Stimulated eosinophils were analyzed by flow cytometry. Supernatants were harvested, and secreted cytokines were quantified by using Bio-plex assay (Bio-Rad). To determine whether eosinophils support plasma cell survival, 6 days of secondary immunization eosinophils were isolated from the BM and plasma cells from the spleen. Using an isolation kit (Miltenyi Biotec), the purity of CD138+ splenic plasma cells was more than 90%. Cultures were set up as described previously 9. Briefly, 100 plasma cells were co-cultured together with eosinophils in U-bottomed 96-well plates for 24 h. After transfer of cells to anti-mouse Ig-coated

plates and further Roxadustat incubation at 37°C for 24 h, wells were washed and incubated with secondary antibody (Southern Biotech). The number of spots was counted by ELISPOT. To determine the viability of plasma cells, 104 plasma cells were co-cultured together with eosinophils isolated from BM of naïve, late primary (late 1°) or early secondary (late 2°) immunized mice for 48 h, and the percentage of living plasma cells was measured by staining with Annexin-V and PI. For cytospins, 1×105 sorted find more BM eosinophils in 150 μL complete medium were deposited into 24-well plates (Costar) fitted with cover slips. After

3 h, plates were centrifuged, washed with PBS and cells fixed with 100% cold ethanol for 10 min. Slides were stained with Alexa-546 conjugated monoclonal rat anti-MBP-specific antibody 28 together with rabbit anti-APRIL (Stressgen) or digoxigenin-conjugated monoclonal rat anti-IL-6-specific antibodies. Alexa-647-conjugated

Ergoloid goat anti-rabbit IgG (Invitrogen) and Cy5-conjugated anti-digoxigenin (DRFZ) antibodies were used as secondary antibodies. Staining was controlled by using rabbit IgG and rat IgG1 antibodies. Total RNA was extracted from 5×105 BM eosinophils using NucleoSpin®RNA II (Macherey-Nagel) according to the manufacturer’s instruction. Total RNA was reverse transcribed into cDNA using a Sensiscript RT kit (Qiagen). The levels of IL-4, APRIL, IL-6, IL-10 and TNF-α expression were determined by RT-PCR and real-time PCR as previously described 9, 27, 29. Primer sequences (TibMolBiol) for the amplification of IL-4, IL-6, APRIL and β-actin were described previously 9. The primers 5′seq: 5′-ctgactggcatgaggatcagc and 3′seq: 5′-ggcttggcaacccaagtaacc were used to amplify IL-10, the primers 5′seq: 5′-ggccaccacgctcttctgtct and 3′seq: 5′-ccagctgctcctccacttggt to amplify TNF-α. Real-time PCR was performed with the LightCycler system (Roche Diagnostics) using the Light-Cycler FastStart DNA Master SYBR Green I kit (Roche Diagnostics). Each sample was run in triplicate. Values were normalized against β-actin, and the expression level was determined as the relative unit (RU) in comparison to non-activated normal eosinophils. For statistical analysis, a paired two-tailed Student’s t-test and two-way ANOVA was performed.

After one wash with PBS, slides were analyzed by fluorescent microscopy using a Nikon eclipse E400 microscope (Nikon, Tokyo, Japan) with a ×20 or ×60 plan objective. For flow cytometry evaluation, staining was performed

without DAPI. Cells were analyzed selleck compound using BD FACSCalibur software (Becton Dickinson, San Jose, CA, USA). A plasmid containing the human NF-κB promoter upstream to the luciferase reporter gene, kindly provided by Y. Ben-Neriah (The Hebrew University), was purified using the Qiagen EndoFree Plasmid Kit (Qiagen, Düsseldorf, Germany) according to the manufacturer’s instructions. Highly purified plasmid DNA, 3 μg, was used to electroporate 0.5–2×106 DC, which were introduced with the Human Dendritic Cell Nucleofector Kit (Amaxa Biosystems, Cologne, Germany). Cells then were incubated

