, 2009) as well as by fluorocalcone A staining in sputum samples from CF patients in whom mucoid P. aeruginosa has been identified by culturing (Yang et al., 2008). An alginate-overproducing strain (PDO300) [isogenic mucoid variant Alg+ PAOmucA22 of the reference P. aeruginosa strain (PAO1) (Mathee et al., 1999)] formed thicker and rougher flow-cell biofilms and exhibited enhanced microcolony formation compared with PAO1 (Hentzer et al., 2001). It has also been established that the structural difference

between the architecture of biofilms formed by a mucoid CF isolate and the nonmucoid revertant is due to alginate (Nivens et al., 2001). Recently, it has been check details shown that in addition to alginate, other polysaccharides such as Psl play an important role in the matrix of mucoid biofilms. Overproduction of alginate causes biofilms which occupy more space, while the Psl causes dense packed biofilms (Ma et al., 2012). The distribution of live and dead cells within PAO1 and PDO300 biofilms during tobramycin treatment suggested that enhanced microcolony formation

creates an antimicrobial-resistant zone in the interior of the microcolony and that this is an important element Selisistat of the increased tolerance of mucoid biofilms. In addition, the differential expression of a large number of genes as a consequence of mutations in the global regulator mucA (Rau et al., 2010) probably also play a role. Treatment

of mucoid and nonmucoid biofilms with tobramycin showed that mucoid biofilms were up to 1000 times more resistant to tobramycin than were the nonmucoid Epothilone B (EPO906, Patupilone) biofilms in spite of similar planktonic MICs (Fig. 1). The exact mechanism of the higher tolerance to antibiotics of mucoid biofilms is not clearly understood. Two of the contributing factors to this tolerance are that the matrix represents a physical and chemical barrier and that due to nutritional gradients, cells buried in a biofilm are reduced in metabolic activity, making them less susceptible to antibiotics which primarily target the metabolically active cells (Stewart, 1996). Dosage optimization based on the pharmacokinetics (PK) and pharmacodynamics (PD) of antimicrobial agents is extremely important to maximize the effect of antibiotics at the infection site and to prevent further development of antimicrobial resistance (Craig, 1998; Safdar et al., 2004). Recently, in vitro studies of the PK and PD on nonmucoid and mucoid biofilm-growing bacteria have been reported (Hengzhuang et al., 2011). In accordance with the results of tobramycin treatment of flow-cell biofilms (Hentzer et al.

41 Mesenchymal stem cells have been found to exert a therapeutic effect in a wide array of diseases, acting through their unique immunomodulatory abilities that can alter the pro-inflammatory course of injury. This may involve the secretion of paracrine factors that dampen inflammation and in turn promote tissue remodelling and repair.39 Their ability to modulate the immune response www.selleckchem.com/products/Imatinib-Mesylate.html in vivo was first reported by Bartholomew et al.42 who demonstrated that the intravenous administration of allogeneic MSC to baboons resulted in prolonged skin-graft survival. MSC have also been reported to be beneficial in an autoimmune disease setting. In a mouse model of multiple sclerosis termed autoimmune encephalomyelitis (EAE), the administration

of MSC at the onset of disease induced peripheral T-cell anergy against the pathogenic peptide myelin oligodendrocyte glycoprotein (MOG), resulting in the amelioration of the progression of injury.43 Furthermore, the administration

of MSC to mice with diabetes type 1 resulted in the recovery of damaged insulin producing pancreatic islets and β-cells and decreased blood glucose levels.44 Two mechanisms appear to be aiding this recovery. In addition to the production of trophic growth factors, MSC also inhibit the β-cell specific T-cell immune reaction.45 selleck chemicals llc In a mouse model of lung fibrosis, MSC reduced local inflammation, collagen accumulation and consequently fibrosis.46 Subsequent studies demonstrated that MSC conferred this protection by inhibiting the release of interleukin (IL)-1α and tumour necrosis factor (TNF)-α through the secretion of IL-1 receptor antagonist (IL-1RA).47 The local injection of MSC to mice following coronary ligation induced the regeneration of cardiac tissue and improved myocardial function.48 Following intravenous administration, MSC preferentially homed to the infarct site where they promoted angiogenesis and myogenesis and mediated myocardial repair

