, 2005). Although opioid analgesia attenuates the sensory aspects of pain, Afatinib a major component of the analgesic response involves a blunting of the negative affective component of pain (Zubieta et al., 2001). An “anti-stress” activity of endogenous opioids may be specifically mediated by the μ-opioid receptor (MOR), the receptor that shows greater selectivity for β-endorphin, endomorphin and the

enkephalins (Akil et al., 1984, Sora et al., 1997 and Drolet et al., 2001). In contrast, a stress-like aversion has been associated with the dynorphin-κ-opioid receptor system (Chavkin, 2013). Support for an anti-stress function of endogenous opioids comes from studies showing evidence for stress-elicited opioid release. In animal studies, many stressors, including those that are non-noxious, produce an analgesia that is cross tolerant with morphine and is antagonized by naloxone (Girardot and Holloway, 1984, Lewis et al., 1980, Miczek et al., 1982 and Rodgers and Randall, 1985). This is also apparent in humans. For example, the presentation of combat-related stimuli to PTSD patients produces naloxone-sensitive analgesic responses (Pitman et al., 1990 and van der Kolk et al., 1989). Stress also increases preproenkephalin mRNA

in certain brain regions and β-endorphin in plasma (Ceccatelli and Orazzo, 1993, Dumont et al., 2000, Mansi et al., 2000, Lightman and Young, AZD6738 research buy 1987 and Rossier et al., 1977). One mechanism by which endogenous opioids can counteract stress is through actions that oppose those of CRF. Enkephalin and CRF are whatever co-localized in many hypothalamic neurons, in the medial preoptic nucleus and in the bed nucleus of the stria terminalis (Sakanaka et al., 1989). The cellular targets of these neurons are potential sites of interaction between CRF and enkephalin. Additionally, CRF and enkephalin distribution overlaps in brain regions

underlying behavioral and autonomic components of the stress response including the CEA, parabrachial nucleus and nucleus tractus solitarias (Swanson et al., 1983, Drolet et al., 2001 and Sakanaka et al., 1989). That these neuromodulators act in an organized fashion to fine-tune neuronal activity in response to stressors is particularly evident in their co-regulation of the LC-NE system during stress (Valentino and Van Bockstaele, 2001). LC neurons are anatomically poised for co-regulation by CRF and enkephalin. Although few axon terminals in the LC and peri-LC region co-localize CRF and enkephalin, LC dendrites receive convergent input from CRF- and enkephalin-containing axon terminals and co-localize MOR and CRF1 (Tjoumakaris et al., 2003 and Xu et al., 2004).

Tout comme l’obésité, les prévalences du SMet et du DT2 s’élèvent avec l’âge. Et fait de nombreuses fois démontré par les études épidémiologiques, elles restent

supérieures chez l’homme à ce qui est observé dans le sexe féminin. A découlé fort logiquement NVP-BKM120 supplier de ce constat, la question du rôle éventuel des stéroïdes sexuels dans cette différence liée au genre. De nombreuses études ont mis en évidence, chez l’homme adulte, un lien indiscutable entre abaissement du taux de testostérone plasmatique et syndrome d’insulino-résistance. Insulino-résistance et hypotestostéronémie sont par ailleurs impliqués dans la physiopathologie de plusieurs facteurs de risque vasculaire : hypertension artérielle, trouble de l’équilibre glycémique, dyslipidémie [1], [2], [3] and [4]. Deux constations supplémentaires ont amené à évaluer plus précisément l’équilibre androgénique des hommes suivis pour obésité, SMet ou DT2 : • la fréquence de ces anomalies métaboliques s’élève avec l’âge tandis que parallèlement la sécrétion testiculaire endocrine décline ; Chez l’homme, une baisse de la testostéronémie a été démontrée dans chacun des Proteasome inhibitor trois cadres pathologiques que constituent obésité, SMet et DT2. Il s’agit donc bien

là d’un point commun supplémentaire à ces trois entités, point commun dont l’identification a amené à s’interroger sur son implication physiopathologique, sa valeur pronostique et l’intérêt thérapeutique d’un rééquilibrage du statut androgénique. Une réduction du taux de testostérone plasmatique, dont l’ampleur est inversement corrélée à l’index de masse corporelle (IMC), a été mise en évidence chez l’homme adulte en surcharge pondérale. Dans le surpoids simple ou l’obésité non morbide, le taux de testostérone libre reste others situé dans les limites de la normale pour la tranche d’âge considérée. Dans ces deux situations, l’abaissement de la testostérone totale est en effet liée à la diminution du taux de la Sex Hormone-Binding Globulin (SHBG), protéine porteuse des stéroïdes sexuels encore dénommée Testosterone-estradiol-Binding Globulin (TeBG) dont le taux est négativement corrélé

