Methods:  Skin tissues from Tg mice were collected for immunostaining against PDPN, LYVE-1, CD11b and VEGF-C. The regulation of specific lymphatic biomarkers and growth factors were determined using qPCR and Fulvestrant datasheet Western Blot analyses. Dermal lymphatic uptake and drainage were assessed using intradermal EB dye micro-injections. Total RNA from IL-4-stimulated HaCaT cells was analyzed in a PCR array to evaluate the regulation of lymphangiogenic-related genes. Results:  Prominent

dermal microvascular lymphangiogenesis occurs in the Tg mice, characterized by a significant increase in number and caliber of the vasculature. The extent of both lymphatic proliferation and drainage parallels the progression of lesion severity, as does the up-regulation of pro-lymphangiogenic factors VEGF-C, VEGFR-3, ANG-1, and ANG-2. IL-4-stimulated HaCaT cells express high levels of MCP-1, a strong macrophage chemo-attractant. Additionally, Tg mice show significantly increased number of dermal CD11b+ macrophages expressing VEGF-C in the skin. Conclusions:  Our results provide

the first demonstration of inflammation-mediated lymphangiogenesis in AD and that compound screening assay IL-4 triggered macrophage recruitment may be closely linked to this phenomenon. “
“Please cite this paper as: Vital, Terao, Nagai and Granger (2010). Mechanisms Underlying the Cerebral through Microvascular Responses to Angiotensin II-Induced Hypertension. Microcirculation17(8), 641–649. Angiotensin II (AngII) and AngII type-1 receptors (AT1r) have been implicated in the pathogenesis of hypertension and ischemic stroke. The objectives of this study was to determine

if/how chronic AngII administration affects blood-brain barrier (BBB) function and blood cell adhesion in the cerebral microvasculature. AngII-loaded osmotic pumps were implanted in wild type (WT) and mutant mice. Leukocyte and platelet adhesion were monitored in cerebral venules by intravital microscopy and BBB permeability detected by Evans blue leakage. AngII (two week) infusion increased blood pressure in WT mice. This was accompanied by an increased BBB permeability and a high density of adherent leukocytes and platelets. AT1r (on the vessel wall, but not on blood cells) was largely responsible for the microvascular responses to AngII. Immunodeficient (Rag-1−/−) mice exhibited blunted blood cell recruitment responses without a change in BBB permeability. A similar protection pattern was noted in RANTES−/− and P-selectin−/− mice, with bone marrow chimeras (blood cell deficiency only) yielding responses comparable to the respective knockouts.

1; Nikon). The light source was a 488 nm solid-state laser (Sapphire 488-30; Coherent, Dieburg, Germany). Between 2 × 105 and 5 × 105 CHO cells were seeded on glass cover slips 2–3 days before the experiments. ZD1839 molecular weight Immediately before Ca2+ imaging, the cells were incubated with the particular concentration of fusion proteins in 50 μl culture medium and washed afterwards with culture medium with 10 mm HEPES added. Glass cover slips were mounted on the stage of an Olympus IX 70 microscope equipped with a 20 × (UApo/340, N.A. 0·75) objective in a self-made

recording chamber, which allowed a complete solution exchange < 1 second. In parallel, T cells were loaded at 22–23° for 30 min with 2 μm fura-2/acetoxymethyl ester (AM) (Invitrogen) in culture medium with 10 mm HEPES added, washed with fresh medium, and immediately used. T cells

were then added and cells were alternately illuminated at 340 and 380 nm with the Polychrome IV monochromator (TILL Photonics, Gräfelfing, Germany) and with an infrared light source using SP 410 as excitation filter and DCLP 410 as dichroic mirror. The fluorescence emissions at λ > 440 nm (LP 440) were captured with Akt inhibitor a CCD camera (TILL Imago), digitized, and analysed using TILL Vision software. Ratio images were recorded at intervals of 5 seconds. In some experiments thapsigargin (TG, 1 μm) was used to completely empty the stores. Excel, Igor Pro and TILL Vision were used for data analysis. An unpaired, two-sided Students t-test was used to test for significance. All fusion proteins were generated as single chain molecules to prevent any false pairing or degradation (Fig. S1). The extracellular domains of CD80 and CD86 were cloned at the N-terminal end of the scFv anti-CD33 to ensure correct binding to their respective receptors.52

