Recombinant GM-CSF Research Articles

A Cell Free Assay System Estimating the Neutralizing Capacity of GM-CSF Antibody using Recombinant Soluble GM-CSF Receptor

Previously, we demonstrated that neutralizing capacity but not the concentration of GM-CSF autoantibody was correlated with the disease severity in patients with autoimmune pulmonary alveolar proteinosis (PAP)¹⁻³. As abrogation of GM-CSF bioactivity in the lung is the likely cause for autoimmune PAP⁴⁻⁵, it is promising to measure the neutralizing capacity of GM-CSF autoantibodies for evaluating the disease severity in each patient with PAP. Until now, neutralizing capacity of GM-CSF autoantibodies has been assessed by evaluating the growth inhibition of human bone marrow cells or TF-1 cells stimulated with GM-CSF⁶⁻⁸. In the bioassay system, however, it is often problematic to obtain reliable data as well as to compare the data from different laboratories, due to the technical difficulties in maintaining the cells in a constant condition. To mimic GM-CSF binding to GM-CSF receptor on the cell surface using cell-free receptor-binding-assay. Transgenic silkworm technology was applied for obtaining a large amount for recombinant soluble GM-CSF receptor alpha (sGMRα) with high purity⁹⁻¹³. The recombinant sGMRα was contained in the hydrophilic sericin layers of silk threads without being fused to the silk proteins, and thus, we can easily extract from the cocoons in good purity with neutral aqueous solutions¹⁴(,)¹⁵. Fortunately, the oligosaccharide structures, which are critical for binding with GM-CSF, are more similar to the structures of human sGMRα than those produced by other insects or yeasts. The cell-free assay system using sGMRα yielded the data with high plasticity and reliability. GM-CSF binding to sGMRα was dose-dependently inhibited by polyclonal GM-CSF autoantibody in a similar manner to the bioassay using TF-1 cells, indicating that our new cell-free assay system using sGMRα is more useful for the measurement of neutralizing activity of GM-CSF autoantibodies than the bioassay system using TF-1 cell or human bone marrow cells. We established a cell-free assay quantifying the neutralizing capacity of GM-CSF autoantibody.

Identification of CCR9− murine plasmacytoid DC precursors with plasticity to differentiate into conventional DCs

AbstractWhereas the final differentiation of conventional dendritic cells (CDCs) from committed precursors occurs locally in secondary lymphoid or peripheral tissues, plasmacytoid dendritic cells (PDCs) are thought to fully develop in the bone marrow from common DC progenitors before migrating to the periphery. In our study, we define, for the first time, a subpopulation of CCR9− major histocompatibility complex class IIlow PDCs in murine bone marrow, which express E2-2 and are immediate precursors of CCR9+ fully differentiated PDCs. However, CCR9− PDCs have the plasticity to acquire the phenotype and function of CD11b+ CD8α− major histocompatibility complex class IIhigh CDC-like cells under the influence of soluble factors produced by intestinal epithelial cells or recombinant GM-CSF. This deviation from the PDC lineage commitment is regulated on the level of transcription factors reflected by down-regulation of E2-2 and up-regulation of ID2, PU.1, and BATF3. Thus, CCR9− PDCs are immediate PDC precursors that can be reprogrammed to differentiate into CDC-like cells with higher antigen-presenting and cytokine-producing capacity under the influence of the local tissue microenvironment.

Different cytokines induce the maturation of human monocyte-derived dendritic cells

Objective To investigate the immune effect of different cytokines on immature dendritic cells (DC) in vitro. Methods The human monocyte-derived DCs were induced in the presence of recombinant human GM-CSF (rhGM-CSF, 1000 U/ml) and interleukin (IL)-4 (500 U/ml). The effects of tumor necrosis factor (TNF) -α (1000 U/ml) , lipopolysaccharide (LPS) (1 mg/L), the cocktail of cytokines (TNF-α, IL-6, IL-1β, PGE2) , and sodium butyrate (1 mmol/L) on the DCs were detected by flow cytometry (FCM), endocytic activity, T cells stimulatory proliferation capacity, and IL-12 production. Results The cocktail of cytokines could up-regulate the major histocompatibility complex (MHC) class Ⅱ and costimulatory molecules of DCs, decrease the endocytic activity 186. 74 ±38. 66, induce a stage of Tcell stimulation 18. 23 ± 2. 22 and promote the T helper cell type 1-skewing factor IL-12 production (656. 18 ±38. 52) ng/L. But the sodium butyrate induced reverse result on DCs compared to cocktail of cytokines. Conclusion The most effective method for inducing dendritic cells maturation was cocktail of cytokines. The sodium butyrate could inhibit the development of mature DCs, resulting in impaired DC function, and modify the outcome of the subsequent immune response. Key words: Dendritic cell; Cytokines; Sodium butyrate