à l’IMC ( figure 1) [5]. L’obésité massive s’accompagne, par contre, d’une réduction de l’ensemble des fractions, libre et liée, de la testostérone plasmatique [6]. L’obésité androïde s’associe à une insulino-résistance. Testostéronémie totale et taux de SHBG plasmatique en représenteraient des marqueurs, susceptibles également d’être impliqués dans son développement et, à un stade évolutif ultérieur, à celui d’un DT2. Il a été montré que le taux de testostérone plasmatique était fréquemment plus bas dans la population d’hommes atteints d’insulino-résistance que dans une population du même âge indemne de pathologie quelconque [2], [7] and [8]. Les résultats de ces études font même l’hypothèse qu’un taux bas de testostérone plasmatique exposerait à un risque plus élevé de développement d’un DT2.

Experimental results were expressed as mean ± SD. The data were analyzed for statistical significance by Analysis of Variance.22 Data were considered significant at p < 0.05. The DPPH radical scavenging activity of silver nanoparticles Onalespib nmr synthesized by M. pubescens was studied. The decolorization from purple DPPH radical to yellow DPPHH molecule by the sample in a dose-dependent manner with an IC50 value of 84 ± 0.25 μg/ml indicated the sample’s high radical scavenging activity which was closer to that of the standard whose IC50 value was found to be 80 ± 0.69 μg/ml as shown in Fig. 1. Superoxide anion derived from

dissolved oxygen by PMS-NADH coupling reaction reduced NBT. The decrease of absorbance at 560 nm with antioxidants indicated the consumption learn more of superoxide anion in the reaction mixture. The silver nanoparticles (100 μg/ml) exhibited superoxide

radical scavenging activity of 34 ± 1.21% comparable to that of the standard which showed 43 ± 1.06% activity. Absorbance values of the sample and the standard were higher than that of control as in Fig. 2. The scavenging capacity of the silver nanoparticles from leaf extract of M. pubescens was shown in Fig. 3. At a concentration of 100 μg/ml, the silver nanoparticles showed 37 ± 2.01% hydroxyl radical scavenging activity with the standard tocopherol activity being 42 ± 2.22%. The radical scavenging capacity of the sample might be attributed to phenolic compounds in the sample with the ability to accept electrons, which could combine with free radical competitively to decrease hydroxyl radical. The presence of

chelating agents in the sample disrupted the Ferrozine-Fe2+ complex Chlormezanone formation. Thus the decrease in the absorbance at 562 nm indicated high levels of iron binding potential and antioxidant activity of the nanoparticles (Fig. 4). The sample of 100 μg/ml concentration possessed 56 ± 1.36% metal chelating activity with EDTA expressing 62 ± 1.78% activity. The assay was based on the reduction of Mo (VI) to Mo (V) by the sample and subsequent formation of a green phosphate-Mo (V) complex at acidic pH. The silver nanoparticles exhibited powerful antioxidant activity of 57 ± 1.65% compared to that of the standard with 69 ± 1.22% activity, in the reduction of phosphomolybdenum complex as shown in the Fig. 5. The FTC method was used to measure the peroxide levels during the initial stage of lipid peroxidation. Silver nanoparticles successfully inhibited the oxidation of linoleic acid. Low absorbance values of the sample compared to the control indicated high levels of antioxidant activity. The absorbance of the control increased till 6th day and then decreased entering into the secondary stage of lipid peroxidation. Fig. 6 detailed the absorbance values with respect to days of incubation.