Soluble proteins were produced in HEK-293 cells by transient gene expression with a yield of 0·5–2 mg total protein/l of cell culture supernatant, purified by IMAC and checked by Coomassie and Western blot analysis for purity and integrity. Proper binding for all fusion proteins was tested by enzyme-linked immunosorbent assay on recombinant CD33 antigen (data not shown) and flow cytometry Dichloromethane dehalogenase (Fig. S2) on either CD33-transfected CHO or Jurkat T cells. Binding of the scFv anti-CD33 was not altered in any of the fusion proteins when compared with the parental scFv anti-CD33. The scFv anti-CD3 and the extracellular domains of CD80 and CD86 showed a moderate to weak binding affinity to their respective receptors. The dscFv anti-CD3/anti-CD19 were used as control. The dscFv anti-CD33/anti-CD3 construct induced proliferation of naïve T cells in the presence of the CD33 antigen in a dose-dependent manner (Fig. 1).

01; group P vs MR, P < 0.05). Nephrin, podocin and podocalyxin staining was preserved in the glomeruli of groups MR and AR compared with group P. Conclusion:  The MR and AR blockers decreased proteinuria in the acute model of nephrotic syndrome with preserved expression of glomerular podocyte protein independently of blood pressure. "
“Aim:  Adverse cardiovascular events resulting from accelerated atherosclerosis are the leading cause of mortality in uraemic

patients on maintenance haemodialysis (MHD). Chronic inflammation due to antigen-specific Buparlisib datasheet responses is an important factor in the acceleration of atherosclerosis. The balance between CD4+ CD25+ forkhead/winged helix transcription factor (Foxp3)+ regulatory T cells (Treg) and T helper (Th)17 cells has been reported to play an important role in the development of inflammatory and autoimmune diseases. The aim of the present

study was to assess the Treg/Th17 pattern in uraemic patients on MHD and to explore the significance of Treg/Th17 imbalance in the development and outcome of acute cardiovascular events. Methods:  A total of 42 uraemic patients on MHD were evaluated. Of the 42, 22 patients with a history of acute cardiovascular events served as the MHD1 group and 20 patients without acute cardiovascular events served as the MHD2 group. Thirty patients with advanced chronic kidney disease (CKD) without acute cardiovascular events just before haemodialysis FDA approved Drug Library therapy served as the CKD control group and 30 healthy volunteers as the normal control group. The Treg and Th17 frequencies were measured by flow cytometry. The retinoic acid receptor-related orphan receptor γt (RORγt) and Foxp3 expressions very were measured by real-time reverse transcription polymerase chain reaction. Serum cytokines and C-reactive protein were detected by enzyme-linked immunosorbent assay and immunoturbidimetry. Results:  Patients with uraemia exhibited an obvious imbalance

of Treg/Th17 function when compared to the normal control group, displaying increased peripheral Th17 frequency, Th17-related cytokines (interleukin [IL]-17, IL-6 and IL-23) and RORγt mRNA levels. These patients also displayed decreased Treg frequency, Treg-related cytokines (IL-10, transforming growth factor-β1) and Foxp3 mRNA levels. This imbalance was more pronounced in the MHD2 group, while there was no significant difference between the MHD1 and CKD control group (P < 0.01 between normal control and uraemic patients; P > 0.05 between CKD control and MHD1; and P < 0.05 between MHD1 and MHD2). It was also observed that the imbalance of Treg/Th17 was not only consistent with the cardiovascular disease but also correlated with a microinflammatory state. Conclusion:  The Treg/Th17 balance was disturbed by uraemia, especially in patients with adverse cardiovascular events.

