LPS is a component of gram-negative bacteria outer-membrane that binds TLR-4. Well known for its pro-inflammatory properties it also dampens immune responses in various experimental setups (e.g. [39, 46, 47]). To test whether Treg are directly involved in the mechanism at the basis of the ‘hygiene hypothesis’, we first tested various protocols of LPS administration selleck chemical for their capacity to prevent diabetes occurrence in NOD mice. Next, by conducting cellular analysis we revealed that LPS treatment enhances Treg numbers and activity. Finally, by performing adoptive transfer experiments we demonstrated that CD25-expressing Treg are involved in the beneficial effect of LPS

in NOD mice, thus providing evidence that CD25+ Treg may play a central role

in the cellular mechanism at the basis of the ‘hygiene hypothesis’. Mice.  Non-obese diabetic (NOD)/Lt and NOD/SCID mice were originally purchased from The Jackson Laboratory (Bar Harbor, ME, USA). All animals were bred and maintained under specific pathogen-free conditions in our animal facilities. Mice experimental protocols were approved by the Instituto Gulbenkian de Ciência ethics committee and by the national authority Direcção Geral de Veterinária. LPS treatment.  In most experiments, 6- to 8-week-old NOD mice were injected i.p. with 10 μg LPS from Salmonella typhimurium (Sigma, Sintra, Portugal) diluted in PBS, once per week until the buy GSK458 time of analysis. Other experimental groups were: 12-week-old NOD females injected weekly with 10 μg LPS i.p. until time of analysis; 7.5 weeks of age NOD females injected once with 10 μg LPS and 4 weeks of age NOD females injected every 3 days with 10 μg LPS i.p., Astemizole during 1 month. In all experimental groups, PBS-injected age-matched animals served as controls. Diabetes detection.  Diabetes was monitored weekly or biweekly, according to the experiment, by measuring blood glucose levels using ACCU-CHEK Sensor Comfort strips (Roche, Mannheim, Germany). Mice that had values ≥250 mg/dl on two consecutive occasions were deemed diabetic. Cell purification and FACS analysis.  For flow cytometry purification,

thymus, pancreatic (p)LN or spleen cells, according to the experiment, were obtained by forcing the organs through a 100 μm nylon mesh. For isolation of pancreas-infiltrating lymphocytes, whole pancreas (after careful removal of pLN used in the same experiment for FACS staining) were cut into small pieces and incubated in OptiMEM medium (Invitrogen, Madrid, Spain) containing 5% FCS and 450 U of collagenase (Sigma) for 20 min at 37 °C. After filtering through 100 μm nylon mesh, lymphoid cells were isolated on a 40% Percoll gradient. The cells were then washed for posterior FACS staining. For FACS staining, 1 × 106 cells (whenever possible) were preincubated for 20 min with unlabelled mAb to the Fc-γ receptor (clone 2.

OPG, which as has been noted is a soluble decoy receptor for RANKL, is also expressed by mTECs 19. OPG-deficient mice exhibit an increased number of mTECs and enlarged thymic medulla containing many Aire-expressing mTECs 19. Thus, RANKL

plays a major role in promoting the proliferation of mTECs, and OPG expressed by mTECs fine-tunes the RANKL-mediated mTEC proliferation and thymic medulla formation. In addition to RANKL, two other TNFSF cytokines are known to be involved in the formation of the thymic medulla. Using transgenic mice, the effect of CD40L (CD154, TNFSF5) in the thymus was first noted in that the forced expression of CD40L induced the formation of an enlarged thymic medulla 37, 38. In those CD40L-transgenic mice, T-cell development was perturbed PD-1/PD-L1 cancer and lethal wasting disease with mononuclear infiltrates accumulating in multiple organs induced 37. On the other hand, mice deficient for CD40 exhibit only a mild

