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MSCs derived from the umbilical cord tissue, termed UCX, were investigated for their immunomodulatory properties and compared to bone marrow-derived MSCs (BM-MSCs), the gold-standard in immunotherapy. Immunogenicity and immunosuppression were assessed by mixed lymphocyte reactions, suppression of lymphocyte proliferation and induction of regulatory T cells. Results showed that UCX were less immunogenic and showed higher immunosuppression activity than BM-MSCs. Further, UCX did not need prior activation or priming to exert their immunomodulatory effects. This was further corroborated in vivo in a model of acute inflammation. To elucidate the potency differences observed between UCX and BM-MSCs, gene expression related to immune modulation was analysed in both cell types. Several gene expression profile differences were found between UCX and BM-MSCs, namely decreased expression of HLA-DRA, HO-1, IGFBP1, 4 and 6, ILR1, IL6R and PTGES and increased expression of CD200, CD273, CD274, IL1B, IL-8, LIF and TGFB2. The latter were confirmed at the protein expression level. Overall, these results show that UCX seem to be naturally more potent immunosuppressors and less immunogenic than BM-MSCs. We propose that these differences may be due to increased levels of immunomodulatory surface proteins such as CD200, CD273, CD274 and cytokines such as IL1ß, IL-8, LIF and TGFß2.

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The blood and lymphatic vascular system of the gut plays an important role in tissue fluid homeostasis, nutrient absorption and immune surveillance. To obtain a better understanding of the anatomic basis of these functions, the blood and lymphatic vasculature of the lower segment of mouse gut and several constituents of gut-associated lymphoid tissue (GALT) including Peyer's patch, specialized lymphoid nodules in the caecum, small lymphoid aggregates and lymphoid nodules in the colon were studied by using confocal microscopy. Additionally, the innervation and nerve/immune cell interactions in the gut and Peyer's patch were investigated by using cell surface marker PGP9.5 and Glial fibrillary acidic protein (GFAP). In the gut and Peyer's patch, the nerves have contact with B cell, T cell and B220CD3 double-positive cells. Dendritic cells, the most important antigen-presenting cells, were closely apposed to some nerves. Some dendritic cells formed membrane-membrane contact with nerve terminals and neuron cell body. Many fine nerve fibres, which are indirectly detected by GFAP, have contact with dendritic cells and other immune cells in the Peyer's patch. Furthermore, the expression of Muscarinic Acetylcholine receptor (subtype M2) was characterized on dendritic cells and other cell population. These findings are expected to provide a route to understand the anatomic basis of neuron-immune regulation/cross-talk and probably neuroinvasion of prion pathogens in the gut and GALT.

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The immune system is challenged by randomly generated immune receptors that by chance can recognize self-antigens. Immunological tolerance functions as a fundamental concept in the control of a broad spectrum of immune responses not only to autoantigens but also to foreign antigens. During the past decade, CD4+ CD25+ regulatory T-cells (Tregs) have emerged as key players in the development of immunological tolerance. This review will present an update on the current knowledge about the phenotype, function, and clinical relevance of this regulatory T-cell population. The therapeutical potential of Tregs to specifically suppress immune responses in autoimmunity and transplantation and their inhibitory effects in anti-tumor immune responses will be discussed.

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AIMS/HYPOTHESIS: In the NOD mouse model, attempts to show MHC class II expression by pancreatic beta cells were unsuccessful so far. We readdressed this question by analysing I-A(g7) expression in single pancreatic beta cells. METHODS: Single-cell multiplex RT PCR and single-cell immunofluorescence were used to study MHC class II expression in NOD and NOD/SCID beta cells. RESULTS: Pancreatic beta cells from NOD mice express the I-A(g7) protein as well as the corresponding mRNA. The frequency of MHC class II mRNA-expressing beta cells is drastically increased during the progression to overt diabetes. MHC class II protein is accumulated intracellularly, and invariant chain is co-expressed. Beta cells from 9- to 10-week-old NOD/SCID mice express MHC class II at the same low frequency as beta cells from 3-week-old NOD mice. CONCLUSION/INTERPRETATION: NOD beta cells express I-A(g7) and could be a direct target of autoreactive CD4+ T cells. This MHC class II expression is triggered by infiltrating lymphocytes.