[98] demonstrates the successful HDAC inhibitor review use of caspofungin in the treatment of invasive candidiasis in neonates. The study suggests that caspofungin may be an effective alternative treatment with fewer adverse effects than amphotericin B. However, amphotericin B is still the drug of choice in the treatment of systemic candidiasis in children,

as observed by Pappas et al. [99]. A more detailed investigation of the mechanisms of pathogenicity of Candida spp. and their relationship with resistance to antifungal agents has become indispensable due to the rise in resistant isolates.[100] The ability of a microorganism to adapt depends on its skills and varies according to exposure conditions, such as the presence or absence of drugs that can stimulate the expression 5-Fluoracil research buy of its virulence attributes.[101] Prophylactic treatment, which is very common in immunocompromised individuals, promotes exposure of Candida spp. to low concentrations of systemic antifungals, such as azoles, over long periods of time. This may lead to the selection of isolates resistant to these drugs.[102] When exposed to subinhibitory antifungal concentrations, yeast like Candida spp. are able to promote their pathogenic potential through the stimulation of virulence factors,[103, 104] therefore increasing the production and secretion of hydrolytic enzymes to improve adherence to tissues and ensuring their survival.[76, 105] Therefore,

the reaction of the pathogen to the stimulus can result in an increase in tissue destruction, which may lead to death in animal models.[105, 106] Patients infected by fluconazole-resistant C. albicans, who are undergoing therapy with clinical doses of fluconazole, may develop a persistent infection due to the increased production of Sap among other virulence–related factors.[100] According to Wu et al. [100], the increased production of Sap by isolates cultivated in subinhibitory

concentrations of fluconazole corresponds to the development of increased resistance to this drug. In this study, a dose-dependent reduction of Sap activity in isolates susceptible to fluconazole was observed, whereas resistant isolates showed increased Sap activity depending on the dose of fluconazole to which they were subjected. Tangeritin According to Graybill et al. [101], isolates that were exposed to fluconazole over a prolonged period of time and which developed resistance were initially more virulent (MIC values higher) but then developed treatable infections, while less virulent isolates (MIC values lower) were refractory to treatment. According to Costa et al. [107], isolates resistant to azoles presented increased Sap activity in the presence of the drug, which did not occur with susceptible isolates. However, in all susceptible and resistant isolates, the presence of SAP1–SAP7 genes was detected thanks to methods with improved specificity.[107] Kumar et al. [108] indicate that the proteolytic activity of Sap is more intense in Candida spp.

flexneri and in a T3SS-dependent manner. Next, we evaluated whether ShET-2 is delivered into cells by intracellular Shigella. We used a reporter assay system based on translational fusion of the secreted proteins with mature TEM-1 β-lactamase (Charpentier & Oswald, 2004). Plasmids carrying translational fusions with sen gene (pTB-ShET-2–TEM-FLAG), ipaH9.8 gene (pTB-IpaH9.8–TEM-FLAG; positive control) or gst gene (pTB-GST–TEM-FLAG) were transferred into S. flexneri wild-type

strain 2457T or BS547 (T3SS-defective mutant). We confirmed the ability of ShET-2–TEM-FLAG to be secreted via Small molecule library the TTSS (data not shown). HEp-2 cells infected with S. flexneri wild-type strain 2457T expressing the translational fusions were loaded with CCF2-AM

and examined with a fluorescence microscope (Fig. 2). As we expected, uninfected cells and cells infected with 2457T/pTB-GST–TEM-FLAG (negative control) emitted green fluorescence as well as cells infected with BS547/pTB-IpaH9.8–TEM-FLAG or Caspase inhibitor BS547/pTB-ShET-2–TEM-FLAG, indicating the absence of β-lactamase activity in these cells (Fig. 2). However, cells infected with 2457T/pTB-ShET-2–TEM-FLAG or 2457T/pTB-IpaH9.8–TEM-FLAG (positive control) emitted blue fluorescence. These data indicated that ShET-2–TEM-FLAG is delivered into the host cells by the intracellular Shigella. The ShET-2 coding gene sen is located downstream of the ospC1 gene (Fig. 3), which has been shown to be coexpressed with other genes related to T3SS function (Mavris et al., 2002).