decrease in mTEC cellularity 19, 20. Unlike T cells from RANKL-deficient mice, the transfer of T cells from CD40-deficient mice does not induce autoimmune symptoms in the recipient nude mice 20. Interestingly, mice deficient for both RANKL and CD40 exhibit a more severe decrease in mTEC cellularity than RANKL-deficient mice, and T cells from RANKL and CD40 doubly deficient mice induce severe autoimmune symptoms 20. Thus, like RANKL, CD40L affects the cellularity of mTECs; however, unlike the major contribution of RANKL, the involvement find more of CD40L in mTECs and the thymic medulla is minor, although RANKL and CD40 cooperate to optimize thymic medulla formation. It has also been reported that autoantigen-specific interactions between CD4+CD8− SP thymocytes and mTECs control mature mTEC cellularity through CD40L–CD40 signals 39. The role of LT in the thymus was first noted by the

analysis of mice deficient for the LT-β receptor (LTβR, TNFRSF3). LTβR-deficient mice exhibit aberrant differentiation of mTECs and autoimmune phenomena 40, 41. LTβR Niclosamide is expressed by both mTECs and cTECs 19, whereas LT-α (TNFSF1) and LT-β (TNFSF3), which together form the ligand for LTβR, are strongly expressed by positively selected SP thymocytes 19, 40, 42. LIGHT (CD258, TNFSF14), another ligand for LTβR, is not clearly detected by DP or SP thymocytes 19 and seems to play a minor, if any, role in mTEC development 40. LTβR regulates the Aire-independent expression of promiscuously expressed genes and chemokine genes in mTECs 43–46. A recent study has shown that the LT-LTβR interaction is involved in the terminal differentiation of mTECs to form involucrin-expressing Hassall’s corpuscle-like structures, whereas RANKL-RANK interaction regulates the initial phase of development of mTECs to become Aire-expressing mTECs 21.

IgA1 HR has up to 6 of the 9 potential O-glycosylation sites occupied; some Gal-deficient glycans consist of terminal N-acetylgalactosamine (GalNAc). IgA1 HR O-glycosylation was reported

to be initiated by GalNAc-T2. However, the expression of GalNAc-T2 does not differ between cells from patients and those from healthy controls (HC). In contrast, expression of GalNAc-T14, the enzyme with highest similarity to GalNAc-T2, is 5-fold greater in IgA1-producing cells derived from IgAN patients than in those from HC. Here, we analyzed kinetics and site-specificities of GalNAc-T2 and -T14 on HR using high-resolution mass spectrometry (MS). Methods: We produced recombinant soluble GalNAc-T2 and -T14 enzymes. A synthetic HR peptide (sHR) and a panel of synthetic MG-132 in vivo HR glycopeptides (sGP) with a single GalNAc residue at different sites were used as acceptors.

Results: GalNAc-T2 showed higher activity i.e., faster rate of glycosylation of sHR, than did GalNAc-T14. Up to 8 sites were glycosylated in sHR by GalNAc-T2, whereas GalNAc-T14 added GalNAc to up to 5 sites in HR of IgA1. Distinct sHR O-glycoforms generated by GalNAc-T2 and -T14 were subjected to tandem MS to localize glycosylated sites. The sites of glycosylation on sHR catalyzed by GalNAc-T2 and -T14 were the same for the variants with up to 5 sites and buy EPZ-6438 appeared predominantly in an ordered fashion: GalNAc was attached to T7 first and then to T15, followed by S11 and T4. Localization of GalNAc on sGP did not affect kinetics of the GalNAc-T2. GalNAc-T14 effectively glycosylated sGP variant with a GalNAc at S9, the site that corresponds to S230 on IgA1 HR, the dominant site with terminal GalNAc in Gd-IgA1 proteins. GalNAc-T2 and -T14 have similar site-specificity on IgA1 HR, but differ in kinetics and how they are affected by preexisting glycosylation. Conclusion: Elevated