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although the isolation methods of liver macrophages have been well-described4-6, most of these methods require sophisticated equipment, such as a centrifugal elutriator and technical skills. Here, we provide a novel method to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult rat liver cells, as schematically illustrated in Figure 1.After dissociation of the liver cells by two-step perfusion method7,8,a fraction mostly composed of parenchymal hepatocytes is prepared and seeded into T75 tissue culture flasks with culture medium composed of DMEM and 10% FCS.Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells (Figure 2). As the culture proceeds, around day 6, phase contrast-bright, round macrophage-like cells start to proliferate on the fibroblastic cell sheet (Figure 2). The growth of the macrophage-like cells continue and reach to maximum levels around day 12, covering the cell sheet on the flask surface. By shaking of the culture flasks, macrophages are readily suspended into the culture medium. Subsequent transfer and short incubation in plastic dishes result in selective adhesion of macrophages(Figure 3), where as other contaminating cells remain suspended. After several rinses with PBS, attached macrophages are harvested. More than 106 cells can be harvested repeatedly from the same T75 tissue culture flask at two to three day intervals for more than two weeks(Figure 3).The purities of the isolated macrophages were 95 to 99%, as evaluated by flow cytometry or immunocytochemistry with rat macrophage-specific antibodies (Figure 4).The isolated cells show active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells9.In conclusion, we provide a simple and efficient method to obtain liver macrophages in sufficient number and purity without complex equipment and skills.This method might be applicable to other mammalian species.

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although the isolation methods of liver macrophages have been well-described4-6, most of these methods require sophisticated equipment, such as a centrifugal elutriator and technical skills. Here, we provide a novel method to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult rat liver cells, as schematically illustrated in Figure 1. After dissociation of the liver cells by two-step perfusion method7,8,a fraction mostly composed of parenchymal hepatocytes is prepared and seeded into T75 tissue culture flasks with culture medium composed of DMEM and 10% FCS.Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells (Figure 2). As the culture proceeds, around day 6, phase contrast-bright, round macrophage-like cells start to proliferate on the fibroblastic cell sheet (Figure 2). The growth of the macrophage-like cells continue and reach to maximum levels around day 12, covering the cell sheet on the flask surface. By shaking of the culture flasks, macrophages are readily suspended into the culture medium. Subsequent transfer and short incubation in plastic dishes result in selective adhesion of macrophages(Figure 3), where as other contaminating cells remain suspended. After several rinses with PBS, attached macrophages are harvested. More than 106 cells can be harvested repeatedly from the same T75 tissue culture flask at two to three day intervals for more than two weeks(Figure 3).The purities of the isolated macrophages were 95 to 99%, as evaluated by flow cytometry or immunocytochemistry with rat macrophage-specific antibodies (Figure 4).The isolated cells show active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells9. In conclusion, we provide a simple and efficient method to obtain liver macrophages in sufficient number and purity without complex equipment and skills.This method might be applicable to other mammalian species.

HIV-1–Infected Peripheral Blood Mononuclear Cells Enhance Neutrophil Survival and HLA-DR Expression Via Increased Production of GM-CSF: Implications for HIV-1 Infection

