Con A-stimulated Spleen Cells Research Articles

Cyclosporine A Loaded Electrospun Poly(D,L-Lactic Acid)/Poly(Ethylene Glycol) Nanofibers: Drug Carriers Utilizable in Local Immunosuppression.

The present study aims to prepare poly(D,L-lactic acid) (PLA) nanofibers loaded by the immunosuppressant cyclosporine A (CsA, 10wt%). Amphiphilic poly(ethylene glycol)s (PEG) additives were used to modify the hydrophobic drug release kinetics. Four types of CsA-loaded PLA nanofibrous carriers varying in the presence and molecular weight (MW) of PEG (6, 20 and 35kDa) were prepared by needleless electrospinning. The samples were extracted for 144h in phosphate buffer saline or tissue culture medium. A newly developed and validated LC-MS/MS method was utilized to quantify the amount of released CsA from the carriers. In vitro cell experiments were used to evaluate biological activity. Nanofibers containing 15wt% of PEG showed improved drug release characteristics; significantly higher release rates were achieved in initial part of experiment (24h). The highest released doses of CsA were obtained from the nanofibers with PEG of the lowest MW (6kDa). In vitro experiments on ConA-stimulated spleen cells revealed the biological activity of the released CsA for the whole study period of 144h and nanofibers containing PEG with the lowest MW exhibited the highest impact (inhibition). The addition of PEG of a particular MW enables to control CsA release from PLA nanofibrous carriers. The biological activity of CsA-loaded PLA nanofibers with PEG persists even after 144h of previous extraction. Prepared materials are promising for local immunosuppression in various medical applications.

Anthelmintic constituents from ginger (Zingiber officinale) against Hymenolepis nana

This study investigated the anthelmintic activity of gingerenone A, [6]-dehydrogingerdione, [4]-shogaol, 5-hydroxy-[6]-gingerol, [6]-shogaol, [6]-gingerol, [10]-shogaol, [10]-gingerol, hexahydrocurcumin, 3R,5S-[6]-gingerdiol and 3S,5S-[6]-gingerdiol, a constituent isolate from the roots of ginger, for the parasite Hymenolepis nana. The cestocidal activity or ability to halt spontaneous parasite movement (oscillation/peristalsis) in H. nana of above constituents was reached from 24 to 72h in a time- and dose-dependent manner, respectively. The [10]-shogaol and [10]-gingero1 have maximum lethal efficacy and loss of spontaneous movement than the others at 24–72h. In addition, worms treated with 1 and 10μM [10]-gingero1, more than 30% had spontaneous movement of oscillation at 72h but [10]-shogaol at 72h only about 15–20% of oscillation. This showing that [10]-gingero1 had less loss of spontaneous movement efficacy than [10]-shogaol. After exposure to 200μM [10]-shogaol, 100% of H. nana had died at 12h rather than died at 24h for [10]-gingerol, showing that [10]-gingero1 had less lethal efficacy than [10]-shogaol. In addition, these constituents of ginger showed effects against peroxyl radical under cestocidal activity. In order to evaluate the cestocidal activity and cytokine production caused by ginger's extract R0 in the H. nana infected mice, we carried out in vivo examination about H. nana infected mice BALB/c mice were inoculated orally with 500 eggs. After post-inoculation, R0 (1g/kg/day) was administered orally for 10 days. The R0 exhibited cestocidal activity in vivo of significantly reduced worms number and cytokines production by in vitro Con A-stimulated spleen cells showed that INF-γ and IL-2 were significantly increases by R0. IL-4, IL-5, IL-6, IL-10 and IL-13 were significantly decreases and Murine KC and IL-12 were not significantly changes by R0. Together, these findings first suggest that these constituents of ginger might be used as cestocidal agents against H. nana.