expression of a specific GalNAc-T is a possible mechanism Y-27632 2HCl for production of Gd-IgA1 in IgAN. TAKAHASHI KAZUO1,2, RASKA MILAN1,3, STEWART TYLER J.1, STUCHLOVA HORYNOVA MILADA1,3, VRABLIKOVA ALENA1,3, HALL STACY D.1, HIKI YOSHIYUKI4, YUZAWA YUKIO2, MOLDOVEANU ZINA1, JULIAN BRUCE A.1, RENFROW MATTHEW B.1, NOVAK JAN1 1University of Alabama at Birmingham; 2Fujita Health University School of Medicine; 3Palacky University in Olomouc; 4Fujita Health University School of Health Sciences Introduction: Patients with IgAN have elevated serum levels of galactose (Gal)-deficient IgA1; some hinge-region (HR) O-glycans consist of terminal N-acetylgalactosamine (GalNAc) with or without N-acetylneuraminic acid (NeuAc, sialic acid). Sialylation of GalNAc blocks subsequent galactosylation. IgA1-producing cells from IgAN patients have increased activity of α2,6-sialyltransferase (ST6GalNAc) that sialylates GalNAc.

However, the exact role played by astrocytes during the development of EAE is still debated. In the present study, we demonstrate that astrocytes are capable of inducing and suppressing lymphocyte functions during different phases of EAE. During the initial phases, astrocytes probably inhibit the activity of myelin oligodendrocyte glycoprotein (MOG)35–55-specific lymphocytes in part by secreting IL-27, which contributes to inhibition of proliferation

and lymphocyte secretion. During EAE progression, lymphocyte-derived IFN-γ might induce the up-regulation of major histocompatibility complex (MHC)-II on astrocytes, thereby promoting lymphocyte proliferation and activation and resulting in disease progression. These findings indicate that the changing physiological role of astrocytes is important to EAE development. The study contributes to a clearer understanding of EAE and adds new insights into the field of EAE research. Female C57BL/6 mice (6–8 weeks selleck compound of age) were purchased from the Beijing Vital River AZD6244 mw Laboratory Animal Ltd (Beijing, China). All mice were bred and housed in a specific pathogen-free animal facility at the Harbin Medical University. Neonatal C57BL/6 mice aged 1–3 days were used for the isolation of astrocytes. All animal experiments were performed in compliance with the principles and procedures outlined in the Care and Use of Laboratory Animals guidelines, which is published by the China National

Institute of Health and approved by the Institutional Animal Care and Use Committee. C57BL/6 mice were immunized subcutaneously in the axillary

fossa with the MOG35–55 (MEVGWYRSPFSRVVHLYRNGK) peptide (200 μg) emulsified in complete Freund’s adjuvant (CFA) at a final volume of 100 μl. Mice were then injected intravenously (i.v.) with 200 ng pertussis toxin (PT) on days 0 and 2. The behavioural performance was assessed by a 0–5-point scale as follows: 0, no clinical signs; 1, floppy tail; 2, hind limb weakness; 3, full hind limb paralysis; 4, quadriplegia; and 5, death as described [34]. Astrocytes were isolated from newborn mice as described previously [35, 36]. Briefly, following removal of the meninges, Thiamet G brains were minced with a Pasteur pipette and passed through a 150 μm nylon filter to remove debris. Cells were then seeded onto 10 μg/ml poly-D-lysine precoated flasks and cultures were incubated at 37°C in 5% CO2. After 72 h, non-adherent cells were removed by changing the media every 3–4 days. When cultures were 70–80% confluent, mixed glia were agitated rigorously for 2 h in an orbital incubator shaker at 0.23 g at 37°C to detach microglia. Cells were then shaken again at 0.23 g at 37°C overnight to ablate oligodendrocytes. Suspended cells were trypsinized [0·25% trypsin and 0·02% ethylenediamine tetraacetic acid (EDTA)] and replated onto flasks. Subcultured astrocytes were 92% positive for glial fibrillary acidic protein (GFAP) by immunofluorescence staining.

