Proliferation Of Spleen Cells Research Articles

Serum-derived MHC class II molecules: Potent regulators of the cellular and humoral immune response

Soluble MHC antigens are detected in body fluids but their role and origin are still unclear. This study examined whether serum IA antigens, isolated from BALB/c mice (sIA d), could modulate the immune response. Specific purification procedures isolated intact IA molecules, which were thereafter applied to functional assays. Thus, sIA d were shown to stimulate spleen cell proliferation and the major target was identified to be the CD4 + cell population. Inhibition of the CD4 co-receptor using specific neutralizing antibodies destroyed the sIA d-mediated proliferative activity, while sIA d successfully antagonized surface IA d antigens for binding to anti-IA d antibody. Serum-IA d stimulated BALB/c versus C3H/HeN but not C3H/HeN versus BALB/c mixed lymphocyte reactions, while increasing responsiveness to Legionella pneumophila. However, sIA d displayed an inhibitory activity during the effector phase of the humoral response, since they inhibited the anti-DNP-specific IgM production to a DNP–HSA hapten–carrier system. Furthermore, sIA d molecules increased Th1/Th2 cytokines during the L. pneumophila stimulus, while decreasing IL-2, GM-CSF and increasing IL-4, IL-15 during the DNP–HSA stimulus. These results suggest that sIA d, following steps similar to surface class II antigen binding mechanisms, stimulate the initiation of a humoral or cellular immune response but rather inhibit the effector phase of the reactions, attributing thus soluble class II MHC antigens important immunomodulatory roles.

Mycobacterial di-O-acyl trehalose inhibits Th-1 cytokine gene expression in murine cells by down-modulation of MAPK signaling

Protection against tuberculosis (TB) is based on cell-mediated immune responses. TB is often characterized by immunological dysfunction of peripheral blood mononuclear cells, especially at chronic stages. Lipids from the Mycobacterium tuberculosis cell wall have been shown to produce various suppressive effects on cell-mediated immunity. The cell-surface lipid di-O-acyl-trehalose (DAT) is able to inhibit T-cell proliferation and cytokine secretion in cells from naïve mice. In the present study, we addressed the mechanisms involved in the suppressive effect caused by DAT. We found that DAT decreased the proliferation of spleen cells induced with PMA-ionomycin, suggesting that the suppressive mechanisms target intracellular functions just after phospholipase C-gamma activation. Addressing this possibility, the effect of DAT was found to involve down-modulation of the di-acyl glycerol-dependent activation of the MAPK-ERK1/2 pathway, one of the crucial signaling pathways leading to adaptive cell immune response against TB. Moreover, the inhibitory effect of DAT on proliferation was reproduced in antigen-stimulated T cells from M. tuberculosis-infected mice, involving the lowering of Th1-type cytokine transcription levels. The present findings thus reveal a new kind of bioactivity for a long-known M. tuberculosis cell wall lipid, DAT.

Immunomodulation by blockade of the TRANCE co-stimulatory pathway in murine allogeneic islet transplantation

We explore herein the effect of TNF-related activation-induced cytokine (TRANCE) co-stimulatory pathway blockade on islet survival after allograft transplantation. Expression of TRANCE on murine C57Bl/6 (B6) CD4+ T cells after allogeneic activation was analyzed by fluorescence-activated cell sorter (FACS). The effect of a TRANCE receptor fusion protein (TR-Fc) and anti-CD154 antibody (MR1) on B6 spleen cell proliferation after allogeneic activation was assessed by mixed lymphocyte reaction (MLR). Three groups of B6 mice were transplanted with allogeneic islets (DBA2): Control; short-term TR-Fc-treatment (days 0-4); and prolonged TR-Fc-treatment (days -1 to 13). Donor-specific transfusion (DST) was performed at the time of islet transplantation in one independent experiment. Transplantectomy samples were analyzed by immunohistochemistry. TRANCE expression was upregulated in stimulated CD4+ T cells in vitro. In MLR experiments, TR-Fc and MR1 both reduced spleen cell proliferation, but less than the combination of both molecules. Short-course TR-Fc treatment did not prolong islet graft survival when compared with controls (10.6 +/- 1.9 vs. 10.7 +/- 1.5 days) in contrast to prolonged treatment (20.7 +/- 3.2 days; P < 0.05). After DST, primary non function (PNF) was observed in half of control mice, but never in TR-Fc-treated mice. Immunofluorescence staining for Mac-1 showed a clear decrease in macrophage recruitment in the treated groups. TRANCE-targeting may be an effective strategy for the prolongation of allogeneic islet graft survival, thanks to its inhibitory effects on co-stimulatory signals and macrophage recruitment.

