profitability the employee would have gen- erated for the firm. As data defining many of these parameters are absent, we simplified by assuming that the firm’s cost due to the short term illness of one of its employees is equal to the average wages paid to that employee in Canada while they are off work sick. We note that there are two estimates of the hourly labor costs in Canada: $24.46/hour reported by Statistics
The results presented in the this study must be interpreted in light of the methods and data limitations. A time series ecological method was used to estimate the seasonal P&I excess hospitalization rates in the study period. As such, the excess P&I hospitalization rate cannot be considered fully attributable toinfluenza epidemics but only associ- ated with the occurrence of the epidemics. Other covariates as the cir- culation of other respiratory virus, like the respiratory syncytial virus, were not considered in the model. This fact could have overestimated the impact of the influenza epidemics reported in the present report, mainly for the younger age groups (<2 years). 41,42 Nevertheless, when stratified by influenza virological profile, there were differences be- tween A(H3), A(H1)pmd09, or B predominant seasons, data consistent with previous reports, which suggests that P&I excess hospitalizations were sensible and probably specific in capturing the impact of the in- fluenza epidemics. In this study, an ARIMA model was used and no information about virus type or subtype circulation was considered. Unlike previous studies, 12,42 the weekly distribution of the influenza
3 hr in the presence of ROS-sensitive fluorescent compound ’, ’-dichlorofluorescin diacetate. Basal levels of free radicals produced in untreated cells were set at 100%. Cisplatin treatment resulted in a dose- dependent increase in DCF fluorescence in wild-type cells (Figure 4). DCF fluorescence increased by 2 and 3.5-fold with 60 and 100 M cisplatin, respectively when compared to untreated wild-type cells. These cisplatin concentrations did not induce DCF fluorescence to the same extent in Rho0 cells, compared to the untreated Rho0 strain (Figure 4). These results were further substantiated by a more sensitive electron paramagnetic resonance (EPR) spectroscopy (Figure 5). No detectable EPR signal was observed with 0 M CDDP, both for WT and Rho° cells. The EPR spectrum observed with 25 M and higher concentration of CDDP in both WT and Rho0 cells exhibited a 2x3x3x2 splitting which usually results from the interaction of an uncoupled electron with a primary nitrogen atom along with the secondary beta and gamma-protons. Based on hyper fine splitting and coupling constants, we observed the free radicals as superoxide free radicals. The hyperfine splitting constants of the signal (a N = 13.1
We collected nasal secretion samples from nasal cavities of all 42 subjects, 14 with PAR, 14 with NARES, and 14 healthy subjects, using the absorption technique. We used cotton- wool sticks (length 10 mm, diameter 4 mm; Institute of Virology, Vaccines and Sera, Torlak, Belgrade, Serbia). We inserted them for 5 minutes into the middle meatus, under the endoscopic guidance, as previously described. 11,12 We placed all samples in a 2 mL Eppendorf tube containing 1 mL of transfer medium (phosphate-buffered saline with genta- micin 50 μg/mL, penicillin G 340 IU/mL, fungizone 500 μg/mL) for 30 minutes. It allowed the diffusion of cytokines into the medium and then stored at 4°C for a maximum of 2 hours until processed. We centrifuged nasal ﬂuid at 1000 g for 10 minutes to separate the cellular components. After centri- fugation, we portioned supernatants and stored at -70°C for no more than two months, pending cytokine determination. We measured levels ofGM-CSF in all of the 42 samples using the commercial human ELISA kit (Thermo Fisher Scientiﬁc Inc., Waltham, MA, U.S.A.). We expressed the concentrations ofGM-CSF in picograms per milliliter (pg/mL). The sensitivity
Abstract The vaccine againstinfluenza is the main preventative intervention in public health for this disease. The aim of this study was to es- tablish the prevalence ofinfluenza vaccination in senior citizens according to indicators for their functional capacity, frailty, social support and in- volvement and state of health. This cross-sectional study was conducted in Campinas in 2008-2009 (FIBRA network, Unicamp center) with a prob- ability sampling of the elderly population( ≥ 65 years old).The dependent variable was immu- nization againstinfluenza in the twelve months prior to the research. The adjusted prevalence ratios were estimated by means of Poisson mul- tiple regression analysis. Of the six hundred and seventy-nine senior citizens involved, 74.4% stat- ed they had been vaccinated during the previous year. The prevalence of the vaccination was sig- nificantly higher among men and lower among those with a higher level of education. Slow gait speed is positively associated with immunization, as are most of the social involvement indicators. This can contribute towards improving immuni- zation adherence against seasonal influenza and should be widely acknowledged in order to broad- en immunization coverage in Campinas. Key words Aged, Influenza immunization, Prev- alence, Frailty in the elderly