7%) after 2 weeks. In more than half of these high-risk Stem Cell Compound Library manufacturer patients, enalapril was ceased because of an increase in serum creatinine. In

all cases, however, renal function recovered after enalapril was ceased. A good correlation was observed between the increase in serum creatinine and the severity of renovascular disease (r = 0.53, P < 0.001). The authors of this study concluded that controlled exposure to ACE inhibitors in this population was safe, and that ACE inhibitor-induced increases in serum creatinine are a sensitive detector of severe bilateral renovascular disease in a high-risk population. In patients with renal artery stenosis, an additional concern is the risk of long-term loss of renal mass and function in the post-stenotic kidney. Data on whether or not renin–angiotensin system blockade increases the risk of this event are inconsistent. In a prospective study performed by Caps et al. 204 kidneys with renal artery stenosis were followed prospectively

for the development of renal atrophy by ultrasound performed every 6 months for 2 years.39 The predictors of increased risk of developing renal atrophy were found to be the severity of the renal artery stenosis observed by duplex ultrasound, a systolic blood pressure greater that 180 mmHg, a renal artery peak systolic velocity > 400 cm/s, and a renal cortical end diastolic volume ≤ 5 cm/s. Interestingly, the use of ACE inhibitors did

PLX4032 not appear in this study to impact on the risk of developing renal atrophy (relative risk (RR) 1.1, 95% CI: 0.5–2.5). In contrast, others have reported that in patients with unilateral renal artery stenosis, ACE inhibitors improve renal function in the unaffected kidney, while hastening ischaemic atrophy on the stenotic side.40–43 This is consistent with some animal studies on the subject.44 In summary, there are variable data suggesting that in renal artery stenosis, renin–angiotensin system inhibition could accelerate renal atrophy in the post-stenotic kidney. In unilateral disease, this appears to be counterbalanced, however, by protection to the non-stenosed kidney, with no net adverse effect on renal function overall. The beneficial effects of renin–angiotensin Baf-A1 system blockade in unilateral renal artery stenosis on blood pressure control and cardiovascular risk potentially, however, outweigh this possible adverse effect of renin–angiotensin system blockade on the function of the post-stenotic kidney. In contrast to the situation of unilateral renal artery stenosis, in the case of severe bilateral renal artery stenosis or severe renal artery stenosis to a solitary functioning kidney, there is a more clinically relevant risk of an overall loss of renal function resulting from reduced perfusion to the total functioning renal mass.

Additionally decorin has been reported to act directly on neurones to increase neurite extension in an inhibitory environment in vitro [197]. However, no additive benefits of recombinant decorin administration were observed on the functional improvements and increased tissue-sparing demonstrated following transplantation of human mesenchymal stem cells alone, although donor cell loss was delayed [198]. In principal, preventing the synthesis or deposition of scaffold matrix which holds inhibitory substances dispenses with a requirement to target specific molecules. On a gross scale, duraplasty (closure of the open dura by surgical patch or re-apposition and suture) after

spinal laceration has been suggested to limit meningeal fibrosis and reduce connective tissue scar deposition [199]. Suppression of glial and fibrotic scar formation coupled with reduced CSPG secretion has also

been BMN 673 research buy reported after low-dose X-irradiation of the spinal injury site to reduce cell proliferation [200]. Furthermore, the growth factor TGFβ is known to partly regulate scar formation and administration of the small proteoglycan decorin after CNS injury was shown to suppress scar deposition by inhibiting TGFβ after cortical [201] and spinal [195] stab lesions, although antibodies to TGFβ did not promote repair in an earlier study [202]. Direct inhibition of collagen synthesis using alpha, alpha’-dipyridyl,