via paracrine mechanisms.49 The first phase I clinical trial in humans involved the intravenous infusion of MSC into patients with hematologic malignancies in complete remission resulting in no adverse events.50 Subsequent trials in breast cancer Protein Tyrosine Kinase inhibitor patients showed that MSC infusion, following high dose chemotherapy and peripheral-blood progenitor-cell infusion resulted in enhanced hematopoietic engraftment and recovery.51 The immunosuppressive effects of MSC have also effectively been used to treat a leukaemia patient with severe treatment-resistant grade IV acute graft-versus-host disease (GvHD).52 Following the promising results obtained from these trials, MSC have since been clinically trialled in a diverse range of other conditions. Numerous phase I–II and III clinical trials exploring the therapeutic potential of MSC in conditions such as diabetes type 1, myocardial infarction, ischemic stroke, Crohn’s disease, cirrhosis and osteoarthritis have been completed or are currently in progress (see http://www.

The starter culture was diluted at 1 : 100 in HI broth and grown with shaking at 33 °C to an OD610 nm of ∼0.5 (exponential growth phase). Bacteria were collected by centrifugation, washed once with phosphate-buffered saline (PBS) (Sigma, St. Louis, MO), suspended in PBS and inactivated by overnight incubation at 25 °C with neutral buffered formalin (0.5% final concentration) (Sigma). The cells were washed twice with PBS and stored at 4 °C. Formalin treatment was used to inactivate V. vulnificus because growth would confound assay results due to cytotoxicity (Shin et al., 2004). Vibrio vulnificus (CFU mL−1) were quantified by plating aliquots of serial

dilutions on HI agar before formalin treatment. Blood was collected aseptically from two to three male mice (10–13 weeks of age) per each genotype (i.e. WT, TLR4 KO, and MyD88 KO) in heparin-flushed ABC294640 molecular weight selleck chemicals llc syringes and pooled to minimize variability. Mouse blood (25 μL) was diluted to 200 μL with Roswell Park Memorial Institute

(RPMI) medium 1640 (Invitrogen Corp., Grand Island, NY) (negative control), RPMI medium containing formalin-inactivated V. vulnificus ATCC 27562 cells, or RPMI medium containing 20 ng (100 ng mL−1) Escherichia coli 0111 : B4 purified lipopolysaccharide (Sigma) (positive control). Duplicate samples were incubated at 34 °C with gentle agitation for 6 and 24 h. Cell-free supernatants were collected following centrifugation and assayed in duplicate for mouse TNFα with a commercial enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems Inc., Minneapolis, MN) at the UNC-CH Immunotechnologies Core Facility. Whole blood assays were repeated at least once. Statistical significance of results was evaluated with the unpaired, two-tailed t-test for analysis of two groups or anova for analysis of more than two groups (graphpad prism 4, GraphPad Software Inc., San Diego, CA). A P-value of <0.05 was considered significant. Splenocytes were prepared from pooled spleens of two male mice (10–12 weeks of age) per each genotype

(i.e. WT, MyD88 KO, and TLR4 KO). Following lysis of red blood cells, splenocytes were washed, suspended in RPMI medium containing 5% heat-inactivated fetal bovine serum (Fisher Scientific, Pittsburgh, PA), and seeded at 5 × 105 cells in 200 μL per well. ADAMTS5 After a 24-h incubation at 37 °C in 5% CO2 with RPMI medium only, 1 × 106 formalin-inactivated V. vulnificus ATCC 27562 cells, or 20 ng E. coli lipopolysaccharide, cell-free supernatants from duplicate samples were collected and assayed in duplicate for TNFα by ELISA. Splenocyte assays were repeated an additional time. Statistical significance of results was evaluated with the unpaired, two-tailed t-test for analysis of two groups or anova for analysis of more than two groups (graphpad prism 4). A P-value of <0.05 was considered significant. Vibrio vulnificus ATCC 27562 was grown with shaking in HI broth at 33 °C to exponential phase.