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Telomere shortening limits the regenerative capacity of primary cells in vitro by inducing cellular senescence characterized by a permanent growth arrest of cells with critically short telomeres. To test whether this in vitro model of cellular senescence applies to impaired organ regeneration induced by telomere shortening in vivo, we monitored liver regeneration after partial hepatectomy in telomerase-deficient mice. Our study shows that telomere shortening is heterogeneous at the cellular level and inhibits a subpopulation of cells with critically short telomeres from entering the cell cycle. This subpopulation of cells with impaired proliferative capacity shows senescence-associated beta-galactosidase activity, while organ regeneration is accomplished by cells with sufficient telomere reserves that are capable of additional rounds of cell division. This study provides experimental evidence for the existence of an in vivo process of cellular senescence induced by critical telomere shortening that has functional impact on organ regeneration.

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CXCL12 (SDF-1), a CXC-chemokine, and its specific receptor, CXCR4, have recently been shown to be involved in tumourgenesis, proliferation and angiogenesis. Therefore, we analysed CXCL12alpha/CXCR4 expression and function in four human kidney cancer cell lines (A-498, CAKI-1, CAKI-2, HA-7), 10 freshly harvested human tumour samples and corresponding normal kidney tissue. While none of the analysed tumour cell lines expressed CXCL12alpha, A-498 cells were found to express CXCR4. More importantly, real-time RT-PCR analysis of 10 tumour samples and respective adjacent normal kidney tissue disclosed a distinct and divergent downregulation of CXCL12alpha and upregulation of CXCR4 in primary tumour tissue. To prove that the CXCR4 protein is functionally active, rhCXCL12alpha was investigated for its ability to induce changes of intracellular calcium levels in A-498 cells. Moreover, we used cDNA expression arrays to evaluate the biological influence of CXCL12alpha. Comparing gene expression profiles in rhCXCL12alpha stimulated vs unstimulated A-498 kidney cancer cells revealed specific regulation of 31 out of 1176 genes tested on a selected human cancer array, with a prominent stimulation of genes involved in cell-cycle regulation and apoptosis. The genetic changes reported here should provide new insights into the developmental paths leading to tumour progression and may also aid the design of new approaches to therapeutic intervention.

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The facultative intracellular, Gram-positive bacterium Listeria monocytogenes invades phagocytic and non-phagocytic cells from the tissues and organs of a wide variety of animals and humans. Here, we report the use of these bacteria as vehicles for gene transfer. Eukaryotic expression plasmids were introduced into the nucleus of host cells following lysis of the intracytosolic, plasmid-carrying bacteria with antibiotics. Cell lines of different tissues and species could be transfected in this way. We examined bacterial properties required for delivery of the expression plasmids and found that this was strictly dependent on the ability of these bacteria to both invade eukaryotic cells and egress from the vacuole into the cytosol of the infected host cells. Macrophage-like cell lines or primary, peritoneal macrophages proved to be almost refractory to Listeria-mediated gene transfer. Thus, attenuated L. monocytogenes represents a serious candidate for consideration as a DNA-transfer vehicle for in vivo somatic gene therapy. The potential for oral administration of L. monocytogenes and the ease in producing and cultivating recombinant strains are further attributes that make its use as a gene transfer vehicle attractive.

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An important feature of microbial infections is the ability of the microorganisms to interfere with and modulate the induction of host immune reactions. However, little is known about the effects of broad host range pathogens such as Listeria monocytogenes on similar cell types in different hosts. Here we examine the effects of the human and animal pathogen L. monocytogenes on human dendritic cells (DC) since this type of cells is essential for the initiation of immune responses. Listeria are phagocytosed efficiently by immature human DC and the bacteria escape from the phagolysosome quickly. Lack of the pore-forming activity of listeriolysin, which was found to be essential for the vacuolar escape of this bacterium in other cell types, retarded but did not prevent egress from the vacuole. Treatment of cultures of immature DC with L. monocytogenes resulted in rapid changes in morphology and cellular constitution followed by maturation of the DC. This could be judged by the appearance of maturation-specific cell surface markers. Antigen presentation to CD4 T cells was apparently not impaired by the infection. These results are in clear contrast to results obtained previously in the mouse system (Guzman et al., Mol. Microbiol. 1996. 20: 119 - 126; Darji et al., Eur. J. Immunol. 1997. 27: 1696 - 1703.).

