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The genetic make-up of the host has a major influence on its response to combat pathogens. For influenza A virus, several single gene mutations have been described which contribute to survival, the immune response and clearance of the pathogen by the host organism. Here, we have studied the influence of the genetic background to influenza A H1N1 (PR8) and H7N7 (SC35M) viruses. The seven inbred laboratory strains of mice analyzed exhibited different weight loss kinetics and survival rates after infection with PR8. Two strains in particular, DBA/2J and A/J, showed very high susceptibility to viral infections compared to all other strains. The LD(50) to the influenza virus PR8 in DBA/2J mice was more than 1000-fold lower than in C57BL/6J mice. High susceptibility in DBA/2J mice was also observed after infection with influenza strain SC35M. In addition, infected DBA/2J mice showed a higher viral load in their lungs, elevated expression of cytokines and chemokines, and a more severe and extended lung pathology compared to infected C57BL/6J mice. These findings indicate a major contribution of the genetic background of the host to influenza A virus infections. The overall response in highly susceptible DBA/2J mice resembled the pathology described for infections with the highly virulent influenza H1N1-1918 and newly emerged H5N1 viruses.

CD4(+)CD25(high) regulatory T cells (T(reg)) have the potent ability to suppress host immune responses, thus preventing autoimmune diseases. However, increased T(reg) frequencies have also been found in cancer patients implicating their involvement in tumor escape from immunological control. We investigated the frequency, functional effects and gene expression pattern of T(reg) in patients with renal cell carcinoma (RCC). Therefore, T(reg) were isolated from the peripheral blood of 11 treatment-naïve RCC patients and 11 healthy donors applying a magnetic cell separation system. Frequency, purity after isolation and function were evaluated using FACS and suppression assays, respectively. Gene expression patterns were compared applying a self-developed customized oligonucleotide microarray and by quantitative RT-PCR. T(reg) frequencies were significantly increased in RCC patients; suppression assays proved that the isolated CD4(+)CD25(high) cells had the functional characteristics of T(reg) cells. Comparing gene expression profiles between T(reg )of RCC patients and healthy controls revealed significant differences in the expression levels of 49 genes. Gene ontology identified an association of significantly up-/downregulated genes to six functional classes, particularly genes involved in apoptosis control such as LGALS1, LGALS3, BAX, IL7R and TNFRSF25. In RCC patients, frequencies of functionally active T(reg) cells were elevated; the T(reg) gene expression pattern differed significantly between patients and controls. As several anti-apoptotic genes were upregulated and pro-apoptotic genes were downregulated in RCC patients, we conclude that T(reg) cells derived from RCC patients might be less responsive to apoptotic stimuli, possibly promoting their accumulation in tumor patients., (Copyright 2009 S. Karger AG, Basel.)

Janus kinase 2 (JAK2)V617F-activating mutations (JAK2mu) occur in myeloproliferative disorders (MPDs) and myelodysplastic syndromes (MDSs). Cell lines MB-02, MUTZ-8, SET-2 and UKE-1 carry JAK2V617F and derive from patients with MPD/MDS histories. Challenging the consensus that expression of JAK2V617F is the sole precondition for cytokine independence in class I cytokine receptor-positive cells, two of four of the JAK2mu cell lines were growth factor-dependent. These cell lines resembled JAK2wt cells regarding JAK2/STAT5 activation: cytokine deprivation effected dephosphorylation, whereas erythropoetin or granulocyte colony-stimulating factor induced phosphorylation of JAK2 and STAT5. Cytokine independence correlated with low expression and cytokine dependence with high expression of the JAK/STAT pathway inhibitor suppressor of cytokine signaling 2 (SOCS2) suggesting a two-step mechanism for cytokine independence of MPD cells: (i) activation of the oncogene JAK2V617F and (ii) inactivation of the tumor suppressor gene SOCS2. Confirming that SOCS2 operates as a negative JAK2V617F regulator, SOCS2 knockdown induced constitutive STAT5 phosphorylation in JAK2mu cells. CpG island hypermethylation is reported to promote SOCS gene silencing in malignant diseases. Accordingly, in one of two cytokine-independent cell lines and in two of seven MPD patients, we found SOCS2 hypermethylation associated with reduced promoter access to transcription factors. Our results provide solid evidence that SOCS2 epigenetic downregulation might be an important second step in the genesis of cytokine-independent MPD clones.

Filopodia are rod-shaped cell surface protrusions composed of a parallel bundle of actin filaments. Since filopodia frequently emanate from lamellipodia, it has been proposed that they form exclusively by the convergence and elongation of actin filaments generated in lamellipodia networks. However, filopodia form without Arp2/3-complex, which is essential for lamellipodia formation, indicating that actin filaments in filopodia may be generated by other nucleators. Here we analyzed the effects of ectopic expression of GFP-tagged full length or a constitutively active variant of the human formin mDia2/Drf3. By contrast to the full-length molecule, which did not affect cell behaviour and was entirely cytosolic, active Drf3 lacking the C-terminal regulatory region (Drf3DeltaDAD) induced the formation of filopodia and accumulated at their tips. Low expression of Drf3DeltaDAD induced rod-shaped or tapered filopodia, whereas over-expression resulted in multiple, club-shaped filopodia. The clubs were filled with densely bundled actin filaments, whose number but not packing density decreased further away from the tip. Interestingly, clubs frequently increased in width after protrusion beyond the cell periphery, which correlated with increased amounts of Drf3DeltaDAD at their tips. These data suggest Drf3-induced filopodia form and extend by de novo nucleation of actin filaments instead of convergent elongation. Finally, Drf3DeltaDAD also induced the formation of unusual, lamellipodia-like structures, which contained both lamellipodial markers and the prominent filopodial protein fascin. Microarray analyses revealed highly variable Drf3 expression levels in different commonly used cell lines, reflecting the need for more detailed analyses of the functions of distinct formins in actin cytoskeleton turnover and different cell types.

