Peritoneal Cavity Of Mice Research Articles

Differential development of oil granulomas induced by pristane injection in galectin-3 deficient mice.

BackgroundGalectin-3 is known to be a lectin that plays an important role in inflammatory processes, acting as pro-inflammatory mediator in activation and migration of neutrophils and macrophages, as well as in the phagocytic function of these cells. The injection of mineral oils into the peritoneal cavity of mice, such as 2, 6, 10, 14-tetramethylpentadecane (pristane), induce a chronic granulomatous inflammatory reaction which is rich in macrophages, B cells and peritoneal plasma cells known as oil granuloma. In addition, this inflammatory microenvironment provided by oil granulomas is also an important site of plasmacytoma induction, which are dependent on cytokine production and cellular mobilization.Here, we have analyzed the role of galectin-3 in inflammatory cells mobilization and organization after pristane injection characterizing granulomatous reaction through the formation of oil granulomas.ResultsIn galectin-3 deficient mice (gal-3−/−), the mobilization of inflammatory cells, between peritoneal cavity and bone marrow, was responsible for the formation of disorganized oil granulomas, which presented scattered cells, large necrotic areas and low amounts of extracellular matrix. The production of inflammatory cytokines partially explained the distribution of cells through peritoneal cavity, since high levels of IL-6 in gal-3−/− mice led to drastically reduction of B1 cells. The previous pro-inflammatory status of these animals also explains the excess of cell death and disruption of oil granulomas architecture.ConclusionsOur data indicate, for the first time, that the disruption in the inflammatory cells migration in the absence of galectin-3 is a crucial event in the formation and organization of oil granulomas.Electronic supplementary materialThe online version of this article (doi:10.1186/s12865-015-0133-9) contains supplementary material, which is available to authorized users.

Transcriptomics Identify CD9 as a Marker of Murine IL-10-Competent Regulatory B Cells.

Regulatory B cells (Breg) have immune suppressive functions in various autoimmune/inflammation models and diseases and are found to be enriched in diverse B cell subsets. The lack of a unique marker or set of markers efficiently identifying Breg cells impedes detailed investigation into their origin, development, and immunological roles. Here, we perform transcriptome analysis of IL-10-expressing B cells to identify key regulators for Breg biogenesis and function and identify CD9, a tetraspanin-family transmembrane protein, as a key surface marker for most mouse IL-10(+) B cells and their progenitors. CD9 plays a role in the suppressive function of IL-10(+) B cells in ex vivo T cell proliferation assays through a mechanism that is dependent upon B/T cell interactions. CD9(+) B cells also demonstrate inhibition of Th1-mediated contact hypersensitivity in an in vivo model system. Taken together, our findings implicate CD9 in the immunosuppressive activity of regulatory B cells.

The Anticandidal and Toxicity Properties of Lampranthus francisci

Fungal infections have been rising due to the increasing number of immunocompromised patients and intensive use of some antifungal agents. Lampranthus francisci is an ornamental succulent plant. In Zimbabwe, the fresh sap from the leaves is used to treat fungal scalp infections. The activity of L. francisci fresh and dry acetone, ethanol, hydroethanolic, and aqueous extracts against Candida albicans and Candida krusei was determined. Mouse peritoneal cavity cells and sheep red blood cells were used to investigate L. francisci’s toxicity profile. The hydroethanolic extracts were the most effective extracts against C. albicans. The fresh ethanol extract was the most effective extract against C. krusei. The dry acetone extract, dry ethanol extract, and the fresh and dry aqueous extracts promoted the growth of C. krusei. The hydroethanolic extracts caused haemolysis of sheep cells. The hydroethanolic extracts promoted the growth of the mouse peritoneal cavity red blood cells. Both aqueous extracts increased the density of the mouse cells, but only the fresh extract increased the metabolism of the mouse cells. L. francisci has some fungicidal activity and boosts the growth of immune cells, thus validating its use in ethnomedicine. L. francisci extracts are potential leads for the isolation of immune stimulatory compounds.