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In order to analyse the expression of the non-structural (ns) protein p80/p125 in cells infected with different pestiviruses at the protein level, radioimmunoprecipitations with the pestivirus-specific monoclonal antibody (MAb) BVD/C16 were performed. Cell lysates infected with cytopathic (cp) and non-cytopathic (ncp) bovine viral diarrhoea (BVD) virus strains and isolates, and with hog cholera (HC) virus strains were analysed. From cpBVD virus-infected cells, the MAb precipitated one or more proteins corresponding to ns p125, displaying a marked size heterogeneity. In contrast, the lower Mr ns p80 proteins from all cpBVD virus strains and isolates analysed had identical electrophoretic motility. The ncpBVD virus strains displayed either one single band or a doublet of the p125 protein and no p80 cleavage products. The p125 proteins precipitated from HC virus-infected cells showed no size heterogeneity. The possibility is discussed that multiple recombination events, including both insertions or deletions in the genomes of ncpBVD viruses, may lead to the heterogeneous expression of the ns p125 in cpBVD virus populations.

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The expression of biotype-specific epitopes in cells infected with cytopathic (cp) and non-cytopathic (ncp) bovine viral diarrhoea virus (BVDV) was analysed by immunofluorescence. Four monoclonal antibodies (MAbs) directed against different epitopes on the viral glycoprotein gp48 were used. With cells infected with cpBVDV strain NADL, the four MAbs yielded a strong and granular cytoplasmic fluorescence. The same pattern was observed when cells were infected with ncpBVDV 7443 with two of the MAbs (BVD/C12, BVD/C42). In contrast, reactivity with the other two MAbs (BVD/C38, BVD/C46) was restricted to a narrow perinuclear zone. These biotype-specific differences were not observed either with a gp53-specific MAb, or with an MAb specific for the non-structural protein p125/p80. Double immunofluorescence staining of living cells with a polyclonal BVDV-specific serum and with the MAbs revealed that expression of viral proteins on the surface of cells infected with cp- or ncpBVDV, respectively, was not detectable.

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This essay is an attempt to point up the gap between, on the one hand, the methods currently available to the biologist in the laboratory and, on the other, the kind of data that he or she would need in order to characterise genetically engineered proteins of topical biological interest in such a way as to make use of the techniques of protein engineering.

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The similarity or identity of O-glycosylation in glycoproteins from natural sources or produced in heterologous cell lines, a central problem for the development of many biotechnologically relevant production processes, was examined using interleukin-2 (IL-2) as a model. Human interleukin-2 was constitutively expressed in several mammalian cell lines in high amounts. The recombinant proteins were purified to homogeneity and their carbohydrate structures were analyzed. Only the NeuAc alpha 2-3Gal beta 1-3[NeuAc alpha 2-6]GalNAc oligosaccharide structure or the NeuAc alpha 2-3Gal beta 1-3GalNAc were found in all IL-2 preparations secreted from recombinant Ltk-, Chinese hamster ovary, and baby hamster kidney cell lines. The O-linked chains were exclusively linked to Thr in position 3 of the polypeptide chain which is the carbohydrate attachment site in natural human IL-2. The proportions of O-glycosylated versus nonglycosylated forms of the protein secreted by each recombinant cell line were independent of productivity or of cell culture conditions. Our results show that O-glycosylated human IL-2 can be produced by applying recombinant DNA technology in heterologous cell lines with the same type of post-translational modification that is observed for the protein secreted from natural T lymphocytes.

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Myxothiazol, a potent inhibitor of the cytochrome bc1 oxidoreductase, was shown by the use of flow cytometry to block reversibly the late G1/S phase of the cell cycle of human lymphoblastic T-cell line Jurkat (clone 886) at concentrations of 0.5 microgram/ml. These observations are compared to those of other drugs, such as antimycin, which effect the respiratory chain, and with O2-deficiency.