HIV-1 bound to intact neutrophils efficiently infects activated peripheral blood mononuclear cells (PBMC). Here, we evaluated the effect of the local milieu created by activated PBMC before and after HIV-1 infection on neutrophil survival and HLA-DR expression, with emphasis placed on a role for GM-CSF. PBMC of healthy adult individuals were activated by phytohemagglutinin (PHA) or anti-CD3/anti-CD28 and were subsequently cultured without (HIV-1⁻) or with HIV-1 (HIV-1+). The effects of the culture supernatants or recombinant GM-CSF on survival and HLA-DR expression by neutrophils of healthy adult individuals and of HIV-1-infected individuals were evaluated using flow cytometry. Conditioned medium from PHA-activated PBMC (HIV-1⁻ and HIV-1+) increased neutrophil survival and induced HLA-DR expression by neutrophils of healthy individuals in a GM-CSF dependent fashion. HIV-1 infection variably, but consistently, increased GM-CSF production by PHA-activated PBMC but not GM-CSF production by anti-CD3/anti-CD28-activated PBMC. The latter was correlated with a loss of CD3+GMCSF+ cells after infection. Neutrophils of elite controllers exhibited a diminished HLADR response to GM-CSF in culture, whereas neutrophils of HIV-1+ subjects having a low viral load on anti-retroviral therapy or subjects with a high viral load exhibited a range of HLA-DR responses. GM-CSF production within the mucosa or draining lymph nodes may promote HIV-1 infection by facilitating sustained contact between viable neutrophils with bound HIV-1 and CD4 lymphocytes. The minimal effect of GM-CSF on HLA-DR expression by neutrophils of elite controllers provides indirect support for this conclusion.

Maturation-Resistant Dendritic Cells Ameliorate Experimental Autoimmune Uveoretinitis

BackgroundEndogenous uveitis is a chronic inflammatory eye disease of human, which frequently leads to blindness. Experimental autoimmune uveoretinitis (EAU) is an animal disease model of human endogenous uveitis and can be induced in susceptible animals by immunization with retinal antigens. EAU resembles the key immunological characteristics of human disease in that both are CD4+ T-cell mediated diseases. Dendritic cells (DCs) are specialized antigen-presenting cells that are uniquely capable of activating naïve T cells. Regulation of immune responses through modulation of DCs has thus been tried extensively. Recently our group reported that donor strain-derived immature DC pretreatment successfully controlled the adverse immune response during allogeneic transplantation.MethodsEAU was induced by immunization with human interphotoreceptor retinoid-binding protein (IRBP) peptide1-20. Dendritic cells were differentiated from bone marrow in the presence of recombinant GM-CSF.ResultsIn this study, we used paraformaldehyde-fixed bone marrow-derived DCs to maintain them in an immature state. Pretreatment with fixed immature DCs, but not fixed mature DCs, ameliorated the disease progression of EAU by inhibiting uveitogenic CD4+ T cell activation and differentiation.ConclusionApplication of iBMDC prepared according to the protocol of this study would provide an important treatment modality for the autoimmune diseases and transplantation rejection.

Development of a successful antitumor therapeutic model combining in vivo dendritic cell vaccination with tumor irradiation and intratumoral GM-CSF delivery

Vaccination of dendritic cells (DC) combined with GM-CSF secreting tumor cells has shown good therapeutic efficacy in several tumor models. Nevertheless, the engineering of GM-CSF secreting tumor cell line could represent a tedious step limiting its application for treatment in patients. We therefore developed in rats, an "all in vivo" strategy of combined vaccination using an in vivo local irradiation of the tumor as a source of tumor antigens for DC vaccines and an exogenous source of GM-CSF. We report here that supplying recombinant mGM-CSF by local injections or surgical implantation of osmotic pumps did not allow reproducing the therapeutic efficacy observed with in vitro prepared combined vaccines. To bypass this limitation possibly due to the short half-life of recombinant GM-CSF, we have generated adeno-associated virus coding for mGM-CSF and tested their efficacy to transduce tumor cells in vitro and in vivo. The in vivo vaccines combining local irradiation and AAV2/1-mGM-CSF vectors showed high therapeutic efficacy allowing to cure 60% of the rats with pre-implanted tumors, as previously observed with in vitro prepared vaccines. Same efficacy has been observed with a second generation of vaccines combining DC, local tumor irradiation, and the controlled supply of recombinant mGM-CSF in poloxamer 407, a biocompatible thermoreversible hydrogel. By generating a successful "all in vivo" vaccination protocol combining tumor radiotherapy with DC vaccines and a straightforward supply of GM-CSF, we have developed a therapeutic strategy easily translatable to clinic that could become accessible to a much bigger number of cancer patients.