Nevertheless, these results are inconsistent with the theories that hold that it is not the difference between values and other group features that leads to dis- crimination, but, on the contrary, it is the similarity between groups that leads to differentiation and, in some situations, to discrimination (Tajfel & Turner, 1979). Our findings also contradict the contentions of Coenders, Scheepers, Sniderman, and Verberk (2001). Partially reanalyzing the data used by Pettigrew and Meertens (1995, 2001), Coenders et al. (2001) maintained that general prejudice (explicit cultural inferiorization and explicit racial inferiorization) is independent of the “perception of cultural differences.” In fact, Coenders, Lubbers, Scheepers, and Verkuyten (2008) assumed that the “perception of cultural difference items in Pet- tigrew and Meertens’ scale ( . . .) reflect not so much evaluative prejudices, but rather cognitive perceptions” (p. 295). They also argue that when, for instance, Dutch respondents say that a Turk is “very different” in religion, they are simply “acknowledging a social reality” (p. 298), and not necessarily expressing an eval- uation. As plausible as the argument might seem at first, our results do not support it.
Pacientes com H1N1 foram pareados com casos con- secutivos de PAC grave admitidos na UTI na proporção de 2:1. Dessa forma, foram usados para comparação os dados de pacientes admitidos na UTI com PAC grave no mesmo período, e nos quais o diagnóstico de infecção por vírus inluenza A H1N1 não foi cogitado ou apre- sentavam RT-PCR da secreção nasofaríngea ou em es- pécimes do trato respiratório negativos. PAC grave com necessidade de internação em UTI foi caracterizada pela presença de um critério maior de gravidade (necessidade de ventilação mecânica ou de vasopressor) ou dois crité- rios menores (pressão sistólica <90 mmHg ou pressão ar- terial média <70 mmHg, pressão arterial de oxigênio/fra- ção inspirada de oxigênio - PO 2 /FiO 2 - <250, frequência respiratória ≥30/minuto, ureia >19,6 mg/dL, confusão mental, pneumonia multilobar, plaquetas <100.000 cel/ mm 3 , leucopenia - ≤4 x 10 9 , ou hipotermia - temperatura
Biopharmaceuticals are medicinal products comprising biotechnology3derived recombinant proteins as active substances. Like all other medicines, they are regulated by the U.S. agency FDA (Food & Drugs Administration) and the European Agency for the Evaluation of Medicinal Products (EMEA), which create and establish standards and scientific mechanisms that ensure safety, efficacy and quality of biopharmaceutical drugs. EMEA has approved the first biosimilar (Sandoz’s Omnitrope, somatropin, somatrophin) in April 2006 whereas the FDA has not approved any yet (Pisani and Bonduelle., 2006). Although the regulatory pathway for approval of biosimilars has not been completely finalized yet, these follow3on protein products should at least shown to be pharmaceutically equivalent (that is, it contains the same active ingredient in the same strength, dosage form and route of administration) and bioequivalent (Woodcock et al., 2007). Recombinant Granulocytes and Macrophages Colony Stimulating Factor (rGM3CSF) has been produced by Schering3Plough in (Leucomax®, Molgramostim) and by Berlex in (Leukine®, Sargramostim), the former had its patent expired in 2006. Leucomax® was co3developed by Novartis and Schering3Plough and has been co3marketed by the two companies in various
Introduction and Objectives: Increased free radical activity and high pro- inflammatory cytokine production by monocytes may be implicated in the pathogenesis of preeclampsia. Silibinin is a major active component of silymarin (Silybum marianum), a polyphenolic plant flavonoid that has antioxidant and anti-inflammatory effects. This study investigated the in vitro effect of silibinin on monocyte production of pro- and anti-inflammatory cytokines, as well as on superoxide anion (O2-) release in pregnant women with preeclampsia. Methods: Thirty preeclamptic (PE), 30 healthy pregnant (HP) and 30 healthy non-pregnant (NP) women were included. Peripheral blood monocytes were incubated with or without silibinin at 5 and 50ug/mL for 60 min, and stimulated with or without phorbol myristate acetate (PMA) for the assessment of O2-. These cells were also cultured with or without lipopolysaccharide (LPS) for 18h and tumor necrosis factor alpha (TNF-alpha), interleukin (IL)-6, IL-10, IL-12p40, IL-15, granulocyte-macrophage colony-stimulating factor (GM-CSF) and transforming growth factor-beta 1 (TGF- 1 ) in monocyte culture supernatants