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The production of glycosylated forms of the human T cell growth factor (interleukin-2, IL-2) has been studied after transfection of a mouse L cell line and a chinese hamster ovary cell line with a plasmid containing the human chromosomal interleukin-2 gene. Both cell lines produced IL-2 constitutively. Based on their behavior in reversed-phase l.c. and their sodium dodecyl sulfate-gel-electrophoresis pattern, human IL-2 protein secreted by L cells showed a similar distribution of glycosylated (Mr 16 500) and nonglycosylated (Mr 14 500) forms as the natural protein secreted by human peripheral lymphocytes, whereas the hamster cell line secreted preponderantly the glycosylated forms. Exoglycosidase digestion of the 16 500 Mr IL-2 protein shifted the gel electrophoretic mobility towards the low-molecular weight form as is true for the natural glycosylated IL-2, which contains the usual tetrasaccharide alpha-NeuAc-(2----3)-beta-D-Galp-(1----3)-[alpha-NeuAc-(2----6)]-D-GalNAc (IL-2 N2) and the trisaccharide alpha-NeuAc-(2----3)-beta-D-Galp-(1----3)-D-GalNAc (IL-2 N1) as the major carbohydrate constituents. These results support the applicability of recombinant DNA technology as a tool for studying glycoprotein biosynthesis in mammalian cells.

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A method has been developed that allows the isolation of genomic clones from a cosmid library by homologous recombination in vivo. This method was used to isolate a human genomic interleukin 2 (IL2) gene. The genomic cosmid library was packaged in vivo into lambda phage particles. A recombination-proficient host strain carrying IL2 cDNA sequences in a non-homologous plasmid vector was infected by the packaged cosmid library. After in vivo packaging and reinfection, recombinants carrying the antibiotic resistance genes of both vectors were selected. From a recombinant cosmid clone the chromosomal IL2 gene was restored. After DNA mediated gene transfer into mouse Ltk- cells human IL2 was expressed constitutively.

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A proteolytic activity is associated with structural protein p15 in avian RNA tumor viruses. Its effect on the known intracellular viral polyprotein precursors obtained by immunoprecipitation was investigated. Cleavage of Pr76gag resulted in the sequential appearance of p15, p27, and p19. The intracellular precursor Pr180gag-pol was also cleaved by p15, whereas the intracellular glycoprotein precursors of avian RNA tumor viruses, Pr92env, remained unaffected by p15 under all conditions tested. The specificities of the antibodies used to precipitate the precursors influenced the pattern of intermediates and cleavage products obtained by p15 treatment. If virus harvested from the the Prague strain of Rous sarcoma virus, subgroup C-transformed cells at 15-min intervals was incubated at 37 degrees C for further maturation, RNA-dependent DNA polymerase activity showed an optimum of DNA synthesis with 70S viral RNA or synthetic template-primers after short incubation periods. The presence of additional p15 during incubation resulted in a shift of the enzyme activity peak toward earlier time points. Virus harvested at 3-h intervals contained significant amounts of Pr180gag-pol and Pr76gag. The addition of p15 resulted in the cleavage of Pr180gag-pol and Pr76gag, but only a few distinct low-molecular-weight polypeptides appeared. Treatment of purified RNA-dependent DNA polymerase with p15 in vitro resulted in a disappearance of the beta subunit and an enrichment of the alpha subunit. In addition, a polypeptide of 32 x 10(3) molecular weight was generated. The cleavage pattern observed differed from the one obtained by trypsin treatment.

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From the same batch of virus, the four major avian viral structural proteins p27, p19, p15, and p12, the reverse transcriptase, the envelope glycoprotein gp85, and the high molecular weight 70 S RNA have been recovered. All proteins, except for gp85, have been purified by use of column chromatography procedures to apparent homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gels and isoelectric focusing. A new isolation procedure for p12 by affinity column chromatography takes advantage of its nucleic acid binding properties. The recovery of nondenatured viral structural proteins is demonstrated by the proteolytic activity revealed by p15. The purified proteins were used for the production of monospecific antibodies. The 70 S RNA served as source for the isolation of 35 S RNA subunits.

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