Comparison of the mouse spleen cell assay and a radioimmunoassay for the measurement of serum erythropoietin

The mouse spleen cell assay (MSCA) has been compared with a radioimmunoassay for the measurement of serum erythropoietin (Ep). In 20 normal subjects the serum values ranged from 15 to 73 mU/ml for the MSCA compared with 5-30 mU/ml for the RIA. For normal sera there was no correlation between the results of the two assays. In 37 patients with anaemias of differing aetiologies and at various stages of treatment values ranged from 10 to 3645 mU/ml for the MSCA and 13-10,000 mU/ml for the RIA. Although patient values from the two assays were highly correlated (r = 0.98, P less than 0.001), the MSCA results were generally lower. These discrepancies can be largely accounted for by two factors. Firstly the MSCA is sensitive to non-specific matrix effects. Secondly, heat inactivation of serum, a prerequisite for the MSCA, but not for the RIA, destroys a variable and unpredictable proportion of the Ep in the test sera leading to an underestimation of Ep in the MSCA. We conclude that the RIA is more reliable than the MSCA which, in its present form, cannot be recommended for the accurate measurement of serum erythropoietin.

Identification of mouse langerin/CD207 in Langerhans cells and some dendritic cells of lymphoid tissues

Human (h)Langerin/CD207 is a C-type lectin of Langerhans cells (LC) that induces the formation of Birbeck granules (BG). In this study, we have cloned a cDNA-encoding mouse (m)Langerin. The predicted protein is 66% homologous to hLangerin with conservation of its particular features. The organization of human and mouse Langerin genes are similar, consisting of six exons, three of which encode the carbohydrate recognition domain. The mLangerin gene maps to chromosome 6D, syntenic to the human gene on chromosome 2p13. mLangerin protein, detected by a mAb as a 48-kDa species, is abundant in epidermal LC in situ and is down-regulated upon culture. A subset of cells also expresses mLangerin in bone marrow cultures supplemented with TGF-beta. Notably, dendritic cells in thymic medulla are mLangerin-positive. By contrast, only scattered cells express mLangerin in lymph nodes and spleen. mLangerin mRNA is also detected in some nonlymphoid tissues (e.g., lung, liver, and heart). Similarly to hLangerin, a network of BG form upon transfection of mLangerin cDNA into fibroblasts. Interestingly, substitution of a conserved residue (Phe(244) to Leu) within the carbohydrate recognition domain transforms the BG in transfectant cells into structures resembling cored tubules, previously described in mouse LC. Our findings should facilitate further characterization of mouse LC, and provide insight into a plasticity of dendritic cell organelles which may have important functional consequences.

Pierre Bourdieu as a Sociologist of the Economy and Critic of 'Globalisation'

This article examines Pierre Bourdieu's sociology of the economy and his more recent politically engaged interventions on 'globalisation'. Many scholars regard these as not being in the same academic league as his classic studies on taste, academia, and state elites, etc., and, instead, dismiss them as a private matter or even, as the spleen of Pierre Bourdieu, the individual. This paper questions this disjunction of the 'academic' and 'politically engaged' sides of Pierre Bourdieu's work. First, it argues that his most recent interventions against a neo-liberal globalisation were the logical result of a particular definition of intellectual practice that had been outlined before in his sociology of the intellectual field. It then demonstrates that Bourdieu's economic sociology and critique of contemporary capitalism not only does not contradict his earlier research, but that it provides valuable and original insights into the current transformation of the political economy of the advanced capitalist countries. The paper concludes with a suggestion of how to strengthen the theoretical foundation of Bourdieu's analysis of contemporary capitalism by relating it to and making it compatible with alternative approaches in the tradition of critical political economy.

Epidermal Langerhans Cells are Dispensable for Humoral and Cell-Mediated Immunity Elicited by Gene Gun Immunization.

Gene gun immunization, i.e., bombardment of skin with DNA-coated particles, is an efficient method for the administration of DNA vaccines. Direct transfection of APC or cross-presentation of exogenous Ag acquired from transfected nonimmune cells enables MHC-I-restricted activation of CD8(+) T cells. Additionally, MHC-II-restricted presentation of exogenous Ag activates CD4(+) Th cells. Being the principal APC in the epidermis, Langerhans cells (LC) seem ideal candidates to accomplish these functions. However, the dependence on LC of gene gun-induced immune reactions has not yet been demonstrated directly. This was primarily hampered by difficulties to discriminate the contributions of LC from those of other dermal dendritic cells. To address this problem, we have used Langerin-diphtheria toxin receptor knockin mice that allow for selective inducible ablation of LC. LC deficiency, even over the entire duration of experiments, did not affect any of the gene gun-induced immune functions examined, including proliferation of CD4(+) and CD8(+) T cells, IFN-gamma secretion by spleen cells, Ab production, CTL activity, and development of protective antitumor immunity.