(an inhibitor of collagen triple helix formation) hypoxia-inducible factor pathway was found to promote axon growth after post-commisural fornix transection in the brain [203,204] but successful blockade of fibrillar collagen III and basal lamina (collagen IV and laminin) formation with 2,2′-bipyridine (a cAMP calcium chelator) injections failed to enhance corticospinal tract (CST) axon regeneration or sprouting following spinal cord dorsal column transection [21], unless combined with cAMP [205] and/or an inhibitor of prolyl 4-hydroxylase, a key enzyme of collagen biosynthesis [206]. This discrepancy between brain and spinal cord was suggested to result from lesion proximity to meningeal fibroblasts, but encouragingly the combined approach did result in significant functional recovery in three motor/sensorimotor tasks and long distance CST axon regeneration [205]. Due to the protective and healing roles of the scar [135,136] more discrete approaches have been taken to target biosynthesis of specific inhibitory ECM components, with promising functional effects. Deoxyribozyme (an RNA-cleaving DNA enzyme)-mediated knock-down of xyosyltransferase-1 (XT-1), the enzyme which initiates construction of the CSPG (and HSPG) linker tetrasaccharide to result in GAG attachment, is associated with increased length and density of regenerating fibres from a DRG microtransplantation [207] or a peripheral nerve graft [208].

oryzae compared to CAS or ABLC monotherapy.[26] Furthermore, based on preclinical studies, Reed et al. showed that patients with rhino-orbital-cerebral mucormycosis treated with CAS and ABLC therapy had superior success and survival time compared with patients who received ABLC monotherapy.[73] The same group of investigators[74] showed that the enhanced efficacy of LAmB with micafungin (MFG) or anidulafungin combination therapy

in treating DKA mice with disseminated mucormycosis is a class effect. Triple therapy for mucormycosis consisting of LAmB, MFG and the iron chelator deferasirox was superior to monotherapy or dual therapy treatments. Triple therapy improved survival of mice by 40% compared to 0–11% for selleck chemicals all other

treatments.[75] Given the resistant phenotype of Mucorales with conventional drugs, the potential for triple therapy in mucormycosis should be further investigated in preclinical and clinical studies. Although PSC shows good in vitro susceptibilities against Zygomycetes, the in vivo efficacy of PSC in immunosuppressed murine models of disseminated mucormycosis is substantially variable as well as species- and dose-dependent.[44-48] selleck chemicals llc In order to evaluate its role in combination therapy, Rodriguez et al.[76] investigated the efficacy of PSC in combination with AmB. Findings showed that low doses of AmB (0.3 mg/kg, once daily) combined with PSC (40 mg/kg, once daily) prolonged survival, but it was not superior to the high-dose of administered AmB (0.8 mg/kg, once daily), allowing reduction of the AmB dose and similar efficacy levels with AmB monotherapy. A most recent in vivo combination study, using a non-lethal murine model of cutaneous mucormycosis caused by R. oryzae, showed that TAC

combined with PSC reduced significantly cutaneous lesions and fungal burden compared to the animals administered VRC alone.[77] To date, there is no adequate clinical evidence on the use of VRC as a single agent or in combination therapy. For this reason, additional studies are required to explore further the role of VRC to improve the prognosis and outcome of the patients who develop invasive mucormycosis. Beta-glucan is an essential cell wall component of fungi that lies beneath a Etomidate dense layer of mannan coat. The inner beta-glucan layer is targeted by the dectin-1 receptor of immune cells, mediating the innate immune response, and by the echinocandin class of antifungal drugs. Lamaris et al. [78] showed that the beta-glucan unmasking effect of CAS enhanced the activity of PMN against A. fumigatus and R. oryzae as well as other non-Aspergillus hyphae. The effect of PMN against A. corymbifera, R. microsporus and R. oryzae under the influence of LAmB and ABLC was also investigated in another in vitro study. While LAMB exhibited synergistic activity with PMN in inducing hyphal damage only to R.

Enhanced maternal anti-fetal immunity contributes to the severity of hypertensive disorder complicating pregnancy. Am J Reprod Immunol 2010 Problem  The aim of this study was to evaluate how fetal monocyte activation and maternal anti-fetal antigen-specific antibody-secreting cells (ASC) affect the severity of hypertensive disorder complicating Selleck BMN 673 pregnancy (HDCP).