Physical properties, lung deposition modeling, and bioactivity of recombinant GM-CSF aerosolised with a highly efficient nebulizer

Pulmonary alveolar proteinosis (PAP) is a rare condition characterized by the accumulation of lipoproteinaceous material within air spaces. Although whole lung lavage is the current standard of care, recent advances in our understanding of PAP pathophysiology suggest that the disorder may benefit from inhalation of recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF). The aim of this study was to determine the physical properties and bioactivity of rGM-CSF aerosolised by the highly efficient AKITA 2 APIXNEB ® nebulizer system. The physical properties of aerosolised rGM-CSF were investigated in terms of droplet size, output and output rate by laser diffraction and gravimetrical analysis. Lung deposition was assessed using deposition modeling (ICRP). Molecular mass before and after aerosolisation was determined by SDS-PAGE, while the bioactivity of rGM-CSF was evaluated by measuring the GM-CSF-stimulated increase in pSTAT5 using mAM-hGM-R cells. Ninety-six % of the rGM-CSF filling dose was aerosolised with the Akita 2 Apixneb ® nebulizer system. Particle size was highly reproducible, and the amount deposited within the lung was 80.35% of the delivered dose. The aerosolisation did not alter the molecular structure of rGM-CSF, nor its ability to stimulate the pSTAT5, which increased by 99.5%, similar to values for rGM-CSF prior to aerosolisation. We conclude that the highly efficient AKITA 2 APIXNEB ® nebulizer system is likely to efficaciously deliver rGM-CSF to the airways of patients with autoimmune PAP.

GM-CSF up-regulated in reumatoid arthritis reverses cognitive impairment and amyloidosis in Alzheimer mice

Rheumatoid arthritis (RA) is a negative risk factor for the development of Alzheimer's disease (AD). While it has been commonly assumed that RA patients' usage of non-steroi dal anti-inflammatory drugs (NSAIDs) helped prevent onset and progression of AD, NSAID clinical trials have proven unsuccessful in AD patients. To determine whether intrinsic factors within RA pathogenesis itself may underlie RA's protective effect, we investigated the activity of colony-stimulatin g factors, upregulated in RA, on the pathology and behavior of transgenic AD mice. 5 µg bolus injections of macrophage, granulocyte, and granulocyte-macrophage colony-stimulating factors (M-CSF, G-CSF, or GM-CSF) were administered unilaterally into the hippocampus of aged cognitively-impaired AD mice and the resulting amyloid load reductions determined one week later, using the artificial cerebrospinal fluid-injected contrala teral sides as controls. G-CSF and more significantly, GM-CS F reduced amyloidosis throughout the treated brain hemisphere one week following bolus administration to AD mice. 20 daily subcutaneous injections of 5 µg of GM-CSF (the most amyloid-reducing CSF in the bolus experiment) were administered to balanced cohorts of AD mice after assessment in a battery of cognitive tests. Reductions in amyloid load and improvements in cognitive function were assessed. Subcutaneous GM-CSF administration significant ly reduced brain amyloidosis and completely reversed the cognitive impairment, while increasing hippocampal synaptic area and microglial density. These findings, along with two decades of accrued safety data using Leukine, recombinant human GM-CSF, in elderly leukopenic patients, suggest that Leukine should be tested as a treatment to reverse cerebral amyloid pathology and cognitive impairment in AD.

A novel isolation method for macrophage-like cells from mixed primary cultures of adult rat liver cells

We report a simple and efficient method to obtain macrophage-like cells from the mixed primary cultures of adult rat liver cells. A parenchymal hepatocyte enriched fraction was prepared from adult rat livers and seeded into culture flasks. After 7 to 10 days of culture, when most hepatocytes were degenerated or transformed into fibroblastic cells, macrophage-like cells vigorously proliferated on the cell sheet. By shaking the flasks, macrophage-like cells were readily detached. Subsequent transfer and incubation in plastic dishes resulted in quick and selective adhesion of macrophage-like cells, while other contaminating cells remained suspended in the medium. After rinsing with saline, attached macrophage-like cells were harvested with 95 to 99% purity, as evaluated by flow cytometry or immunocytochemistry. These cells showed typical macrophage morphology and were strongly positive for markers of rat macrophages, such as ED-1, ED-3, and OX-41, but negative for cytokeratins and α-smooth muscle actin. They possessed functional properties of typical macrophages, including active phagocytosis of latex beads, proliferative response to recombinant GM-CSF, secretion of inflammatory and anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells. As more than 10 6 cells can be recovered repeatedly from a T75 culture flask at two to three day intervals for more than two weeks, our procedure might implicate a novel alternative to obtain Kupffer cells in sufficient number and purity without complex equipment and skills.