Abstract A02: The art of omission: Low fucose antibodies enhance tumor cell clearance in vivo

Abstract The use of monoclonal antibodies (mAbs) in treating cancer patients has become an important addition to chemo- and radiotherapy. Depending on the type of antibody, these mAbs induce a variety of different effector functions, including induction of apoptosis and growth inhibition that can lead to eradication of the tumor. Additionally, tumor targeting mAbs can activate the immune system resulting in induction of complement dependent cytotoxicity (CDC), antibody dependent cellular cytotoxicity (ADCC) or phagocytosis (ADCP) of tumor cells by natural killer cells or myeloid cells (monocyte/macrophages and neutrophils). Currently most clinically used mAbs are primarily chimeric or humanized Immunoglobulin G1 (IgG1) mAbs, which bind to IgG Fc receptors (FcγRs). Efficient binding to FcγRs requires the IgG-Fc-glycan moieties at position 297. In humans, core-fucosylation of IgG is particularly important for binding to human FcγRIIIa and FcγRIIIb, resulting in enhanced uptake of opsonized particles and enhanced ADCC. These properties are increasingly exploited in the next generation of therapeutic anti-tumor IgG. In this study we investigated whether removal of the core fucose also enhanced killing of tumor cells by mouse effector cells, and whether this prevented tumor outgrowth in vivo. Therefore a chimeric IgG1 (TA99), targeting gp75 on B16F10 mouse melanoma cells, was generated with- and without core-fucose, and their effect was assessed in both in vitro and in vivo models. In vitro mouse immune cells were incubated with B16F10-gp75 cells in the presence or absence of wildtype or low fucose TA99 and tumor killing was analyzed. In vivo the effect of the TA99 IgG1 low fucose on tumor outgrowth was assessed by injecting both B16F10-gp75 cells and either TA99 IgG1 wt or low fucose in the peritoneal cavity of mice. Fourteen days post injection tumor outgrowth was analyzed. Mice treated with TA99 low fucose had significantly less tumors in comparison to the wildtype TA99 IgG1, showing that changing the fucosylation of the Fc domain has a remarkable effect on the effectiveness of an antibody therapy. These results emphasize that when the core fucose of therapeutic IgG antibodies is removed, efficacy of monoclonal antibody therapies will be enhanced significantly. Additionally, the effect works cross species, suggesting mouse models are also suitable to study this phenomenon and to screen antibodies in vivo for enhanced effector functions. Ultimately, this may lead to improved clinical outcome of cancer patients. Citation Format: Rens Braster, Remco Visser, Gestur Vidarsson, Marjolein van Egmond. The art of omission: Low fucose antibodies enhance tumor cell clearance in vivo. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A02.

KARAKTERISASI KULTUR MAKROFAG HASIL ISOLASI Mouse Peritoneum Makrofag (MPM)

<p><em>The </em><em>aims </em><em>of t</em><em>his study </em><em>is </em><em>to determine the MPM isolation techniques</em><em>, </em><em>results of </em><em>the </em><em>isolation and identification, so it can be used as a reference for the studies using macrophage cell cultures. The most appropriate source for culturing macrophages are the primary culture of MPM. Resident peritoneal macrophages (RPM) is a free-living phagocytic cells in the peritoneal cavity. Millions of resident macrophages can be taken from the mice. The peritoneal cavity is an easy place to harvest the resident macrophages.</em><em> I</em><em>dentification of</em><em> macrophages was conducted using flow</em><em>cy</em><em>tometr</em><em>y</em><em> </em><em>with</em><em> specific marker CD 11B. While the morphology of macrophages in culture was observed microscopically</em><em> The results </em><em>of the study </em><em>showed the average number of macrophages 1.03 x 106 cells / ml were isolated from peritoneal cavity of mice. The result of identification with flow</em><em>cy</em><em>tometr</em><em>y</em><em> with marker CD 11 B shows positive results macrophages. While the observation of morphological characteristics of macrophages cultures show the same characteristics as the result of research conducted by Kim in 1997 and Wang et al, 2010 so that it can be concluded that macrophages can be isolated from the peritoneal cavity and can be used as a source of primary cultures of macrophages with good results and can be applied to studies related to the function and activation of macrophages in vitro.</em></p> <em>Keyword; Kultur Makrofag, Isolasi Mouse Peritoneum Makrofag (Mpm)</em>

A method for microbial decontamination of Acanthamoeba cultures using the peritoneal cavity of mice