for varying times, under varying conditions, as indicated. The iDC were then harvested, washed, and lysed. Luciferase activity was measured by the Floustar luminometer, using the Luciferase Assay Kit (Promega, Madison, WI, USA). Statistical significance was assessed using the learn more Student’s t-test for unpaired data comparisons unless indicated otherwise. Kolmogorov−Smirnov analysis was used for flow cytometry analysis. The authors wish to thank Shifra Fraifeld for her editorial assistance with the preparation of this article. Conflict of interest: The authors declare no financial or commercial conflict of interest. “
“MHC class I-restricted CD8 T-lymphocyte epitopes comprise anchor motifs, T-cell Methane monooxygenase receptor (TCR) contact residues and the peptide backbone. Serial variant epitopes with substitution of amino acids at either anchor motifs or TCR contact residues have been synthesized for specific interferon-γ responses to clarify the TCR recognition mechanism

as well as to assess the epitope prediction capacity of immunoinformatical programmes. CD8 T lymphocytes recognise the steric configuration of functional groups at the TCR contact side chain with a parallel observation that peptide backbones of various epitopes adapt to the conserved conformation upon binding to the same MHC class I molecule. Variant epitopes with amino acid substitutions at the TCR contact site are not recognised by specific CD8 T lymphocytes without compromising their binding capacity to MHC class I molecules, which demonstrates two discrete antigen presentation events for the binding of peptides to MHC class I molecules and for TCR recognition. The predicted outcome of immunoinformatical programmes is not consistent with the results of epitope identification by laboratory experiments in the absence of information on the interaction with TCR contact residues.

Cells were incubated with fluorescent mAbs at 4°C for 1 h, then washed twice in phosphate-buffered saline (PBS) containing 2·0% fetal bovine serum (FBS) and fixed in 1·0% paraformaldehyde. Data were collected using FACSCalibur (BD Biosciences), and data analysis was performed using CellQuest software (BD Biosciences). FcαRIR209L/FcRγ Tg mice genomic DNA was extracted from mouse tails. PCR was performed using puReTaq Ready-To-Go PCR Beads (Amersham

Maraviroc nmr Bioscience, Amersham, UK). The following groups were studied. In group 1, mice received 80 µl normal saline once daily intraperitoneally. In group 2, mice were injected with 4 mg of horse spleen apoferritin (HAF; Sigma Aldrich Chemicals) in 80 µl of 0·1 M sodium chloride once daily intraperitoneally for 14 consecutive days. Mice in this group received 100 µl of normal saline intraperitoneally

at 8 h after the selleckchem HAF injection at days 7 and 8. In group 3, HAF was administered once daily as above. At days 7 and 8, 40 µg of endotoxin-free CpG-ODN 1668 (Invitrogen) in 100 µl of saline was administered intraperitoneally. In group 4, HAF was administered once daily as above. At days 7 and 8, 20 µg of MIP-8a in 200 µl of saline was administered via the caudal

vein after 40 µg of endotoxin-free CpG-ODN administered intraperitoneally. In group 5, HAF was administered once daily as above. At days 7 and 8, 20 µg of control IgG in 200 µl of saline was administered via the caudal vein after CpG-ODN intraperitoneally. At day 14, samples and renal tissues were collected. Urine samples were collected at days 0, 7, 9 and 14 in the morning. Urinary albumin was tuclazepam measured by immunoassay (DCA 2000 system; Bayer Diagnostics, Elkhart, IN, USA). Measurement of albuminuria is useful for detection of beginning of glomerular injury. This occurs before increasing of blood urea nitrogen (BUN) or creatinine values that sometimes mean renal failure. Blood samples were collected from each mouse at the end of the study from the retro-orbital venous plexus under general anaesthesia with inhaled ether. TNF-α, MCP-1 and RANTES levels were measured by ELISA (R&D Systems), according to the manufacturer’s protocol. For light microscopy, the sections were cut at 3 µm and then stained with periodic acid-Schiff (PAS) reagent after paraffin embedding.