Identification of immunologically relevant genes in mare and foal dendritic cells responding to infection by Rhodococcus equi

Rhodococcus equi is a facultative intracellular bacterial pathogen of horses; infected foals develop pyogranulomatous pneumonia, however adult horses are largely unaffected. R. equi infects and proliferates within host macrophages and dendritic cells (DCs). DCs initiate the appropriate adaptive immune response, thereby playing a critical role in determining the outcome of infection. Our aim was to identify genes that are differentially expressed in R. equi infected monocyte-derived DCs (mdDCs). Peripheral blood monocytes from mares and foals were used to derive mdDCs by culturing with recombinant equine IL-4 and recombinant human GM-CSF. RNA harvested 24 h after infection with R. equi (ATCC 33701+) was used to perform suppression subtractive hybridization (SSH) experiments. Approximately 38 unique sequences were obtained from these experiments. Differential expression of 19 immunologically relevant genes was validated by PCR. These genes are characterized by the following functions: cell adhesion, chemotaxis/migration, immune/inflammatory response, ion transport, signal transduction, T-cell regulation, and vesicular transport. In summary, we identified several novel genes that are differentially expressed in foal and adult mdDCs in response to R. equi infection. These genes provide promising targets for further research into the host response to R. equi, and the susceptibility of foals to this disease.

A multicenter clinical trial of recombinant human GM-CSF hydrogel for the treatment of deep second-degree burns

A multicenter clinical trial of recombinant human GM-CSF hydrogel for the treatment of deep second-degree burns

A multicenter clinical trial of recombinant human GM-CSF hydrogel for the treatment of deep second-degree burns

A multicenter clinical trial of recombinant human GM-CSF hydrogel for the treatment of deep second-degree burns

Pegylated Recombinant Murine GM-CSF Is a Potent Mobilizer of Murine Bone Marrow Progenitors, Synergizes with BIO5192 and Plerixafor (AMD3100), and Skews Mobilized Cells to a Tolerogenic Phenotype.