The external primers used were 5′-CACGGTACCTCTTTCTTTATCG-3′ (KpnI restriction site underlined) and 5′-GGTTCTCTGCAGAGACATGC-3′ (PstI restriction site underlined). The internal primers responsible for introducing the mutation leading to the amino acid replacement

G33D were 5′-GAATCGATGGCAGATAAAAG-3′ and 5′-CTCTTTTATCTGCCATCGAT-3′. The amplification reactions were performed Gemcitabine as described previously [39]. The resulting fragment was purified using a gel purification kit (Ilustra™ GFX™ PCR DNA and Gel Band Purification Kit, GE Healthcare), digested with restriction enzymes and then ligated into the corresponding KpnI and PstI sites of the linearized pBSPKS (−) vector [41], generating the recombinant plasmid pKSLTG33D. The pKSLTG33D plasmid was subsequently introduced into chemically competent E. coli DH5α bacteria. One bacterial clone carrying the correct plasmid was named LDVLTG33D. The correct sequence of the etxG33D gene was confirmed by DNA sequencing. LTG33D was purified by galactose-affinity

chromatography following a standard LT purification procedure [40]. Briefly, the LDVLTG33D lineage was cultivated in Terrific Broth (TB) [42], click here containing 200 μg/ml of ampicillin, overnight at 37 °C in an orbital shaker set at 200 rpm. Cells were suspended at a 10% (w/v) concentration in TEAN buffer (50 mM Tris; 1 mM EDTA; 3 mM azide-Na and 200 mM NaCl; pH 7.5) and lysed by mechanical shearing in an APLAB-10 homogenizer (ARTEPEÇAS, Brazil). The soluble extract was applied into

a XK 16/20 column (GE Amershan Biosciences) containing immobilized d-galactose gel (Pierce), extensively washed with TEAN buffer prepared with pyrogen-free water, and subsequently eluted with TEAN buffer containing 0.3 M else galactose. The final amount of LTG33D was determined in GeneQuant spectrophotometer (GE Amershan Biosciences). The purification of the DENV2 NS1 recombinant protein was achieved after denaturation/refolding steps of the protein expressed in bacterial cells and affinity chromatography, as previously reported [36]. Endotoxin levels in LTG33D and NS1 preparations were determined with the Chromogenic Limulus Amebocyte Lysate assay (Cambrex Bio Science) [43]. The recombinant NS1 and LTG33D proteins were analyzed for purity and antigenicity by SDS-PAGE and Western blot. Protein aliquots (2 μg) were sorted in 15% polyacrylamide gels after heat treatment (100 °C for 10 min) or kept at room temperature with sample buffer [36] and [44]. Standard ELISA assays were performed as previously described [36] and [45]. The recombinant NS1 protein was tested in the non-heated or in heat-denatured state with serum samples collected from a DENV2-infected individual (kindly supplied by Dr. Bergman M. Ribeiro, Brasília University, FD, Brazil). A serum sample generated after immunization of mice with heat-denatured (100 °C for 10 min) NS1 in FA after the same immunization regimen described bellow (Fig.

The seven group X strains were isolated in Burkina Faso, Ghana and Uganda. Two strains from Burkina Faso expressed fHbp ID73, the other isolates expressed ID74. The strains from Burkina Faso were sequence type 751 and 181, respectively. The two strains from Uganda were ST5403 and expressed PorA subtype P1.19,26, while the other five group X strains were P1.5-1,10-1. The two strains from Uganda differed

from each other by the level of fHbp expression. Strain Ug11/07 had 4% and Ug9/06 has 200% of the fHbp expression level compared to the reference strain (Table 1). GMMA with MLN8237 supplier or without fHbp over-expression elicited high bactericidal titres that were not significantly different from each other against the three W strains this website expressing either fHbp v.1 or v.2 (Fig. 3A). This is consistent with previous observations that bactericidal activity against strains sharing the same PorA as the GMMA-production strain is predominantly mediated by anti-PorA antibodies [26]. GMMA from the Triple KO, OE fHbp strain induced antibodies that were

able to kill six out of seven serogroup A strains (geometric mean titres [GMT] ranging from 20 to 2500) (Fig. 3B). The only isolate that was resistant to killing was readily killed by a mouse serum raised against group A polysaccharide conjugate vaccine. The antibodies induced by the GMMA from the Triple KO, OE fHbp strain were able to kill all serogroup X strains tested (GMT = 18–5500) (Fig. 3C). GMMA produced from the W strain which lacked fHbp v.1 over-expression (Triple KO), induced antibodies that were only able to kill one X strain (BF7/07), consistent with the majority of bactericidal antibodies induced by the GMMA vaccine being directed against fHbp. Antibodies made against the