The CD4+ T cells were incubated with magnetic beads conjugated with an anti-CD25 monoclonal PLX3397 antibody to separate CD4+ CD25+ and CD4+ CD25− T-cell subpopulations. The purity of the resulting T-cell subpopulations was higher than 95% by flow cytometry. To determine the suppressive capacity of hASC-induced Treg cells, proliferation assays were performed in triplicate by culturing CD4+ CD25− cells (responder, 5 × 104 from splenocytes of EAHL mice), CD4+ CD25+ T cells (suppressor, 5 × 104 from splenocytes of β-tubulin-immunized mice treated with either hASCs or PBS) in 96-well plates with irradiated antigen-presenting cells (5 × 104 from splenocytes

of normal BALB/c mice) for 72 hr at 37° in complete medium. Cultures were stimulated by β-tubulin (10 μg/ml), and some co-cultures were treated with anti-IL-10 antibody (10 μg/ml). After 72 hr, the proliferation of autoreactive T cells was assayed by measuring bromodeoxyuridine-substituted DNA incorporation. Data were analysed using analysis of variance or Student’s t-test to compare differences between the treatment AZD2281 groups. In the present study, we investigated the potential therapeutic effect of hASCs in an experimental model of murine autoimmune hearing loss. Mice were examined weekly for ABRs for hearing capacity. After three injections (Fig. 1a), the hASC administration

group showed that the ABR threshold to click stimulus and wide range of specific frequencies, in comparison with the PBS control group, significantly decreased. After six injections of hASCs (Fig. 1b), ABR click

and pure tone thresholds of the hASC administration group showed improved hearing level at all frequencies tested from 8 to 32 kHz. The ABRs detected threshold levels similar to those in naive mice that received no treatment (Fig. 1b), and the hASC administration completely restored hearing in deaf mice, whereas the PBS control group developed EAHL. Therefore, electrophysiology tests demonstrated recovery of hearing to click stimulus and a wide range of specific frequencies after six injections of hASCs. We investigated the possible immune-modulating effect of hASCs on T-cell priming and differentiation in vivo by examining the recall CYTH4 response to β-tubulin in isolated splenocytes from hASC-treated or PBS-treated mice with EAHL in vitro. To determine the ability of hASC treatment to suppress the ongoing inflammatory process, mice with EAHL were treated with PBS or hASCs once a week for 6 consecutive weeks after β-tubulin immunization, and splenocytes that were isolated 10 days after the last treatment with the hASCs were assessed for proliferative responses to β-tubulin. T cells from hASC-treated mice exhibited a significantly decreased stimulation index compared with that in cells from PBS-treated mice (Fig. 2a). Moreover, T cells from hASC-treated non-immunized mice did not develop a xenogenic response to the hASCs in those non-immunized animals (data not shown).

With regard to ALI alveolar fluid transport Talazoparib manufacturer can be up- or down-regulated [45]. Hypoxia inhibits transepithelial sodium transport in ex-vivo lungs [16], while endotoxin A from

Pseudomonas aeruginosa stimulates alveolar fluid clearance in rats [46], probably by cytokine-induced stimulation of sodium uptake. Conversely, intratracheal application of endotoxin-impaired alveolar fluid clearance in adult rats at 6 h of injury [26,47]. Evidence from previous studies indicates that a complex network of inflammatory cytokines and chemokines mediate and modify the inflammatory process in lung injury, including oedema formation [48–50]. It is known that inflammation in AEC is mitigated by application of sevoflurane [25]. Our in-vitro investigations in AECII reveal that LPS-induced impairment of both ENaC and Na+/K+-ATPase is reversed upon co-exposure to sevoflurane. These data suggest that active sodium transport and thus water transport can be increased functionally in injured AECII by administration of sevoflurane. So far, only type II cells were considered as the important regulators for salt and water buy LDK378 transport

[51]. However, as both types I and II AEC cells express sodium transport channels [52,53], AECI might also play an important role in water and salt homeostasis in the lung [52]. Therefore, after the positive findings in AECII, in-vitro experiments regarding sodium transport were reassessed in a mixture of types I and II cells, a set-up which more probably reflects the in-vivo situation with only 5% of type II and 95% of type I cells in the lungs. With this mixture of AEC (mAEC), no LPS-induced change or significant

influence of sevoflurane was observed for functionality of ENaC. For Na+/K+-ATPase we could demonstrate increased activity upon LPS exposure, while sevoflurane did not have any significant impact on its function. Therefore, we conclude that AECI are not involved actively in water reabsorption with regard to sodium channels. A previous study showed evidence that oxygenation improved significantly using sevoflurane in a post-conditioning set-up in an LPS-induced 4-Aminobutyrate aminotransferase ALI model (intratracheally applied LPS, followed 2 h later by application of sevoflurane compared to propofol anaesthesia) [26]. The present promising in-vitro results from AECII encouraged us to elucidate the question of to what extent sevoflurane may influence either oedema resolution or oedema formation. We were able to demonstrate that wet/dry ratio in the sevoflurane-treated animals was significantly lower compared to the propofol/LPS group, linking better oxygenation to less alveolar oedema. However, when blocking the activity of ENaC using amiloride, the wet/dry ratio remained unchanged.