ObjectiveTo evaluate whether the inoculation of contaminated cultures in the peritoneal cavity of mice could implement decontamination of Acanthamoeba cultures. MethodsSuspensions of Acanthamoeba, Acanthamoeba polyphaga ATCC 30461, or Acanthamoeba spp. isolated from soil (UnB13 strain) were inoculated in the peritoneal cavity of Swiss mice (n = 24). After 1, 6, 12, or 24 h of exposure the peritoneal cavity was washed and assessed for the presence of bacteria, fungi, and Acanthamoeba. ResultsAfter 1 h of intraperitoneal inoculation at least 97% of the bacteria and 96% of the fungi (P < 0.05) and 99% of the bacteria (P < 0.05) were successfully eliminated from the ATCC 30461 strain and from the soil isolate UnB13 strain, respectively. This method also allowed the recovery of most trophozoites and cysts from both Acanthamoeba cultures at the end of 24 h. ConclusionsOur data demonstrated that this technique has great potential for decontamination of Acanthamoeba cultures in a short period of time.

Levels of Transforming Growth Factor-Beta After Immunization of Mice With in vivo prepared Toxoplasma gondii Excretory/Secretory Proteins.

Background:Zoonotic parasite Toxoplasma gondii has a high prevalence in human populations. A suitable vaccine for animals can stop the transmission of infection between animal and human.Objectives:The aim of this study was to evaluate in vivo prepared excretory/secretory antigens (E/SA) as a potential candidate for immunization against the parasite and its effect on the production of transforming growth factor-beta (TGF-β).Materials and Methods:Toxoplasma gondii tachyzoites were inoculated in the peritoneal cavity of mice and E/SA was harvested and used in animal immunization with and without adjuvant. Serum levels of anti-E/SA antibodies and TGF-β were measured in days 0, 3, 7, 14, 28 and 56 after immunization using ELISA technique. The measurements were statistically analyzed.Results:Our results showed that the serum levels of anti-E/SA immunoglobulins significantly increased in all of the immunized groups. The differences of the serum levels of TGF-β between the groups were statistically significant at days 28 and 56 after immunization with E/SA.Conclusions:Based on our study, in vivo prepared E/SA may be considered as a good candidate for animal immunization.

Revisiting mouse peritoneal macrophages: heterogeneity, development, and function.

Tissue macrophages play a crucial role in the maintenance of tissue homeostasis and also contribute to inflammatory and reparatory responses during pathogenic infection and tissue injury. The high heterogeneity of these macrophages is consistent with their adaptation to distinct tissue environments and specialization to develop niche-specific functions. Although peritoneal macrophages are one of the best-studied macrophage populations, recently it was demonstrated the co-existence of two subsets in mouse peritoneal cavity (PerC), which exhibit distinct phenotypes, functions, and origins. These macrophage subsets have been classified, according to their morphology, as large peritoneal macrophages (LPMs) and small peritoneal macrophages (SPMs). LPMs, the most abundant subset under steady state conditions, express high levels of F4/80 and low levels of class II molecules of the major histocompatibility complex (MHC). LPMs appear to be originated from embryogenic precursors, and their maintenance in PerC is regulated by expression of specific transcription factors and tissue-derived signals. Conversely, SPMs, a minor subset in unstimulated PerC, have a F4/80lowMHC-IIhigh phenotype and are generated from bone-marrow-derived myeloid precursors. In response to infectious or inflammatory stimuli, the cellular composition of PerC is dramatically altered, where LPMs disappear and SPMs become the prevalent population together with their precursor, the inflammatory monocyte. SPMs appear to be the major source of inflammatory mediators in PerC during infection, whereas LPMs contribute for gut-associated lymphoid tissue-independent and retinoic acid-dependent IgA production by peritoneal B-1 cells. In the previous years, considerable efforts have been made to broaden our understanding of LPM and SPM origin, transcriptional regulation, and functional profile. This review addresses these issues, focusing on the impact of tissue-derived signals and external stimulation in the complex dynamics of peritoneal macrophage populations.

