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Cutaneous leishmaniasis is an infectious disease that is endemic especially in tropical and desert regions with an incidence of 1.5 million cases per year and a prevalence of 12 million people infected worldwide. The infection can be caused by the intracellular parasite Leishmania major. The disease has been studied extensively in the murine model. It has become apparent that the induction of a class of interferon (IFN)--producing CD4+ T helper cells (TH1 cells) that activate macrophages to kill the parasites they harbor is desicive for the establishment of immunity. The redirection of the host’s immune response towards a protective TH1 phenotype will also be the key to an effective vaccine. Dendritic cells (DC) loaded with leishmanial antigens ex vivo were lately described as vaccines against L. major infections. One single recombinant Leishmania antigen, LeIF (Leishmania homologue of eukaryotic ribosomal initiation factor 4a), which was identified as a protein that stimulates DC to secrete interleukin (IL)-12 and discussed as a pattern-associated molecular pattern (PAMP), was found to mediate a protective TH1-dependent effect when used for pulsing of DC. The application of recombinant proteins is tied to many disadvantages, which is why other methods of antigen administration have been developed. RNA electroporation of DC has recently emerged from tumor research as a safe and versatile method of antigen delivery, by which a large number of RNA molecules encoding a specific antigen gains access to the cytosol of DC by an electrical impulse. The present study describes, for the first time, transfection of DC with RNA encoding a molecularly defined parasite antigen. Initially, a standardized protocol for RNA transfection was established, using the enhanced green fluorescent protein (EGFP) as reporter antigen. EGFP-RNA was well translatable in an in vitro translation system, and both a DC cell line (fetal skin-derived DC; FSDC) and murine primary bone marrow-derived DC (BMDC) could be transfected efficiently, with a yield of up to 90% and 75%, respectively. In both cell types, maximal transfection efficiency was attained with 20 µg RNA and could not be further increased with larger amounts of RNA. The level of antigen expression, measured as the mean fluorescence intensity (MFI) by flow cytometry, was directly proportional to the amount of RNA used for transfection. In FSDC, transfection efficiency and MFI were generally higher than in BMDC when the same amounts of RNA were used. Furthermore, the kinetics was shown to be sensitive to treatment with lipopolysaccharide (LPS): the expression peak was higher and was reached sooner, followed by a more rapid decline. In transfection experiments with LeIF, two variants of LeIF-RNA were used: LeIF(fl)-RNA, encoding the complete LeIF sequence, and LeIF(226)-RNA, encoding only the aminoterminal half of the LeIF sequence (226 amino acids), the immunogenic part of LeIF. Only LeIF(fl) was detectable by Western Blot in whole cell lysates of BMDC after LeIF(fl)-RNA transfection, whereas LeIF(226) could never be detected in LeIF(226)-transfected BMDC. However, as both constructs were well translatable in a cell-free system, the failure to detect LeIF(226) in BMDC lysates did not represent a failure in RNA translation, but rather a rapid antigen degradation. It was therefore expected that LeIF(226)-transfected BMDC should nevertheless be able to present LeIF(226)-derived antigenic peptides to T cells from BALB/c mice primed with recombinant LeIF (rLeIF). This hypothesis was confirmed by measuring IFN- production in BMDC-T cell co-incubation assays, showing that rLeIF-pulsed, LeIF(226)- and LeIF(fl)-transfected day 7 BMDC did indeed activate T cells from LeIF-immunized mice in an antigen-specific manner. In contrast, IL-4 was not produced, which was consistent with the fact that T cells found in lymph nodes from LeIF-primed mice are primarily of the TH1 type. In the supernatants of LeIF-transfected BMDC cultures, in contrast to rLeIF-pulsed BMDC, the proinflammatory cytokines IL-1β, IL-6, IL-10 and IL-12 were not detected. This effect was not due to the electroporation procedure, as cytokine production by BMDC electroporated with rLeIF was only partially impaired. Also, the expression levels of CD86 were lower upon LeIF transfection than after pulsing with rLeIF. Thus, LeIF transfection did not induce maturation of DC. In conclusion, LeIF-transfected BMDC may have acted as semi-mature antigen-specific tolerance inducers, with regulatory T cells as responders. The effect of LeIF transfection on the immunostimulatory capacity of BMDC was not significantly increased when day 8 or 9 BMDC were used. However, day 8, and even more day 9 BMDC pulsed with rLeIF mounted a vigorous T cell response. Day 9 BMDC were able to activate naïve T cells. In conclusion, before a strong T cell response against LeIF can be induced, DC need to – besides presenting antigen and expressing co-stimulatory molecules – exhibit a susceptibility to the innate signaling molecule LeIF which is linked to their maturation age. This third signal is provided by extracellular rLeIF, but it is not conveyed – or is suppressed – by intracellular LeIF after LeIF-RNA transfection. Furthermore, electroporation of rLeIF abrogated IL-12 production by BMDC completely, the production of IL-1 was reduced with higher antigen doses, and the production of IL-10 was partially increased. The IL-6 production was unaffected. This altered cytokine profile suggests that LeIF as a PAMP might have a bipartite nature: besides exhibiting the capacity to stimulate IL-12 production upon extracellular presence, thereby enhancing host resistance against L. major, LeIF could also contribute to parasitic host evasion mechanisms from intracellular compartments of DC, possibly by interfering with mitogen-activated protein (MAP) kinase signaling pathways. Thus, the adjuvant properties of LeIF depend both on its mode of delivery (transfection with RNA vs. pulsing with the recombinant protein) and the targeted compartment (extra- vs. intracellular). From this work, it can be summarized that BMDC are well transfectable with a parasite antigen. The antigen is processed and presented, but it is not recognized as a PAMP by DC. Hence, transfection with antigen-encoding mRNA by itself does not convey all necessary signals for the elicitation of a potent immune response.