Although type I NKT cells seem to recognize lipids of symbiotic https://www.selleckchem.com/products/Deforolimus.html commensal bacteria,[120-122] the nature of microbial lipids that activate type II NKT cells is not yet known. Recent findings suggest that both pathogenic and non-pathogenic microbes may modulate intestinal immune responses in healthy and diseased conditions. Evidence from several animal models of experimental inflammatory bowel disease demonstrates that type I NKT cells can be both protective and pathogenic in inflammatory bowel disease.[9] In

contrast, type II NKT cells seem to promote intestinal inflammation and may be pathogenic in inflammatory bowel disease when both CD1d expression and the frequency of type II NKT cells are increased in mice as well as patients with ulcerative colitis. However, adoptive transfer studies need to be carried out to substantiate these effects and cross-regulation of NKT cell subsets may further influence the disease outcomes at these sites. As mentioned above, activation of type II NKT cells with self-glycolipid sulphatide induces a novel regulatory mechanism that may protect from autoimmune disease and inflammatory tissue damage. This unique pathway involves cross-regulation see more of type I NKT cells and inhibition of

pathogenic Th1/Th17 cells through tolerization of conventional DCs (cDCs). It has been shown to be effective in the control of EAE[19, 98, 109-112], type 1 diabetes,[89] liver diseases,[19, 62] and systemic lupus erythematosus (R. Halder, unpublished data). Interestingly, while activation of type I NKT cells predominantly activates hepatic cDCs, sulphatide-mediated activation of type II NKT cells predominantly activates hepatic plasmacytoid DCs (pDCs). Additionally, type II NKT–DC interactions result in a rapid (within hours) recruitment of type

I NKT cells into liver in an IL-12 and macrophage inflammatory protein 2-dependent fashion. However, recruited type I NKT cells are neither activated nor secrete cytokines, and consequently become anergic. Hence, anergy in type I NKT cells leads to reduced levels of IFN-γ followed by reduced recruitment of myeloid cells and NK cells and protection from liver damage.[123] Furthermore, tolerized cDCs further inhibit NADPH-cytochrome-c2 reductase conventional pathogenic CD4+ effector T cells that can elicit autoimmunity.[27] Hence, adoptive transfer of cDCs from sulphatide-treated but not control-treated mice into naive recipients leads to protection against inflammation. Furthermore, activation of sulphatide-reactive type II NKT cells leads to the tolerization of tissue-resident APCs, such as microglia in the CNS. Importantly, this tolerization impairs the development of pathogenic Th1 and Th17 cells.[27] A recent study has suggested that the inducible T-cell co-stimulator and programmed death-1 ligand pathways are required for regulation of type 1 diabetes in NOD mice by CD4+ type II NKT cells.

Each group was boosted with EG95 protein at 4 weeks post-immunization. Antibody levels were determined in 2-weekly collections of serum, Erlotinib mw by ELISA. The results are presented in Figure 3. All sheep infected with VV399 showed evidence of seroconversion. Animals primed with EG95 showed higher levels of priming with EG95 protein compared with animals immunized with VV399. However, there was no difference in the serological response of both groups of sheep to the booster injection of EG95 protein. Poxviruses are proven delivery vehicles for a wide range of antigens against various diseases (11–13). The secreted EG95 protein, produced during the oncosphere and post-oncospheral life stage

prior to metacestode formation, affords protection against hydatid disease in the intermediate host, and in this study, we explored the use of VACV as a delivery system for this antigen. Mice are a well-established species for testing the immune