The OspC1 protein has been implicated in Shigella-induced MEK/ERK pathway activation and PMN transepithelial migration (Zurawski et al., 2006). Expression of the ospC1 gene is controlled by the MxiE regulator via binding of the protein to a 17-bp MxiE-binding motif located in the promoter upstream region (Kane et al., 2002). Le Gall et al. (2005) suggested that both the ospC1 and sen genes might be part of the same operon based on macroarray analysis. We performed RT-PCR to determine whether sen was Fossariinae cotranscribed with ospC1. Pairing primers downstream of ospC1 and upstream of sen, we found that the amplified products were consistent with the presence of a polycistronic ospC1-sen mRNA transcript (Fig. 3). The role of putative promoter sequences in the region between ospC1 and sen that might drive the expression of ShET-2 cannot be ruled out. Considering that ospC1 is regulated by MxiE, a regulator proposed to control the expression of virulence factors after internalization of the bacterium in the eukaryotic cell (Kane et al., 2002; Mavris et al., 2002), the data presented here suggest that ShET-2 might be regulated by MxiE and could also play a role in the intracellular stage of Shigella infection. Vaccine trials in humans using attenuated Shigella strains with mutations in the ShET showed a diminution of reactogenicity, defined as less diarrhea and fever (Kotloff et al., 2004, 2007).

Consistent with this, splenocytes from Camp−/− mice that had been administered with a T-cell-dependent antigen were also found to have increased IL-4 mRNA expression and increased numbers of CD4+IL-4+ T cells as compared with those from similarly treated WT mice. The connections between mCRAMP and IL-4 open up intriguing possibilities for the role of cathelicidins in adaptive

immunity. In the mice given TNP-OVA/alum and in the in vitro T cells, the responses indicate that mCRAMP suppresses both the development of a Th2 response and the Th2-mediated class switching to IgG1 through IL-4 17, 19. In contrast, LY2606368 cost the results from isolated B cells stimulated with CD40L/IL-4 indicated that mCRAMP see more promoted IgG1 production by increasing transcription 17. Kurosaka et al. 13 showed that mCRAMP administered as an adjuvant with OVA increases IL-4 and OVA-specific IgG1 in splenocytes, although the response

was not Th2-mediated. Similarly, An et al. 16 found that LL-37 acts as an effective adjuvant in a vaccine against tumor cells, while Davidson et al. 8 found a bias towards a Th1 response in human DCs. The conflicting reports may reflect methodological differences, such as using Camp−/− mice versus injecting cathelicidin into WT mice, or the timing and nature of other stimuli applied. Nonetheless, these studies indicate that mCRAMP likely mediates its effects on adaptive immunity through many other factors in addition to IL-4. The work by Kin et al. 17 shows that mCRAMP alters B- and T-cell responses, highlighting the novel role of mCRAMP in the T-cell-dependent activation of B cells, and thus providing evidence that mCRAMP and other cathelicidins have a greater role in the adaptive immune response than previously appreciated. However, many questions still remain, particularly whether

mCRAMP acts directly on components of the adaptive immune system or if intermediates are involved. It is also of interest to determine whether the changes seen by Kin et al. 17 in response to T-cell-dependent antigen are due to mCRAMP altering Flavopiridol (Alvocidib) both T and B cells or whether only one cell type is directly involved. The use of conditional knockouts or adoptive transfer to examine when Camp is absent from either T or B cells will help resolve these issues. Similar models could also be used to clarify the functions of APCs in shaping the Camp−/− effects on lymphocytes. Determining the specific cells and pathways altered by mCRAMP will provide further insight into the roles of cathelicidins in bridging innate and adaptive immunity. Funding from the Canadian Institutes for Health Research for the authors own peptide research is gratefully acknowledged. REWH holds a Canada Research Chair. Conflict of interest: The authors have declared no financial or commercial conflict of interest. See accompanying article: http://dx.doi.org/10.1002/eji.