The beta-(1--

The stimulatory effects of the synthetic beta-(1-->6)-branched beta-(1-->3) glucohexaose and its analogues containing an alpha-(1-->3)-linked bond on the mouse spleen were studied for elucidation of the mechanism of their antitumor activity, and their stimulatory effects were compared with Lentinan. The mouse spleen's weight was increased after the intraperitoneal (i.p.) injection of the oligosaccharides compared with the saline group. In addition, routinely hematoxylin and eosin (HE)-stained spleen sections showed that the injection also changed the spleen's histopathology. RNA samples were isolated from splenocytes of oligosaccharides, Lentinan or saline-injected mice. Reverse transcription-polymerase chain reaction (RT-PCR) and Northern blot showed that the administration of the oligosaccharides or Lentinan enhanced mouse spleen mRNA production of TNF-alpha but not IL-2. The injection also enhanced Concanavalin A (Con A)-induced mouse splenocytes proliferation, but the in vitro administration of the oligosaccharides did not have the proliferation-enhancing effect. Taken together, these results suggest that the synthetic beta-(1-->6)-branched beta-(1-->3) glucohexaose and its analogues containing an alpha-(1-->3)-linked bond have similar stimulatory effects as Lentinan. Additionally, they may exert their antitumor effects through the induction of splenocytes mediated immune responses.

Nitric oxide synthase gene therapy enhances the toxicity of cisplatin in cancer cells

BACKGROUND AIMS: Cell-based gene therapy is an alternative to viral and non-viral gene therapy. Emerging evidence suggests that mesenchymal stem cells (MSC) are able to migrate to sites of tissue injury and have immunosuppressive properties that may be useful in targeted gene therapy for sustained specific tissue engraftment. METHODS: In this study, we injected intravenously (i.v.) 1x10(6) MSC, isolated from green fluorescent protein (GFP) transgenic rats, into Rif-1 fibrosarcoma-bearing C3H/HeN mice. The MSC had been infected using a lentiviral vector to express stably the luciferase reporter gene (MSC-GFP-luci). An in vivo imaging system (IVIS 200) and Western blotting techniques were used to detect the distribution of MSC-GFP-luci in tumor-bearing animals. RESULTS: We observed that xenogenic MSC selectively migrated to the tumor site, proliferated and expressed the exogenous gene in subcutaneous fibrosarcoma transplants. No MSC distribution was detected in other organs, such as the liver, spleen, colon and kidney. We further showed that the FGF2/FGFR pathways may play a role in the directional movement of MSC to the Rif-1 fibrosarcoma. We performed in vitro co-culture and in vivo tumor growth analysis, showing that MSC did not affect the proliferation of Rif-1 cells and fibrosarcoma growth compared with an untreated control group. Finally, we demonstrated that the xenogenic MSC stably expressing inducible nitric oxide synthase (iNOS) protein transferred by a lentivirus-based system had a significant inhibitory effect on the growth of Rif-1 tumors compared with MSC alone and the non-treatment control group. CONCLUSIONS: iNOS delivered by genetically modified iNOS-MSC showed a significant anti-tumor effect both in vitro and in vivo. MSC may be used as a target gene delivery vehicle for the treatment of fibrosarcoma and other tumors

Differential Effect of IL-27 on Developing versus Committed Th17 Cells

IIL-27 counters the effect of TGF-beta+IL-6 on naive CD4(+) T cells, resulting in near complete inhibition of de novo Th17 development. In contrast, little is known about the effect of IL-27 on already differentiated Th17 cells. A better understanding of how IL-27 regulates these cells is needed to evaluate the therapeutic potential of IL-27 in Th17 cells-associated diseases. In this study, we show that IL-27 had surprisingly little effect on committed Th17 cells, despite its expression of a functional IL-27R. Contrary to de novo differentiation of Th17 cells, IL-27 did not suppress expression of retinoid-related orphan receptor (ROR)gammat or RORalpha in committed Th17 cells. Consistent with this finding, the frequency of committed Th17 cells and their cytokine secretion remained unaffected by IL-27. Both memory Th17 cells (CD4(+)CD25(-)CD62L(low)) that developed in vivo and encephalitogenic Th17 cells infiltrating the CNS of mice developing experimental autoimmune encephalomyelitis produced similar amounts of IL-17A when reactivated with IL-23 in the absence and presence of exogenous IL-27. Finally, IL-27 failed to suppress encephalitogenicity of Th17 cells in an adoptive transfer of experimental autoimmune encephalomyelitis. Analysis ex vivo of transferred Th17 cells in the spleen and CNS of recipient mice showed that cells retained similar phenotype irrespective of whether cells were treated or not with IL-27. Our data demonstrate that in contrast to inhibition of de novo differentiation of Th17 cells, IL-27 has little or no effect on committed Th17 cells. These findings indicate that therapeutic applications of IL-27 might have a limited efficacy in inflammatory conditions where aggressive Th17 responses have already developed.

