Total Spleen Cells Research Articles

Evaluation of the Genotoxicity of the Aerial Parts of Iraqi Euphorbia cyathophora on Bone Marrow and Spleen Cells in Mice

The aim of the study was extraction of arial part of Euphorbia cyathophora constituents with methanol and evaluate its effect on mitotic index and total chromosomal aberration bone marrow cell and spleen cell in mice
 200 gm of E. cyathophora fine powder was defatted then extracted by cold maceration 80% ethanol for seven days. The extract was filtered and dried in a rotary evaporator then the dried extract was suspended with water and consecutively extracted using chloroform, ethyl acetate for each. The aqueous layer was then mixed with 100ml methanol. These fractions are dried under reduced pressure to obtain the dry extract. Twenty-four Albino mice were used for the experiment. The animals were divided into four groups: Group 1: Mice were treated with distilled water. The dose was given daily for seven successive days. Group 2: Mice were treated with a single dose (20mg/kg) of methotrexate (positive control). Group 3: Mice were treated with (100mg/kg) of menthol fraction for seven successive days. Group 4: Mice were treated with (200mg/kg) of methanol fraction for seven successive days. Mice were sacrificed by (spinal dislocation). Samples of bone marrow cells and spleen cells were taken and genotoxic analyses.
 Methanol fraction of Euphorbia cyathophora at a dose of 100mg/kg demonstrated a significant decrease in mitotic index and a significant increase in total chromosomal aberrations as compared to distilled water in both bone marrow cells and spleen cells
 (p<0.05). 100 mg/kg and 200 mg/kg of methanol fraction of Euphorbia cyathophora that showed to be significantly higher in mitotic index and significantly lower in total chromosomal aberration as compared to methotrexate (p<0.05).
 In conclusion the current study revealed that the methanol fraction of aerial parts of Euphorbia cyathophora is genotoxic but its genotoxicity is less than that of methotrexate.

Comprehensive study of health effects of plasma technology occupational environment: Exposure to high frequency and intensity noise and toxic gases

ObjectivesTo evaluate on animal models the health effects of the combined or separate exposure to main chemical and physical hazards of plasma-based material processing technology environment. Materials and methodsMale Wistar rats were exposed to actual levels of hazardous factors in plasma technology occupational environment: i.e., ozone and nitrogen oxides (O3 and NOx) in respective concentrations of 0.5 mg/m3 and 1.0 mg/m3 and high-frequency (1000–1600 Hz) of 112 dB intensity noise for 3 h/day, 5 days/week for 12 weeks, with a recovery period of 1 month. ResultsExposure to noise or its combination with chemical factors (ozone, nitrogen oxides) causes non-specific CNS changes testifying for significant excitation dominance, especially in the case of joint exposure. Histological examination of rats’ brain in experimental revealed a pronounced increase in blood filling of small vessels on the tenth day of the experiment, with subsequent intensification of vascular alterations and eventually to cerebral edema. The exposure to noise significantly reduced total thymus, bone marrow and spleen cell numbers and these was also more pronounced under the joint impact of noise and toxic gases. Thymus, but not bone marrow or spleen, mitotic activity was as well reduced under the same modes of exposure. Cytological investigation of film preparations of subcutaneous connective tissue revealed that joint exposure led to microcirculatory disorders, increased number of dark mast cells and reduced degranulation processes indicative of increased autoregulatory processes effective at microvasculature level. ConclusionsHigh-frequency and intensity noise is main stressor factor that has negative impact on CNS and immune system, morphology and functioning of hematopoietic organs (spleen, bone marrow, thymus) and connective tissue. Its negative impact is significantly potentiated by concurrent exposure to ozone and nitrogen oxide, while exposure only to these toxic gases has no significant effect on the above targets.

P982: MYELOID-DERIVED SUPPRESSOR CELLS: CHARACTERIZATION IN PERIPHERAL BLOOD AND SPLEEN TISSUE OF PATIENTS WITH MYELOFIBROSIS