response against foreign antigens expressed by recombinant VACV (11,19) and were used in this study to examine the antibody response against EG95 expressed from VACV and to examine the ability of the mouse antiserum produced to kill E. granulosus oncospheres in vitro. Whilst all groups infected with VV399 showed little antibody response to EG95 at 2 weeks post-infection, it was observed that the antibody response continued to increase with time, and by 42 days post-infection, antibody levels were comparable with mice learn more immunized intraperitoneally with EG95 protein at 2 weeks post-immunization. Furthermore, it was clear that all animals were primed with VV399 as significant responses were detected in all animals either boosted with VV399 or EG95 protein. The primary antibody response observed was consistent with the immune response seen in foxes and skunks fed orally a sponge bait containing recombinant VACV expressing the rabies virus glycoprotein (20–22). In their experiments, neutralizing antibody next increased up to day 28–30, but decreased thereafter. In addition, the priming effect of VV399 was also observed by

the antibody levels produced in animals boosted intraperitoneally with EG95 protein (Figure 1). We found that a second immunization with VV399 enhanced the antibody response generated from previous exposure, albeit not to the same level as that seen with EG95 protein combined with alum adjuvant. The priming and boosting effect of EG95/HIS with alum is interesting. Previous work (16) has shown that in sheep, alum is a poor adjuvant compared with QuilA. It was necessary to use alum because QuilA was shown to be lethal in our mice. The level of immunity generated by the double exposure to VV399 intranasally prompts the suggestion that this recombinant virus could be used for immunizing grazing animals in the field.

It has been Doxorubicin molecular weight suggested that multifunctional CD4+T cells, able to produce simultaneously IFN-γ, IL-2 and TNF-α, are associated with protective immunity or a beneficial outcome in chronic infectious diseases, such as HIV [25–28] and HCV [29]. We therefore evaluated the quality of Th1 responses

induced by LbAg and LaAg in healed CL patients, based on their ability to secrete these three major Th1-related cytokines at the single-cell level. Using multiparametric flow cytometry, seven distinct populations of cytokine-producing cells can be delineated based on any of the possible combinations of IFN-γ+, IL-2+ and TNF-α+ producers, and the relative frequency of these distinct populations defines the quality of the Th1 response. The percentages of cytokine-producing cells were shown to be higher in the healed CL patient group than in healthy controls, and we were able to observe statistically significant differences between those groups for triple-positive (3+) multifunctional PI3K Inhibitor Library price T cells (with both LbAg and LaAg), IFN-γ single-positive cells after LaAg stimulation and for IFN-γ+IL-2+ cells stimulated with LbAg (Fig. 2a). When comparing the quality of the Th1 response elicited by each Leishmania antigen evaluated we could observe that LbAg induces significantly higher percentages of multifunctional CD4+T cells and

IFN-γ+IL-2+ cells than LaAg stimulation in the healed CL patient group (Fig. 2a). The quality of the Th1 response was also evaluated by analysing the contribution of each phenotype in the total Th1 response, and is represented pictorially by pie charts (Fig. 2b). This kind of representation demonstrates clearly that LbAg induced a major proportion of multifunctional CD4+T cells (in red – 28% of the total Th1 response evaluated) and double-positive CD4+T cells Sclareol (in blue – comprising 44% of the total Th1 response), while LaAg induced

predominantly single-positive cells (68%). More than half of the single-positive cells induced by LaAg were IFN-γ single-positive. In the control group, the majority of responsive cells were single-positives (>60%), and no major differences were observed concerning LbAg and LaAg stimulation. Having shown that LbAg induced higher cytokine production by CD4+T cells than LaAg in healed CL patients (Fig. 1b), we also investigated the relative cytokine concentrations produced by all distinct Th1 phenotypes induced by LbAg and LaAg, measured as the geometric MFIs. The highest MFI values for all three cytokines were found among triple-positive multifunctional CD4+T cells (both after LbAg and LaAg stimulation) (Fig. 2c) and a progressive decrease in the MFIs for all cytokines was observed as the degree of functionality decreased (3+ to single-positives). MFIs for IFN-γ and IL-2 from multifunctional T cells stimulated with LbAg were significantly higher than those obtained after LaAg stimulus (Fig. 2c).