Saha Suppresses Peritoneal Fibrosis in Mice

Long-term peritoneal dialysis causes peritoneal fibrosis in submesothelial areas. However, the mechanism of peritoneal fibrosis is unclear. Epigenetics is the mechanism to induce heritable changes without any changes in DNA sequences. Among epigenetic modifications, histone acetylation leads to the transcriptional activation of genes. Recent studies indicate that histone acetylation is involved in the progression of fibrosis. Therefore, we examined the effect of suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, on the progression of peritoneal fibrosis in mice. Peritoneal fibrosis was induced by the injection of chlorhexidine gluconate (CG) into the peritoneal cavity of mice every other day for 3 weeks. SAHA, or a dimethylsulfoxide and saline vehicle, was administered subcutaneously every day from the start of the CG injections for 3 weeks. Morphologic peritoneal changes were assessed by Masson's trichrome staining, and fibrosis-associated factors were assessed by immunohistochemistry. In CG-injected mice, a marked thickening of the submesothelial compact zone was observed. In contrast, the administration of SAHA suppressed the progression of submesothelial thickening and type III collagen accumulation in CG-injected mice. The numbers of fibroblast-specific protein-1-positive cells and α-smooth muscle actin α-positive cells were significantly decreased in the CG + SAHA group compared to that of the CG group. The level of histone acetylation was reduced in the peritoneum of the CG group, whereas it was increased in the CG + SAHA group. Our results indicate that SAHA can suppress peritoneal thickening and fibrosis in mice through up-regulation of histone acetylation. These results suggest that SAHA may have therapeutic potential for treating peritoneal fibrosis.

In vivo administration of the frog skin peptide frenatin 2.1S induces immunostimulatory phenotypes of mouse mononuclear cells

Host-defense peptides secreted by epithelial cells exhibit cytotoxic and immunoregulatory effects in order to protect the organism against invading microorganisms. Antimicrobial peptides derived from frog skin display both immunostimulatory and immunosuppressive actions as demonstrated by in vitro cytokine production by macrophages. Frenatin 2.1S, first isolated from skin secretions of the frog, Sphaenorhynchus lacteus (Hylidae), enhances the in vitro production of pro-inflammatory IL-1β, TNF-α and IL-23 by mouse peritoneal cells. In order to test whether the immunostimulatory action of frenatin 2.1S may be reproduced in vivo, effects of intraperitoneal injections of this peptide on mononuclear cells in the peritoneum and spleen were determined 24h after administration. The data indicate that frenatin 2.1S enhances the activation state and homing capacity of Th1 type lymphocytes and NKT cells in the mouse peritoneal cavity, as evaluated by increased expression of early activation marker CD69 among T and NKT cells and chemokine receptor CXCR3 among T cells. Frenatin 2.1S significantly increases the percentage of (F4/80+CD11c+CD206+) pro-inflammatory M1 macrophages and enhances the expression of MHC class II molecules on F4/80+CD11c+ macrophages in the mouse peritoneal cavity. Additionally, injection of frenatin 2.1S, in the presence or absence of lipopolysaccharide, increases the percentage of peritoneal B cells of the (CD19+CD11b+CD5+) B1a phenotype thus contributing to an inflammatory milieu. We suggest that the immunostimulatory effect of frenatin 2.1S may have therapeutic relevance in disease states, such as certain types of cancer, in which an enhanced inflammatory response may be beneficial.

AMA1-deficient Toxoplasma gondii parasites transiently colonize mice and trigger an innate immune response that leads to long-lasting protective immunity.

The apical membrane antigen 1 (AMA1) protein was believed to be essential for the perpetuation of two Apicomplexa parasite genera, Plasmodium and Toxoplasma, until we genetically engineered viable parasites lacking AMA1. The reduction in invasiveness of the Toxoplasma gondii RH-AMA1 knockout (RH-AMA1(KO)) tachyzoite population, in vitro, raised key questions about the outcome associated with these tachyzoites once inoculated in the peritoneal cavity of mice. In this study, we used AMNIS technology to simultaneously quantify and image the parasitic process driven by AMA1(KO) tachyzoites. We report their ability to colonize and multiply in mesothelial cells and in both resident and recruited leukocytes. While the RH-AMA1(KO) population amplification is rapidly lethal in immunocompromised mice, it is controlled in immunocompetent hosts, where immune cells in combination sense parasites and secrete proinflammatory cytokines. This innate response further leads to a long-lasting status immunoprotective against a secondary challenge by high inocula of the homologous type I or a distinct type II T. gondii genotypes. While AMA1 is definitively not an essential protein for tachyzoite entry and multiplication in host cells, it clearly assists the expansion of parasite population in vivo.