Serine protease activity of Per a 10 augments allergen‐induced airway inflammation in a mouse model

We recently reported an immunodominant serine protease allergen (Per a 10) from Periplaneta americana. This study investigates the role of its proteolytic activity in driving the immune responses towards self and other allergens. Groups of Balb/c mice were sensitized intraperitoneally and subcutaneously with proteolytically active Per a 10 or inactivated Per a 10 (using aminoethyl benzenesulfonyl fluoride hydrochloride) or whole body P. americana extract and subsequently challenged intranasally with the respective antigens. Mice were also sensitized and challenged with ovalbumin (OVA) alone or co-administered with active or inactive Per a 10. The immune-inflammatory responses were measured by airway hyperresponsiveness (AHR) and cellular infiltration of lungs i.e. eosinophil counts, eosinophil peroxidase (EPO) activity, myeloperoxidase activity (MPO), lung histopathology, serum levels of specific-antibodies and levels of Th1/Th2 interleukins in bronchoalveolar lavage fluid (BALF) and in spleen cells culture supernatant. Mice challenged with active Per a 10/P. americana extract showed a significant airway inflammation demonstrated by enhanced AHR and increased cellular infiltration of lungs as evidenced by high eosinophil counts, EPO activity, IL-4 and IL-5 in BALF. Active Per a 10 also induced a significant proliferation of spleen cells, increased secretion of IL-4 and IL-5 in the spleen cells culture supernatant and systemic production of specific-IgE and IgG1. However, exposure with inactive Per a 10 elicited a low cellular infiltration and systemic antibody production. Exposure to OVA with active Per a 10 demonstrated a significantly high cellular infiltration and production of OVA-specific IgE and IgG1, than exposure to OVA alone or with inactive Per a 10. Proteolytic activity of Per a 10 plays an important role in driving the allergic immune response by providing an adjuvant effect, towards self and other potential allergens present in the same microenvironment.

Cytokine Imbalance after Measles Virus Infection Has No Correlation with Immune Suppression

Measles virus infection leads to immune suppression. A potential mechanism is the reduction of interleukin 12 (IL-12) secretion during acute measles, resulting in a TH2 response. Studies in humans have reported conflicting results, detecting either a TH2 or a TH1 response. We have investigated the correlation between a TH2 response and immune suppression in specific-pathogen-free inbred cotton rats which were infected with measles vaccine and wild-type viruses. After infection of bone marrow-derived macrophages with wild-type virus, IL-12 secretion was reduced in contrast to the level for vaccine virus infection. In bronchoalveolar lavage cells, IL-12 secretion was suppressed after infection with both wild-type and vaccine virus on days 2, 4, and 6 and was detectable on days 8 and 10. After stimulation of mediastinal lymph node and spleen cells with UV-inactivated measles virus at various time points after infection, gamma interferon but no IL-4 was found. After stimulation with phorbol myristate acetate-ionomycin, high gamma interferon and low IL-4 levels were detected. To investigate whether the secretion of IL-4 contributes to immune suppression, a recombinant vaccine virus was created which secretes cotton rat IL-4. After infection with this recombinant virus, IL-4 secretion was enhanced. However, neither inhibition of concanavalin A-stimulated spleen cells nor keyhole limpet hemocyanin-specific proliferation of spleen cells was altered after infection with the recombinant virus in comparison to the levels with the parental virus. Our data indicate that measles virus infection leads to a decrease in IL-12 secretion and an increase in IL-4 secretion, but this does not seem to correlate with immune suppression.

Isolation, purification and immunobiological activity of a new water-soluble bee pollen polysaccharide from Crataegus pinnatifida Bge.