1d). Concentration of lidocaine, bupivacaine and ropivacaine has a significant effect on cell death (for lidocaine P < 0·001, bupivacaine P < 0·001 and ropivacaine P = 0·001). Group arrangement also influences cell survival significantly: P = 0·001 for lidocaine, P = 0·029 for bupivacaine and P = 0·01 for ropivacaine.

Cell viability determined in fibroblasts from group 1 showed a similar pattern to trypan blue assays: only minor impairment over time was observed for the three signaling pathway LA with the 0·3 mg/ml concentration (Fig. 2a). While viability was not diminished after incubation with lidocaine and ropivacaine at a 0·6 mg/ml concentration, MTT decreased time-dependently after incubation with bupivacaine (Fig. 2b). In group 2, MTT did not change upon incubation with lidocaine and ropivacaine with the lower concentration. However, no cells survived after 9 days of bupivacaine exposure (Fig. 2c). With the higher concentration, fibroblasts experienced serious impairment of viability with increasing exposure time. The most pronounced effect was observed in the bupivacaine group (Fig. 2d). Correlation analysis revealed a time- and concentration-dependent effect on cell viability for all three LA with the following values: lidocaine time P = 0·019, concentration P < 0·001; bupivacaine time P = 0·05, concentration P < 0·001; ropivacaine time P = 0·004, concentration P < 0·001. An effect based on the type

of stimulation (group 1 or 2) was not observed. Thymidine incorporation over time upon incubation click here with each of the three LA was not changed after exposure to a low concentration of LA (Fig. 3a). With the 0·6 mg/ml concentration, again the proliferation rate was decreased only in the bupivacaine Metabolism inhibitor group (Fig. 3b). In group 2, with continued incubation with the low LA concentration, the proliferation rate decreased to 80% in the lidocaine and ropivacaine groups (Fig. 3c). This effect

was more pronounced with the 0·6 mg/l concentration. Bupivacaine had a more pronounced effect on thymidine incorporation with both concentrations compared to the two other LA (Fig. 3d). LA concentration had a statistically significant impact on proliferation rate (lidocaine: P < 0·001, bupivacaine: P < 0·001, ropivacaine P = 0·001), as did the group constellation (lidocaine: P < 0·001, bupivacaine: P = 0·009, ropivacaine P = 0·001). Fibroblast apoptosis was determined upon exposure to lidocaine, bupivacaine and ropivacaine. In group 1, apoptosis rate was diminished for all three LA in a similar manner for both concentrations (Fig. 4a and b). With permanent incubation with LA, the apoptosis rate decreased in a time- and concentration-dependent fashion for lidocaine. An increase in the apoptosis rate was observed at 3 days of incubation with the 0·3 mg/ml (bupivacaine, ropivacaine) and 0·6 mg/ml (ropivacaine) concentrations (Fig. 4c and d).

1. To study the differences in cytokine production between CD25+ and CD25− B cells, we used the TLR selleck chemicals ligands, Pam3Cys, LPS and CpG stimulating TLR 2, 4 and 9, respectively. The results are summarized in Table 1. The levels of IL-6 in supernatants from CD25+ B cells were significantly higher when compared with

CD25− B cells following stimulation for 12 h with CpG-PS, LPS or Pam3Cys (P < 0.05, respectively). In addition, CD25+ B cells secreted significantly higher levels of INF-γ as well as IL-10 following 72 h stimulation with CpG-PS, LPS and Pam3Cys (P < 0.05, respectively). Finally, CD25+ B cells produced significantly higher levels of IL-4 following 72 h of stimulation with CpG-PS (P < 0.05) when compared with CD25− B cells. The levels of IL-2 and TNF were analysed at the different time points (24 and 72 h); however, no secretion was detected (data not shown). The increased cytokine production after TLR stimulation was not because of a higher proliferation rate within the CD25+ B-cell subset compared with CD25− B cell as we did not detect any difference in the proliferative ability of these cell populations (data not shown). To BI 6727 manufacturer investigate if there was any difference in the ability of CD25+ B cells to present antigens to CD4+ T cells, we used a mixed lymphocyte reaction (MLR) as