Molecular control of PtdIns(3,4,5)P3 signaling in neutrophils.

Neutrophils play critical roles in innate immunity and host defense. However, excessive neutrophil accumulation or hyper-responsiveness of neutrophils can be detrimental to the host system. Thus, the response of neutrophils to inflammatory stimuli needs to be tightly controlled. Many cellular processes in neutrophils are mediated by localized formation of an inositol phospholipid, phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3), at the plasma membrane. The PtdIns(3,4,5)P3 signaling pathway is negatively regulated by lipid phosphatases and inositol phosphates, which consequently play a critical role in controlling neutrophil function and would be expected to act as ideal therapeutic targets for enhancing or suppressing innate immune responses. Here, we comprehensively review current understanding about the action of lipid phosphatases and inositol phosphates in the control of neutrophil function in infection and inflammation.

Ciclamilast ameliorates adjuvant-induced arthritis in a rat model.

We assessed the effect of a novel and selective phosphodiesterase 4 (PDE4) inhibitor, ciclamilast, on chronic inflammation in adjuvant-induced arthritis (AIA), a rat model of rheumatoid arthritis (RA), and acute inflammation in the rat and mouse model of carrageenan-induced paw edema and peritonitis. Our results showed that daily oral administration of ciclamilast at 1, 3, and 10 mg/kg dose-dependently inhibited the increase in hind paw volume of rats with AIA. The inhibition of paw edema was associated with inhibition of both the production of cytokines such as TNF-α, IL-1β, and IL-6 and cell infiltration assessed in subcutaneous paw tissue. Moreover, there was significantly less tissue destruction in the ciclamilast-treated rats compared to the vehicle-treated rats, as assessed by radiographic analysis and histopathological evaluation. In the two acute inflammation models, ciclamilast inhibited carrageenan-induced paw edema in rats and inflammatory cell migration into the peritoneal cavity in mice in a dose-dependent manner. These results not only suggest that ciclamilast, as a disease-modifying antirheumatic drug (DMARD), can attenuate RA but also provide proof of principle that a PDE4 inhibitor may be useful for the treatment of arthritis.

Opposite Effects of M1 and M2 Macrophages on Hematopoietic Stem Cell Self-Renewal and Ex Vivo Expansion