Abstract 2432Poster Board II-409Granulocyte-macrophage colony stimulating factor (GM-CSF) is a naturally occurring acidic glycoprotein growth factor that regulates proliferation and differentiation of hematopoietic stem and progenitor cells. Inflammation results in the release of GM-CSF from numerous cell types including T-cells, macrophages, and neutrophils. Murine GM-CSF (mGM-CSF), unlike human GM-CSF, has a very short half-life in vivo. Pegylation of mGM-CSF (peg-mGM-CSF) has been shown to prolong its half-life.There are numerous reports of GM-CSF use in humans to mobilize stem cells. It was observed by our group that patients who were transplanted with stem cell products mobilized by GM-CSF had a lower incidence of acute GVHD (Devine et al. BBMT 2005; 36:531). The reason for this is unclear. A recent report suggested that regulatory T-cells (T-regs) express the low affinity alpha subunit of the mGM-CSF receptor and can be expanded ex vivo in the presence of GM-CSF and independent of IL-2 (Kared et al. Blood 2008;112:2575). We hypothesized that peg-mGM-CSF may expand T-regs in vivo and act synergistically with the CXCR4 antagonist Plerixafor (AMD3100) and a small molecule inhibitor of VLA-4, BIO5192, to generate optimal peripheral blood stem cell products enriched in hematopoietic progenitors and T-regs.Peg-mGM-CSF was manufactured in a bioreactor as previously described (Sainathan et al. Prot Express and Purification 2005;44:94). C57/Bl6 mice were injected with peg-mGM-CSF 5ug IP, daily x 4 days then bled and analyzed for CFU progenitor assays (CFU-C) and FACS analysis. The same mice were then injected with AMD3100 (5mg/kg SC) with or without BIO5192 (1mg/kg IV) and serially bled at 30 minutes, 1 hour, 3 hours, and 6 hours. CFU-C were performed and blood and spleen were analyzed for T-cell and dendritic cell subsets by FACS. We observed that peg-mGM-CSF was a potent mobilizer of hematopoietic progenitors and resulted in leukocytosis (mean difference 17.98 K/uL +/- 2.15, 95%CI 13.4 - 22.53, p<0001, n=6) and splenomegally. Peg-mGM-CSF increased CFU-C by 26.5 fold compared to PBS treated mice (n=6, 95% CI = 10.25-42.73). Peg-mGM-CSF had synergistic effects on progenitor mobilization when combined with AMD3100 (89.8 fold, 95% CI 61.6-118, n=6) or BIO5192 (94 fold, 95% CI 46.5-141.6, n=6) compared to PBS treated mice. The combination of peg-mGM-CSF, AMD3100 and BIO5192 resulted in 119.7 fold (95% CI 63.7-175.6, n=6) increase in progenitor mobilization over PBS treated mice and peaked at 30 minutes. In addition, peg-mGM-CSF increased circulating CD4+ FoxP3+ T-regs from a mean of 10.66% +/- 2.65 to 23.86% +/- 4.7 at 5 days (n=15, p=0.001 by paired t-test). Splenic T-regs were also increased from 16.09% +/- 2.7 to 31.56% +/- 4.34 (n=9, p=0.001 by paired t-test). This effect was sustained out to 4 days after the last dose of peg-mGM-CSF. Furthermore, there was no significant change in thymic T-regs, and this effect was not significantly affected by the addition of AMD3100 or BIO5192. The function of these peg-mGM-CSF T-regs, as measured in a MLR was found to be equivalent to T- regs from PBS treated control mice (n=3, p=0.27, by paired t-test). Interestingly, analysis of the spleens from animals on day 5 after peg-mGM-CSF demonstrated an increase in absolute CD11c+ cells, as expected, and a 4 fold increase in the absolute number of plasmacytoid dendritic cells as determined by CD11c+ SiglecH+ staining. Additional studies are underway to assess the functional impact of murine peripheral blood stem cells mobilized with peg-mGM-CSF in combination with small molecule inhibitors of the CXCR4 and VLA-4 axes on engraftment and graft versus host disease in a murine acute GVHD parent to F1 model. Disclosures:DiPersio:Genzyme Corporation: Honoraria.

Human NKT cells promote monocyte differentiation into suppressive myeloid antigen-presenting cells

NKT cells have been shown to promote peripheral tolerance in a number of model systems, yet the processes by which they exert their regulatory effects remain poorly understood. Here, we show that soluble factors secreted by human NKT cells instruct human peripheral blood monocytes to differentiate into myeloid APCs that have suppressive properties. NKT instructed monocytes acquired a cell surface phenotype resembling myeloid DCs. However, whereas control DCs that were generated by culturing monocytes with recombinant GM-CSF and IL-4 had a proinflammatory phenotype characterized by the production of IL-12 with little IL-10, NKT-instructed APCs showed the opposite cytokine production profile of high IL-10 with little or no IL-12. The control DCs efficiently stimulated peripheral blood T cell IFN-gamma secretion and proliferation, whereas NKT-instructed APCs silenced these T cell responses. Exposure to NKT cell factors had a dominant effect on the functional properties of the DCs, since DCs differentiated by recombinant GM-CSF and IL-4 in the presence of NKT cell factors inhibited T cell responses. To confirm their noninflammatory effects, NKT-instructed APCs were tested in an in vivo assay that depends on the activation of antigen-specific human T cells. Control DCs promoted substantial tissue inflammation; however, despite a marked neutrophilic infiltrate, there was little edema in the presence of NKT-instructed APCs, suggesting the inflammatory cascade was held in check. These results point to a novel pathway initiated by NKT cells that can contribute to the regulation of human antigen-specific Th1 responses.