IL-12R2 promotes the development of CD4+CD25+ regulatory T cells

We have previously shown that mice lacking the IL-12-specific receptor subunit ß2 (IL-12Rß2) develop more severe experimental autoimmune encephalomyelitis than wild-type (WT) mice. The mechanism underlying this phenomenon is not known; nor is it known whether deficiency of IL-12Rß2 impacts other autoimmune disorders similarly. In the present study we demonstrate that IL-12Rß2-/- mice develop earlier onset and more severe disease in the streptozotocin-induced model of diabetes, indicating predisposition of IL-12Rß2-deficient mice to autoimmune diseases. T cells from IL-12Rß2-/- mice exhibited significantly higher proliferative responses upon TCR stimulation. The numbers of naturally occurring CD25+CD4+ regulatory T cells (Tregs) in the thymus and spleen of IL-12Rß2-/- mice were comparable to those of WT mice. However, IL-12Rß2-/- mice exhibited a significantly reduced capacity to develop Tregs upon stimulation with TGF-ß, as shown by significantly lower numbers of CD25+CD4+ T cells that expressed Foxp3. Functionally, CD25+CD4+ Tregs derived from IL-12Rß2-/- mice were less efficient than those from WT mice in suppressing effector T cells. The role of IL-12Rß2 in the induction of Tregs was confirmed using small interfering RNA. These findings suggest that signaling via IL-12Rß2 regulates both the number and functional maturity of Treg cells, which indicates a novel mechanism underlying the regulation of autoimmune diseases by the IL-12 pathway. Copyright © 2008 by The American Association of Immunologists, Inc.

Overexpression of endoplasmic reticulum protein 29 regulates mesenchymal-epithelial transition and suppresses xenograft tumor growth of invasive breast cancer cells

Endoplasmic reticulum protein 29 (ERp29) is a novel endoplasmic reticulum ( ER) secretion factor that facilitates the transport of secretory proteins in the early secretory pathway. Recently, it was found to be overexpressed in several cancers; however, little is known regarding its function in breast cancer progression. In this study, we show that the expression of ERp29 was reduced with tumor progression in clinical specimens of breast cancer, and that overexpression of ERp29 resulted in G(0)/G(1) arrest and inhibited cell proliferation in MDA-MB-231 cells. Importantly, overexpression of ERp29 in MDA-MB-231 cells led to a phenotypic change and mesenchymal-epithelial transition (MET) characterized by cytoskeletal reorganization with loss of stress fibers, reduction of fibronectin (FN), reactivation of epithelial cell marker E-cadherin and loss of mesenchymal cell marker vimentin. Knockdown of ERp29 by shRNA in MCF-7 cells reduced E-cadherin, but increased vimentin expression. Furthermore, ERp29 overexpression in MDA-MB-231 and SKBr3 cells decreased cell migration/invasion and reduced cell transformation, whereas silencing of ERp29 in MCF-7 cells enhanced cell aggressive behavior. Significantly, expression of ERp29 in MDA-MB-231 cells suppressed tumor formation in nude mice by repressing the cell proliferative index (Ki-67 positivity). Transcriptional profiling analysis showed that ERp29 acts as a central regulator by upregulating a group of genes with tumor suppressive function, for example, E-cadherin (CDH1), cyclin-dependent kinase inhibitor (CDKN2B) and spleen tyrosine kinase (SYK), and by downregulating a group of genes that regulate cell proliferation (eg, FN, epidermal growth factor receptor ( EGFR) and plasminogen activator receptor ( uPAR)). It is noteworthy that ERp29 significantly attenuated the overall ERK cascade, whereas the ratio of p-ERK1 to p-ERK2 was highly increased. Taken together, our results showed that ERp29 is a novel regulator leading to cell growth arrest and cell transition from a proliferative to a quiescent state, and reprogramming molecular portraits to suppress the tumor growth of MDA-MB-231 breast cancer cells. Laboratory Investigation (2009) 89, 1229-1242; doi: 10.1038/labinvest.2009.87; published online 21 September 2009

Innate immunogenicity and in vitro protective potential of Schistosoma mansoni lung schistosomula excretory-secretory candidate vaccine antigens