Macrophages (MΦ) are professional phagocytes in the innate immune system. They are not only involved in regulation of various immune functions and inflammation, but also exhibit plasticity in modulation of tissue regeneration and repair after being polarized into M1 and M2 MΦ by different inflammatory cytokines. In addition, several recent studies show that MΦ are a new constituent of the hematopoietic stem cells (HSCs) niche and play a role in regulation of HSCs maintenance and mobilization in bone marrow (BM). However, it is not known whether MΦ can regulate HSCs self-renewal and whether the effects of MΦ on HSCs can be influenced by differential MΦ polarization. This was investigated using an ex vivo HSCs expansion model consisting of mouse bone marrow LSK (Lin-sca-1+c-Kit+) cells cultured with or without MΦ in a mouse HSCs expansion medium (StemSpanTM serum-free medium supplemented with 20ng/ml of stem cell factor [SCF] and thrombopoietin [TPO]). We found that LSK cells were expanded about 20-, 15-, and 30-fold after 6 days of co-culture with MΦ harvested from mouse BM, spleen, and peritoneal cavity, respectively, whereas there was no significant expansion after culture without MΦ or with BM Gr-1high or Gr-1low monocytes. In addition, we found that M1-MΦ polarized by INFγ were more effective than IL4-polarized M2-MΦ in promoting LSK cells expansion ex vivo (45-fold vs. 15-fold). However, the promotion of LSK cells expansion by M1-MΦ resulted in about 88% reduction in HSCs as judged by 5-week cobblestone area forming cell (CAFC) assay. In contrast, M2-MΦ significantly promoted HSCs expansion. A greater expansion of HSCs was achieved after LSK cells were co-cultured with M2-MΦ for 9 days than for 6 days (20-fold vs. 6-fold). These findings suggest that M1-MΦ are more effective than M2-MΦ in promoting LSK cells or hematopoietic progenitor cells (HPCs) expansion, at the expense of HSCs self-renewal, whereas M2-MΦ can promote HPCs expansion as well as HSCs self-renewal. This suggestion is supported by results of serial transplantation and competitive repopulation unit (CRU) assays. CRU assay showed that LT-HSCs (e.g. 4-month CRU) were increased about 13 folds relative to the starting numbers of CRU in the input after LSK cells were co-cultured with M2-MΦ for 9 days, but were barely detectable after the cells were cultured without MΦ or with M1-MΦ. The inhibitory effect of M1-MΦ on HSCs self-renewal and expansion was attenuated by inhibition of inducible nitric oxide synthase (iNOS) activity with an inhibitor or knockout iNOS. Inhibition of arginase and/or cyclooxygenase activities with an inhibitor attenuated the promotion of HSCs self-renewal and expansion by M2-MΦ. More importantly, we found that human CD34+ cells, 8-week CAFC, and SCID mice repopulating cells (SRCs) were increased 42±14, 8±2.1, and 4 folds over the input values, respectively, after human cord blood CD34+ cells were co-cultured with M2-MΦ generated from human cord blood CD34- cells for 7 days in a human HSCs expansion medium (StemSpanTM serum-free medium supplemented with 50 ng/ml of SCF, TPO, and FLT-3 ligand). These findings demonstrate that M1-MΦ and M2-MΦ have opposite effects on HSCs self-renewal, which may be important for regulation of hematopoiesis under various pathological conditions in which MΦ are differentially polarized to M1 or M2 by diverse inflammatory cytokines. In addition, M2-MΦ may be used to promote human cord blood HSCs ex vivo expansion to make human cord blood transplantation available to more patients. DisclosuresNo relevant conflicts of interest to declare.

Therapeutic adenoviral gene transfer of a glycosyltransferase for prevention of peritoneal dissemination and metastasis of gastric cancer.

Increased expression of sialyl Lewis(x/a) carbohydrates, ligands for E-selectin, correlates with clinically advanced stages and metastasis of gastric and colon cancers. In contrast, Sd(a) carbohydrate is abundantly detected in the normal gastrointestinal mucosa but dramatically reduced or lost in cancer tissues. A glycosyltransferase, β1,4N-acetylgalactosaminyltransferase 2 (B4GALNT2) that catalyzes Sd(a) carbohydrate synthesis, is silenced in cancer. In the present study, we aimed at reducing the expression of sialyl Lewis(x/a) of cancer cells in vivo by forced expression of B4GALNT2 and Sd(a), thereby preventing dissemination/metastasis, especially metastasis triggered by surgical maneuvers. We used a fiber-modified adenovirus (Ad) vector that contained a chimeric construct with a serotype 5 shaft and a serotype 3 knob. Using this Ad5/3 vector, we successfully introduced the B4GALNT2 gene into a human gastric cancer cell line KATO III in vitro and confirmed replacement of sialyl Lewis(x) to Sd(a) with a decrease in E-selectin-dependent adhesion. Administration of Ad5/3-B4GALNT2 vectors into the peritoneal cavity of mice after inoculation of KATO III cells with laparotomy significantly reduced the incidence of metastasis. Our results indicate that the transfer of a single gene encoding B4GALNT2 modified carbohydrate chains of cancer cells in vivo and decreased tumor dissemination and metastasis.

Characterization of the inflammatory response during Ehrlich ascitic tumor development