Background: Myeloid-derived suppressor cells (MDSCs) are immature myeloid cells that accumulate in patients with malignancies, sepsis or chronic inflammation. Features of MDSCs are: myeloid origin, immature state, strong ability to reduce cytotoxic functions of T/NK cells, potential to differentiate into endothelial cells favoring neoangiogenesis. MDSCs are conventionally divided into polymorphonuclear (PMN)- and monocytic (M)-MDSCs. PMN-MDSCs are characterized by the production of reactive oxygen species and arginase-1 (Arg-1), that directly inhibits T-cell proliferation and activation, while M-MDSCs predominantly express inducible nitric oxide synthase and produce nitric oxide. Myelofibrosis (MF) is characterized by clonal expansion of a hematopoietic stem cell, extramedullary hematopoiesis, bone marrow (BM) fibrosis, splenomegaly, BM and splenic neoangiogenesis and an acquired mutation of JAK2, CALR or MPL gene. Inflammation plays a relevant role in MF pathogenesis, as proven by high levels of inflammatory cytokines with prognostic significance and by a state of chronic oxidative stress. Aims: To characterize circulating MDSCs and assess their localization and quantification in spleen samples from MF patients. Methods: Peripheral blood mononuclear cells (PBMCs) from MF patients (n=15) and healthy subjects (HDs; n=10) were stained with surface antibodies (CD11b, CD14, HLA-DR, CD15, CD33) and intracellular Arg-1 and analysed by flow cytometry. Formalin-fixed paraffin-embedded samples of spleen specimens (MF n=41; HDs n=20) were immunostained with anti-Arg-1 antibody using the automated platform Dako Omnis Envision Flex with the EnVision FLEX, High pH, HRP Rabbit/Mouse High pH [GV800] revelation kit. Results: In PBMCs, the percentage of PMN-MDSCs (identified as CD11b+HLA-DRlow/-CD33dimCD14-CD15+) was higher (p=0) in patients (median 9.2, range 1-59) than HDs (median 1.7, range 0.1-4.5) and directly correlated with the allelic burden (R=0.86, p=0.006) in JAK2-mutated patients. Patients in pre-fibrotic phase (n=7) had levels of PMN-MDSCs (median 9.2, range 1-31.4) comparable to HDs; on the contrary, the percentage of PMN-MDSCs in patients with BM fibrosis (n=8; median 13.3, range 3.5-59) was higher (p=0) than in HDs. All PMN-MDSCs were Arg-1+, both in MF and in HDs. Gene expression studies are ongoing. The expression of the CXCR4 receptor on PMN-MDSCs was lower, although not statistically significant, in MF (median 1.1, range 0-9.3) than in HDs (median 2.5, range 0-11.9). PMN-MDSCs in MF inversely correlated (R=-0.89 p=0.007) with CXCR4 expressed on CD34+ cells. The percentage of M-MDSCs (identified as CD11b+HLA-DRlow/-CD33hiCD14+CD15-) was higher (p=0.015) in HDs (median 0.49, range 0.25-1.5) than in MF (median 0.29, range 0.02-0.76). Immunohistochemical staining of spleen sections showed a higher (p=0) percentage of Arg-1+ cells (calculated on total spleen cells) in MF (median 20, range 5-60) than in HDs (median 7, range 2-20); Arg-1+ cells were present in all patients and predominantly located in the red pulp in MF patients, while in HDs were detected in the white pulp. Summary/Conclusion: The identification of MDSCs and their relation with the mutational status suggest a new mechanism of MF pathogenesis, either related to the chronic inflammatory status of patients or as players of the neoangiogenesis that characterizes the disease. Moreover, the low expression of CXCR4 on PMN-MDSCs could be, on one hand, related to the low expression of this receptor on CD34+ cells or suggest a MF-specific recruitment mechanism, other than that observed in solid tumors.

Mild hypobaric hypoxia influences splenic proliferation during the later phase of stress erythropoiesis

Tissue trauma and hemorrhagic shock are common battlefield injuries that can induce hypoxia, inflammation, and/or anemia. Inflammation and hypoxia can initiate adaptive mechanisms, such as stress erythropoiesis in the spleen, to produce red blood cells and restore the oxygen supply. In a military context, mild hypobaric hypoxia-part of the environmental milieu during aeromedical evacuation or en route care-may influence adaptive mechanisms, such as stress erythropoiesis, and host defense. In the present study, healthy (control), muscle trauma, and polytrauma (muscle trauma and hemorrhagic shock) mice were exposed to normobaric normoxia or hypobaric hypoxia for ∼17.5 h to test the hypothesis that hypobaric hypoxia exposure influences splenic erythropoiesis and splenic inflammation after polytrauma. This hypothesis was partially supported. The polytrauma + hypobaric hypoxia group exhibited more splenic neutrophils, fewer total spleen cells, and fewer splenic proliferating cells than the polytrauma+normobaric normoxia group; however, no splenic erythroid cell differences were detected between the two polytrauma groups. We also compared splenic erythropoiesis and myeloid cell numbers among control, muscle trauma, and polytrauma groups. More reticulocytes at 1.7 days (40 h) post-trauma (dpt) and neutrophils at 4 dpt were produced in the muscle trauma mice than corresponding control mice. In contrast to muscle trauma, polytrauma led to a reduced red blood cell count and elevated serum erythropoietin levels at 1.7 dpt. There were more erythroid subsets and apoptotic reticulocytes in the polytrauma mice than muscle trauma mice at 4 and 8 dpt. At 14 dpt, the red blood cell count of the polytrauma + normobaric normoxia mice was 12% lower than that of the control + normobaric normoxia mice; however, no difference was observed between polytrauma + hypobaric hypoxia and control + hypobaric hypoxia mice. Our findings suggest muscle trauma alone induces stress erythropoiesis; in a polytrauma model, hypobaric hypoxia exposure may result in the dysregulation of splenic cells, requiring a treatment plan to ensure adequate immune functioning.