SIRNA-MEDIATED INHIBITION OF INTERLEUKINE-13 PRODUCTION IN VITRO

Background. According to current views, one of the major mediators involved in the development of allergic process is IL-13. The goal of this work was to design small interfering RNA molecules to effectively inhibit il-13 gene expression of mice in experiments in vitro. Methods. For the expression of IL-13 in in vitro gene coding sequence il-13 was amplified using cDNA ConA-stimulated spleen cells from BALE / c mice as a template and cloned into the expression vector pUCHR IRES GFP. Using a computer analysis were designed six variants of siRNA, directed against mRNA-il-13. To test the efficiency of siRNA a co-transfection of 1x 105 cells HEK293T mixture (0,5 mg and 1 mg of plasmid siRNA) coupled with Lipofectamine 2000 reagent was carried out. Twenty-four hours later, the gene expression changes in il-13 recorded by flow cytometry on the fluorescence intensity of GFP+-cells. Gene expression of il-13 mRNA was assessed by quantitative PCR, and the level of the protein product by ELISA. results. As a result, siRNA molecules were obtained and three of them were able to effectively inhibit the gene expression of il-13. Conclusion. Thereby variants of siRNA, which can effectively inhibit the production of mice’s IL-13 in vitro; can be used later in experiments in vivo so to understand the role of IL-13 in the pathogenesis of allergic conditions as to develop new therapy approaches.

Immunomodulatory Properties of Highly Viscous Polysaccharide Extract from the Gagome Alga (Kjellmaniella crassifolia).

Marine brown algae are rich in sulfated polysaccharides, which have the ability to form gels and viscous solution. Sulfated polysaccharides exhibit many biological activities; however, little is known whether the viscoelastic property in the polysaccharide extract is correlated with biological activities. We examined the immunomodulatory properties of highly viscous polysaccharide extract (HVPE) from Gagome Kjellmaniella crassifolia in a murine model, and the effects were compared with those of a less viscous polysaccharide extract (LVPE). HVPE or LVPE (10, 30, and 100mg/kg/day) were orally administered to C57BL/6 mice for 14days. Secretions of cytokine and IgA in Con A-stimulated spleen and Peyer's patch (PP) cells and phagocytic activity of peritoneal macrophages was determined. IFN-γ, IL-12, IL-6, and IgA secretions showed high levels in spleen cell cultures from mice administered HVPE, whereas these effects were diminished in the LVPE-administered mice. The phagocytic activity of peritoneal macrophages was enhanced by the continuous oral administration of HVPE, and these effects were higher than those of LVPE. Furthermore, an increase in IgA secretion by administration of HVPE was observed in Con A-stimulated PP cells. These results suggest that the polysaccharide extract from K. crassifolia has immunomodulatory activities, which depend on the viscosity.

Effects of anticoccidial and antibiotic growth promoter programs on broiler performance and immune status

This study investigated the effects of various coccidiosis control programs in combination with antibiotic growth promoters (AGPs) on growth performance and host immune responses in broiler chickens. The coccidiosis programs that were investigated included in ovo coccidiosis vaccination (CVAC) with Inovocox or in-feed medication with diclazuril as Clinacox (CLIN) or salinomycin (SAL). The AGPs were virginiamycin or bacitracin methylene disalicylate plus roxarsone. As a negative control, chickens were non-vaccinated and fed with non-supplemented diets (NONE). All animals were exposed to used litter from a commercial broiler farm with confirmed contamination by Eimeria parasites to simulate in-field exposure to avian coccidiosis. Broiler body weights in the CVAC group were greater at 14 and 32days of age, but not at day 42, compared with the NONE, CLIN, and SAL groups. At day 14, the SAL group showed decreased body weight and reduced ConA-stimulated spleen cell proliferation compared with the CLIN and SAL groups. In contrast, at days 34 and 43, splenocyte proliferation was greater in the CVAC and CLIN groups compared with the NONE and SAL groups. Lymphocyte subpopulations and cytokine mRNA expression levels in the intestine and spleen were also altered by the denoted treatments. Collectively, these results suggest that in ovo coccidiosis vaccination or coccidiostat drug medication programs in combination with AGPs influences chicken growth and immune status in an Eimeria-contaminated environment.