A novel water-soluble polysaccharide was obtained from bee pollen of Crataegus pinnatifida Bge. Two fractions of this polysaccharide, CPP-1 and CPP-2, were first extracted by hot-water and purified. The average molecular weight of CPP-1 and CPP-2 were approximately 3.7 × 10 5 Da and 7.8 × 10 4 Da, and their chemical structures were studied by gas chromatography (GC), Fourier transform-infrared (FT-IR) spectroscopy, methylation analysis. We evaluated the effects of CPP-1 and CPP-2 on the basis of phagocytosis of macrophage assay, natural killer cells cytotoxicity assay and spleen lymphocyte proliferation assay. The results showed CPP-1 and CPP-2 significantly induced phagocytic rates and phagocytic indexes by peritoneal macrophages. Moreover, these two fractions caused a significant stimulation of rat spleen cell proliferation. At 50 μg/mL, CPP-2 activated NK cells more significantly than CPP-1. These findings suggest that they should be explored as a novel potential immunostimulants.

Yam Storage Protein Dioscorins from Dioscorea alata and Dioscorea japonica Exhibit Distinct Immunomodulatory Activities in Mice

The aim of this study was to elucidate the effect of the major storage protein dioscorin isolated from two different yam species, Tainong No. 1 (TN1-dioscorins) and Japanese yam (Dj-dioscorins), on the immune activities of mice. Dj-dioscorins, like TN1-dioscorins, could induce expression of the pro-inflammatory cytokines and stimulate phagocytosis of RAW 264.7. Intraperitoneal injection of the TN1-dioscorins into mice stimulated phagocytosis of bone marrow, spleen, and thymic cells. In contrast, the T and B cells in bone marrow, spleen, and thymus isolated from mice injected with Dj-dioscorins had higher proliferative responses to mitogens. Furthermore, Dj-dioscorins enhanced proliferation of CD4(+), CD8(+), and Tim3(+) (Th1) cells in spleen and CD19(+) cells in both spleen and thymus. Supplement of Dj-dioscorins in the lymphoid cells isolated from Dj-dioscorins primed mice induced cell proliferation of both spleen and thymic cells. These findings indicated that TN1-dioscorins have a higher ability to stimulate the phagocytic activity of the lymphoid cells than Dj-dioscorins, whereas Dj-dioscorins possess more abilities than TN1-dioscorins to enhance the proliferation of the lymphoid cells.

Neonatal Tolerization with Bacterial Antigens Stimulates an Expansion of CD11b+ Leukocytes with Inhibitory Activity in IL-10 Gene-Deficient Mice (39.38)

Abstract Tolerance to antigens of the endogenous flora can be induced by intra-peritoneal injection of 100 microliter of a sterile lysate solution (2mg/ml protein) prepared from fecal material of conventionally-raised mice with enteric micro flora into neonates. In IL-10 gene-deficient mice this treatment results in delayed onset of intestinal inflammation and reduced levels of pro-inflammatory cytokine release with a concomitant increase in CD11b+ leukocytes in spleen. To investigate whether these CD11b+ leukocytes play an active role in the reduction of bacteria-induced immune responses we isolated CD11b+ leukocytes from spleens of 20 week old mice that were tolerized to bacterial antigens as neonates and added these cells in limited dilutions to spleen cell cultures stimulated with bacterial antigens. We found that in bacterial antigen-tolerized, Il-10 gene-deficient mice the amount of CD11b+ leukocytes in spleen doubled compared to the amount found in untreated mice and mice injected with germ-free lysates. Sorted CD11b+ spleen cells from tolerized mice were able to inhibit antigen-stimulated spleen cell proliferation and release of pro-inflammatory cytokines in a concentration-dependent manner suggesting that CD11b+ cells play a regulatory role in the response to bacterial antigens in this IBD model. This work is supported by the Crohn's and Colitis Foundation of Canada.

Taenia crassiceps infection and its antigens abrogate Experimental Autoimmune Encephalomyelitis, a role for alternatively activated macrophages (AAMφ) (99.51)

Abstract Helminth infections induce strong immunoregulation and may modulate subsequent pathogenic challenges. Taenia crassiceps causes chronic infections, induces Th2-biased responses and recruits AAMΦ with suppressive ability. To test the influence of this cestode on autoimmunity, we infected C57BL/6 mice with 40 T. crassiceps metacestodes, 8 weeks later Experimental Autoimmune Encephalomyelitis (EAE) was induced with MOG35-55 on infected and uninfected mice. It was found that T. crassiceps-infected mice delayed the onset of EAE; only 50% of them displayed symptoms and those were significantly less severe than uninfected mice. They also had decreased MOG-specific spleen cell proliferation, low IL-17 production and low leukocyte infiltration in the spinal cord. Levels of TNFα in sera and transcripts for TNFα and iNOS in CNS were lower in previously infected mice. AAMΦ markers Ym1, Relm-α and Arg1 were over-expressed in CNS of EAE T. crassiceps-bearing mice, whereas T-bet was expressed in EAE mice. Injection of 50μg of soluble T. crassiceps Ag at the onset of EAE abrogated the symptoms. Infection and Ag induced AAMΦ expressing PDL1 &amp; PDL2 which suppressed anti-MOG specific cell proliferation; such effects were inhibited in mice receiving chlodronate-loaded liposomes. Thus, T. crassiceps' immune-regulation decreases EAE severity by dampening T cell proliferation and migration to CNS with a key role for AAMΦ.