TGF-βR inhibitor SB431542 restores immune suppression induced by regulatory B-T cell axis and decreases tumour burden in murine fibrosarcoma.

The contribution of immune cells in soft tissue sarcomas (STS) is not completely known and understanding their role is very essential for employing immunotherapy strategies. Here, we show that murine fibrosarcoma-conditioned medium promoted total spleen cell proliferation but inhibited T cell responses to mitogenic and allo-antigen-mediated stimulation. This increased proliferation was found to be in B cells resulting in generation of Breg further leading to Treg population. This was found to be the same in vitro and in vivo. The phenotype of these B cells was CD19+CD81+CD27+CD25+PD-L1hi and they secreted both IL-10 and TGF-β. These tumor evoked Bregs (tBreg), when co-cultured with B depleted T cells, suppressed their proliferation in response to anti-CD3/CD28 stimulation. tBreg-induced suppression of T cell responses wasnot abrogated by the inhibition or neutralization of IL-10 but by the small molecule inhibitor of TGFβ Receptor type I, SB431542. While SB531542 per se was not cytotoxic to tumor cells, administration of SB431542 in tumor-bearing mice (TBM) significantly reduced the tumor burden. In addition, the treatment significantly reduced Treg cells and rescued proliferation of T cells in response to mitogen and allo-antigen. Collectively, our results identify that tumor evoked Breg cells mediate T cell immune suppression through TGFβ-mediated pathway and that targeting the Breg-Treg axis can be potentially used as an immunotherapy agent.

Antitumor Reactive T-Cell Responses Are Enhanced In Vivo by DAMP Prothymosin Alpha and Its C-Terminal Decapeptide.

Prothymosin α (proTα) and its C-terminal decapeptide proTα(100–109) were shown to pleiotropically enhance innate and adaptive immune responses. Their activities have been broadly studied in vitro, focusing primarily on the restoration of the deficient immunoreactivity of cancer patients’ leukocytes. Previously, we showed that proTα and proTα(100–109) act as danger-associated molecular patterns (DAMPs), ligate Toll-like receptor-4, signal through TRIF- and MyD88-dependent pathways, promote the maturation of dendritic cells and elicit T-helper type 1 (Th1) immune responses in vitro, leading to the optimal priming of tumor antigen-reactive T-cell functions. Herein, we assessed their activity in a preclinical melanoma model. Immunocompetent mice bearing B16.F1 tumors were treated with two cycles of proTα or proTα(100–109) together with a B16.F1-derived peptide vaccine. Coadministration of proTα or proTα(100–109) and the peptide vaccine suppressed melanoma-cell proliferation, as evidenced by reduced tumor-growth rates. Higher melanoma infiltration by CD3+ T cells was observed, whereas ex vivo analysis of mouse total spleen cells verified the in vivo induction of melanoma-reactive cytotoxic responses. Additionally, increased levels of proinflammatory and Th1-type cytokines were detected in mouse serum. We propose that, in the presence of tumor antigens, DAMPs proTα and proTα(100–109) induce Th1-biased immune responses in vivo. Their adjuvant ability to orchestrate antitumor immunoreactivities can eventually be exploited therapeutically in humans.

Aryl hydrocarbon receptor activation does not impact the number of myeloid-derived suppressor cells in the spleens of mice immunized with cholera toxin