Oxidative damage to diaphragm lipids and proteins is a hallmark of VIDD (reviewed in ). Importantly, previous work indicates that prevention of MV-induced oxidative stress via anti- oxidants can defend against MV-induced diaphragm atrophy and contractile dysfunction [9, 24, 39, 41]. More specifically, recent studies reveal that increased mitochondria ROS produc- tion during MV is a required upstream signal to promote diaphragm dysfunction in both ani- mals and humans [21, 51]. Further, inhibition of MV-induced mitochondrial ROS emission via administration of the mitochondrial-targeted antioxidant SS-31 can protectagainst both diaphragm atrophy and contractile dysfunction in rats . This previous study reveals that SS-31 protects the diaphragm against increased proteolysis by inhibiting the activation of sev- eral proteolytic systems (i.e. calpain, caspase-3 and the ubiquitin-proteasome system) . Moreover, it is feasible that prevention of oxidative stress in the diaphragm can preserve the rate of in vivo protein synthesis in the diaphragm during CMV. This prediction is based on evi- dence indicating that increased oxidative stress is sufficient to depress the rates of protein syn- thesis [27–29]. The current results reveal that treatment of animals with SS-31 successfully prevented MV-induced oxidative stress in the diaphragm and protected against the MV- induced decrease in diaphragm protein synthesis during 12 hours of CMV.
(50 mg/kg). The animals were then incubated and ventilated by a volume- regulated respirator during surgery. After a left lateral thoracotomy and pericardectomy, the left coronary artery was identified and gently ligated with a 6.0 prolene suture. Successful AMI was confirmed by the typical ST segment elevation in electrocardiography. Myocardial ischemia lasted for 30 mins and reperfusion for 2 hours. Freshly prepared melatonin (Sigma-Aldrich, St. Louis, MO, USA) was administered intraperitoneally at a dose of 20 mg/kg 12 hours prior to MI. PD98059 (ERK1 inhibitor, Sigma,USA) was administered with intraperitoneal injection at a dose of 5 mg/kg 4 hours prior to melatonin treatment. At the end of the reperfusion period, the hearts were excised for the preparation of sections (4 µm thickness) to detect the expression of SERCA2a and IP3R by immunofluorescence staining.
This study also provides the first study of global gene expression in healthy, unstimulated and cytokine-stimulated human neutro- phils using RNA-seq technology. Whilst several published studies have used microarray technology to investigate changes in neutrophil gene expression induced by agonists such as GM- CSF  and LPS [38,39,40], our investigation provides the first analysis of neutrophils using RNA-seq, and our data have been made publically available via GEO. Both microarray and RNA- seq are established, robust technologies for the study of global gene expression, and have been shown to correlate well when the same biological samples have been analysed by both technologies [41,42,43,44]. However, RNA-seq offers several advantages over microarray, as it allows estimation of absolute gene expression levels, and in particular, is not biased by signal saturation from high abundance genes. It also provides greater sensitivity for low abundance transcripts. Our study also provides the first direct comparison of the changes induced by two different cytokines on global gene expression in human neutrophils. Neutrophil studies have previously characterised the effect of single cytokines or agonists on global gene expression [36,38,40], and have then utilised real-time PCR to confirm changes in gene expression on a small sample of genes of interest with a larger number of agonists. The functional effects of TNF-a and GM-CSF priming on healthy neutrophils in vitro have been described previously by ourselves and others [15,16,17,18,19,20], and include delayed apoptosis, priming of the respiratory burst, altered expression of Fcc receptors and increased expression/affinity of adhesion molecules. Priming involves both molecular re-arrangements to change the activity and/or sub-cellular localisation of pre-existing molecules, and also activation of gene expression. Examples of the former processes include rapid phosphorylation of the cytosolic phox components of the NADPH oxidase  and cytoskeletal rearrangements to mobilise intracellular granules and secretory vesicles containing membrane proteins from the cytoplasm to the plasma membrane . Priming also results in activation of de novo biosynthesis, for example for the generation of cytokines and chemokines. Many of the functional effects of TNF-a and GM- CSF are similar, and yet our data show that these two cytokines activate different sets of transcription factors resulting in significant differential expression of several hundred genes.