Method of study  Forty-six healthy third-trimester pregnant women and 20 patients with gestational hypertension, 20 with mild pre-ecalmpsia and another 20 with severe pre-eclampsia were included in the study. Interleukin-6 (IL-6) release from cord blood monocytes was examined by intracellular cytokine staining and flow cytometric analysis. Moreover, the maternal anti-fetal antigen-specific ASC were detected by enzyme-linked immunospot assay. Results  A significantly increased percentage of IL-6-positive monocytes were detected in the cord blood of study

groups compared with the controls (P < 0.01). The percentage of IL-6-positive monocytes was increased as the disease progressed (P < 0.05). There were more anti-fetal antigen-specific ASC in the study groups than those MG-132 concentration in the controls (P < 0.001). Furthermore, the anti-fetal antigen-specific ASC showed difference in gestational hypertensive and severe pre-eclamptic groups (P < 0.05). Conclusion  We conclude that the fetal monocyte activation and the increase in maternal anti-fetal antigen-specific ASC were related to the incidence and severity of HDCP. These results provide both indirect and direct evidence for the occurrence of exaggerated maternal humoral immunity against the fetal antigens in HDCP. "
“Many pathogens are initially encountered in the gut, where the decision is made to mount an immune response or induce tolerance. The mesentric lymph node (mLN) science has been shown to be involved in immune response and much more in oral tolerance induction. Furthermore, using an in vivo transplantation model, we showed recently that lymph node (LN) stromal cells can affect T-cell function and influence the IgA response by supporting a site-specific environment. To elucidate the importance

of LN stromal cells for tolerance induction, mLN or peripheral LN were transplanted into mice (mLNtx or pLNtx) and oral tolerance was induced via ovalbumin. A reduced delayed-type hypersensitivity (DTH) response was detected in pLNtx compared to mLNtx mice. Reduced IL-10 expression, reduced percentages of Tregs, and increased proportions of B cells were identified within the pLNtx. The increase of B cells resulted in a specific immunoglobulin production undetectable in mLNtx. Moreover, transferred IgG+ cells of tolerized peripheral LN induced a strong reduction of the delayed-type hypersensitivity response, whereas CD4+ cells were less efficient. Thus, stromal cells have a high impact on creating a unique environment.

g. 3-monthly after a treatment duration of > 24 months). Pathogenesis of PML – the most click here feared potential SADR of NAT – is multi-factorial, comprising cellular immunity of the host [48], reactivation of latent John Cunningham virus (JCV) infection or new infection combined with genetic variation of the virus. Both viral and host factors predisposing for PML development are under investigation. The differentiation

between virulent and non-virulent JCV variants may be helpful, but relies on viraemia [49] and so far is not sufficiently validated. Epidemiological risk factors for PML development are previous use of immunosuppressants, a positive anti-JCV antibody status and treatment duration [45, 50-52]. Hence, the estimated PML incidence ranges from ≤ 0·09/1000 to 11·1/1000 [45]. A total of 418 NAT-PML cases have been reported (as of November 2013 [53]). PML must be suspected when new neurological symptoms occur

in individuals on NAT therapy. In particular, neuropsychological symptoms and seizures are highly suspicious, whereas spinal or optic nerve symptoms are uncommon. Its diagnosis is based on clinical findings, MRI [47] and the detection of JCV DNA in cerebrospinal fluid (CSF) [35, 54], although there are JCV DNA-negative NAT–PML reports [55, 56]. In uncertain cases, biopsy of suspicious lesions has to be discussed. In the course of PML, immune reconstitution inflammatory syndrome (IRIS) can occur with a mean of about 1 month after NAT removal via plasma exchange [57]. This inflammatory reaction directed against JCV can cause additional tissue damage selleck kinase inhibitor with neurological deterioration after initial improvement after PML diagnosis.