Abstract The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that regulates immune responses. Previous studies in this laboratory have found that AhR activation with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mice suppresses the anti-cholera toxin (CT) IgA response, but fecal antibody levels recover at least two weeks earlier than serum levels after repeated immunization. Studies by others have shown that TCDD exposure increases splenic myeloid-derived suppressor cell (MDSC) numbers in mice, and MDSC have been associated with enhanced IgA responses. In this study, we hypothesized that TCDD exposure would increase MDSC numbers in CT-immunized mice. Female C57Bl/6 mice were administered peanut oil (vehicle) or TCDD (40 μg/Kg) orally on day 0, and each animal was orally immunized with cholera toxin (CT, 10 μg/Kg) on days 1 and 8. On day 14, CT-specific serum IgA levels (ELISA) were significantly suppressed by TCDD exposure to 21.6% of control (P < 0.05), but fecal IgA levels were not significantly different. Total spleen cell numbers were significantly reduced by TCDD exposure to 72.9% of control (P < 0.05), but neither the percent nor number of MDSC (CD11b+ Gr1+, by flow cytometry) per spleen was significantly altered: 2.21% ± 0.48% and 1.91% ± 0.23% (450,000 ± 98,000 and 290,000 ± 34,000) for control and TCDD-treated, respectively (mean ± SEM). These results suggest that AhR activation differentially impacts antigen-specific IgA responses between serum and feces in cholera toxin-immunized mice, but not the number of MDSCs.

Modular MLV-VLPs co-displaying ovalbumin peptides and GM-CSF effectively induce expansion of CD11b+ APC and antigen-specific T cell responses in vitro

The development of novel vaccination strategies is a persistent challenge to provide effective prophylactic treatments to encounter viral infections. In general, the physical conjugation of selected vaccine components, e.g. antigen and adjuvant, has been shown to enhance the immunogenicity and hence, can increase effectiveness of the vaccine. In our proof-of-concept study, we generated non-infectious, replication deficient Murine Leukemia Virus (MLV)-derived virus-like particles (VLPs) that physically link antigen and adjuvant in a modular fashion by co-displaying them on their surface. For this purpose, we selected the immunodominant peptides of the model antigen ovalbumin (OVA) and the cytokine granulocyte macrophage-colony stimulating factor (GM-CSF) as non-classical adjuvant. Our results show that murine GM-CSF displayed on MLV-VLPs mediates expansion and proliferation of CD11b+ cells within murine bone marrow and total spleen cells. Moreover, we show increased immunogenicity of modular VLPs co-displaying OVA peptides and GM-CSF by their elevated capacity to induce OVA-specific T cell-activation and -proliferation within OT-I and OT-II splenocyte cultures. These enhanced effects were not achieved by using an equimolar mixture of VLPs displaying either OVA or GM-CSF. Taken together, OVA and GM-CSF co-displaying MLV-VLPs are able to target and expand antigen presenting cells which in turn results in enhanced antigen-specific T cell activation and proliferation in vitro. These data suggest MLV-VLPs to be an attractive platform to flexibly combine antigen and adjuvant for novel modular vaccination approaches.

Tarin, a Potential Immunomodulator and COX-Inhibitor Lectin Found in Taro (Colocasia esculenta).

Taro (Colocasia esculenta) corm is a rustic staple food, rich in small starch granules, fibers, and bioactive phytoconstituents such as flavonoids, alkaloids, sterols, tannins, phytates, micronutrients, and proteins, including tarin, a GNA‐related lectin. Tarin exhibits recognized biocide activities against viruses and insects, has antitumoral properties and is an immunomodulator molecule candidate. It has been isolated in highly purified form (>90%) from taro corms through low‐cost and single‐step affinity chromatography. It comprises 2‐domain 27 to 28 kDa protomer, posttranslational cleaved into 2 nonidentical monomers, 11.9 and 12.6 kDa, held by noncovalent binding. At least 10 tarin isoforms sharing over 70% similarity have been described. The monomers assume the β‐prism II fold, consisting of 3 antiparallel β‐sheets formed by 4 β‐strands each. Tarin exhibits an expanded‐binding site for complex and high‐mannose N‐glycan chains 49, 212, 213, 358, 465, and 477 found on cell surface antigens of viruses, insects, cancer, and hematopoietic cells, explaining its broad biological activities. Tarin may stimulate innate and adaptive immune responses, enabling hosts to recover from infections or immunosuppressed status inherent to several pathological conditions. In a murine model, tarin stimulates the in vitro and in vivo proliferation of total spleen and bone marrow cells, especially B lymphocytes. Granulocyte repopulation has also been demonstrated in long‐term mice bone marrow cell cultures. As a potential immunomodulator, tarin, administered to immunosuppressed mice, attenuated cyclophosphamide‐induced leukopenia. We propose a molecular model that unites the potential prophylactic and therapeutic action of tarin on hematopoietic and cancer cells, as a potential immunomodulator.