The images include a wealth of macroscopic images, light

microscopy images depicting numerous staining methods and electron microscopy images. Where applicable there are useful tables and schematic drawings for easier understanding and recall. Last but not least the index is very detailed and comprehensive making a search for the basic definitions or findings for a topic of interest speedy and rewarding. The preface states that the general intention of this edition, similar to the previous editions, is to provide a concise introductory text covering the basic morphology of lesions underlying diseases of the nervous system, limiting pathophysiological considerations to essential principles and purposefully excluding historical, clinical, neurological,

radiological imaging data and reference listings. In my SCH772984 solubility dmso opinion, this is exactly what the book provides. Although the information provided in the book is a concise and ‘basic’ introduction to the various diseases of the nervous system and their underlying pathology; this edition, similar to previous editions, will surprise the reader with how much valuable information, covering nearly whole spectrum of neuropathological processes, can be included in just over 400 pages. There is no online check details access or accompanying CD-ROM for image download. However, in my opinion this is an insignificant downside for a practical diagnostic manual providing up to date information on

a broad range of nervous system and skeletal muscle pathologies for a price of £65.00. As a concise easily readable introductory text, with numerous high quality illustrations supplemented with short clear figure legends, this book is a ‘must-have’ for anyone wishing to learn or revise the basics of neuropathology; be they a student, trainee, experienced specialist or scientist. The spectrum of readers who would find the book of value is broad. In addition to pathologists it would provide an excellent introduction Arachidonate 15-lipoxygenase to neuropathology for those in clinical specialties, such as neurology, neurosurgery, psychiatry, neuroradiology, neuroendocrinology and neuroscience. In view of the valuable updates, I am very glad to add this new edition on the bookshelf right next to my old well-loved, hence very much worn-out blue book. I would recommend you to do the same! “
“This chapter contains sections titled: Introduction Modeling Specific Functions or Behaviors Experimental Manipulations: Consequences of Drugs, Toxicants, and Lesions Relevance to Humans References “
“It is an honour to be appointed as the new Editor-in-Chief of Neuropathology and Applied Neurobiology and I look forward to the challenge of following in the footsteps of five distinguished editors to lead the journal forward in the coming years.

Due to the amount

of IgE sensitization and low antigen doses used in our model, we could not detect syk phosphorylation. Our findings indicate that the mast cell-activating machinery was intact for a non-desensitizing antigen action, since no mediator depletion occurred with desensitization, calcium flux was restored in desensitized cells when challenged with a non-desensitizing antigen and microscopic analysis confirmed that rapid desensitization is antigen specific and does not induce anergy 27. While we do not know the exact mechanism that could explain this inhibition of receptor internalization during desensitization, it is possible that the mobility of antigen/IgE/FcεRI complexes and membrane re-arrangement could prevent their internalization, as shown by others with low doses of multivalent antigen H 89 25. In addition, receptors engaged with low doses of antigen could be segregated into different compartments, preventing access to phosphorylating

molecules. Inhibitory phosphatases such as SHP-1 may not be excluded from those compartments, thus preventing phosphorylation of key molecules required for signal transduction. A time course study of SHP-1 phosphorylation in RBL-2H3 cells 28 has shown a peak at 1 min of FcεRI crosslinking and a gradually decline within 10 min. Our initial results indicated a lack of phosphorylation at 100 min. (data not shown). Further studies are planned to look for phosphorylation of SHP-1 and other mafosfamide ITIM-bearing molecules 29, 30 at each step of the desensitization Selleckchem LY2835219 protocol since it may be transient. In conclusion, this model of rapid IgE desensitization is effective

and reproducible and provides an optimal dose–time relationship, leading to almost complete abrogation of early- and late-phase activation events. This model of antigen-specific desensitization disables the specific response to one antigen but keeps the cell machinery unaffected, unlike non-specific desensitization. Most importantly, we show here that specific rapid desensitization inhibits internalization of the antigen/IgE/FcεRI complexes. The lack of severe anaphylactic reactions in our previous clinical reports 4, 5, including hundreds of desensitizations using a modified protocol, illustrates a profound inhibition of acute and delayed mast cell activation. These studies provide proof of concept for the effectiveness and specificity of human desensitizations. BMMCs derived from femurs of male BALB/c mice 8–12 wk old (Jackson Laboratory) were cultured in RPMI 1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 1% Penicillin-Streptomycin, 0.1 mM MEM nonessential amino acids (all from Sigma-Aldrich) and 10 ng/mL of IL-3. IL-3 was obtained from supernatants of 293T cells expressing mouse IL-3 31, 32.