In tyrosinemia type 1 (HT1), accumulation of toxic metabolites results in oxidative stress and DNA damage, leading to a high incidence of hepatocellular carcinomas. Nuclear factor erythroid-2 related factor 2 (Nrf2) is a key transcription factor important for cellular protection against oxidative stress and chemical induced liver damage. To specifically address the role of Nrf2 in HT1, fumarylacetoacetate hydrolase (Fah)/Nrf2(-/-) mice were generated. In acute HT1, loss of Nrf2 elicited a strong inflammatory response and dramatically increased the mortality of mice. Following low grade injury, Fah/Nrf2(-/-) mice develop a more severe hepatitis and liver fibrosis. The glutathione and cellular detoxification system was significantly impaired in Fah/Nrf2(-/-) mice, resulting in increased oxidative stress and DNA damage. Consequently, tumor development was significantly accelerated by loss of Nrf2. Potent pharmacological inducers of Nrf2 such as the triterpenoid analogs 1[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole have been developed as cancer chemoprevention agents. Pretreatment with 1[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole dramatically protected Fah(-/-) mice against fumarylacetoacetate (Faa)-induced toxicity. Our data establish a central role for Nrf2 in the protection against Faa-induced liver injury; the Nrf2 regulated cellular defense not only prevents acute Faa-induced liver failure but also delays hepatocarcinogenesis in HT1.

A reduction in the cellular levels of the cyclin kinase inhibitor p27(kip1) is frequently found in many human cancers and correlates directly with patient prognosis. In this work, we identify argyrin A, a cyclical peptide derived from the myxobacterium Archangium gephyra, as a potent antitumoral drug. All antitumoral activities of argyrin A depend on the prevention of p27(kip1) destruction, as loss of p27(kip1) expression confers resistance to this compound. We find that argyrin A exerts its effects through a potent inhibition of the proteasome. By comparing the cellular responses exerted by argyrin A with siRNA-mediated knockdown of proteasomal subunits, we find that the biological effects of proteasome inhibition per se depend on the expression of p27(kip1).

Stress is a powerful modulator of neuroendocrine, behavioral, and immunological functions. After 4.5-d repeated combined acoustic and restraint stress as a murine model of chronic psychological stress, severe metabolic dysregulations became detectable in female BALB/c mice. Stress-induced alterations of metabolic processes that were found in a hepatic mRNA expression profiling were verified by in vivo analyses. Repeatedly stressed mice developed a hypermetabolic syndrome with the severe loss of lean body mass, hyperglycemia, dyslipidemia, increased amino acid turnover, and acidosis. This was associated with hypercortisolism, hyperleptinemia, insulin resistance, and hypothyroidism. In contrast, after a single acute stress exposure, changes in expression of metabolic genes were much less pronounced and predominantly confined to gluconeogenesis, probably indicating that metabolic disturbances might be initiated already early but will only manifest in repeatedly stressed mice. Thus, in our murine model, repeated stress caused severe metabolic dysregulations, leading to a drastic reduction of the individual's energy reserves. Under such circumstances stress may further reduce the ability to cope with new stressors such as infection or cancer.

Background: Although uterine macrophages are thought to play an important regulatory role at the maternal-fetal interface, their global gene expression profile is not known., Methodology/principal Findings: Using micro-array comprising approximately 14,000 genes, the gene expression pattern of human first trimester decidual CD14+ monocytes/macrophages was characterized and compared with the expression profile of the corresponding cells in blood. Some of the key findings were confirmed by real time PCR or by secreted protein. A unique gene expression pattern intrinsic of first trimester decidual CD14+ cells was demonstrated. A large number of regulated genes were functionally related to immunomodulation and tissue remodelling, corroborating polarization patterns of differentiated macrophages mainly of the alternatively activated M2 phenotype. These include known M2 markers such as CCL-18, CD209, insulin-like growth factor (IGF)-1, mannose receptor c type (MRC)-1 and fibronectin-1. Further, the selective up-regulation of triggering receptor expressed on myeloid cells (TREM)-2, alpha-2-macroglobulin (A2M) and prostaglandin D2 synthase (PGDS) provides new insights into the regulatory function of decidual macrophages in pregnancy that may have implications in pregnancy complications., Conclusions/significance: The molecular characterization of decidual macrophages presents a unique transcriptional profile replete with important components for fetal immunoprotection and provides several clues for further studies of these cells.