recombinant fHbp ID1 were only bactericidal against serogroup X strain Ug9/06 with the highest fHbp expression. We investigated the dose-dependent bactericidal antibody response against one W (1630), A (N2602) and X (BF7/07) isolate (Fig. 4A). Sera raised against GMMA with over-expressed fHbp were bactericidal against Thalidomide these strains in a dose-dependent manner (Spearman Rank P = 0.001 for group A and P < 0.0001 for group W and X) with killing occurring at all three doses (0.2, 1 and 5 μg). GMMA from the triple KO, OE fHbp mutant was prepared from a mutant with deleted capsule expression in order to attenuate virulence of the vaccine strain and reduce serogroup-specific antibody production. To test the latter, we investigated whether maintaining capsule expression in the GMMA-producing strain affects the bactericidal antibody response. Sera from mice immunised with GMMA prepared from the Triple KO, OE fHbp vaccine strain had significantly higher SBA activity against three of five A and X strains tested than GMMA from the isogenic mutant that expressed the capsule ( Fig. 4B).

The specific research

questions were: 1. Does electrical stimulation increase strength after stroke? Are any benefits maintained beyond the intervention period or carried over to activity? In order to make recommendations based on a high level of evidence, this review included only randomised or controlled trials. Subgroup analyses based on time after stroke and initial level of strength were planned. Searches were conducted in MEDLINE (1946 to December 2012), CINAHL (1986 to December 2012), EMBASE (1980 to December 2012) and PEDro (to December 2012) for relevant studies without date or language restrictions. Search terms included: words related to stroke; words related to randomised, quasi-randomised or controlled trials; and words related to electrical stimulation (such as electric stimulation, neuromuscular stimulation, nerve stimulation and INCB28060 functional stimulation) (see Appendix 1 on the eAddenda for the full search strategy). Title and abstracts

were displayed and screened by two reviewers in order to identify relevant studies. Full text copies of peer-reviewed relevant papers were retrieved and their reference lists were screened to identify further relevant studies. The method section of the retrieved papers was extracted and reviewed independently by two reviewers using predetermined criteria ( Box 1). Both reviewers buy JQ1 were blinded to authors, journals and results. Disagreement or ambiguities were resolved by consensus after discussion with a third reviewer. Design  • Randomised or controlled trial Participants  • Adults (>18 years old)  • Diagnosis of stroke  • Muscle weakness (Manual Muscle Test < Grade 4) Intervention  • Electrical stimulation in order to increase strength (ie, it is clearly stated that the aim of the intervention is to increase strength or strength is an outcome measure) Outcomes measures  • Strength measured as peak force/torque and congruent with the stimulated muscle/s Comparisons  • Electrical stimulation versus placebo/nothing or non-strengthening intervention  • Electrical stimulation versus

any other strengthening intervention  • Electrical stimulation versus different dose/mode of electrical stimulation Full-size table Table options View medroxyprogesterone in workspace Download as CSV The quality of the included trials was assessed by extracting PEDro scores from the Physiotherapy Evidence Database26. The PEDro scale is a 11-item scale designed for rating the methodological quality (internal validity and statistical information) of randomised trials. Each item, except for Item 1, contributes one point to the total PEDro score (range: 0–10 points). Where a trial was not included in the database, it was scored by a reviewer who had completed the PEDro Scale training tutorial. Trials involving adult participants of either gender at any time following stroke were included.

Significant benefits in functional exercise capacity have also been identified after six weeks to six months of home-based training in people with chronic heart

failure (Corvera-Tindel et al 2004, Evangelista et al 2006, Harris et al 2003) and in a meta-analysis of these studies (Chien et al 2008). The improvement in six-minute walk distance in our study was somewhat smaller than that reported in studies related to supervised or centre-based training (Rees et al 2004, van Tol et al 2006). This BIBW2992 may be related to the clinical characteristics of our subjects (who tended to have less severe disease), the low to moderate intensity of the exercise, and the relatively short period of exercise training. Some other strategies of reinforcement, such as a personalised workbook, an interactive video, or an intervention of longer duration