Regulation of B lymphocyte terminal differentiation and death by the transcription factor Blimp-1
(2005)
B lymphocyte induced maturation protein-1 (Blimp-1) and X-box-binding protein-1 (XBP-1) are indispensible transcription factors required for B lymphocyte terminal differentiation into Ig secreting plasma cells. Occurrence of an unfolded protein response (UPR) and XBP-1 splicing, due to elevated Ig levels, are critical events during plasma cell generation. However, the upstream molecule sufficient to trigger these events remain elusive. Because ectopic expression of Blimp-1 in B cells is sufficient to generate plasma cells, it is plausible that Blimp-1 might be the upstream molecule, sufficient for the induction of UPR and XBP-1 splicing. The results from the current study indicate that ectopic expression of Blimp-1 or its N-terminal domain, in B cells, is sufficient to induce XBP-1 splicing, UPR and Ig (immunoglobulin) secretion. Further more Blimp-1 is able to directly repress the antiapoptotic gene A1, by binding to specific DNA elements in A1 promoter. This repression of A1 by Blimp-1 seems to be an important prerequisite for Plasma cell differentiation because ectopic expression of A1 in primary B cells resulted in reduced immunoglobulin secretion.
Expression of the neural cell adhesion molecule and polysialic acid during early mouse embryogenesis
(1994)
The expression of the neural cell adhesion molccule (N-CAM) and a 2-8 linked polysialic acid (PSA), whieh is believed to be predominantly expressed on N-CAM, was investigated during early embryonie development ofthe mouse (embryonic days 7.5 to 10.0). By immunoeytoehemistry, in tissue sections, N-CAM and PSA were not detectable at embryonie day 7.5 but were expressed in the prominent body regions such as somites, unsegmented mesoderm, developing heart, and neuroectoderm at embryonie day 8.0 N-CAM and PSA immunoreaetivities were always predominantly associated with tbe plasma membrane. No tissue could be detected which was positive for PSA but negative for N-CAM. In Western blot analysis of whole embryos, by contrast, only the lightly sialylated and PSA-negative 180 and 140 kD isoforms of N-CAM werc present at embryonie day 8.0 and strong expression of PSA-bearing, heavily sialylated N-CAM was not detectable before embryonie day 10.0. In Western blot analysis of N-CAM immunoaffinity purifled from whole embryos and digested with neuraminidase as weil as in Northern blot analysis, the 120 kD isoform of N-CAM or its eorresponding mRN A were not expressed in detectable amounts during the time period investigated.
Measles virus is a highly contagious virus causing acute and persistent diseases in man, the receptor of which is still not weil characterized. We have isolated a monoclonal antibody (mAb), designated mAb 119, which specifically inhibits measles virus infection of susceptible celllines in a dosa-dependent manner. This antibody precipitates a protein with an apparent molecular mass of 75 kDa from 1251 surface-labeled cells and its epitope is present on human peripheral blood mononuclear cells, human celllines, and the African green monkey cellline Vero. Affinity chromatography of detergent-solubilized cell membrane proteins over a Sepharose column with covalently bound mAb 119 led to the partial purification of the 75-kOa protein. Preincubation of measles virus with this affinity-purified protein inhibited measles virus infection dose dependently. Aminoacid microseq,uencing of this protein revealed its identity with the human membrane-organizing extension spike protein moesin, a protein intra- and extracellularly associated with the plasma membrane of cells. Subsequently, an antibody raised against purified moesin (mAb 38/87) was also found to specifically inhibit measles virus infection of susceptible cells and confirmed our data obtained with mAb 119. Our data suggest that moesin is acting as a receptor for measles virus.
As critical steps in the life cycle oJ measles virus (Mfl), the e.fficiency of uptake into and replication in susceptible host cells are governed by cellular determinants. Measles virus infections of cells of the human CNS are characterized by particular constraints imposed on v1:ral transcription and translation attenuating viral gene Junctions and thus contributing to the pathogenesis oJ MV persistence in these cells.