Severe aplastic anemia (SAA): Response to cyclosporin A (CyA) in vivo and in vitro

The aim of the present study was to test the effect of cyclosporin A (CyA) in vitro on CFU-GM growth from patients with severe aplastic anemia (SAA). For this purpose, bone marrow (BM) cells from 9 SAA patients and 5 healthy individuals were incubated with or without CyA and then cultured for CFU-GM growth in the presence of exogenous recombinant human GM-CSF (30 ng/ml). SAA patients were tested before or after treatment with CyA, or after treatment with antilymphocyte globulin (ALG). In 3 patients responding to CyA, the addition of CyA in vitro enhanced colony growth from 13 +/- 10 to 40 +/- 20/10(5) BM cells (p = 0.01) - the median increment of colony formation was 2.4-fold. In 5 ALG responders, CyA produced no increment of CFU-GM growth (from 14 +/- 26 to 15 +/- 16/10(5) BM cells, p = 0.1). CyA did not enhance significantly CFU-GM growth in normal controls (from 57 +/- 45 to 58 +/- 81/10(5) BM cells, p = 0.9). In conclusion, it would appear that some patients with SAA can respond to CyA in vivo and in vitro, and ALG responders are not necessarily among these. This is in keeping with different mechanisms of action of CyA and ALG and possibly with the existence of distinct pathogenetic pathways in SAA.

Endogenous Galectin-3 Is Localized in Membrane Lipid Rafts and Regulates Migration of Dendritic Cells

This study reveals a function of endogenous galectin-3, an animal lectin recognizing beta-galactosides, in regulating dendritic cell motility both in vitro and in vivo, which to our knowledge is unreported. First, galectin-3-deficient (gal3(-/-)) bone marrow-derived dendritic cells exhibited defective chemotaxis compared to gal3(+/+) cells. Second, cutaneous dendritic cells in gal3(-/-) mice displayed reduced migration to draining lymph nodes upon hapten stimulation compared to gal3(+/+) mice. Moreover, gal3(-/-) mice were impaired in the development of contact hypersensitivity relative to gal3(+/+) mice in response to a hapten, a process in which dendritic cell trafficking to lymph nodes is critical. In addition, defective signaling was detected in gal3(-/-) cells upon chemokine receptor activation. By immunofluorescence microscopy, we observed that galectin-3 is localized in membrane ruffles and lamellipodia in stimulated dendritic cells and macrophages. Furthermore, galectin-3 was enriched in lipid raft domains under these conditions. Finally, we determined that ruffles on gal3(-/-) cells contained structures with lower complexity compared to gal3(+/+) cells. In view of the participation of membrane ruffles in signal transduction and cell motility, we conclude that galectin-3 regulates cell migration by functioning at these structures.

Stability of the recombinant GM-CSF produced by baculovirus gene expression system

Stability of the recombinant GM-CSF produced by baculovirus gene expression system

Effects of Alcohol in the Lung

Alcohol abuse is a systemic disorder and increases the risk of lung injury. After ingestion, alcohol freely diffuses from the bronchial circulation directly through the ciliated epithelium where it vaporizes as it moves into the conducting airways. Some of vaporized alcohol can deposit back into the airway lining fluid and results in repeated exposure of the airway epithelium to high local concentrations of alcohol. Chronic alcohol ingestion is associated with increased renin-angiotensin system, induces oxidative stress through angiotensin (Ang) II stimulated NADPH oxidase expression and activity, and superoxide production. Alcohol metabolism within the lung through the cytochrome P 450 system may also be sufficient to exert significant oxidative stress. Alcohol inhibits the expression of GM-CSF and impairs immune system. Chronic ingestion of ethanol altered cellular functions and viability such as decreased surfactant processing, decreased barrier integrity, increased sensitivity to cytotoxin-induced apoptosis in vitro and in vivo in alveolar type II cells, and decreased phagocytosis of microorganisms in alveolar macrophages. The lung becomes more susceptible when a second hit such as sepsis occurs. Though dietary supplementation with glutathione precursors or selective Ang II type 2 receptor inhibition limits lung injury in animal model, the most attractive candidate for treating the alcoholic lung in the acute setting is recombinant GM-CSF. Whether any of these therapeutic strategies will prove to be effective in decreasing the consequences of alcohol abuse on acute pulmonary diseases is at present unknown. Keywords: Alcohol, alveolar epithelial barrier, glutathione, lung, matrix metalloproteinases, mucocilliary clearance, oxidative stress, rennin-angiotensin system, transforming growth factor-beta, tumor necrosis factor-alpha