Immune-enhancing activities of low molecular weight β-glucan depolymerized by gamma irradiation

β-glucans are structural cell wall polymers of many microorganisms and cereals which possess immunomodulatory properties and have been used in the food, cosmetic and medical industry. In our previous study, β-glucan was depolymerized by gamma irradiation and leads to improve the solubility and viscosity. This study was carried out to evaluate the functional properties, mainly immune-enhancing activities of low molecular weight β-glucan fragmented by gamma irradiation. The results showed that RAW 264.7 macrophage cell stimulation activities of irradiated β-glucan were higher than that of non-irradiated β-glucan. In addition, the oral administration of gamma-irradiated β-glucan significantly increased the proliferation and cytokine (IFN-γ and IL-2) release of spleen and Peyer's patch cells compared with non-irradiated β-glucan. In conclusion, gamma irradiation could be used as an effective method for the production of depolymerized β-glucan improved functional property such as immunomodulatory activity.

게르마늄을 함유한 상추의 단일 경구투여 독성 검사

유기게르마늄과 무기게르마늄을 첨가하여 양액 재배한 상추의 안전성을 평가하기 위하여 게르마늄 함유 상추의 생쥐(C57BL/6)에 대한 단회 경구 독성시험을 수행하였다. 실험전 기간 동안 사망한 실험동물은 없었으며, 일반증상과 임상증상도 관찰되지 않았고, 부검 결과 어떠한 육안적 병변도 관찰되지 않았다. 그리고 대조군과 시료 투여군 간의 체중 차이도 관찰되지 않았다. 암컷의 사료 소비량과 물 섭취량은 수컷에 비해 약간 적은 것으로 나타났다. 암컷의 경우에 대조군에 비해 각 실험군의 장기 무게가 모두 약간 감소하는 경우(흉선, 오른쪽 난소)와 악간 증가하는 경우(왼쪽 난소)가 있었고, 수컷의 경우에도 흉선, 심장, 오른쪽 신장, 왼쪽 부고환의 무게가 대조군에 비해 모든 실험군에서 약간 감소하였지만 유의한 차이는 아니었고, 왼쪽 신장과 좌우 고환의 무게가 약간 증가하였지만 역시 유의한 차이는 아니었다. 암수 간의 몸무게에 대한 상대적인 장기 무게의 차이는 모두 정상적인 범위내에 속하였다. 혈액 생화학적 검사 결과, 수컷의 경우에는 GPT와 GOT 측정값이 대조군에 비해 <TEX>$Ge_{132}$</TEX>를 함유한 상추를 먹인 실험군에서 유의적으로 감소하였고, LDH 측정값이 대조군에 비해 실험군에서 각각 유의적으로 감소하였다. 혈액학적 검사에서는 수컷의 혈소판(PLT) 수치가 대조군에 비해 각 실험군이 증가한 것으로 나타났지만 유의적인 변화는 아니었다. LPS와 Con A 자극에 대해 비장세포의 증식반응과, 비장세포 내 B세포, 보조 T세포, 세포독성 T세포의 비율은 유의한 파이가 없었다. 이상의 실험 결과로 <TEX>$$GeO_2</TEX>와 <TEX>$Ge_{132}$</TEX> (60 mg/kg)출 함유한 상추 분말을 식약청 고시에 명시된 최고농도인 2,000 mg/kg을 단회 결구 투여하였을 때, 시료와 관련된 특기할만한 독성증상이 관찰되지 않았다. Single-dose toxicity test of germanium-fortified lettuce was investigated in mice. Both sexes of C57BL/6 mice were orally administered once at a dose of 2,000 mg/kg. No death, clinical signs and pathological findings related to the treatment were observed. In addition, no significant changes in feed consumption and body weight gain were obtained during the treatment period, in spite of day-to-day fluctuation of water consumption. There were no considerable changes in hematology and serum biochemistry, except a significant decrease in GPT, GOT and LDH. Several alterations were observed in organ weight and blood biochemistry, including thymus, ovaries, heart, kidney and platelet in male or female mice. The ability of spleen cells proliferation was almost same level as shown in control group. However the population of B cells, helper T cells and cytotoxic T cells was not comparably changed in all groups. Taken together, it is suggested that single oral dose of germanium-fortified lettuce to C57BU6 mice did not cause apparent toxicological change at the dose of 2,000 mg/kg body weight.