Immunotoxic and hepatotoxic effects of perfluoro-n-decanoic acid (PFDA) on female Harlan Sprague–Dawley rats and B6C3F1/N mice when administered by oral gavage for 28 days

Poly- and perfluoroalkyl substances (PFAS) are chemically and thermally stable, hydrophobic, lipophobic compounds used in stain repellants and water and oil surfactants, and associated with immunosuppression and peroxisome proliferator activity. Perfluoro-n-decanoic acid (PFDA, (CF3(CF2)8COOH), a fluorinated straight chain fatty acid compound, is reported to induce thymic atrophy and reversible bone marrow hypocellularity in rodent models. The objective of this study was to assess potential immunotoxicity of PFDA, due to its structural similarity to other immunosuppressive PFASs. Female Harlan Sprague–Dawley rats were exposed to 0–2.0 mg PFDA/kg by oral gavage daily for 28 d. Female B6C3F1/N mice were exposed once/week to 0–5.0 mg PFDA/kg by gavage for 4 weeks. Animals were evaluated for effects on immune cell populations in spleen and bone marrow, and innate, humoral-, and cell-mediated immunity. Mice were also evaluated for resistance to Influenza virus. Treatment-related hepatocyte necrosis and hepatomegaly were observed in rats treated with 0.5 mg PFDA/kg/d. In mice, hepatomegaly (26–89%) was observed following exposure to ≥0.625 mg PFDA/kg/week, while splenic atrophy (20%) was observed at 5.0 mg PFDA/kg/week. At 5.0 mg PFDA/kg/week, total spleen cells, and Ig + and NK + cells were decreased (17.6–27%). At ≥ 1.25 mg PFDA/kg/week the numbers of splenic CD3+, CD4+, CD8+, and Mac3+ cells were decreased (10.5–39%). No changes were observed in leukocyte subpopulations in PFDA-exposed rats. Phagocytosis by fixed-tissue macrophages was decreased in liver (specific activity, 24–39%) at ≥0.25 mg PFDA/kg/d in rats. PFDA-induced effects on humoral- and cell-mediated immunity, host resistance, and bone marrow progenitor cells were limited. These data suggest that exposure to PFDA may induce adverse effects in rat liver in a manner consistent with the PFAS class, and may also alter the balance of immune cell populations in lymphoid tissues in mice.

Sex differences in regulatory cells in experimental stroke

Stroke is the leading cause of disability in the United States. Sex differences, including smaller infarcts in females and greater involvement of immune-mediated inflammation in males may affect the efficacy of immune-modulating interventions. To address these differences, we sought to identify distinct stroke-modifying mechanisms in female vs. male mice. The current study demonstrated smaller infarcts and increased levels of regulatory CD19+CD5+CD1dhi B10 cells as well as anti-inflammatory CD11b+CD206+ microglia/macrophages in the ipsilateral vs. contralateral hemisphere of female but not male mice undergoing 60min middle cerebral artery occlusion followed by 96h of reperfusion. Moreover, female mice with MCAO had increased total spleen cell numbers but lower B10 levels in spleens. These results elucidate differing sex-dependent regulatory mechanisms that account for diminished stroke severity in females and underscore the need to test immune-modulating therapies for stroke in both males and females.

Secreted heat shock protein gp96-Ig vaccine for malaria prophylaxis

Abstract Proposed vaccine principle relies on secreted gp96-Ig chaperoning Plasmodium falciparum (Pf) AMA1 and PfCSP sporozoite proteins that are efficiently taken up and cross presented by activated DC via MHC I to CD8 CTL, thereby stimulating an avid, antigen specific, cytotoxic T cell response. The generation of a powerful, cytotoxic anti-sporozoite CD8 CTL response by the vaccine is expected to provide prophylactic immunity against malaria by killing infected liver cells, thereby preventing blood stage infection. We performed mouse immunogenicity experiments using 293-gp96-IgPfAMA1-PfCSP vaccine cells that produce gp96-Ig and express PfAMA1 and PfCSP. One million 293-gp96-IgPfAMA1-PfCSP cells were injected in B6 mice by intraperitoneal (IP), subcutaneous (SC), intramuscular (IM) and intradermal (ID) routes. Control mice received 293-gp96-Ig. Five days later, mice were sacrificed. Total spleen cells were cultured overnight in medium only or with pool of overlapping PfAMA1 and PfCSP peptides. Frequency of PfAMA1- and PfCSP-specific CD8 T cell responses in the spleen was measured by intracellular cytokine staining assay and analyzed by flow cytometry. We found that vaccine cells delivered by IP and SC routes induced the strongest PfAMA1- and PfCSP-specific immune response systemically (1.8±0.4% and 1.3±0.3% PfAMA1- and PfCSP-specific spleen CD8 T cells, respectively). In addition, after 293-gp96-IgPfAMA1-PfCSP vaccination frequency of liver-infiltrating CD8+ T cell was significantly increased (70% of all liver CD3+ cells were CD8+CD44+CD62L− T cells). Our findings are strongly supportive of the novel gp96-Ig malaria vaccine as unique systemic and liver-homing, sporozoite specific CD8 CTL vaccine strategy.