Similar to , we used additive generalized estimating equation (GEE)  models with a Poisson distribution to attribute all-cause mortality to ILI-incidence. We decided to use the overall (instead of age-specific) ILI-incidence, because the coverage in the GP-ILI-sentinel may have been insufficient for some of the age categories included in the study. Thus we considered the overall ILI-incidence as a measure ofinfluenza activity on the national level, associated with all-cause mortality by age category. As fluctuations in ILI-incidence may to some extent reflect activity of pathogens other than influenza, we only included the ILI-incidence within a 23/+3 week window around influenza epidemic episodes (defined as two or more successive weeks that the overall weekly ILI-incidence was above the threshold for influenza epidemics; i.e. 5.1 per 10,000 population ); in other weeks the ILI incidence was truncated at zero. First, we regressed mortality time series – separately for each age category – on (lagged) overall ILI-incidence, using a generalized linear model with a Poisson distribution and an identity link. To correct for mortality due to seasonal factors other than influenza, we started with a constant basic mortality level plus sine and cosine variables (sine(k2pweek/52) and cosine(k2pweek/52), k = 1,2,3,4). We also added the temperature variables as covariables to correct for mortality due to hot (mean daily temperature.17uC) and/or cold (mean daily temperature,0uC) weather conditions. We then added the ILI-incidence and the RSV counts to the model, selecting the lagged ILI and RSV variable that contributed most to the model fit (-4 up to +2 week lags were considered for inclusion; e.g., in step 1, ILI was included with a 1-week lag if that exhibited a better model fit than all other ILI/RSV-lag combinations, assessed with Akaike’s information criterion ). We included lagged GP-ILI-incidence and the RSV counts in the model only once (i.e. for one lag). We used year-specific ILI regression coefficients as described in  so that the modeled association of mortality with ILI can vary among years. Essentially, these year- specific ILI regression coefficients are scaling factors for the number of deaths associated with a one-case increase in ILI- incidence per 10,000 population. Finally, we used GEEs  to correct the model outcomes for autocorrelation between observa- tions. See Appendix S1 for details on the regression models.
It is possible that the greatest determinant of non- adherence to vaccination is not lack of access. We suggest that increasing divulgation of the vaccine among healthcare professionals, as well as by spe- cific campaigns aimed at persons with chronic dis- eases, may increase coverage among groups at higher risk of developing complications of respiratory in- fections. Likewise, adherence of subjects older than 65 years may be increased by means of divulgation campaigns aimed at specific age groups.
However, the first results demonstrated that liposomes were physico-chemically and biologically unstable and drug encapsulation was not efficient. Fortunately, great advances in liposome technology allowed researchers to make significant improvements in its stability, as well as in the understanding of its characteristics and how they interact with biological fluids. Several factors have shown direct influence on the behavior of liposomes in a biological environment, such as preparation, vesicle size, composition, rate stability and drug encapsulation (Lasic, 1998). Thus, methods of characterization and controlling these factors became of extreme importance to produce these nanocarriers for drug delivery purposes. Similarities between the lipid bilayer structure and the cell membrane, make liposomes capable of interacting with the cells, allowing the targeting of the drug to reach the specific site, and therefore, with less toxicity than free drugs (McPhail, Tetley, Dufes, & Uchegbu, 2000).