Effect of immunological adjuvants: GM-CSF (granulocyte-monocyte colony stimulating factor) and IL-23 (interleukin-23) on immune responses generated against hepatitis C virus core DNA vaccine

The use of cytokines as adjuvants has been shown to be a promising approach for enhancing DNA vaccine induced-immune responses. In this report, we investigate the administration of cytokines to modulate both humoral and cell-mediated immune responses elicited by an HCV-core plasmid DNA vaccine in Balb/c mice. Our studies indicate that the HCV-core DNA vaccine has been able to induce both antibody and cellular immunity in a DNA prime-protein boost regimen. GM-CSF (granulocyte-monocyte colony stimulating factor) which is considered to be a cytokine displaying both Th1 and Th2 characteristics, and plays an important role in augmenting antibody and cell-mediated immunity was also administered. The induction of cellular immunity was not as striking as humoral immunity in this case. To obtain a stronger cellular response, IL-23, a Th1 cytokine belonging to the IL-12 family, was also included in the regimen. Spleen cell proliferation, IFN-γ production from spleen cells and specific serum IgG2a, all demonstrate the enhancement of cell-mediated immunity without any observable suppressive effect on antibody and humoral immune responses. We also examined the timing of plasmid IL-23 administration on the phenotype of the resultant T cell responses in a 3 day interval, before and after plasmid GM-CSF administration. The results did not indicate any change in theTh1/Th2 balance as compared with simultaneous IL-23 administration.

Cancer Metastasis Is Accelerated through Immunosuppression during Snail-Induced EMT of Cancer Cells

Epithelial-mesenchymal transition (EMT) is a key step toward cancer metastasis, and Snail is a major transcription factor governing EMT. Here, we demonstrate that Snail-induced EMT accelerates cancer metastasis through not only enhanced invasion but also induction of immunosuppression. Murine and human melanoma cells with typical EMT features after snail transduction induced regulatory T cells and impaired dendritic cells in vitro and in vivo partly through TSP1 production. Although Snail(+) melanoma did not respond to immunotherapy, intratumoral injection with snail-specific siRNA or anti-TSP1 monoclonal antibody significantly inhibited tumor growth and metastasis following increase of tumor-specific tumor-infiltrating lymphocytes and systemic immune responses. These results suggest that inhibition of Snail-induced EMT could simultaneously suppress both tumor metastasis and immunosuppression in cancer patients.

Effects of 5-Aza-dcR and TSA on methylation of transcription regulation of Syk gene in colorectal cancer cell line

Objective To explore the effects of5-Aza-2-deoxyeytidine (5-Aza-dcR) and Tricho-statin A (TSA) on Spleen tyrosine kinase (Syk) gene expression, cell proliferation and infiltration in colo-rectal cancer cell line HCT-15. Methods The HCT-15 cells were treated with 2.5 μmol/L 5-Aza-dcR, 1 μmol/L TSA or together. Syk gene methylation status and expression level were detected by MSP, RT-PCR and/or Western blot. HCT-15 cell proliferation and in-vitro cell infiltration ability were detected by 3H in-corporation assay and in-vitro cell infiltration assay. Results (1) Compared with the control group, Syk gene was demethylated and restored expression in the presence of 5-Aza-dcR and/or TSA and TSA en-hanced the effect of 5-Aza-dcR-induced demethylation; (2) Compared with Syk (-) HCT-15 cell group in the absence of 5-Aza-dcR and/or TSA,the cell proliferation ability in groups of 5-Aza-deR,TSA, com-bined treatment,or Syk stable expression was reduced by 30% ,25% ,45% and 54% respectively. Mean-while,cell infiltration ability was reduced by 22% ,27% ,43% and 64% respectively, Conclusion Ad-ministration of 5-Aza-dcR and/or TSA can restore the re-expression of methylated Syk gene, and inhibit the proliferation, invasion and metastasis of HCT-15 cells. Key words: 5-Aza-2-deoxycytidine ; Trichostatin A; Spleen; Tyrosine kinase ; Methyla-tion