NAT and JCV elimination as well as mafosfamide control of IRIS evolution must be covered by PML treatment strategies which comprise plasma exchange, mefloquine, mirtazapine and corticosteroid pulses [35, 58]. However, due to relatively low patient numbers, none of these treatment options are evidence-based. Although the outcome of NAT–PML seems to be better than HIV-associated PML [57], it is associated with disability [45, 57]. Seizures occur in more than 50% of patients [59] and are often linked to the appearance of IRIS, explaining the higher rate than in other PML cases; preventive anti-convulsive therapy may thus be beneficial [59]. Routine anti-JCV antibody testing is established in clinical practice. However, false negative rates have to be considered for both first- and second-generation anti-JCV antibody testing. There is also a considerable proportion of seroconverters and – possibly linked to fluctuating antibody titres at the detection threshold – patients reverting from seropositive to seronegative [45, 52, 60, 61]. The prevalence of anti-JCV antibodies differs in patient groups according to age and gender [52]. Two studies reported antibody titres rather than mere serostatus.

Cells were maintained in culture for 6 days before their use. After 6 days, human macrophages (hMDMs) were detached by incubation with Accutase (Sigma Aldrich) for 30 min at 37°C and then plated on fibronectin- or Gelatin-FITC-coated coverslips for 24 h in the above medium with a FCS concentration of 1%. Mouse wild-type fibroblasts were isolated from 15–18 days embryos

by standard procedures and SYF (src–/–yes–/–fyn–/–) fibroblasts were https://www.selleckchem.com/products/U0126.html obtained from ATCC. Fibroblasts were cultured in DMEM supplemented with 10% FCS, 100 U/mL penicillin, and 100 μg/mL streptomycin. For immunofluorescence experiments, cells were detached with trypsin and then plated for 24 h on fibronectin-coated coverslips in the above medium with a FCS concentration of 1%. Transfection of BMDMs was carried out by electroporation

using the NucleofectorTM technology of Amaxa (Koel, Germany) according to proposed protocols. Cells were transfected with control nonsilencing siRNA pool or mouse-specific ON-TARGET plus siRNA Reagents targeting Abl or Arg (Dharmacon, Lafayette, CO). For fluorescence CH5424802 in vivo microscopy (confocal analysis of podosome formation) and assays of gelatin degradation, matrigel migration, and trans-endothelial migration, cells were detached after 48 h from transfection and plated on fibronectin- or gelatin-coated coverlips for further 24 h. For assays of migration in 2D and immunoblotting, cells were assayed after 72 h of culture as above described. An aliquot of BMDMs used for the different assays was lysed to control for

the efficacy of Abl silencing by the siRNA-specific reagent. Mean per cent of Abl expression in BMDM filipin treated with siRNA targeting Abl was 37.8% ± 11 compared to control siRNA-treated ones. Cells were fixed with 4% (w/v) paraformaldehyde (PFA) for 30 min. PFA was quenched with 50 mM NH4Cl. Cells were then permeabilized with PBS-0.1% Triton X-100, blocked with 1% BSA for 30 min and stained with primary Ab for 1 h. Cells were stained with secondary Ab and rhodamine-phalloidin for 30 min, followed by DAPI (Sigma Aldrich) for 10 min. Images were collected using the SP5 confocal microscope from Leica Microsystems (Wetzlar, Germany) with a 63× objective. Images were processed for brightness and contrast with Adobe Photoshop. Controls were done by staining cells with secondary Abs only or, in the case of Abl, by staining BMDMs in which Abl was silenced with anti-Abl and secondary Abs. In either cases we did nondetect any signal. For gelatin degradation assays, coverslips were incubated with poly-L-Lysine for 20 min, washed with PBS and then incubated with 0.5% glutaraldehyde for 15 min. After washing with PBS, coverslips were put on a drop of 0.2 mg/mL Gelatin-FITC in PBS/2% sucrose, left for 10 min and washed again with PBS. BMDMs and hMDMs were plated for 24 h on gelatin-FITC-coated coverslips.