Immunoglobulin G-dependent enhancement of the infection with Coxsackievirus B4 in a murine system

ABSTRACTIt was demonstrated that specific IgG can enhance the infection with CV-B4, in vitro, in the human system. This enhancement could be involved in the pathophysiology of CV-B4 induced diseases. To investigate further the role of enhancing IgG in the infection with CV-B4 E2 in vivo, animal models are needed. Therefore, it was decided to assess whether inoculation of CV-B4 E2 to mice results in the appearance of IgG able to enhance the infection with this virus. Swiss albino mice were inoculated with CV-B4 E2 intraperitoneally. Serum samples were obtained from tail vein blood collected from day 0 to day 80 p.i. IgG were isolated by Protein G affinity chromatography. Seroneutralisation assays were carried out. In total murine spleen cells cultures inoculated with CV-B4 E2 mixed with various dilutions of serum or IgG samples, the enhancing activity was assayed through i) the antiviral activity titer of supernatants ii) the detection of intracellular viral RNA by RT-PCR iii) the level of infectious particles in supernatants.In most serum samples (76/105), neutralizing and enhancing activities were detected peaking between days 14 and 30 p.i and were higher in sera from mice inoculated with 2.106 TCID50 units than with lower doses. The enhancing activity was due to the IgG-enriched fraction of serum from CV-B4 E2 infected animals but not from control animals.These data show that IgG from immune mice can enhance the infection of splenocytes with CV-B4 E2 in vitro and open the way to explore whether such an enhancing activity can play a role in vivo.

Patency of Litomosoides sigmodontis infection depends on Toll‐like receptor 4 whereas Toll‐like receptor 2 signalling influences filarial‐specific CD4+ T‐cell responses

BALB/c mice develop a patent state [release of microfilariae (Mf), the transmission life-stage, into the periphery] when exposed to the rodent filariae Litomosoides sigmodontis. Interestingly, only a portion of the infected mice become patent, which reflects the situation in human individuals infected with Wuchereria bancrofti. Since those individuals had differing filarial-specific profiles, this study compared differences in immune responses between Mf(+) and Mf(-) infected BALB/c mice. We demonstrate that cultures of total spleen or mediastinal lymph node cells from Mf(+) mice produce significantly more interleukin-5 (IL-5) to filarial antigens but equal levels of IL-10 when compared with Mf(-) mice. However, isolated CD4(+) T cells from Mf(+) mice produced significantly higher amounts of all measured cytokines, including IL-10, when compared with CD4(+) T-cell responses from Mf(-) mice. Since adaptive immune responses are influenced by triggering the innate immune system we further studied the immune profiles and parasitology in infected Toll-like receptor-2-deficient (TLR2(-/-)) and TLR4(-/-) BALB/c mice. Ninety-three per cent of L. sigmodontis-exposed TLR4(-/-) BALB/c mice became patent (Mf(+)) although worm numbers remained comparable to those in Mf(+) wild-type controls. Lack of TLR2 had no influence on patency outcome or worm burden but infected Mf(+) mice had significantly lower numbers of Foxp3(+) regulatory T cells and dampened peripheral immune responses. Interestingly, in vitro culturing of CD4(+) T cells from infected wild-type mice with granulocyte-macrophage colony-stimulating factor-derived TLR2(-/-) dendritic cells resulted in an overall diminished cytokine profile to filarial antigens. Hence, triggering TLR4 or TLR2 during chronic filarial infection has a significant impact on patency and efficient CD4(+) T-cell responses, respectively.

Crude extract from taro (Colocasia esculenta) as a natural source of bioactive proteins able to stimulate haematopoietic cells in two murine models

Besides its nutritional value, taro (Colocasia esculenta) is traditionally used as a medicinal plant and provides bioactive compounds with important biological properties. This study evaluated the in vitro and in vivo immunomodulatory potential of the protein extract from taro corms on the haematopoietic cells of C57BL/6 and BALB/c mice. The crude taro extract (CTE) stimulated the in vitro proliferation of mice splenocytes in a dose-dependent manner in both mice strains. The intraperitoneal inoculation of CTE induced splenomegaly and the proliferation of total spleen and bone-marrow cells. Additionally, CTE promoted in vivo B220+ splenocyte proliferation, which was accompanied by a reduction of mature (B220+ IgM+) and immature (B220+ IgM−) B cells in the bone marrow. The production of Antibody by B220+ splenocytes indicated the activation of B1 cells by the CTE only in C57BL/6. CTE represents a powerful source of immunostimulatory proteins, new candidates as additives for food and pharmaceutical industries.