Development and characterization of mouse monoclonal antibodies reactive with chicken CD80

This study was carried out to develop and characterize mouse monoclonal antibodies (mAbs) against chicken CD80 (chCD80). A recombinant plasmid containing a chCD80/horse IgG4 fusion gene was constructed and expressed in CHO cells to produce recombinant chCD80/IgG4 protein. Chicken CD80 was purified from the chCD80/IgG4 fusion protein following enterokinase digestion, and used to immunize BALB/c mice, resulting in 158 hybridomas that produced mAbs against chCD80. Three mAbs with high binding specificity for recombinant chCD80/IgG4-transfected CHO cells were identified by flow cytometry, and one of these (#112) was selected for further characterization. Immunoprecipitation of CD80/IgG4-CHO cell extract, or lipopolysaccharide (LPS)-treated monocytes identified 35.0kDa proteins. Immunohistochemical analysis revealed chCD80-expressing cells exclusively in the bursal follicles at the outer portion of the cortex, and throughout the red pulp and the outer boundary of the white pulp in the spleen. By immunofluorescence microscopy, chCD80 was observed on intestinal dendritic cells. LPS treatment of bursa or spleen monocytes for 24 or 48h increased chCD80 expression. Finally, addition of chCD80 mAb to Con A-stimulated spleen cells inhibited the expression of major histocompatibility complex class II antigens and IL-2-driven proliferation of lymphoblast cells. In summary, these chCD80 mAbs will serve as valuable immunological reagents for basic and applied poultry immunology research.

An increase in mouse tumor growth by an in vivo immunomodulating effect of titanium dioxide nanoparticles

Here, we investigated whether titanium dioxide (TiO2) nanoparticles affect in vivo tumor growth through the modulation of mononuclear leukocytes. In vitro lymphocyte proliferation by lipopolysaccharide (LPS) or concanavalin A (ConA) was reduced by < 25 nm TiO2 with a dose-dependent manner. Similarly, TiO2 nanoparticles inhibited nitric oxide (NO) production from bone marrow-derived macrophages obtained from naïve mice. When mice were intraperitoneally (IP) injected with < 25 or < 100 nm TiO2 once a day for 7 days, total cell number of splenocytes was reduced in the spleen of TiO2 nanoparticle-exposed mice. Both CD4+ and CD8+ T-lymphocyte numbers were significantly decreased and B-lymphocyte development was retarded by host exposure to the TiO2 nanoparticles. LPS-stimulated spleen cell proliferation was significantly reduced by host exposure to < 25 or < 100 nm TiO2, but no changes were detected in ConA-stimulated spleen cell proliferation. Further, LPS-stimulated cytokine production by peritoneal macrophages and the percentage of NK1.1+ natural killer cells among splenocytes was reduced by the host exposures to the TiO2 nanoparticles. When mice were IP injected with TiO2 nanoparticles once a day for 28 days prior to the subcutaneous implantation of B16F10 melanoma cells, tumor growth was subsequently significantly increased. Collectively, these results show that TiO2 nanoparticles may damage the development and proliferation of B- and T-lymphocytes, reduce the activity of macrophages, and decrease natural killer (NK) cell population levels, outcomes that appear to lead to an increase in tumor growth in situ. These studies allow us to suggest that TiO2 nanoparticles might have the potential to enhance tumor growth through immunomodulation of B- and T-lymphocytes, macrophages, and NK cells.

T cells cooperate with palmitic acid in induction of beta cell apoptosis

BackgroundDiabetes is characterized by progressive failure of insulin producing beta cells. It is well known that both saturated fatty acids and various products of immune cells can contribute to the reduction of beta cell viability and functionality during diabetes pathogenesis. However, their joint action on beta cells has not been investigated, so far. Therefore, we explored the possibility that leukocytes and saturated fatty acids cooperate in beta cell destruction.ResultsRat pancreatic islets or insulinoma cells (RIN) were co-cultivated with concanavalin A (ConA)-stimulated rat lymph node cells (LNC), or they were treated with cell-free supernatants (Sn) obtained from ConA-stimulated spleen cells or from activated CD3+ cells, in the absence or presence of palmitic acid (PA). ConA-stimulated LNC or Sn and PA cooperated in inducing caspase-3-dependent RIN cell apoptosis. The observed effect of PA and Sn on RIN cell viability was mediated by p38 mitogen-activated protein kinase (MAPK)-signaling and was achieved through auto-destructive nitric oxide (NO) production. The cooperative effect of Sn was mimicked with the combination of interleukin-1β, interleukin-2, interleukin-6, interleukin-17, interferon-γ and tumor necrosis factor-α.ConclusionThese results imply that stimulated T cells produce cytokines that cooperate with saturated free fatty acids in beta cell destruction during diabetes pathogenesis.