Background & Aims: The transcription factor nuclear factor-eythroid 2-related factor 2 (Nrf2(-/-)) is essential for protecting cells against xenobiotic and oxidative stress. Increased oxidative stress has been implicated in the pathophysiology of many diseases including ethanol-induced liver disease. Therefore, the role of Nrf2(-/-) in ethanol-induced liver injury was investigated., Methods: Wild-type and Nrf2(-/-) mice were fed with the ethanol diet, followed by examination of liver pathology, mortality, and ethanol metabolism., Results: Nrf2(-/-) mice displayed a dramatically increased mortality associated with liver failure when fed doses of ethanol that were tolerated by WT mice. Nrf2(-/-) mice showed a significantly reduced ability to detoxify acetaldehyde, leading to an accumulation of the toxic metabolite. Loss of Nrf2(-/-) caused a marked steatosis in livers of ethanol-fed mice, and Srebp1 was identified as a candidate transcription factor responsible for lipogenic enzyme induction. Furthermore, ethanol consumption led to a progressive depletion of total and mitochondrial reduced glutathione, which was associated with more pronounced structural and functional changes to mitochondria of Nrf2(-/-) mice. In addition, ethanol feeding elicited an aggravated inflammatory response mediated by Kupffer cells in Nrf2(-/-) mice as shown by an increased tumor necrosis factor-alpha secretion and activation of the interleukin-6/Stat-3 pathway. Together these changes lead to a vicious cycle of accumulating hepatocellular damage, ultimately leading to liver failure and death of Nrf2(-/-) mice., Conclusions: Our data establish a central role for Nrf2(-/-) in the protection against ethanol-induced liver injury.

Keloid disease is a significant clinical problem, especially in black populations, with an estimated incidence of 4-16%. Keloids are fibroproliferative dermal tumors developing as a result of deregulated wound healing. The dynamic nature of keloids is illustrated by clinical regression in the center, while the margin remains active growing into the surrounding healthy skin. Therefore, the gene expression profiles of fibroblasts from different sites of the keloids were characterized using Affymetrix microarrays covering the whole human genome. This study revealed 105 genes that were differentially regulated (79 genes were up-regulated and 26 down-regulated) in a unique gene expression profile in different sites of keloids where progression or regression of the process was in progress. The apoptosis inhibitor AVEN was found to be up-regulated at the active margin of keloids, while apoptosis-inducing genes such as ADAM12 and genes inducing extracellular matrix (ECM) degradation such as matrix metalloproteinase-19 were up-regulated in the regressing keloid center. We identified genes previously not described in the development of keloids. Activating proapoptotic genes or inhibiting ECM-inducing genes as INHBA or monocyte chemoattractant protein-1 might be possible target genes for new treatment strategies for keloid disease.

Autoinducer 2 (AI-2) is the only species-nonspecific autoinducer known in bacteria and is produced by both gram-negative and gram-positive organisms. Consequently, it is proposed to function as a universal quorum-sensing signal for interaction between bacterial species. AI-2 is produced as the by-product of a metabolic transformation carried out by the LuxS enzyme. To separate the metabolic function of the LuxS enzyme from the signaling role of AI-2, we carried out a global transcriptome analysis of a luxS null mutant culture of Streptococcus mutans UA159, an important cariogenic bacterium and a crucial component of the dental plaque biofilm community, in comparison to a luxS null mutant culture supplemented with chemically pure 4,5-dihydroxy-2,3-pentanedione, the precursor of AI-2. The data revealed fundamental changes in gene expression affecting 585 genes (30% of the genome) which could not be restored by the signal molecule AI-2 and are therefore not caused by quorum sensing but by lack of the transformation carried out by the LuxS enzyme in the activated methyl cycle. All functional classes of enzymes were affected, including genes known to be important for biofilm formation, bacteriocin synthesis, competence, and acid tolerance. At the same time, 59 genes were identified whose transcription clearly responded to the addition of AI-2. Some of them were related to protein synthesis, stress, and cell division. Three membrane transport proteins were upregulated which are not related to any of the known AI-2 transporters. Three transcription factors were identified whose transcription was stimulated repeatedly by AI-2 addition during growth. Finally, a global regulatory protein, the delta subunit of the RNA polymerase (rpoE), was induced 147-fold by AI-2, representing the largest differential gene expression observed. The data show that many phenotypes related to the luxS mutation cannot be ascribed to quorum sensing and have identified for the first time regulatory proteins potentially mediating AI-2-based signaling in gram-positive bacteria.

Mycobacterium avium subspecies (ssp.) paratuberculosis (MAP) is the etiological agent of paratuberculosis, a chronic, non-treatable granulomatous enteritis of ruminants. MAP is the only mycobacterium affecting the intestinal tract, which is of interest since it is presently the most favoured pathogen linked to Crohn's disease (CD) in humans due to its frequent detection in CD tissues. MAP is genetically closely related to other M. avium ssp. such as M. avium ssp. avium (MAA) and M. avium ssp. hominissuis (MAH) which can cause mycobacteriosis in animals and immunocompromised humans. We have recently shown that murine macrophage cell lines represent suitable systems to analyse M. avium ssp. patho-mechanisms and could show that MAP, but not MAA, specifically inhibited the antigen-specific stimulatory capacity for CD4(+) T-cells. In the present study, we compared gene expression profiles of murine RAW264.7 macrophages in response to infections with MAP or MAA using murine high-density oligonucleotide Affymetrix microarrays. A comparison of MAP and MAA infection revealed 17 differentially expressed genes. They were expressed at a much lower level in MAP-infected macrophages than in MAA-infected macrophages. Among these were the genes for IL-1beta, IL-1alpha, CXCL2, PTGS2 (COX2), lipocalin (LCN2) and TNF, which are important pro-inflammatory factors. The microarray data were confirmed for selected genes by quantitative real-time reverse transcription PCR and, by protein array analyses and ELISA. Similar to MAA, infection with MAH also showed robust induction of IL-1beta, CXCL2, COX2, LCN2 and TNF. Taken together, our results from M. avium ssp.-infected murine macrophages provide evidence that MAP in contrast to MAA and MAH specifically suppresses the pro-inflammatory defence mechanisms of infected macrophages.