may be considered in future studies to gain better adherence and thereby to maximise improvement. Nevertheless, home-based exercise can be recommended when all the physical and psychological benefits are considered. Health-related quality of life showed an overall between-group difference of 7 points on the 105-point Minnesota questionnaire. This exceeds the minimum clinically important difference of 5 Integrin inhibitor points proposed by Riegel et al (2002). However, the lower limit of the confidence interval around this result may not be clinically worthwhile. Exercise training might improve quality of

life by Mannose-binding protein-associated serine protease ameliorating the fatigue, shortness of breath, oedema, and other common symptoms in chronic heart failure. The improved quality of life could also be related to the improvement in functional exercise capacity and, hence, in disability. Our finding that home-based exercise improves quality of life in people with chronic heart failure is consistent with past research in this area (Harris et al 2003, McKelvie et al 2002, Oka et al 2000). Anxiety and depression are of multi-factorial origin and may be bi-directionally related to the cardiac dysfunction, functional disability, and prognosis in subjects with chronic heart failure (Haworth et al 2005, Rutledge et al 2006, Tousoulis et al 2010). Antidepressant effects of exercise have previously been attributed to social contact and changes in stress hormones and brain-derived neurotrophic factors (Herring et al 2010, Tousoulis et al 2010). Previous studies have demonstrated some beneficial effects of exercise training on reducing anxiety and depression in people with chronic heart failure, although the effect sizes were relatively small (Koukouvou et al 2004, Kulcu et al 2007). Subjects in our study were relatively stable, with predominantly low levels of anxiety and depression and less dependence with the activities of daily living.

In absence of a convenient and truly representative model of the alveolar epithelium, bronchial systems have been favoured [3] and [4]. Among these, the human bronchial cell line Calu-3 and normal human bronchial epithelial (NHBE) cells are gaining in popularity due to their capacity to develop polarised cell layers morphologically similar to the native epithelium and suitability for permeability measurements when

cultured at an air–liquid interface (ALI) [4], [5] and [6]. However, although the presence of active drug transport mechanisms has been confirmed in Calu-3 and NHBE layers [1], [6], [7], [8] and [9], an overview of the range of transporters being expressed and functional in these models is still lacking. P-glycoprotein/multidrug resistance protein 1 (P-gp/MDR1) is a member of the ATP-binding cassette (ABC) efflux transporter family and plays a major role in drug–drug interactions see more [10], limitation of oral drug absorption and poor drug penetration find more in the central nervous system [11]. As it has been reported several drugs administered by the pulmonary route might be MDR1 substrates [1], targeting the transporter present in the epithelium has been envisaged as a strategy to increase the residence time of inhaled drugs in the lung tissue. Consequently, MDR1 is by far the most extensively studied drug

transporter in the lung [1]. Although weakly expressed in the lung as compared to other major organs [12], the presence

of the MDR1 protein has been demonstrated in the bronchial epithelium [1]. However, its actual impact on the pulmonary absorption of established substrates is a matter of debate [1]. Similarly, reports on the expression, localisation and functionality of MDR1 in bronchial epithelial cell culture models are CYTH4 conflicting [1]. Passage number and time in culture have recently been shown to impact on MDR1 expression or activity in ALI bronchial epithelial layers [6], [13] and [14], which may partly explain discrepancies between studies. Identifying the transporter protein involved in carrier-mediated drug trafficking is highly challenging in biological systems expressing multiple transporters with broad and overlapping substrate specificities. For instance, the cardiac glycoside digoxin has been well characterised as an MDR1 substrate and is largely used for evaluating the risk of drug–drug interactions with new chemical entities consequent to their modulation of MDR1 activity [15] and [16]. Accordingly, although not an inhaled drug, digoxin has been used for probing MDR1 activity in bronchial cell culture models [13] and in rodent lungs [13] and [17]. However, digoxin has also been described as a substrate for carriers other than MDR1.