The constituents of Anisomeles indica and their anti-inflammatory activities

Ethnopharmacological relevance Anisomeles indica (L.) Kuntze. (Labiatae), popularly known as ‘yu-chen-tsao’, has been traditionally used as anti-inflammatory agent. Aim of the study Investigate the chemical constituents from the whole plants of Anisomeles indica, and evaluate their in vitro anti-inflammatory activities. Results The combined MeOH extract was successively partitioned with CHCl 3 and n-butanol, then submitted to several column chromatographic, and HPLC purification procedures which led to the isolation of one cembrane-type diterpenoid ( 3), two benzenoids ( 4 and 5), five flavonoids ( 1, 2, 6, 7 and 14), and six phenyl propanoids ( 8– 13). The compounds 1– 14 were examined for their inhibitory effects on inflammatory mediator's enhanced production from LPS/IFN-γ-stimulated macrophages. Among these, ovatodiolide ( 3) exhibited potent inhibition on NO, TNF-α and IL-12 enhanced production at a concentration of 5 μM, followed by pedalitin ( 1), scutellarein 7- O-β- d-glucuronide methyl ester ( 6), and acteoside ( 12) at 40 μM ( P < 0.05). Furthermore, 2 μM of 3, and 20 μM of 1 and 6 significantly ( P < 0.05) arrested the cell cycle of Con A-stimulated spleen cells at the G0/G1 stage. Conclusion This is the first report on the presence of compounds 1 and 4– 13 in this plant and of the potent anti-inflammatory activity of 1, 3, 6 and 12 in vitro. These compounds may account for the use of Anisomeles indica in folk medicine to treat inflammation.

Schistosoma mansoni infection reduces severity of collagen-induced arthritis via down-regulation of pro-inflammatory mediators

Various experimental and epidemiological studies have demonstrated that helminth infections affect outcomes of allergic or autoimmune disorders. Here, we examined the effects of Schistosoma mansoni infection on mouse collagen-induced arthritis, one of the most widely used animal models for rheumatoid arthritis. Male DBA/1 mice were infected with S. mansoni 2 weeks prior to being immunized with type II collagen (IIC). Cytokine mRNA expression in mouse paws, cytokine production by ConA-stimulated spleen cells, and anti-IIC antibodies were evaluated in addition to the severity of arthritis. S. mansoni infection significantly reduced the severity of arthritis. Anti-IIC IgG and IgG2a levels were lower in infected than uninfected mice. With regard to cytokine producing potentials in the infected mice, the down-regulation of Th1 (IFNγ) and pro-inflammatory cytokines (TNFα and IL-17A), and up-regulation of Th2 (IL-4) and an anti-inflammatory cytokine (IL-10) were observed. In addition, real-time PCR revealed that the augmentation of pro-inflammatory mediators such as IL-1β, IL-6 and receptor activator of NFκB in inflamed paws was abrogated by S. mansoni infection. In conclusion, schistosome infection reduced the severity of autoimmune arthritis via systemic and local suppression of pro-inflammatory mediators, suggesting the potential of parasite-derived materials as therapeutic agents against rheumatoid arthritis.