HO-1 levels in monocytes were significantly reduced in patients with SLE compared with healthy controls. These results were confirmed by flow cytometry. No differences were observed in other cell types, such as DCs or CD4+ T cells, although decreased MHC-II levels were observed in DCs from patients with SLE. In conclusion, we found a significant decrease in HO-1 expression, specifically in monocytes from patients with SLE, suggesting RG7420 mw that an imbalance of monocyte function could be partly the result of a decrease in HO-1 expression. Systemic lupus erythematosus (SLE) is a chronic autoimmune disease of unknown aetiology, characterized

by, among other findings, the presence of autoantibodies against double-stranded DNA, nucleosomes, ribonucleoproteins and other nuclear components, as well as by the presence of circulating DNA and nucleosomes in peripheral blood.1–3 Multi-organ compromise may arise as a consequence of the deposition of immune complexes in blood vessels, which leads to macrophage

and complement activation, inflammation and tissue damage.4–7 Abnormalities in almost every component of IWR-1 solubility dmso the immune system have been described in patients with SLE and in mouse models of SLE, including the presence of activated autoreactive CD4+ T cells that drive the subsequent activation of self-reactive B cells, leading to the production of autoantibodies.8–10 In addition, peripheral blood monocytes derived

from patients with SLE display an abnormal phenotype, characterized by deregulated expression of HLA-DR and CD14, which could lead to defects in antigen presentation by monocyte-derived antigen-presenting cells, such as dendritic cells (DCs) or macrophages.11,12 These alterations are likely to contribute to autoreactive T-cell priming during the onset of SLE.12–15 Accordingly, expression of co-stimulatory molecules that are essential for T-cell activation, such as CD86, is significantly increased in monocytes and DCs from patients with SLE, compared with healthy individuals.16 We have previously shown that monocyte-derived DCs from patients with SLE display higher expression ratios of activating over inhibitory Fcγ receptors (FcγRs), promoting the presentation of autoantigens derived from immune complexes to previously activated self-reactive T cells and perpetuating T-cell Resveratrol activation.17 Hence, an unbalanced expression of activator/inhibitory molecules in monocytes and DCs could contribute to maintaining SLE pathogenesis.17,18 Haem oxygenases (HO) are microsomal enzymes that catalyse the degradation of the haem group into biliverdin, free iron and carbon monoxide (CO).19 Biliverdin is rapidly reduced to bilirubin by the enzyme biliverdin reductase and free iron is removed by ferritin, which produces a depletion in the intracellular free iron.20 Until now, three HO isoforms have been described and designated HO-1, HO-2 and HO-3.

Therefore, the levels of FOXP3 were compared with that buy FK506 of established FOXP3+ and

FOXP3− clones (C148.31 and C271.9, respectively) 5. In total, 16 of 59 tested T-cell clones (28%) showed constitutive FOXP3 expression similar to the FOXP3+ reference clone C148.31, as was determined 21 days after last antigen-specific activation in two separate assays 6 months apart (Fig. 2F). Of these 16 FOXP3+ clones, 14 produced IL-10; however there was no correlation between the quantity of IL-10 produced and the level of expression of Foxp3 (data not shown). The cytokine profile of the influenza-specific CD4+ T-cell clones resemble that of Treg specific for chronic infections and tumor antigens 5, 7, 20. Therefore, the isolated clones were further expanded and tested for their suppressive capacity.