an alloantigenic stimulation. CD25+ B cells are significantly better at presenting alloantigen

to CD4+ T cells when compared with CD25− B cells (P < 0.05) (Fig. 2). To evaluate if there were any differences in spontaneous immunoglobulin secretion between naïve CD25+ and CD25− B cells, we performed ELISPOT assays detecting IgA, IgG and IgM secreting B cells and found that the frequency of CD25+ B cells secreting immunoglobulins of IgA, IgG and IgM class was significantly increased compared with CD25− B cells (P < 0.05, respectively) (Fig. 3A). To analyse the Galactosylceramidase ability of CD25+ B cells to produce antigen-specific antibody, mice were immunized with OVA. At day 14 after immunization, we found that the frequency of CD25+ B cells secreting OVA-specific IgM antibodies were significantly (P < 0.01) increased compared with CD25− B cells (Fig. 3B), whereas the difference regarding the IgG response was less pronounced (P < 0.05). The levels of IgA secretion were very low in both groups, and there was no significant difference in the number of IgA OVA-specific secreting cells between the populations. We found that CD25+ B cells migrated significantly better both spontaneously and towards the recombinant mouse chemokine CXCL13 (P < 0.05, respectively) than CD25− B cells (Fig. 4). The number of CD25+ B cells expressing homing receptors was significantly increased compared with CD25− B cells with respect to α4β7, CD62L, CXCR4 and CXCR5 (P < 0.01, and P < 0.05, respectively) (Fig. 5A–D).

These results confirm the engagement of Notch signalling and indicate that it should be Delta-like 1 rather than Jagged1 that promotes collagen-specific Th1- and Th17-type expansion. A fundamental feature of T cell-dependent immune responses is the necessity for a very small population of CD4+ T cells to undergo clonal expansion and activation following encounter with a specific antigen. In the present study, we established an in vitro collagen-specific proliferation system in which the percentages of three CD4+ T cell subsets were analysed. The increased

percentage of Th1 cells and Th17 cells after CII restimulation indicates that collagen-specific reactivation tends to Th1- and Th17-type expansion. T cell responses to CII immunization have been studied extensively in mice with the I-Aq haplotype, which are highly Tipifarnib mouse Cabozantinib susceptible

to CIA (e.g. the DBA/1 strain). Intradermal injection of CII emulsified in complete Freund’s adjuvant results in the activation and expansion of antigen-specific CD4+ T cells with the Th1 phenotype, which initiate the harmful response [15]. By using tetrameric human leucocyte antigen D-related 1 (HLA-DR1) with a covalently bound immunodominant CII peptide, Latham et al. also reported that DR1–CII-tetramer+ cells expressed high levels of Th1 and proinflammatory cytokines, including IL-2, IFN-γ, IL-6, tumour necrosis factor (TNF)-α, and especially Olopatadine IL-17 [16]. These data confirm the pathogenic role of CII-specific Th1 and Th17 cells in promoting the development of disease in the arthritis model. Notch signalling plays an essential role in the development of embryonic haematopoietic stem cells and influences multiple lineage decisions of developing lymphoid and myeloid cells. Moreover, recent evidence suggests that Notch