IntroductionEhrlich tumor is a mammary adenocarcinoma with aggressive behavior. Inoculated in mice peritoneal cavity, the Ehrlich tumor grows in ascitic form (EAT). Since inflammation modulates tumor progression we further investigated the inflammatory response during EAT growth. MethodsBalb/C mice were intraperitoneal inoculated with 5×105 Ehrlich cells and after every 2days, blood samples were collected for hemoglobin, hematocrit, platelets and leukocytes counts. The ascitic fluid was collected for protein concentration and cell count. Phenotype analysis of the peritoneal cells was made by FACS, prostaglandin E2 (PGE2) and cytokines by ELISA, nitric oxide (NO) by nitrate conversion protocol, and cyclooxygenase-1 (COX1), COX2 and inducible nitric oxide synthase (iNOS) by immunoblotting. ResultsFollowing EAT inoculation into the peritoneal cavity there was a rapid increase in ascitis volume and protein concentration. The cell number in ascitis remained stable until day 8 (lag phase) followed by a sharp increase. As tumor progressed, blood leukocytes increased and erythrocyte decreased. Phenotypic analysis showed that during the lag phase the percentage of F4/80+ cells remained similar to control levels and around 7% of this population was also positive for the GR1 marker. These double-positive cells (probably inflammatory monocytes) markedly increased at day 6. The percentage of F4/80-GR1+cells (probably neutrophils) was low and did not significantly vary during tumor progression. CD4+ and CD8+ cells were not detected in the time points analyzed. iNOS and COX1 expression increased after day 2 reaching peak levels on day 10. COX2 enzyme expression did not change significantly over time. Sustained increase in PGE2 and NO levels was observed. IL-10 and MCP-1 peaked at day 14 and IL-1β increased progressively till day 10. IFN-γ levels were low till day 10, increasing progressively after that. DiscussionThese data extended the characterization of the inflammatory response during Ehrlich ascitis tumor growth, further validating it as a useful model for antitumor drugs screening.

Therapy of peritoneally disseminated colon cancer by TAP-deficient embryonic stem cell-derived macrophages in allogeneic recipients.

We established a method to generate a large quantity of myeloid lineage cells from mouse embryonic stem (ES) cells, termed ES cell-derived proliferating myeloid cell lines (ES-ML). ES-ML continuously proliferated in the presence of M-CSF and GM-CSF. ES-ML genetically modified to express an anti-HER2 (neu) mAb single-chain V region fragment reduced the number of cocultured mouse Colon-26 cancer cells expressing HER2. Stimulation of ES-ML with IFN-γ plus LPS or TNF resulted in almost complete killing of the Colon-26 cells by the ES-ML, and the cytotoxicity was mediated, in part, by NO produced by ES-ML. When ES-ML were injected into mice with i.p. established Colon-26 tumors, they efficiently infiltrated the tumor tissues. Injection of ES-ML with rIFN-γ and LPS inhibited cancer progression in the mouse peritoneal cavity. Coinjection of TNF-transfected or untransfected ES-ML with rIFN-γ inhibited cancer growth and resulted in prolonged survival of the treated mice. In this experiment, transporter associated with Ag processing (TAP)1-deficient ES-ML exhibited therapeutic activity in MHC-mismatched allogeneic recipient mice. Despite the proliferative capacity of ES-ML, malignancy never developed from the transferred ES-ML in the recipient mice. In summary, TAP-deficient ES-ML with anticancer properties exhibited a therapeutic effect in allogeneic recipients, suggesting the possible use of TAP-deficient human-induced pluripotent stem cell-derived proliferating myeloid cell lines in cancer therapy.

Megakaryocytic potentiating factor and mature mesothelin stimulate the growth of a lung cancer cell line in the peritoneal cavity of mice.

The mesothelin (MSLN) gene encodes a 71 kilodalton (kDa) precursor protein that is processed into megakaryocytic potentiating factor (MPF), a 31 kDa protein that is secreted from the cell, and mature mesothelin (mMSLN), a 40 kDa cell surface protein. The mMSLN binds to CA125, an interaction that has been implicated in the intra-cavitary spread of mesothelioma and ovarian cancer. To better define the role of MPF and mMSLN, growth of the lung cancer cell line A549 was evaluated in immuno-deficient mice with inactivation of the Msln gene. We observed that Msln–/– mice xenografted with intraperitoneal A549 tumors survive significantly long than tumor-bearing Msln+/+ mice. When tumor-bearing Msln–/– mice are supplemented with recombinant MPF (and to a lesser extent mMSLN), most of this survival advantage is lost. These studies demonstrate that MPF and mMSLN have an important role in the growth of lung cancer cells in vivo and raise the possibility that inactivation of MPF may be a useful treatment for lung and other MSLN expressing cancers.