The concept of immune regulation/suppression has been well-established and, besides thymus-derived CD4+CD25+ regulatory T (TR) cells, it became clear that a variety of additional peripherally induced TR cells play vital roles in protection from many harmful immune responses including intestinal inflammation. In the present study, we have analyzed in vivo-induced Ag-specific CD4+ TR cells with respect to their molecular and functional phenotype. By comparative genomics we could show that these Ag-specific TR cells induced by chronic Ag stimulation in vivo clearly differ in their genetic program from naturally occurring thymus-derived CD4+CD25+ TR cells. This distinct population of induced TR cells express neither CD25 nor the TR-associated transcription factor Foxp3. Strikingly, CD25 is not even up-regulated upon stimulation. Despite the lack in Foxp3 expression, these in vivo-induced CD25- TR cells are able to interfere with an Ag-specific CD8+ T cell-mediated intestinal inflammation without significant increase in CD25 and Foxp3 expression. Thus, our results demonstrate that in vivo-induced Ag-specific TR cells represent a distinct population of Foxp3-CD25- TR cells with regulatory capacity both in vitro and in vivo.

The function unknown gene PA2384 of Pseudomonas aeruginosa PAO1 has been previously shown dramatically responsive to iron limitation. In the present study, a bioinformatics analysis showed that PA2384 has a weak similarity to the N-terminus DNA-binding domain of Fur, the well-known ferric uptake regulator. To investigate the potential function of PA2384 in iron regulation a P. aeruginosa PAO1 recombinant (pUCP20::PA2384) over-expressing PA2384 and a PA2384 disrupted mutant PAO1*PA2384 were constructed. Physiological characterization showed that the knockout mutant had a longer lag phase. Genome-scale transcriptional profiles at different growth stages were compared between the wild type and the DeltaPA2384 mutant grown under iron-limiting conditions. The expression of more than 350 genes was affected by the knockout of PA2384. Among them, 71 genes involved in iron uptake were significantly down-regulated in the absence of PA2384. One hundred two quorum sensing (QS) dependent genes displayed differential transcriptions, including genes involved in the biosynthesis of some important virulence factors such as pyocyanin, rhamnolipids and hydrogen cyanide. The transcription of genes responsible for the synthesis of Pseudomonas quinolone signal (PQS) was greatly enhanced by the knockout of PA2384. Furthermore, the knockout of PA2384 also resulted in an altered expression of genes involved in electron transfer, central metabolism, phosphorus starvation and translation. It implies that PA2384 might affect more physiological processes than iron acquisition in P. aeruginosa.

Serum response factor (SRF), is a crucial transcription factor for murine embryonic development and for the function of muscle cells and neurons. Gene expression data show that SRF and its transcriptional cofactors are also expressed in lymphocyte precursors and mature lymphocytes. However, the role of SRF in lymphocyte development has not been addressed in vivo so far, attributed in part to early embryonic lethality of conventional Srf-null mice. To determine the in vivo role of SRF in developing lymphocytes, we specifically inactivated the murine Srf gene during T or B cell development using lymphocyte-specific Cre transgenic mouse lines. T cell-specific Srf deletion led to a severe block in thymocyte development at the transition from CD4/CD8 double to single positive stage. The few residual T cells detectable in the periphery retained at least one functional Srf allele, thereby demonstrating the importance of SRF in T cell development. In contrast, deletion of Srf in developing B cells did not interfere with the growth and survival of B cells in general, yet led to a complete loss of marginal zone B cells and a marked reduction of the CD5+ B cell subset. Our study also revealed a contribution of SRF to the expression of the surface molecules IgM, CD19, and the chemokine receptor 4 in B lymphocytes. We conclude that SRF fulfills essential and distinct functions in the differentiation of different types of lymphocytes.

The complex response of murine macrophages to infection with Streptococcus pyogenes was investigated at the level of gene expression with a high-density oligomer microarray. More than 400 genes were identified as being differentially regulated. Many of the up-regulated genes encode molecules involved in the immune response and in inflammation, transcription, signaling, apoptosis, the cell cycle, electron transport, and cell adhesion. Of particular interest was the up-regulation of proinflammatory cytokines, typical of the classically activated macrophages (M1 phenotype), such as tumor necrosis factor alpha, interleukin 1 (IL-1), and IL-6, and as well as the up-regulation of anti-inflammatory mediators, such as IL-1 decoy receptor and IL-10, associated with alternative macrophage activation (M2 phenotype). Furthermore, the gene encoding inducible nitric oxide synthase (iNOS), an enzyme typically implicated in classical activation, was not induced in infected macrophages. Instead, the gene encoding arginase, a competitor for the iNOS substrate arginine involved in the alternative activation pathway, was up-regulated in S. pyogenes-infected cells. Thus, the microarray-based gene expression analysis demonstrated that S. pyogenes induces an atypical activation program in macrophages, with some but not all features of the classical or alternative activation phenotypes. The microarray data also suggested that the bactericidal activity of macrophages against S. pyogenes is mediated by phagocyte oxidase, as p47phox was up-regulated in infected cells. Indeed, the in vivo and in vitro killing of S. pyogenes was markedly diminished in the absence of functional phagocyte (p47(phox-/-)) but not in the absence of iNOS (iNOS(-/-)). An understanding of how macrophages respond to S. pyogenes at the molecular level may facilitate the development of new therapeutic paradigms.