is an important modulator of T cell-mediated immune responses. One of the most intriguing, and perhaps most controversial, functions assigned recently to Notch proteins is that of a regulator of Th cell differentiation. To assess whether Notch signalling is activated in collagen-specific Th1- and Th17-type expansion, we determined the abundance of the Notch target gene Hes-1. Hes-1 is the most well-characterized, γ-secretase-dependent transcriptional target gene of Notch signalling, and up-regulated expression of Hes-1 may be related to activated Notch signalling. As expected, we observed up-regulated transcript levels of Hes1. When we used γ-secretase inhibitor DAPT to block Notch signalling in SMNCs from CII immunized mice co-cultured with CII, we found that DAPT reduced T cell proliferation and the percentage of Th1 and Th17 cells. Palaga et al. also reported that γ-secretase inhibitor (GSI)-mediated inhibition of Notch signalling in peripheral CD4+ T cells stimulated by CD3- and CD28-specific antibodies resulted in decreased T cell proliferation and reduced IFN-γ production [12].

T helper type 2 development can be influenced by such cytokines as IL-33 and thymic stromal lymphopoietin,54,55 but IL-4 remains the primary signal that drives Th2 commitment from naive precursors.55,56 The Th2 differentiation involves the integration of signals both from

the T-cell receptor and from IL-4 signalling via STAT6, which culminates in the induction of the GATA3 transcription factor. GATA3 subsequently promotes transcription at the Th2 cytokine locus containing the IL-4, IL-5 and IL-13 selleck screening library genes. This pathway also acts acutely to inhibit expression of the IL-12Rβ2 subunit.57 Consequently, induction of GATA3 serves to block Th1 development while positively regulating Th2 commitment. Moreover, while there seems to be some level

of plasticity in Th2 cells,58 GATA3 is involved in an autoregulatory feedback loop VX-765 research buy that maintains Th2 commitment even in the absence of further IL-4 signalling.59,60 Hence, autoregulation by GATA3 represents an important stabilizing mechanism for Th2 commitment. However, early reports demonstrated that IFN-α/β could inhibit IL-5 secretion and eosinophil migration during allergic responses.61,62 Furthermore, IFN-α/β treatment of bulk CD4+ T cells during acute stimulation seemed to inhibit IL-5, but not IL-4 or IL-13. This was somewhat curious considering the dominant role played by IL-4 and GATA3 in Th2 effector function. Yet, despite these and other similar studies, one central question remained: can IFN-α/β regulate the ability of IL-4 to drive Th2 differentiation? Recently, Huber et al.63 found that unlike the Th1-promoting cytokines IL-12 and IFN-γ, IFN-α/β potently and specifically inhibited the ability of IL-4 to drive Th2 differentiation of human cells but not murine cells. Moreover, IFN-α/β destabilized pre-committed Th2 cells and blocked Th2 cytokine expression. Interferon-α/β also reduced expression of the Th2 marker, CRTH2. It appears to do this, at least in part, by suppressing mRNA and protein levels of GATA3, Urease which is critical for expression of CRTH2 as well as Th2-associated cytokines. While the underlying mechanism of GATA3 suppression is not yet clear, there are a few clues. First, as neither IL-12 nor

IFN-γ inhibits Th2 commitment, the effect is not likely to be mediated by STAT4 or STAT1. Furthermore, the inhibition of Th2 cells by IFN-α/β paralleled recent studies demonstrating that type-III interferon (IFN-λ) can also suppress Th2 responses.64 Since both IFN-α/β and IFN-λ activate STAT2 and drive ISGF3 complex formation,65 STAT2 may play a crucial role in suppressing human Th2 development. In addition to Th2 cells, there is increasing evidence that Th17 cells contribute to a variety of inflammatory processes involved in autoimmunity and allergic diseases.66 The Th17 cells are regulated by combined signalling via transforming growth factor-β, IL-6, IL-23 and IL-1β, culminating in the induction of the transcription factor retinoic acid-related orphan receptor γT.