Of the isolated Foxp3 positive and negative, and/or IL-10 positive and negative M1-specific T-cell clones, 69 could be sufficiently expanded to test their suppressive capacity. In total, 26 of 69 clones showed significant suppression (>50%) of the proliferation BYL719 in vivo of anti-CD3 stimulated CD4+CD25− T cells. Categorization of the T-cell clones based on IL-10 production, IL10− (<50 pg/mL) and IL-10+(>50 pg/mL), revealed that the Treg are significantly more found among the population of IL-10-producing T cells (p<0.001; two-tailed Mann–Whitney test), but are not exclusively found within this population (Fig. 4A). Dot plot analysis of the PDK4 quantity of IL-10 produced versus suppression also did not reveal a correlation, suggesting that IL-10 itself is not responsible for the observed suppression. These data are consistent with previous reports showing that IL-10 is not involved in suppression 5, 20, 21. A number of influenza-specific CD4+ Treg clones were studied in more detail. These suppressive clones not only prevented proliferation of CD3-stimulated effector cells, but also their capacity to produce IFN-γ (Fig. 4B). To study whether these clones could also exert their suppressive function when activated through their TCR upon recognition of cognate antigen, the Treg

clones were stimulated with M1 peptide during the assay (Fig. 4C). CFSE-labeled responder cells were stimulated with allogeneic APC in the presence of a PKH-26-labeled influenza-specific T-cell clone (Fig. 4C; upper panels). Consistent with the anti-CD3-based suppression assay, the clones D1.6, D1.52, D4.6 and D1.68 were able to suppress proliferation upon stimulation with M1 peptide. The M1-specific T-cell clones D1.50 and D4.11 did not suppress proliferation (Fig. 4C; lower panels). The fact that proliferation was only inhibited when cognate antigen was added to the co-cultures in which Treg were present ruled out the possibility that physical and immune competition played a role in the assay. As we had noted earlier that an increase in antigen dose could alter the cytokine profile of the Treg clones (Fig.

T-helper (TH1) CD4+ cells expressing INF-γ play a critical role in controlling M. tuberculosis

infection FK506 clinical trial in humans as well as in various animal models [26-28]. However, the protective efficacy of TH1 CD4+ cells might be attenuated by a TH2-cell response. Recently, it was found that antigen-containing exosomes can drive a predominate TH1 immune response against parasite infection or tumor progression in mice [29-31]. To determine whether CFP exosome vaccination generates both a TH1 and TH2 immune response, the expression of IL-4, a marker for TH2-mediated immunity, was investigated by intracellular cytokine staining followed by FACS analysis. BCG but not CFP exosome vaccination induced expression of IL-4 positive CD4+ cells following ex vivo stimulation (Fig. 3). To evaluate this TH1/TH2 balance further, mycobacterial antigen-specific antibody isotypes in serum were defined 2 weeks postvaccination. Both BCG and CFP exosome vaccinated mice produced antigen-specific IgG (Fig. 4A). However, CFP exosomes induced a greater titer

of antigen-specific IgG2c antibody, an indicator of a TH1-mediated immune response, compared with that elicited by BCG (Fig. 4B). In www.selleckchem.com/products/abt-199.html contrast, antibody titers for IgG1, which is an indicator of TH2-mediated immune response, were higher in mice immunized with BCG compared with those receiving CFP exosomes (Fig. 4C). The relative ratio (IgG2c/IgG1) against specific antigens is used as an indicator of the balance between a TH1 or TH2 immune response (Fig. 4D). Our results suggest that mice vaccinated with CFP exosomes produce a more predominant TH1 immune response compared with that generated