Immunization with a Nontoxic Mutant of Staphylococcal Enterotoxin A, SEAD227A, Protects against Enterotoxin‐Induced Emesis in House Musk Shrews

Staphylococcal enterotoxins (SEs) are the most common cause of foodborne diseases and toxic shock throughout the world. However, no vaccine that prevents emesis induced by SEs has been described. A nontoxic mutant of SEA, SEAD227A, was constructed by site-directed mutagenesis and was purified by means of the Escherichia coli expression system. House musk shrews, a small emetic animal model, were immunized with SEAD227A and then challenged with wild-type SEA. SEA-induced emesis was recorded for 3 h. Antibody production was analyzed by gel double-immunodiffusion assay. Neutralizing activities of the antibodies with respect to superantigenic and emetic activities were analyzed in vitro and in vivo. SEAD227A was devoid of both superantigenic and emetic activities but still retained its immunological activity. Immunization with SEAD227A strongly induced specific antibody production and provided significant protection against SEA-induced emesis. Antibodies from immunized shrews markedly inhibited the SEA-induced proliferation of spleen cells and also significantly ablated SEA-induced vomiting in the animals. These results suggest that vaccination with SEAD227A, which is devoid of toxic properties, provides protection against SEA-induced emesis. This nontoxic mutant and its specific antibodies might be useful in the prevention and treatment of staphylococcal food poisoning.

L-Arginine Reverses Radiation-Induced Immune Dysfunction: The Need for Optimum Treatment Window

The aim of the present study was to investigate the protective efficacy of l-arginine in mitigating the injury induced by 2 Gy of total-body gamma radiation (TBI). Mice exposed to radiation (TBI group) had significantly decreased spleen weight, splenocyte numbers and bone marrow cellularity. Administration of l-arginine 2 h after TBI (TBI + l-arginine group) was effective in reducing the radiation-induced depletion of spleen and bone marrow cellularity but was not effective when administered before TBI (l-arginine + TBI group). The radiation-induced decrease in Con A-induced spleen cell proliferation, specific antibody response of spleen B cells to sheep red blood cells, and spleen RNA content was reversed in mice in the TBI + l-arginine group. The radiation-induced increase in serum TNF-alpha levels, serum nitrate/nitrite (NOx) levels, spleen DNA fragmentation, spleen nitric oxide synthase (NOS) activity, spleen inducible NOS (iNOS) activity, and hepatic iNOS activity was reversed in mice in the TBI + l-arginine group. l-Arginine administered before TBI could not reverse these changes. Mice in the TBI + l-arginine group had significantly increased spleen arginase activity compared to mice from either the TBI or l-arginine + TBI group. The results suggest the importance of the time of administration of l-arginine and the l-arginine pathway in mitigating the radiation-induced host immune dysfunction.

Madecassoside attenuates inflammatory response on collagen-induced arthritis in DBA/1 mice

Madecassoside (MA), a triterpenoid product isolated from Centella asiatica, has been described to exhibit antioxidant and anti-inflammatory activities. The present study was undertaken to determine whether madecassoside (MA) is efficacious against collagen-induced arthritis (CIA) in mice and its possible mechanisms. DBA/1J mice were immunized with bovine type II collagen and treated with MA (3, 10 and 30 mg/kg d, i.g.) from days 21 to 42 after immunization. Arthritis was evaluated by hind paw swelling, polyarthritis index, and histological examination. In vitro proliferation of spleen cells was examined using 3-[4,5-dimethylthylthiazol-2-yl]-2, 5-diphenyltetrazoliumbromide (MTT) assay. Plasma levels of cytokines tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), interleukin-10 (IL-10) and the expression of prostaglandin E(2) (PGE(2)), cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) in synovial tissues were also determined. The results showed that comparing with untreated CIA mice, treated with MA dose-dependently suppressed the clinical arthritis score and joints tissues pathological damage, reduced the proliferation of spleen cells, plasma levels of TNF-alpha and IL-6, synovial tissues PGE(2) production and COX-2 protein expression, however, the expression of COX-1 in synovial tissues did not change and the plasma levels of IL-10 were increased. These results suggest that MA can effectively alleviate inflammatory response on CIA, and anti-inflammatory effects of MA can be attributed, at least partially, to the inhibition of pro-inflammatory mediators, including COX-2 expression, PGE(2) production, TNF-alpha and IL-6 levels and the up-regulation anti-inflammatory molecule IL-10.