Cells were incubated at 37 °C in 5% CO2. On the day of tumour challenge, TC-1 cells Barasertib manufacturer were harvested by trypsinization, washed with

phosphate-buffered saline (PBS), counted and finally resuspended in 500 μl of PBS. Plasmid DNA construction.  The generation of pcDNA-E7 (E7 Genebank accession number K02718, 294 bp, kindly provided by Prof. T.C. Wu, John Hopkins Medical Institutions, USA) and pQE-(NT-gp96) has been described previously [27]. For construction of pUC-E7, the E7 fragment was first amplified with PCR using pcDNA-E7 as the template and a set of primers designed as follows: E7F: 5′-GGGGATCCACCATGCATGGAGATACACCT-3 E7R: 5′-ATAAGCTTCCCGGGTGGTTTCTGAGAACA-3 The BamHI restriction site in forward primer and HindIII and SmaI restriction sites in reverse primer were underlined. PCRs were performed under conditions including 95 °C, 30 s; 67 °C, 30 s; 72 °C, 1 min for a total of 30 cycles. The amplified

product was then cloned into the BamHI/SmaI sites of the pUC18 cloning vector (Fermentas). To prepare plasmid DNA pDrive-(NT-gp96) (gp96 gene was kindly provided by Dr. Jacques Robert, University of Rochester Medical Center, USA), PCR was performed using pQE-(NT-gp96) as template and a set of primers (The SmaI in forward primer and KpnI restriction sites in reverse primer were indicated in bold): NTgp96FF: 5′-CGGCCCGGGGAAGATGACGTTGAA-3 gp96RN: 5′-ATGAGCTCGGTACCTTTGTAGAAGGCTTTGTA-3 The amplification program for performing PCR was as follows: 95 °C, 1 min; 62 °C, 2 min; 72 °C for 1.5 min for SAHA HDAC manufacturer a total of 30 cycles. The PCR product

was cloned in pDrive cloning vector according to kit instruction (Qiagen® PCR cloning kit, Hilden, Carbachol Germany). As the PCR product could insert in both direct and reverse orientation, therefore the direct-oriented clone was selected using PstI endonuclease which cut the NT-gp96 gene and also exist in multiple cloning site of pDrive. The PstI digestion resulted in 905 and 2945 bp fragments in direct-oriented pDrive-(NT-gp96) clone. To generate pUC-(E7-NT-gp96), the NT-gp96 fragment was isolated from pDrive-(NT-gp96) and then cloned into the SmaI/SacI sites of pUC-E7. DNA sequencing was performed to confirm the pUC-(E7-NT-gp96). For protein expression, the E7-NT-gp96 gene was digested from pUC-(E7-NT-gp96) and then cloned in BamHI/SacI sites of pQE-30 expression vector (Qiagen, Germany). Expression and purification of the recombinant E7-NT-gp96 [rE7-NT-gp96].  The production and purification of rE7 and rNT-gp96 were carried out as previously described [27]. E. coli strain M15 transformed with the recombinant pQE-(E7-NT-gp96) was grown at 37 °C in LB medium supplemented with 100 μg/ml ampicillin and 25 μg/ml kanamycin (Sigma, Germany).

NK cells are relatively easy to select from apheresis donations, but although typically approximately 5 × 108

cells can be obtained relatively pure, this may not represent a sufficient number for clinical efficacy [94]. Miller and colleagues therefore sought to expand transfused NK cells in vivo. Selected NK cells from HLA identical donors were transfused into 19 patients with high-risk AML after conditioning with low-dose total body irradiation or a combination of fludarabine and cyclophosphamide. The conditioning induced a rise of IL-15 and circulating NK cell numbers which showed enhanced cytotoxicity to leukaemia lasting more than 3 weeks. Five patients see more achieved complete remission [95]. Other investigators have developed clinical-grade strategies to expand NK cells ex-vivo using B cell lines [96] or modified K562 cells [97]. Such techniques can yield 20–200-fold expansion of pure but activated NK cells over several weeks. Expanded cells are fully functional and kill leukaemia and tumour targets. Clinical trials using expanded NK cells have not yet been reported. Future developments may include combined