Background: In spite of the emerging importance of Streptococcus dysgalactiae subspecies equisimilis (human group C streptococci [GCS] and group G streptococci [GGS]) in human health, its molecular makeup remains largely undefined. Apart from sharing a phylogenetic relationship with the human pathogen group A streptococci (GAS), GCS/GGS and GAS colonize the same ecological niche and exhibit considerable overlap in their disease profiles. Such similarities imply that the virulence factors associated with diseases may also be similar., Methods: In this study, we used a targeted microarray containing 216 GAS virulence genes to profile the virulence gene repertoires of 58 S. dysgalactiae subspecies equisimilis isolates recovered during human infections. We performed comparative analyses to investigate the relationship between GAS virulence genes in and the invasive potential of GCS/GGS., Results: Up to one-half of the GAS virulence genes represented in the microarray were identified in GCS/GGS. No statistical differences were observed between isolates harboring the group C versus group G carbohydrates; however, clustering algorithms revealed 2 genetically distinct clusters of S. dysgalactiae subspecies equisimilis isolates. No relationship was observed between the virulence profile of GCS/GGS and the propensity for disease or the tissue site of isolation., Conclusions: This is, to our knowledge, the first comprehensive analysis of the virulence profile of S. dysgalactiae subspecies equisimilis, and it enables novel insights into the pathogen's genetic basis of disease propensity shared with GAS. Human group C and group G streptococci may not be considered to be separate species; in fact, they may constitute 2 distinct lineages. Additional incongruent relationships were observed between virulence profiles and GCS/GGS disease propensity.

The pathogenesis of Lassa fever is poorly understood. As the liver is a major target organ of Lassa virus, gene expression in Lassa virus-infected HuH-7 cells, a differentiated human hepatoma cell line, was studied. Cellular mRNA levels were measured at the late phase of acute infection, when virtually all cells expressed large amounts of nucleoprotein, and virus RNA concentration had reached>10(8) copies (ml supernatant)-1. Two types of transcription array were used: cDNA-based macroarrays with a set of 3500 genes (Atlas Human 1.2 arrays; Clontech) and oligonucleotide-based microarrays covering 18,400 transcripts (Human Genome U133A array; Affymetrix). Data analysis was based on statistical frameworks controlling the false-discovery rate. Atlas array data were considered relevant if they could be verified by U133A array or real-time RT-PCR. According to these criteria, there was no evidence for true changes in gene expression. Considering the precision of the U133A array and the number of replicates tested, potential expression changes due to Lassa virus infection are probably smaller than twofold. To substantiate the array data, beta interferon (IFN-beta) gene expression was studied longitudinally in Lassa virus-infected HuH-7 and FRhK-4 cells by using real-time RT-PCR. IFN-beta mRNA levels increased only twofold upon Lassa virus infection, although there was no evidence that the virus inhibited poly(I:C)-induced IFN-beta gene expression. In conclusion, Lassa virus interferes only minimally with gene expression in HuH-7 cells and poorly induces IFN-beta gene transcription.

Group A streptococcus (GAS) is a human pathogen associated with a wide range of human diseases that vary in symptoms and clinical severity. In this report we describe the use of a targeted low density array representing genes encoding classical virulence factors, purported virulence factors and other extracellular proteins to examine differences in the genetic profiles of 68 clinical GAS isolates. Of the 226 genes on the array (encoding 217 virulence factors or putative extracellular proteins and nine positive control house-keeping proteins) 62 had distributions that were statistically associated with specific GAS M-types. While 32 of these genes were bacteriophage related, the remaining 30 have not previously been described as bacteriophage associated. We show that these 'non-bacteriophage related' genes are found in 11 loci located in five greater chromosomal regions, often near classical GAS virulence factors, and often accompanied by genes associated with mobile genetic elements (MGEs). Many of these loci also demonstrated genetic variation within strains of the same M-type, suggesting these regions to be recombinatorial and mutational hotspots. Evidence for acquisition of genes from other species is also apparent in these loci. Our data suggests that imprecise recombination events involving MGEs not only result in acquisition of new genes, but can also result in deletion of flanking chromosomal genes. Thus MGE related events would appear to be the major contributor to variation of discrete virulence loci, which could account for the disease causing propensity of individual strains. We believe that profiling of the 11 loci could be a meaningful tool in epidemiological GAS typing studies.

Lysosome-related organelles have versatile functions, including protein and lipid degradation, signal transduction and protein secretion. The molecular elucidation of rare congenital diseases affecting endosomal-lysosomal biogenesis has given insights into physiological functions of the innate and adaptive immune system. Here, we describe a previously unknown human primary immunodeficiency disorder and provide evidence that the endosomal adaptor protein p14, previously characterized as confining mitogen-activated protein kinase (MAPK) signaling to late endosomes, is crucial for the function of neutrophils, B cells, cytotoxic T cells and melanocytes. Combining genetic linkage studies and transcriptional profiling analysis, we identified a homozygous point mutation in the 3' untranslated region (UTR) of p14 (also known as MAPBPIP), resulting in decreased protein expression. In p14-deficient cells, the distribution of late endosomes was severely perturbed, suggesting a previously unknown role for p14 in endosomal biogenesis. These findings have implications for understanding endosomal membrane dynamics, compartmentalization of cell signal cascades, and their role in immunity.