A neutral polysaccharide from Glycyrrhiza inflata

A neutral polysaccharide Gi-A1 was isolated from the roots of Glycyrrhiza inflata Bat. It had a molecular mass of over 2000 kDa and showed [α] D 20 + 81.4° (c 1.05, H2O). Acid hydrolysis and methylation analysis indicated that Gi-A1 was mainly composed of α-D-glucose, α-L-arabinose, and α-D-galactose with a molar ratio of 8.0:1.8:1.0. It can significantly stimulate spleen cell proliferation in vitro (P < 0.01).

Increased cell proliferation in spleen and lymph nodes peripheral to contact allergen application site

The local lymph node assay (LLNA) is widely used to identify chemicals that are contact sensitizers. The assay involves dosing mice with the chemical on both ears and pooling the superficial parotid lymph nodes for assessment of lymphocyte proliferation as a marker of sensitization. The present study explored potential reduction in animal usage by dosing one ear with the allergen and the other with vehicle-only. The respective draining lymph nodes were processed separately for tritiated thymidine ( 3H-TdR) incorporation. Cell proliferation in proper axillary and renal nodes, as well as in the spleen was also assessed. Cross-contamination of the chemicals from the dosed ears to other parts of the body via preening was prevented by dosing restrained animals and washing off the residual chemical with saline after 4 h. Dosing the left ear with 0.02% oxazolone (OX) on unrestrained animals resulted in marked cell proliferation in its draining lymph node (stimulation index, SI = 12.8) and in the lymph node draining the contra-lateral vehicle-dosed ear (SI = 6), as well as the proper axillary lymph nodes (SI = 3.3). Increased 3H-TdR incorporation was not observed in the renal lymph nodes (SI = 1.1). Similar stimulation of cells was observed in the lymph node draining the ear contra-lateral to the 30% hexylcinnamaldehyde (HCA)-dosed ear. Increased proliferative activity was observed in contra-lateral draining lymph nodes of restrained mice demonstrating that these results cannot be attributed to cross-contamination of adjacent skin. A significant increase in proliferation of splenocytes was also observed. It is concluded that dermal application of a contact allergen, as exemplified by OX and HCA, may induce cell proliferation in the neighboring lymph nodes and spleen indicative of hapten and/or haptenated proteins diffusing through the skin to peripheral nodes and the blood to produce systemic sensitization. It is also possible that lymphatic capillaries may communicate between the left and right side of the mouse head. Thus the contra-lateral draining superficial parotid node cannot be used as a control for application of contact allergen to a single ear in a modified LLNA.

Investigation of the immunogenicity of p-phenylenediamine and Bandrowski's base in the mouse

p-Phenylenediamine (PPD) exposure is associated with T-cell mediated contact dermatitis. T-cells from allergic patients proliferate following exposure to PPD and the oxido-conjugation product Bandrowski's base (BB). Both compounds are classified as sensitizers in the local lymph node assay; however, because of their instability the nature of the antigenic determinant remains ill-defined. The aim of this study was to explore the immunogenic potential of PPD and BB in mice. Spleen cell proliferation and cytokine secretion was measured ex vivo following antigen recall with soluble PPD or BB and either irradiated or glutaraldehyde fixed, antigen pulsed dendritic cells from syngeneic mice. Glutathione was added to certain incubations. LC–MS analysis and solvent extraction were used to monitor the fate of [ 14C]BB in culture and the extent of BB binding, respectively. Spleen cells from BB exposed, but not PPD- or vehicle-exposed, mice proliferated when stimulated with BB. Proliferating cells secreted high levels of IFN-γ, GM-CSF and IL-2. Stimulation with PPD instigated low levels of proliferation. Irradiated, but not fixed, dendritic cells pulsed with BB stimulated proliferation signifying a classical hapten mechanism involving irreversible BB binding to protein and processing. BB bound preferentially to serum protein when incubated together with cells and serum. Degradation of BB in the presence of glutathione was associated with a stronger stimulation of specific T-cells at higher BB concentrations. These data demonstrate that BB is a potent immunogen in the mouse.