Short-Term Oral Administration of Ginseng Extract Induces Type-1 Cytokine Production

Ginseng radix (Panax ginseng C.A. Meyer) is a popular herbal medicine used as a major ingredient in tonic recipes in eastern Asian countries. In our study, male BALB/c mice were treated orally with various doses of ginseng root extract for 5 consecutive days. The extract reduced the serum level of IgG but elevated the level of IgA. Under in vitro condition, the lipopolysaccharide-stimulated spleen cells from the ginseng-treated mice also showed a significant decrease in IgG production but an increase in IgA production. The serum level and production of IgM was unaffected. The interleukin-2, interferon-γ (Th1-type cytokines), and interleukin-10 (Tr1-type cytokine) production by Con A-stimulated spleen cells from the ginseng-treated mice showed an upregulation relative to the control group. However, the production of interleukin-4 (Th2-type cytokine) showed no significant change. The activity of natural killer cells was increased in the ginseng group, but the percentages of T-lymphocytes (CD3+) and CD4+8−, CD4−8+ subset were reduced. Thus, short-term oral administration of ginseng extract appears to enhance Th1-type cytokine production.

Serglycin-deficient Cytotoxic T Lymphocytes Display Defective Secretory Granule Maturation and Granzyme B Storage

Cytotoxic T lymphocytes eliminate infected and tumor cells mainly by perforin/granzyme-induced apoptosis. Earlier studies suggested that serglycin-proteoglycans form macromolecular complexes with granzymes and perforin in the cytotoxic granule. Serglycin-proteoglycans may also be involved in the delivery of the cytolytic machinery into target cells. We have developed a serglycin-deficient mouse strain, and here we studied the importance of serglycin-proteoglycans for various aspects of cytotoxic T lymphocyte function. 35SO4(2-) radiolabeling of serglycin-deficient cells demonstrated a dramatic reduction of incorporated label as compared with wild type cells, indicating that serglycin is by far the dominating proteoglycan species produced by the cytotoxic T lymphocyte. Moreover, lack of serglycin resulted in impaired ability of cytotoxic T lymphocytes to produce secretory granule of high electron density, although granule of lower electron density were produced both in wild type and serglycin-deficient cells. The serglycin deficiency did not affect the mRNA expression for granzyme A, granzyme B, or perforin. However, the storage of granzyme B, but not granzyme A, Fas ligand, or perforin, was severely defective in serglycin-deficient cells. Serglycin-deficient cells did not display defects in late cytotoxicity toward target cell lines. Taken together, these results point to a key role for serglycin in the storage of granzyme B and for secretory granule maturation but argue against a major role for serglycin in the apoptosis mediated by cytotoxic T lymphocytes.

Cell Cycle Regulators Modulating Con A Mitogenesis and Apoptosis in Low-Dose Radiation-Exposed Mice

Low doses of ionizing radiation (LDR) are reported to induce transient changes in mouse lymphocytes, such as enhanced response to polyclonal T cell mitogens, increased expression of heat shock proteins, and p53. We evaluated the role of cell cycle proteins and apoptosis in lymphocytes of C57BL/6 mice exposed to 20 cGy fractionated LDR. We found an enhanced cell proliferation in response to the mitogen concanavalin A (con A). The expression of several cell cycle and apoptosis-related intracellular and extracellular proteins was analyzed by flowcytometry following labeling with specific antibodies. An increased response to con A was accompanied by an increase in the expression of proliferating cell nuclear antigen (PCNA) and cyclins D and A. The expression of cyclin B did not change significantly. In 20 cGy-exposed C57BL/6 mice, the caspase activity and apoptosis were reduced in con A-stimulated spleen cells as compared to sham controls. The expression of Fas and Fas ligand was analyzed by labeling with specific antibodies followed by flowcytometry. There was no change in the expression of Fas and Fas ligand. The change in the mitochondrial transmembrane potential was followed by labeling the cells with the dye 5,5',6,6',-tetrachloro-1,1,3,3-tetraethylbenzimidazolcarbocyanine iodide (JC-I) and analyzing by flowcytometry. Mitochondrial stability was increased in spleen cells of LDR-treated mice. These data suggest that LDR induces augmentation of mitogenic response by modulation of expression of cyclins and the mitochondrial membrane potential leading to reduced apoptosis.