Most CD4(+)CD25(hi)FOXP3(+) regulatory T cells (T(regs)) from adult peripheral blood express high levels of CD45RO and CD95 and are prone to CD95L-mediated apoptosis in contrast to conventional T cells (T(convs)). However, a T(reg) subpopulation remained consistently apoptosis resistant. Gene microarray and 6-color flow cytometry analysis including FOXP3 revealed an increase in naive T-cell markers on the CD95L-resistant T(regs) compared with most T(regs). In contrast to T(regs) found in adult humans, most CD4(+)CD25(+)FOXP3(+) T cells found in cord blood are naive and exhibit low CD95 expression. Furthermore, most of these newborn T(regs) are not sensitive toward CD95L similar to naive T(regs) from adult individuals. After short stimulation with anti-CD3/CD28 monoclonal antibodies (mAbs), cord blood T(regs) strongly up-regulated CD95 and were sensitized toward CD95L. This functional change was paralleled by a rapid up-regulation of memory T-cell markers on cord blood T(regs) that are frequently found on adult memory T(regs). In summary, we show a clear functional difference between naive and memory T(regs) that could result in different survival rates of those 2 cell populations in vivo. This new observation could be crucial for the planning of therapeutic application of T(regs).

Background: Aplastic anemia (AA) is a bone marrow failure syndrome mostly characterized by an immune-mediated destruction of marrow hematopoietic progenitor/stem cells. The resulting hypocellularity limits a detailed analysis of the cellular immune response. To overcome this technical problem we performed a microarray analysis of CD3+ T-cells derived from bone marrow aspirates and peripheral blood samples of newly diagnosed AA patients and healthy volunteers. Two AA patients were additionally analyzed after achieving a partial remission following immunosuppression. The regulation of selected candidate genes was confirmed by real-time RT-PCR., Results: Among more than 22,200 transcripts, 583 genes were differentially expressed in the bone marrow of AA patients compared to healthy controls. Dysregulated genes are involved in T-cell mediated cytotoxicity, immune response of Th1 differentiated T-cells, and major regulators of immune function. In hematological remission the expression levels of several candidate genes tend to normalize, such as immune regulators and genes involved in proinflammatory immune response., Conclusion: Our study suggests a pivotal role of Th1/Tc1 differentiated T-cells in immune-mediated marrow destruction of AA patients. Most importantly, immune regulatory genes could be identified, which are likely involved in the recovery of hematopoiesis and may help to design new therapeutic strategies in bone marrow failure syndromes.

Background: The factors behind the reemergence of severe, invasive group A streptococcal (GAS) diseases are unclear, but it could be caused by altered genetic endowment in these organisms. However, data from previous studies assessing the association between single genetic factors and invasive disease are often conflicting, suggesting that other, as-yet unidentified factors are necessary for the development of this class of disease., Methods: In this study, we used a targeted GAS virulence microarray containing 226 GAS genes to determine the virulence gene repertoires of 68 GAS isolates (42 associated with invasive disease and 28 associated with noninvasive disease) collected in a defined geographic location during a contiguous time period. We then employed 3 advanced machine learning methods (genetic algorithm neural network, support vector machines, and classification trees) to identify genes with an increased association with invasive disease., Results: Virulence gene profiles of individual GAS isolates varied extensively among these geographically and temporally related strains. Using genetic algorithm neural network analysis, we identified 3 genes with a marginal overrepresentation in invasive disease isolates. Significantly, 2 of these genes, ssa and mf4, encoded superantigens but were only present in a restricted set of GAS M-types. The third gene, spa, was found in variable distributions in all M-types in the study., Conclusions: Our comprehensive analysis of GAS virulence profiles provides strong evidence for the incongruent relationships among any of the 226 genes represented on the array and the overall propensity of GAS to cause invasive disease, underscoring the pathogenic complexity of these diseases, as well as the importance of multiple bacteria and/or host factors.

Decreased expression of the CDK inhibitor p27kip1 in human tumors directly correlates with increased resistance to chemotherapies, increased rates of metastasis, and an overall increased rate of patient mortality. It is thought that decreased p27 expression in tumors is caused by increased proteasomal turnover, in particular activation of the pathway governed by the SCFskp2 E3 ubiquitin protein ligase. We have directly tested the importance of the SCFskp-mediated degradation of p27 in tumorigenesis by analyzing the tumor susceptibility of mice that express a form of p27 that cannot be ubiquitinated and degraded by this pathway (p27T187A). In mouse models of both lung and colon cancer down-regulation of p27 promotes tumorigenesis. However, we found that preventing p27 degradation by the SCFskp2 pathway had no impact on tumor incidence or overall survival in either tumor model. Our study unveiled a previously unrecognized role for the control of p27 mRNA abundance in the development of non-small cell lung cancers. In the colon cancer model, the frequency of intestinal adenomas was similarly unaffected by the p27T187A mutation, but, unexpectedly, we found that it inhibited progression of intestinal adenomas to carcinomas. These studies may guide the choice of clinical settings in which pharmacologic inhibitors of the Skp2 pathway might be of therapeutic value.

Virulence factor production and the development of biofilms in Pseudomonas aeruginosa have been shown to be regulated by two hierarchically organized quorum-sensing systems activated by two types of small acyl-homoserine lactone signal molecules. Recently, a third type of bacterial signal molecule, the Pseudomonas quinolone signal (PQS), has been identified, which positively regulates a subset of genes dependent on the quorum-sensing systems. However, the molecular mechanism underlying PQS signalling has remained poorly understood. In this study the global transcriptional profile of P. aeruginosa in response to PQS revealed a marked upregulation of genes belonging to the tightly interdependent functional groups of the iron acquisition and the oxidative stress response. Remarkably, most of the differentially regulated genes, as well as the induction of rhlR, could be traced back to an iron-chelating effect of PQS. Our results amount to the elucidation of how PQS affects P. aeruginosa and have important implications for the understanding of the complex regulatory circuits involved in P. aeruginosa gene regulation.