The expression of measles virus (MV) in six different permanent human glioma cell lines (D-54, U-251, U-138, U-105, U-373, and D-32) was analyzed. Although all celllines were permissive for productive replication of all MV strains tested, U-251, D-54, and D-32 cells spontaneously revealed restrictions of MV transcription similar to those observed for primary rat astroglial cells and brain tissue. In vitro differentiation of D-54 and U-251 cells by substances affecting tbe intracellular cyclic AMP Ievel caused a significant reduction of tbe expression of tbe viral proteins after 18, 72, and 144 b of infection. This pronounced restriction was not paralleled to a comparable Ievel by an inhibition of tbe syntbesis and biological activity in vitro of virus·specific mRNAs as sbown by quantitative Northem (RNA) blot analyses and in vitro translation. The block in viral protein syntbesis could not be attributed to tbe induction of type I interferon by any of tbe substances tested. Our findings indicate tbat down-regulation of MV gene expression in human brain cells can occur by a cell type-rlependent regulation of tbe viral mRNA transcription and a differentiation-dependent regulation of translation, botb of wbicb may be crucial for the establisbment of persistent MV infections in tbe centrat nervous system.
Quantification of the peripheral nerve myelin glycoprotein PO and antibodies to PO is difficult due to insolubility of PO in physiological solutions. We have overcome this problern by using the water-soluble recombinant form of the extracellular domain of PO (PO-ED) and describe newly developed assays which allow detection and quantitation of PO and antibodies to PO, in serum and cerebraspinal fluid (CSF). These sensitive and specific assays based on the ELISA technique were used to study humoral immune responses to PO during experimental autoimmune ("allergic") neuritis (EAN). In order to establish these tests, monoclonal antiborlies to different epitopes of rodent and human PO-ED were produced. A two-antibody sandwich-ELISA allowing quantitation of PO Oower detection Iimit of 0.5 ngjml or 30 fmoljml) and an antibody-capture ELISA (lower detection Iimit 1 ng specific antibody jml) to detect antiborlies to PO in serum and CSF were developed. EAN was induced in rats by active immunization with bovine myelin or the neuritogenic protein P2 or by adoptive transfer using P2 specific CD4 positive T cells. Serum and CSF were assayed for the presence of PO-ED and antibodies to PO-ED or P2. Antibodies to PO-ED were detected during active myelin-induced EAN, but not during P2-induced or adaptive transfer EAN. The anti-PO-ED antibodies in the CSF showed a correJation with disease activity. In contrast, in the same model antibodies to P2 persisted long after the disease ceased. No soluble PO-Iike fragments could be found in serum or CSF during any of the three types of EAN. We conclude that PO may be a B-eeil epitope in EAN. These findings warrant a screen for antibodies to PO-ED in human immune neuropathies.
The effect of measles virus (MV) infection on mRNA expression and protein synthesis of cytokines in human malignant glioma celllines (0-54 and U-251) was investigated. Primary MV infections led in both celllines to the induction of interleukin-1 fJ (ll-1 (3), interleukin-6 (IL-6), interferon-(3 (IFN-fJ), and tumor necrosis factor-a (TNF-a). ln contrast, persistently infected astrocytoma lines continually produced IL-6 (two out of 12 lines high Ievels) and IFN-ß, whereas only 1 out of 121ines synthesized TNF-a and none IL-1ß. The pathways for induction of IL-1fJ and TNF-a expression were not suppressed by the persistent MV infection, since IL-1ß and TNF-a could be induced by external stimuli Jike diacylglycerol analog plus calcium ionophore. lnterestingly, persistently infected astrocytoma cells synthesized considerably higher Ievels of ll-1ß and TNF-a than uninfected cells afteradditional external induction. These results suggest that in the centrat nervous system (CNS) of SSPE patients a percentage of persistently infected astrocytes may continually synthesize IL-6 and IFN-ß, and in the presence of additional external stimuli, as possibly provided by activated lymphocytes, might ovarexpress the inflammatory cytokines IL-1 ß and TNF-a. This may be of pathogenetic significance in CNS diseases associated with persistent MV infections.
Seven monoclonal antibodies were raised against the immunoglobulin-like extracellular domain of PO (POED), the major protein of peripheral nervous system myelin. Mice were immunized with purified recombinant rat PO-ED. After fusion, 7 clones (POI-P07) recognizing either recombinant, rat, mouse, or human PO-ED were selected by ELlS A and were characterized by Western blot, immunohistochemistry, and a competition assay. Antibodies belonged to the IgG or IgM class, and P04-P07, reacted with PO in fresh-frozen and paraffin-embedded sections of human or rat peripheral nerve, but not with myelin proteins of the central nervous system of either species. Epitope specificity of the antibodies was determined by a competition enzyme-linked immunosorbent assay (ELISA) and a direct ELlS A using short synthetic peptides spanning the entire extracellular domain of PO. These assays showed that POl and P02 exhibiting the same reaction pattern in Western blot and immunohistochemistry reacted with different distant epitopes of PO. Furthermore, the monoclonal antibodies P05 and P06 recognized 2 different epitopes in close proximity within the neuritogenic extracellular sequence of PO. This panel of monoclonal antibodies, each binding to a different epitope of the extracellular domain of PO, will be useful for in vitro and in vivo studies designed to explore the role of PO during myelination and in demyelinating diseases of the peripheral nervous system.