Oral administration of royal jelly inhibits the development of atopic dermatitis-like skin lesions in NC/Nga mice

We have shown previously that in addition to IL-4, IL-5 and IL-10, antigen-specific interferon-γ (IFN-γ) production by spleen cells from ovalbumin (OVA)/Alum-immunized mice is inhibited by the administration of royal jelly (RJ). Since it has been shown that both Th1 and Th2 cytokines play pathogenic roles in the generation of atopic dermatitis (AD), we have examined whether RJ suppresses the development of AD-like skin lesions in NC/Nga mice induced by repeated application of picryl chloride (PiCl) under specific pathogen-free (SPF) conditions. Oral administration of RJ to the PiCl-treated NC/Nga mice inhibited the development of AD-like skin lesions in these mice as exemplified by the significant decrease in the total skin severity scores and the decrease in hypertrophy, hyperkeratosis, and infiltration of the epidermis and corium by inflammatory cells. IFN-γ production by spleen cells from PiCl-treated NC/Nga mice in response to TNP–KLH was partially but significantly inhibited by the oral administration of RJ, while IFN-γ production by Con A-stimulated spleen cells was not affected. Since inducible nitric oxide (NO) synthase (iNOS)-derived NO has been suggested as an important immunoregulatory mediator in inflammatory autoimmune diseases, we have also examined the expression of iNOS in the dorsal skin lesions of PiCl-treated NC/Nga mice. Interestingly, the expression of iNOS was significantly increased in the skin lesions of RJ-administered mice compared with those of control PBS-administered mice. Thus, our results suggest that RJ suppresses the development of AD-like skin lesions in PiCl-treated NC/Nga mice, possibly by a combination of down-regulating TNP-specific IFN-γ production and up-regulating iNOS expression.

Effects of boar seminal immunosuppressive fraction on production of cytokines by Concanavalin A-stimulated spleen cells and on proliferation of B lymphoma cell lines.

The immunosuppressive fraction (ISF) of boar seminal vesicle fluid has recently been demonstrated to inhibit mitogen-stimulated proliferation of lymphocytes and antibody response to corpuscular and soluble antigens. The effects of ISF on in vitro and in vivo production of cytokines as well as its possible inhibitory effect on proliferation of B lymphoma cells remain to be elucidated. The effect of ISF on proliferation of normal mouse spleen cells stimulated by Concanavalin A (Con A) and on mouse B lymphoma cells was measured by 3H-thymidine incorporation. Cytokines were determined in the supernatants of mouse spleen cells stimulated with Con A in the presence or absence of ISF by enzyme-linked immunosorbent assay (ELISA). In vivo cytokine production in the sera samples of mice treated with ISF and immunized with keyhole limpet hemocyanin (KLH) was followed by ELISA, too. We confirmed the inhibitory effect of ISF on Con A-stimulated lymphocyte proliferation. ISF affected cytokine production in the Con A-stimulated spleen cells: production of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) was lowered, but production of IL-4, IL-6, and IL-10 was enhanced. Similarly, in the sera samples of mice immunized with keyhole limpet hemocyanin (KLH), IL-2 and IFN-gamma levels were decreased by ISF. ISF inhibited proliferation of Ag 8 and X 63-IL-2 B lymphoma cells as well. ISF inhibited production of T helper1 (Th1) cytokines (IL-2 and IFN-gamma) and enhanced production of Th2 cytokines (IL-4, IL-6, and IL-10). ISF seems to shift the Th1/Th2 pattern in favor of Th2. ISF exhibited an antiproliferative activity on mouse B lymphoma cells.

Pathogenic and immunosuppressive effects of avian pneumovirus in turkeys.