Here, we report the identification of the ubiquitin-like gene UBD as a downstream element of FOXP3 in human activated regulatory CD4(+)CD25(hi) T cells (T(reg)). Retroviral transduction of UBD in human allo-reactive effector CD4(+) T helper (T(h)) cells upregulates CD25 and mediates downregulation of IL4 and IL5 expression similar to overexpression of FOXP3. Moreover, UBD impairs T(h) cell proliferation without upregulation of FOXP3 and impairs calcium mobilization. In the presence of ionomycin, overexpression of UBD in T(h) cells leads to the induction of IL1R2 that resemble FOXP3-transduced T(h) cells and naturally derived T(reg) cells. A comparison of the transcriptome of FOXP3- and UBD-transduced T(h) cells with T(reg) cells allowed the identification of the gene LGALS3. However, high levels of LGALS3 protein expression were observed only in human CD4(+)CD25(hi) derived T(reg) cells and FOXP3-transduced T(h) cells, whereas little was induced in UBD-transduced T(h) cells. Thus, UBD contributes to the anergic phenotype of human regulatory T cells and acts downstream in FOXP3 induced regulatory signaling pathways, including regulation of LGALS3 expression. High levels of LGALS3 expression represent a FOXP3-signature of human antigen-stimulated CD4(+)CD25(hi) derived regulatory T cells.

Induction of inflammatory bowel (IBD)-like disease in mice by a targeted mutation in the Il10 gene (Il10(-/-)) is inbred strain dependent. C3H/HeJBir (C3) mice are colitis susceptible, whereas C57BL/6J (B6) mice are resistant. Genetic dissection of this susceptibility revealed 10 colitogenic quantitative trait loci (QTL). The aim of this study was to identify valuable candidate genes by a combination of QTL mapping and microarray analyses. Sixteen genes were differentially expressed between B6- and C3-Il10(-/-) mice and were located within the QTL intervals. Three major candidate genes (Cd14, Gbp1, Pla2g2a) showed prominent expression differences between B6- and C3-Il10(-/-) as well as between B6 and C3 wild-type mice, which was confirmed by semiquantitative or real-time RT-PCR. Because strain differences are known for Gbp1 and Pla2g2a, further analyses focused on Cd14. Western blot analysis revealed strain differences also on the protein level. Cd14 expression in animals with defective and intact Toll-like receptor (TLR)4 signaling (C3, C3H/HeN, B6, B6-Tlr4(tm1Aki)) make the TLR4 defect of C3 mice unlikely to be the reason for higher Cd14 expression. Less Cd14 expression in germ-free mice indicates a contribution of the microflora on Cd14 expression. Stimulation of naive peritoneal macrophages with bacterial antigens showed lower CD14 surface expression in B6 than in C3 mice. In conclusion, the large number of candidate genes was reduced to three major candidates that play an important role in inflammatory processes and immune response. Strain differences for them are already known or are shown in this study.

The administration of macrolides such as azithromycin for chronic pulmonary infection of cystic fibrosis patients has been reported to be of benefit. Although the mechanisms of action remain obscure, anti-inflammatory effects as well as interference of the macrolide with Pseudomonas aeruginosa virulence factor production have been suggested to contribute to an improved clinical outcome. In this study we used a systematic approach and analyzed the impact of azithromycin on the global transcriptional pattern and the protein expression profile of P. aeruginosa PAO1 cultures versus those in untreated controls. The most remarkable result of this study is the finding that azithromycin exhibited extensive quorum-sensing antagonistic activities. In accordance with the inhibition of the quorum-sensing systems, virulence factor production was diminished and the oxidative stress response was impaired, whereas the type III secretion system was strongly induced. Moreover, P. aeruginosa motility was reduced, which probably accounts for the previously observed impaired biofilm formation capabilities of azithromycin-treated cultures. The interference of azithromycin with quorum-sensing-dependent virulence factor production, biofilm formation, and oxidative stress resistance in P. aeruginosa holds great promise for macrolide therapy in cystic fibrosis. Clearly quorum-sensing antagonist macrolides should be paid more attention in the management of chronic P. aeruginosa infections, and as quorum-sensing antagonists, macrolides might gain vital importance for more general application against chronic infections.

Objective: Impairment in cell-mediated immunity has long been recognized in classical Hodgkin's lymphoma (cHL). The immunosuppressive environment at the tumor site and/or a primary T-cell defect may contribute to an ineffective immune clearance of Hodgkin/Reed-Sternberg (H/R-S) cells. Here, we analyzed whether circulating T lymphocytes of cHL patients show specific alterations in gene expression with possible impact on anti-tumor immunity., Material and Methods: Gene expression profiles were performed from CD3+ T cells isolated from peripheral blood samples of untreated patients with cHL versus two control groups consisting of healthy donors and patients with sarcoidosis. The regulation of gene expression was confirmed in additional patients for selected genes by real-time RT-PCR., Results and Conclusion: Circulating T cells of cHL show a Th1 immune response likely supporting anti-tumor immunity. However, the molecular profile reveals an association between cell cycle transition/proliferation and induction of immune regulatory genes which may limit an effective anti-tumor immune response of differentiated Th1 cells., (Copyright (c) 2006 S. Karger AG, Basel.)