Aryl hydrocarbon receptor activation enhances total but not antigen-specific IgA secretion in Leishmania major-infected mice (MUC9P.748)

Abstract Secretory IgA plays a critical role in neutralizing enteric toxins and protecting against some mucosal pathogens. In mice, activation of the aryl hydrocarbon receptor (AhR) with a single dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been shown to transiently decrease total fecal IgA levels but increase antigen-specific IgA levels after mucosal antigen challenge. The goal of this study was to determine if TCDD exposure would alter secretion of antigen-specific IgA levels after systemic pathogen challenge. Female C57Bl/6 mice were administered TCDD at 0 or 40 μg/Kg (po) one day prior to infection with Leishmania major promastigotes. Three weeks later, IgA levels were measured by ELISA. L. major-specific IgA levels in serum were readily detected but unchanged by TCDD treatment. L. major-specific lgA was undetected in the feces of any animal. In contrast, total fecal IgA levels (OD) were significantly increased by 34% in TCDD treated animals (p < 0.05). Total spleen and bone marrow cell numbers were reduced significantly in TCDD-treated mice. The expression of IgM heavy chain, IgJ chain, or kappa light chain in these cells (measured by RT-RT-PCR) was unchanged by TCDD treatment. In contrast, the expression of IgA heavy chain was significantly reduced by approximately 50% in TCDD-treated animals. These results suggest that AhR activation enhances bulk IgA secretion but does not enhance secretion of antigen-specific IgA following systemic pathogen challenge.

T cell regulation by Phlomis lanata protein extracts in mice

Context: Phytopharmacology is a complex but very promising research area. The different plant parts and extraction methods may result in opposed effects. Phlomis species have been reported for both anti-inflammatory and tonic properties.Objective: The effect of Phlomis lanata Willd. (Lamiaceae) protein extracts on immune cell reactivity was studied in the experimental mouse model.Materials and methods: Protein extracts from P. lanata aerial parts were fractionated by Q-sepharose ion-exchange chromatography and applied to whole spleen cells or T-cell subsets at 5 μg/ml. Cell growth and cytokine production were evaluated after 4 and 2 d of culture using 3H-thymidine-uptake and ELISA techniques, respectively.Results: Among the protein fractions tested, column wash proteins (W1) and the fraction eluted using 600 mM NaCl (F6) reduced by 76% and increased by 78% spleen cell proliferation, respectively. W1 suppressed proliferation of effector T-cells, but stimulated the growth of suppressor/regulatory cells by 62–148%. Although W1 stimulated IL-2 and IL-10 production from total spleen cells, it significantly increased IL-10 (50%) and reduced IL-2 (30–50%) production from T-cells, while TNF-α release was enhanced in CD25+CD4+ by 92% and reduced by 50% in CD25+CD8+ cells. F6 stimulated whole spleen cell growth, reduced proliferation of CD8+ and CD25+ cells by approximately 50%, while decreasing by 60–80% TNF-α production from CD25− and CD25+CD8+ cells.Discussion and conclusion: The suppressive activity of W1 could be attributed to IL-10 and TNF-α, while the stimulatory effect of F6 could be attributed to the inhibition of T-regulatory cells. In the same plant, coexisting protein fractions induce both immunostimulatory and immunosuppressive activities.

All-trans-retinoic acid and CD38 ligation differentially regulate CD1d expression and α-galactosylceramide-induced immune responses

The MHC class-I like molecule CD1d presents glycolipid antigens and thereby activates invariant natural killer-T (NKT) cells. However, little is understood regarding the regulation of its expression. All-trans-retinoic acid (RA) and CD38, which is itself a target of RA, both independently regulate the differentiation of antigen presenting cells. In the current study, we treated human THP-1 cells and murine splenic cells with RA, with and without antibody-mediated ligation of cell-surface CD38. Whereas a physiological concentration (20nM) of RA alone rapidly and markedly increased CD1d protein in THP-1 cells, there was a marked synergy between RA and ligation of CD38 with antibody to CD38. Moreover, RA and CD38 ligation differentially regulated CD1d protein distribution between the cell surface and intracellular compartments, as, whereas RA mainly increased intracellular CD1d protein, ligation of CD38 increased CD1d protein both at the cell surface and intracellularly. By confocal microscopy, CD1d was located close to the plasma membrane but only partially overlapped with LAMP1, a late endosomes/lysosomal marker. Furthermore, RA and/or CD38 ligation increased splenocyte proliferation and differentiation after treatment with the CD1 ligand α-galactosylceramide (αGalCer), evidenced by an increase in the number of splenic dendritic cells, NKT cells, and germinal center plasmacytes. RA also differentially regulated αGalCer-induced cytokine expression, increasing IL-4 and decreasing IFNγ production by total spleen cells and the NKT cell population. Our results indicate a previously unknown mechanism in which RA and CD38 differentially yet cooperatively regulate CD1d expression and antigen-presenting function, which could be important for the enhancement of immunity.