Fresh lysozyme artificially increased the signal intensity of the PyroGene™ assay. The dry chemical stock of lysozyme possibly harboured Gram-negative microbes or pyrogenic byproducts. Unlike with the LAL assay, high molecular weight carbohydrates such as carrageenan were not found to enhance the PyroGene™ assay [42]. Several of the tested substances (i.e. BSA, HA, lysozyme, and dextran) exhibited apparent enhancement when initially tested. As these liquid samples had been stored non-sterile

at 5 °C for two weeks, fresh stocks were prepared. Using the fresh stocks, no enhancement was observed, highlighting Trametinib solubility dmso the importance of mitigating potential Gram-negative bacteria contamination. None of the tested species consistently interfered

except for those shown in Fig. 9. Utilization of the PyroGene™ assay will necessitate extensive dilution (i.e. 10−3–10−4) to eliminate interference from bacterial feedstreams. The level of dilution will be predicated on the concentration and nature of components in the sample background, with samples upstream in the process requiring greater dilution than the more purified streams found further downstream in the process. Although the magnitude of the inhibition is significant, the PyroGene™ assay is still suitable for measuring endotoxin in impure pools. In polysaccharide process streams derived from Gram negative bacteria, the starting concentrations of endotoxin are high. These values often exceed 20,000,000 EU/mL (personal communication from Dr. Bernie Violand;

Pfizer R&D). However, the linear range Palbociclib cost of the PyroGene™ assay is 0.01–10 EU/mL, necessitating multiple serial dilutions to fall within the standard curve. Because of the large difference between the range of the PyroGene™ assay and typical endotoxin concentrations, Linifanib (ABT-869) it is possible to measure adequate LRV of endotoxin, even when factoring in dilution to eliminate interference (Table 4). With such high amounts of endotoxin present, dilution to 10−3–10−4 should still enable the demonstration of 5–6 log removal value (LRV) of endotoxin clearance for harvest samples and 2–3 LRV of endotoxin clearance for polishing steps. Demonstration of adequate clearance may be hampered in samples taken downstream of polishing steps. The capability to automate assays used to inform purification process development is clearly an important attribute. All of the described assays can be integrated into an automated analytical platform, enabling multi-faceted characterization of impurity clearance and product yield in less than one day by a single scientist. Automation requires an initial upfront investment of effort to refine but can be indispensable when repeat analyses are required. In purification process development, several high throughput screens can be run to evaluate different unit operations or distinct modes within a given unit operation.

Trademarks: Gardasil® is a registered trade mark of Alectinib research buy Merck Sharp & Dohme Corp., Cervarix® is a registered trade mark of the GlaxoSmithKline group of companies. Conflict of interest: ND and GVK are employees of the GlaxoSmithKline group of companies and ND owns stock in the GlaxoSmithKline group of companies. DC has no conflict of interest related to this manuscript. XC has performed consultancy work for the GlaxoSmithKline group of companies. He received funding for board membership and lectures from the GlaxoSmithKline group of companies. None of these

activities was directly related to the current study Author contributions: GVK, XC, DC and ND conceived and designed the study; GVK and ND developed the model; GVK and XC acquired the data; GVK analysed the data; all authors have made substantial intellectual contributions to the manuscript, reviewed and commented on drafts and approved the final manuscript. Role of the funding source: GlaxoSmithKline Biologicals SA was the funding source and was involved in all stages of the study conduct and analysis. GlaxoSmithKline Biologicals SA also funded all costs associated with the development and the publishing of the present manuscript. All authors had full access to the data and agreed with the submission

of the manuscript for publication. “
“Hemorrhagic fever with renal syndrome (HFRS) is a zoonosis caused by Hantaviruses. It is widely distributed in eastern Asia, particularly in China. The number of HFRS cases and deaths in China is the highest in the world and therefore find protocol HFRS is an important public health problem in China [1]. Hu County is one of the main HFRS epidemic areas in China, with the third highest HFRS incidence among all counties of China in

2010 [2]. Both Hantaan virus (carried by Apodemus agrarius mice that thrive in the wild) and Seoul virus (carried by Rattus norvegicus rats that thrive in residential areas) were detected and in this county, with the Hantaan virus as the primary cause. Since 1994, Hu County has offered a free HFRS vaccination program to people between 16 and 60 years of age. The HFRS vaccines were supplied free of charge by the government in October to December of each year to people who had never received this vaccination. An HTNV-inactive vaccine was provided during 1994 to 2003 in Hu County and an inactive bivalent vaccine, consisting of HTNV and SEOV, was provided from 1994 to 2011. People younger than 16 and older than 60 years were suggested to avoid contact with rats and its excreta. However, this county is still severely threatened by HFRS, with an incidence of 48.5 per 100,000, which was 68.3 times higher than that in the rest of China in 2011 [3]. Some important considerations remained, including the effectiveness of the vaccination program and the necessity to continue to provide the HFRS vaccination freely in Hu.