Avian pneumovirus (APV) causes a respiratory disease in turkeys. The virus has been associated with morbidity and mortality due to secondary infections. Our objective was to determine if APV caused immunosuppression in the T-cell or B-cell compartments and to study the pathogenesis of the disease in APV maternal antibody-lacking 2-wk-old commercial turkeys. APV was administered by the eyedrop/intranasal route. Observations were made for gross lesions, viral genome, and T-cell mitogenesis and cytokine secretion at 3, 5, 7, 14, and 21 days postinoculation (DPI). During the acute phase of the disease that lasted for about 1 wk, the turkeys exposed to APV showed clinical signs characterized by nasal discharge and sinus swelling. Virus genome was detected by in situ hybridization in cells of turbinates and trachea at 3 and 5 DPI. At 3 and 5 DPI, spleen cells of the birds infected with APV markedly decreased proliferative response to concanavalin A (Con A). Con A and lipopolysaccharide stimulation of spleen cells from virus-exposed turkeys resulted in accumulation of nitric oxide-inducing factors (NOIF) in the culture fluid. NOIF were not detected in culture fluids of Con A-stimulated spleen cells of virus-free turkeys. APV did not compromise the antibody-producing ability of turkeys against several extraneous antigens such as Brucella abortus and tetanus toxoid.

Effects of granulocyte-macrophage colony stimulating factor (GM-CSF) in vivo on cytokine production and proliferation by spleen cells.

GM-CSF is a potent stimulator of haematopoietic cells as well as some functions of granulocytes and macrophages. GM-CSF has many clinical uses; however, little is known about the effects of GM-CSF treatment in vivo on the responses of tissue lymphocytes in terms of secretion of Th-1 and Th-2 cytokines. We investigated this issue by measuring the responses of spleen cells from mice 24 h after treatment i.p. with saline or rmGM-CSF. GM-CSF at 16.7-50.0 microg/kg significantly increased (P < 0.01) spleen cellularity 2-2.5-fold and enhanced proliferative responses of non-stimulated (no mitogen) as well as concanavalin A (Con A)-stimulated spleen cells. Secretion of IFN-gamma by Con A (2.5 microg/ml)-stimulated spleen cells was significantly (P < 0.01) increased from 1.8 microg/ml by control spleen cells to 5.2 microg/ml by GM-CSF spleen cells. IL-10 production was greater (0.25 microg/ml, P < 0.05) by Con A-stimulated spleen cells from GM-CSF-treated mice compared to control spleen cells (0.06 microg/ml). By contrast, there were no significant differences in IL-4 production by Con A-stimulated spleen cells from the different groups. These results show that GM-CSF treatment increases spleen cellularity and primes lymphocytes for enhanced responses. The enhanced production of Th-1 cytokines by primed lymphocytes may partially explain the beneficial role of in vivo administration of GM-CSF in several clinical situations.

A role for parasite-induced PGE2 in IL-10-mediated host immunoregulation by skin stage schistosomula of Schistosoma mansoni.

Significant quantities of PGE(2) were produced by cercariae of Schistosoma mansoni following incubation with linoleic acid, a free fatty acid found on the surface of the skin. Cyclooxygenase (COX) 2 inhibitors failed to block this PGE(2) production, suggesting that a different biochemical pathway may be involved in the production of PGE(2) by the parasite. In addition, the parasites were also able to induce PGE(2) and IL-10 from human and mouse keratinocytes. Analysis of mouse skin during skin migratory phases of infection confirmed these in vitro observations. COX2 inhibitors blocked the parasite-induced PGE(2) and IL-10 from keratinocytes. Further analysis of the parasite secretions showed that the PGE(2)/IL-10-inducing effect was associated with a fraction <30 kDa molecular size. Addition of this fraction or parasite-stimulated keratinocyte culture supernatant to Con A-stimulated spleen cells resulted in the suppression of cell proliferation. This effect could be blocked by anti-IL-10 treatment. In sharp contrast, attenuation of the parasites with gamma-irradiation significantly abrogated their ability to induce PGE(2) or IL-10 from skin cells. Significance of IL-10 in host immunoregulation by skin stage schistosomula of S. mansoni was further confirmed by using IL-10-deficient mice. In these mice the normal subdued cutaneous reaction to the parasite was absent. Instead, a prominent cellular reaction occurred around the parasite, and there was considerable delay in parasitic migration through the skin. Thus these results suggest a key role for parasite-induced PGE(2) in IL-10-dependent down-regulation of host immune responses in the skin.

Hydrophobic extracts of a Chinese herb (CMX-13) exhibit potent immunosuppressive properties and prevent acute rejection in a highly histoincompatible model of rat lung transplantation.