In vivo anti-inflammatory and analgesic activities of strictosamide from Nauclea officinalis

Context: Strictosamide is the main representative constituent of Nauclea officinalis Pierre ex Pitard (Rubiaceae), which has been used for a long time in China to treat diseases related to infection and inflammation, but its pharmacological activities are not well studied.Objective: This work evaluates the anti-inflammatory and analgesic activities of strictosamide by in vivo experiments.Materials and methods: The anti-inflammatory activity was assessed in mice by models of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ear edema, acetic acid-elevated vascular permeability, and carboxymethylcellulose sodium (CMC–Na)-induced leukocyte migration. The analgesic activity was estimated in mice using acetic acid-induced writhing and hot-plate tests. Compound was injected to mice twice a day for 3 d at doses of 10, 20, and 40 mg/kg.Results: At 20 and 40 mg/kg, strictosamide obviously decreased the TPA-induced mice ear edema (24.7 and 28.1% inhibition, respectively), and significantly inhibited acetic acid-stimulated peritoneal vascular permeability in mice (23.3 and 33.4% inhibition, respectively). It also significantly decreased the leukocytes in the mice peritoneal cavity induced by CMC–Na at all the tested doses (46.0, 49.1, and 58.7% inhibition, respectively). To acetic acid-induced writhing test in mice, strictosamide markedly prolonged the pain latency at 20 and 40 mg/kg and decreased the writhing counts at 40 mg/kg (49.7% inhibition). However, it did not obviously improve the pain threshold of mice in hot-plate test.Discussion and conclusion: Strictosamide may have important effects on inflammation and inflammatory pain. The results provide scientific support for the role of strictosamide in the use of N. officinalis to treat inflammatory diseases.

Blockade of CD47-Signaling Regulatory Protein alpha Signaling Enhances the Macrophage Phagocytic Activity Against Cancer Cells.

Background: Liver transplantation (LT) is the best treatment for unresectable, early-stage hepatocellular carcinoma (HCC). However, HCC recurrence is common, due to either occult metastasis development or circulating tumor cell engraftment. We previously demonstrated that the phagocytic activity of human liver macrophages is inhibited by the binding of signaling regulatory protein alpha (SIRPα) on macrophages to CD47 on target cells, a major regulator of phagocytic responses (2006 PNAS). Using in vitro and in vivo mouse syngeneic models, we examined whether the inhibition of CD47-SIRPα signaling would increase macrophage phagocytic activity against cancer cells. Methods: For the in vitro phagocytosis assay, Hepa1-6 and CMT93 target cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) and incubated with mouse peritoneal cavity (PerC) macrophages and either anti-mouse SIRPα-blocking monoclonal antibodies (mAbs) (SIRPα mAbs) or isotype-matched control antibodies (isotype Abs). The phagocytosis of CFSE-labeled target cells was measured by flow cytometry. For the in vivo phagocytosis assay, CFSE-labeled target cells were injected into the peritoneal cavity, together with SIRPα mAbs, CD47 mAbs, or isotype Abs. CD47 knock-down (KD) was achieved via lentiviral particle-mediated shRNA delivery to Hepa1-6 cells, which were used in both in vitro and in vivo assays. Results: In the in vitro phagocytosis assay, anti-SIRPα mAb addition markedly enhanced the phagocytic activity of PerC macrophages against Hepa1-6 and CMT93, compared with isotype Abs treatment (p < 0.01). In the in vivo phagocytosis assay, a significant increase in phagocytic activity (p < 0.01) was observed with SIRPα mAb treatment, but not with CD47 mAb treatment. CD47 KD Hepa1-6 cells were significantly more sensitive to macrophage phagocytosis than parental Hepa1-6 cells in both in vitro and in vivo assays (p = 0.01), demonstrating the pivotal role of the CD47-SIRPα interaction in restricting tumor cell killing. In vivo CD47 mAb administration led to a remarkable decrease in CD80, CD86, and MHC class II expression on macrophages, implying their functional impairment. This finding might explain why treatment with CD47 mAbs alone did not enhance macrophage phagocytic activity against cancer cells. Conclusions: CD47-SIRPα interaction could be a therapeutic target for preventing HCC recurrence in LT recipients.