109: Granulocyte macrophage-colony stimulating factor and interleukin-3 are not essential for endotoxin-induced extramedullary myelopoiesis

Several studies have reported that splenic extramedullary haematopoiesis is important during certain inflammatory conditions. The haematopoietic growth factor, granulocyte macrophage-colony stimulating factor (GM-CSF), is known to stimulate the growth of in vitro murine macrophages from bone marrow progenitors. Current literature suggests that GM-CSF and interleukin-3 (IL-3) are implicated in regulating the survival and proliferative activity of granulocyte-monocyte progenitors (GMPs) during atherosclerosis. In the current study, we sought to determine the involvement of GM-CSF and/or IL-3 in splenic haematopoiesis following multiple intra-peritoneal (i.p) injections of endotoxin, lipopolysaccharide (LPS). GM-CSF −/− mice and C57BL/6 wild-type (WT) mice were administered with 10 μg Escherichia coli LPS into the peritoneal cavity for four consecutive days. In experiments using neutralizing antibodies against GM-CSF and/or IL-3, 150 μg of the neutralizing antibody was administered on day −1 and 2. Spleen, bone marrow and blood were collected and analysed using flow cytometry. In response to endotoxin challenge, both GM-CSF −/− and WT mice displayed a comparable degree of splenomegaly, with increased total spleen cell numbers, mainly attributable to an increase in splenic myeloid lineage cells, including neutrophils and subsets of monocytes (Ly6C hi and Ly6C lo ). The numbers of splenic GMPs were alsosignificantly increased following multiple endotoxin injections in both WT and GM-CSF −/− mice. The cellular composition and number of bone marrow were unaltered following endotoxin challenge, whereas there was significant leukocytosis in peripheral blood of both WT and GM-CSF −/− mice. Neutralizing GM-CSF and/or IL-3 in WT mice, which received multiple endotoxin injections, failed to inhibit the development of splenomegaly, in which the numbers of GMPs were again increased amongst all treatment groups. Taken together, these results demonstrate that, while splenic extramedullary myelopoiesis may be important in supplying inflammatory cells during certain inflammatory models, it is independent of GM-CSF and IL-3 following multiple endotoxin challenges.

Block Copolymer/DNA Vaccination Induces a Strong Allergen-Specific Local Response in a Mouse Model of House Dust Mite Asthma

BackgroundAllergic asthma is caused by abnormal immunoreactivity against allergens such as house dust mites among which Dermatophagoides farinae (Der f) is a common species. Currently, immunotherapy is based on allergen administration, which has variable effect from patient to patient and may cause serious side effects, principally the sustained risk of anaphylaxis. DNA vaccination is a promising approach by triggering a specific immune response with reduced allergenicity.ObjectiveThe aim of the study is to evaluate the effects of DNA immunization with Der f1 allergen specific DNA on allergic sensitization, inflammation and respiratory function in mice.MethodsMice were vaccinated 28 and 7 days before allergen exposure with a Der f1-encoding plasmid formulated with a block copolymer. Asthma was induced by skin sensitization followed by intra-nasal challenges with Der f extract. Total lung, broncho-alveolar lavage (BAL) and spleen cells were analyzed by flow cytometry for their surface antigen and cytokine expression. Splenocytes and lung cell IFN-γ production by CD8+ cells in response to Der f CMH1-restricted peptides was assessed by ELISPOT. IgE, IgG1 and IgG2a were measured in serum by ELISA. Specific bronchial hyperresponsiveness was assessed by direct resistance measurements.ResultsCompared to animals vaccinated with an irrelevant plasmid, pVAX-Der f1 vaccination induced an increase of B cells in BAL, and an elevation of IL-10 and IFN-γ but also of IL-4, IL-13 and IL-17 producing CD4+ lymphocytes in lungs and of IL-4 and IL-5 in spleen. In response to CD8-restricted peptides an increase of IFN-γ was observed among lung cells. IgG2a levels non-specifically increased following block copolymer/DNA vaccination although IgE, IgG1 levels and airways resistances were not impacted.Conclusions & Clinical RelevanceDNA vaccination using a plasmid coding for Der f1 formulated with the block copolymer 704 induces a specific immune response in the model of asthma used herein.