The potential of higher plants as sources for new immunosuppressive medications is well recognized. In our experiments we investigated the immunosuppressive effect of a highly refined and potent extract of a Chinese herbal preparation, CMX-13, on inhibiting acute allograft rejection (AR) in a highly histoincompatible rat lung transplant model, BN-->LEW, and on lymphocyte activation and cytokine gene expression in vitro. Left lung transplants: the control group (group 1) received only dimethylsulfoxide (DMSO) which is the solvent for CMX-13. Group 2 received intramuscular cyclosporin A (CsA, 25 mg/kg) on day 2 posttransplant. Group 3 and 4 received i.p. CMX-13 (0.5 mg/day, low dose and 5 mg/day, high dose, respectively) on day 1, 2, and 3 posttransplant. All animals were killed on day 6 posttransplant. Several pathological categories of inflammation were examined. In vitro experiments: rat spleen cells were incubated with Con A or irradiated stimulator cells with/without serial dilutions of CMX-13 or CsA. Cell proliferation was measured by 3H-thymidine incorporation. mRNA expression of interleukin-2 and interferon-gamma was examined by reverse transcriptase-polymerase chain reaction. The severity of AR in animals receiving high dose CMX-13 was significantly reduced (stage II, P<0.05) compared with controls (stage IV). Significant differences were also seen when more specific parameters of inflammation were examined (necrosis, 0 vs. 1.7+/-1.0, P<0.05; interalveolar hemorrhage, 0 vs. 3.0+/-0.9, P<0.05). The responses seen in the animals treated with high dose CMX-13 were similar to those in the CsA group. CMX-13 inhibited T cell proliferative responses induced by Con A and alloantigen stimulation in a dose-dependent manner that were similar to CsA. Interleukin-2, and interferon-gamma mRNA expression in Con A-stimulated spleen cells was not inhibited by CMX-13 although CsA showed significant inhibition. 1) CMX-13 significantly reduces the stage of AR and parameters of inflammation in a highly histoincompatible rat lung transplant model. 2) CMX-13 has equal potency to CsA in the inhibition of Con A and alloantigen stimulated rat spleen cell proliferation. 3) CMX-13 showed no inhibitory effects on IL-2 and gamma-IFN mRNA expression, suggesting that its mechanism of action is different from CsA. 4) CMX-13 or derivatives may have potential utility as an immunosuppressive agent(s) in modulation of AR and management of other inflammatory and immunological disorders.

IL-18 prevents the development of chronic graft-versus-host disease in mice.

The development of chronic graft-versus-host disease (GVHD), which is induced by the transfer of DBA/2 spleen cells into (C57BL/6 x DBA/2)F1 (BDF1) mice, is closely related to diminished donor anti-host CTL activity and host B cell hyperactivation. Therefore, an approach which activates donor CD8+ T cells or suppresses donor CD4+ T cell-host B cell interaction may have clinical utility in the treatment of chronic GVHD. We have previously demonstrated that IL-18 induces the development of naive CD8+ T cells into type I effector cells in DBA/2 anti-BDF1 MLC. In this paper we examined the effect of IL-18 administration on the development of chronic GVHD in mice. The treatment was started before or after the onset of clinical evidence of the disease. Regardless of the treatment schedule, IL-18 significantly decreased immunological parameters indicative of chronic GVHD, such as elevated serum IgG antinuclear Abs, IgG1, and IgE levels, and host B cell numbers and their activation. Importantly, IL-18-treated mice did not show the same acute GVHD-like symptoms reported for IL-12 treatment, because there was no weight loss, death, or severe immunodeficiency as indicated by a decrease in IL-2 and IFN-gamma production by Con A-stimulated spleen cells. In contrast, IL-18 treatment partially but significantly restored the production of these cytokines. Data further suggested that these IL-18-mediated therapeutic effects may be due to the induction of donor CD8+ CTL, the decrease in donor CD4+ T cell numbers, and a down-regulation of host B cell MHC class II expression. Thus, our results suggest that IL-18 has beneficial effects in the prevention and treatment of chronic GVHD.