ex-vivo and in vivo expansion approaches. Allogeneic T cells BMS-777607 cell line can be raised against mHag by peptide-pulsed DC or AML cells and are being used in treatment of relapsed leukaemia after stem cell transplantation. Outside the context of SCT, the occurrence in patients of CTL specific for AML supports the possibility

of using expanded autologous antigen-specific CTL to attack AML [3,86]. Adoptive transfer of leukaemia-specific T cells presents different challenges according to whether the transfused T cells are autologous or allogeneic in origin. Treatment with allogeneic T cells requires immunosuppression of the recipient to permit at least the short-term survival of the transfused cells. Two studies of allogeneic T cell transfer in non-transplant recipients have been reported [98,99]. Haploidentical donor lymphocyte transfusions were given to patients with diverse malignancies, including 13 patients with high-risk AML. Transfusion was followed by a cytokine storm without any SB-3CT sustained cellular engraftment, but there were tumour responses including five complete remissions in the AML patients [99]. Future developments will need to focus upon ways to achieve a short controlled engraftment sufficient to confer an anti-leukaemia effect perhaps by engineering T cells to escape immune attack, which may in turn require the co-insertion of a suicide gene as a safety precaution to prevent sustained persistence and expansion of the foreign T cell clone. Autologous T cell infusions can avoid the problems of alloreactivity of patient to donor or donor to patient. Here the problem is to generate sufficient numbers of T cells with powerful anti-leukaemia activity.

[60] Nanotechnology has brought new options for hRSV treatment and prophylaxis,

using the anti-microbial activity of metals, such as silver and gold.[66] Although due to their toxicity, the clinical use of these metals in humans seems unfeasible, the development of silver or gold nanoparticles combined with polyvinylpyrrolidone have been shown to efficiently inhibit hRSV replication, showing low toxicity in cell Selleckchem A769662 lines. Further, gold nanoparticles fused with inhibitor peptides displayed a high inhibitory capacity against hRSV.[66] Human RSV F protein nanoparticle vaccines have recently initiated clinical and preclinical studies to evaluate safety.[67] Another interesting therapeutic approach is the use of interference RNA that targets different steps during the hRSV infective cycle. The small interfering RNA (siRNA) strategy was initially used to target the expression of NS2[68] and the P[69] proteins, the latter showing an efficient capacity to protect mice against hRSV infection. This approach was also used to target the F gene, showing inhibition of hRSV

infection.[70] Nanotechnology has also been applied in combination with the siRNA approach to target the NS1 gene, resulting in the increase of IFN-β production by DCs and stimulated the Th1 differentiation of CD4+ cells.[71] Such a strategy protected mice against RSV infection, because treated mice showed decreased viral loads in lungs and

reduced inflammation in this tissue. Roscovitine A new siRNA specific against NS1(ALN-RSV01) showed high antiviral activity that impaired nucleocapsid expression.[72] Studies in mice reported that administration of this molecule reduces RSV titres in the lungs.[73] This antiviral drug has also been evaluated in human clinical trials, demonstrating their safety and tolerance in healthy adults.[72] In addition, the effectiveness of ALN-RSV01 against hRSV infection was evaluated Orotidine 5′-phosphate decarboxylase in humans, with a 44% reduction of hRSV infection without adverse effects[74] and the phase IIb clinical trial has concluded. Further, this drug has been tested in lung transplant patients, where it has demonstrated safety and effectiveness.[74] Another strategy to combat the disease caused by hRSV is to target the harmful immune response elicited by hRSV infection. The exacerbated Th2 response associated with the hRSV bronchiolitis is characterized by high production of IL-4. Along these lines, a study generated an antisense oligomer to promote local silencing of il4 gene expression, which was delivered intranasally.[75] This approach was evaluated in neonatal murine models, showing a reduction of Th2 response and decreasing the airway damage caused by hRSV.[75] To improve the specificity of siRNA technology as an antiviral approach for hRSV, the use of phosphorodiamidatemorpholino oligomers (PMOs) has been proposed.