in BCG-vaccinated mice. To measure the exosome’s ability to protect against an M. tuberculosis infection, mice were vaccinated with CFP exosomes or exosomes from uninfected macrophages at a dose of 20 μg or 40 μg per mouse as described in the Materials and methods. As a positive control, mice were vaccinated i.n. with M. bovis BCG. Four weeks after the last exosome vaccination, all mice were subjected to a low-dose aerosol challenge with virulent M. tuberculosis H37Rv using the Glas-Col Inhalation Exposure System. Initial infection dose was approximately 100 CFU. After a 6 week infection, mycobacterial load in the lungs and spleens Astemizole were determined. In CFP exosome-vaccinated mice, M. tuberculosis burden decreased significantly in the spleens when compared with unvaccinated mice or mice vaccinated with exosomes from uninfected cells (Fig. 5). We did not observe a statistical difference between the 20 and 40 μg CFP exosome doses. Of note, the CFP exosomes generated a comparable protection to BCG vaccination and showed a half log better protection than BCG in the lung, although this was only statistically different for the 20 μg vaccine dose (Fig. 5). As the primary infection site after aerosol challenge, M.

Heat-killed E. faecalis (5×107 CFU/mL) were prepared by heating bacteria at 65°C for 30 min. No viability of the bacteria was confirmed by plating an aliquot of the heat-killed bacteria on TSB agar plates. Murine rCCL2 was purchased from BD Biosciences (San Jose, CA, USA), and mAb

directed against CCL2 was obtained from BioLegend (San Diego, CA, USA). rCCL5, rCCL17 and mAbs directed against these chemokines were purchased from R&D Systems GPCR Compound Library manufacturer (Minneapolis, MN, USA). Biotin-conjugated anti-CD3, anti-F4/80 and anti-CD19 mAbs were obtained from eBioscience (San Jose, CA, USA). Phosphorothioated CCL2 antisense ODNs (5′-AAGCGTGACAGAGACCTGCATAGTGGTGG-3′) and scrambled ODNs (5′-CCACCACTATGCAGGTCTCTGTCACGCTT-3′) were purchased from Sigma-Genosys (The Woodlands, TX, USA). RPMI-1640 medium supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin was utilized for the cultivation of various Mϕ preparations. Thermally injured mice were created according to our previously reported protocol 23–25. This procedure consistently produced a third degree burn on approximately 25% of total body surface area (TBSA) for a 26-g

mouse. Immediately after thermal injury, physiologic saline (1 mL per mouse, i.p.) was administered for fluid resuscitation. Deaths within 5 days of 25% TBSA flame burn were not demonstrated Trichostatin A clinical trial after our burn procedure. As controls, mice were anesthetized and shaved but were not exposed to the gas flame. They also received physiologic saline (1 mL per mouse, i.p.). Buprenorphine (2 mg/kg) was given s.c. every 12 h during the postburn period. Sham burn animals also received identical regimens of analgesics (buprenorphine) throughout the study period. Mϕs (F4/80+ cells) were prepared from MLNs of various groups of mice, as previously described 24, 25. F4/80+ cells with

94% or more purity were consistently obtained using this technique. Severely burned mice were subjected to CCL2 antisense ODN gene therapy. Thus, burned mice were treated twice with CCL2 antisense ODNs at 2 and 12 h after burn injury. Based on our Sitaxentan preliminary studies, CCL2 antisense ODNs were administered s.c. to burned mice at doses ranging from 0.01 to 100 μg/mouse. Weight loss, reduced appetite and abnormal body temperature were not demonstrated in normal mice treated with 100 μg/mouse of CCL2 antisense ODNs twice a day for 7 days. The effect of the gene therapy was confirmed by measuring CCL2 levels in the sera of these mice 24 h after burn injury, because the maximum level of CCL2 in sera of these mice was reached within 24 h of severe burn injury. CCL2 in serum specimens was assayed by ELISA. To determined the efficacy of CCL2 antisense ODNs on the generation of M2Mϕs, MLN-Mϕs were isolated from severely burned mice treated twice with 10 μg/mouse of CCL2 antisense ODNs (2 and 12 h after burn injury) 1–8 days after burn injury.