Excretory secretory products (ESP) of Schistosoma mansoni developing larvae are ideal potential vaccines as such molecules may readily induce host primary immune responses, and local memory immune response effectors that would target, surround, and pursue the larvae while negotiating the lung blood capillaries. We herein characterized the cytokines response ESP, e.g., SG3PDH, 14-3-3-like protein, TPX, and calpain induce in the natural context of infection, and defined the global cytokine profile conducive to effective schistosome larvae killing. Accordingly, spleen cells (SC) taken from naive, and 7-, or 9-day S. mansoni-infected mice were stimulated in vitro with the selected ESP, in a recombinant or multiple antigen peptide (MAP) form, and examined for production of T helper type (Th) 1, Th2, and Th17 cytokines, and the ability to mediate in vitro attrition of lung-stage schistosomula. The study indicated that larval ESP principally elicit Th1 and Th17 type cytokines. Recombinant SG3PDH was the only test ESP to additionally activate SC from S. mansoni-infected BALB/c mice to release higher IL-4 levels than unstimulated SC and mediate significant (P

PREVENTION OF INFECTION AFTER SPLENECTOMY - WHAT PRECAUTIONS ARE REQUIRED

Removal of the spleen presents a lifelong risk of infection, in particular the syndrome of overwhelming postsplenectomy sepsis. Streptococcus pneumoniae, Haemophilus influenzae and Neisseria meningitides are the most common organisms involved, but malaria, babesiosis and DF-2 also create a problem. Immunisation with pneumococcal vaccine, H. influenzae type b vaccine, influenza vaccine and, if in a high risk area, meningococcal vaccine is recommended. Lifelong phenoxymethylpenicillin 250mg twice daily is also advised, especially in high risk groups such as children and immunocompromised patients. If patients are unwilling to take medicine lifelong, or are unlikely to comply, an antibiotic supply should be made available at all times and administration should commence at the first sign of illness.

The effect of hypobaric hypoxia on misonidazole binding in normal and tumour-bearing mice

The effect of hypobaric hypoxia on the in vivo binding of misonidazole was investigated in normal mice and mice bearing T50/80 or CA NT mammary carcinomas. After the intraperitoneal injection of radiolabelled misonidazole, mice were randomised to breathe either room air or air at 0.5 atmospheres. The distribution of misonidazole in liver, kidney, heart, spleen and tumour tissue, 24 h later, was studied by scintillation counting and by autoradiography. Significantly higher misonidazole binding occurred in the livers (x2.5), kidneys (x2.4), spleens (x2.9) and hearts (x1.8) of hypoxic mice compared to controls. Hypobaric hypoxia was associated with a greater than four-fold increase in misonidazole binding within T50/80 tumours. However, significantly higher binding was not demonstrated within CA NT tumours after exposure of tumour-bearing animals to hypoxic conditions. In autoradiographs of hypoxic liver, labelling was intense in regions near to hepatic veins but sparse in areas surrounding portal tracts. This pattern was striking and consistent. In hypoxic kidney, labelling was most intense over tubular cells, less intense over glomeruli and sparse in the renal medulla. It is likely that the hepatic and renal cortical distributions of misonidazole binding reflect local oxygen gradients.

CD11b+ bone marrow-derived monocytes are the major leukocyte subset responsible for retinal capillary leukostasis in experimental diabetes in mouse and express high levels of CCR5 in the circulation

We investigated the phenotype of cells involved in leukostasis in the early stages of streptozotocin-induced diabetes in mice by direct observation and by adoptive transfer of calcein-AM-labeled bone marrow-derived leukocytes from syngeneic mice. Retinal whole mounts, confocal microscopy, and flow cytometry ex vivo and scanning laser ophthalmoscopy in vivo were used. Leukostasis in vivo and ex vivo in retinal capillaries was increased after 2 weeks of diabetes (Hb A(1c), 14.2 ± 1.2) when either donor or recipient mice were diabetic. Maximum leukostasis occurred when both donor and recipient were diabetic. CD11b(+), but not Gr1(+), cells were preferentially entrapped in retinal vessels (fivefold increase compared with nondiabetic mice). In diabetic mice, circulating CD11b(+) cells expressed high levels of CCR5 (P = 0.04), whereas spleen (P = 0.0001) and retinal (P = 0.05) cells expressed increased levels of the fractalkine chemokine receptor. Rosuvastatin treatment prevented leukostasis when both recipient and donor were treated but not when donor mice only were treated. This effect was blocked by treatment with mevalonate. We conclude that leukostasis in early diabetic retinopathy involves activated CCR5(+)CD11b(+) myeloid cells (presumed monocytes). However, leukostasis also requires diabetes-induced changes in the endothelium, because statin therapy prevented leukostasis only when recipient mice were treated. The up-regulation of the HMG-CoA reductase pathway in the endothelium is the major metabolic dysregulation promoting leukostasis.