Background: Naturally occurring CD4+ CD25+ regulatory T cells (TReg) are involved in the control of autoimmune diseases, transplantation tolerance, and anti-tumor immunity. Thus far, genomic studies on TReg cells were restricted to murine systems, and requirements for their development, maintenance, and mode of action in humans are poorly defined., Results: To improve characterization of human TReg cells, we compiled a unique microarray consisting of 350 TReg cell associated genes (Human TReg Chip) based on whole genome transcription data from human and mouse TReg cells. TReg cell specific gene signatures were created from 11 individual healthy donors. Statistical analysis identified 62 genes differentially expressed in TReg cells, emphasizing some cross-species differences between mice and humans. Among them, several 'old friends' (including FOXP3, CTLA4, and CCR7) that are known to be involved in TReg cell function were recovered. Strikingly, the vast majority of genes identified had not previously been associated with human TReg cells (including LGALS3, TIAF1, and TRAF1). Most of these 'new players' however, have been described in the pathogenesis of autoimmunity. Real-time RT-PCR of selected genes validated our microarray results. Pathway analysis was applied to extract signaling modules underlying human TReg cell function., Conclusion: The comprehensive set of genes reported here provides a defined starting point to unravel the unique characteristics of human TReg cells. The Human TReg Chip constructed and validated here is available to the scientific community and is a useful tool with which to study the molecular mechanisms that orchestrate TReg cells under physiologic and diseased conditions.

1alpha,25-Dihydroxyvitamin D3 (1alpha,25(OH)2D3), the activated vitamin D3 hormone, is a key regulator of calcium homeostasis and thereby indispensable for bone metabolism. In addition, 1alpha,25(OH)2D3 is known to mediate predominantly immunosuppressive responses in vitro and in vivo. It has been demonstrated that macrophages can produce 1alpha,25(OH)2D3 on activation with interferon gamma (IFN-gamma), although little is understood about the biologic significance of this response. We show here that 1alpha,25(OH)2D3 can selectively suppress key effector functions of IFN-gamma-activated macrophages. Among these are the suppression of listericidal activity, the inhibition of phagocyte oxidase-mediated oxidative burst, and the suppression of important IFN-gamma-induced genes, including Ccl5, Cxcl10, Cxcl9, Irf2, Fcgr1, Fcgr3, and Tlr2. The deactivation of IFN-gamma-stimulated macrophages is dependent on a functional vitamin D receptor and 1alpha,25(OH)2D3 acts specifically on IFN-gamma-activated macrophages, whereas the steroid has no effects on resting macrophages. Therefore, the 1alpha,25(OH)2D3-mediated suppression of macrophage functions is distinct from previously described macrophage deactivation mechanisms. In conclusion, our data indicate that the production of 1alpha,25(OH)2D3 by IFN-gamma-stimulated macrophages might be an important negative feedback mechanism to control innate and inflammatory responses of activated macrophages.

Background: The use of live microorganisms to influence positively the course of intestinal disorders such as infectious diarrhea or chronic inflammatory conditions has recently gained increasing interest as a therapeutic alternative. In vitro and in vivo investigations have demonstrated that probiotic-host eukaryotic cell interactions evoke a large number of responses potentially responsible for the effects of probiotics. The aim of this study was to improve our understanding of the E. coli Nissle 1917-host interaction by analyzing the gene expression pattern initiated by this probiotic in human intestinal epithelial cells., Methods: Gene expression profiles of Caco-2 cells treated with E. coli Nissle 1917 were analyzed with microarrays. A second human intestinal cell line and also pieces of small intestine from BALB/c mice were used to confirm regulatory data of selected genes by real-time RT-PCR and cytometric bead array (CBA) to detect secretion of corresponding proteins., Results: Whole genome expression analysis revealed 126 genes specifically regulated after treatment of confluent Caco-2 cells with E. coli Nissle 1917. Among others, expression of genes encoding the proinflammatory molecules monocyte chemoattractant protein-1 ligand 2 (MCP-1), macrophage inflammatory protein-2 alpha (MIP-2alpha) and macrophage inflammatory protein-2 beta (MIP-2beta) was increased up to 10 fold. Caco-2 cells cocultured with E. coli Nissle 1917 also secreted high amounts of MCP-1 protein. Elevated levels of MCP-1 and MIP-2alpha mRNA could be confirmed with Lovo cells. MCP-1 gene expression was also up-regulated in mouse intestinal tissue., Conclusion: Thus, probiotic E. coli Nissle 1917 specifically upregulates expression of proinflammatory genes and proteins in human and mouse intestinal epithelial cells.

Despite the known anti-proliferative and tumor-suppressive effects seen with retinoic acid (RA), treatment of metastatic renal cell carcinoma (RCC) failed to meet the initial expectations. As the exact mechanisms of action of RA and especially the role of the cellular RA binding proteins (CRABP) have not been elucidated yet, we investigated the expression of CRABP-I and its potential influence on RA response in RCC. Real-time RT-PCR analysis disclosed a significant lack of CRABP-I expression in four RCC cell lines and 12 primary RCC samples; in contrast, high expression levels were found in the respective adjacent normal kidney tissue. To further investigate the impact of CRABP-I on RA response in RCC, A-498 RCC cells were employed as a cellular model system. CRABP-I was stably transfected into A-498 cells which consequently displayed substantial resistance to all-trans (ATRA) and 9-cis RA compared to vector controls lacking CRABP-I. Comparison of gene expression profiles of ATRA-treated CRABP-I-expressing A-498 cells and vector controls revealed specific regulation of 54 of approximately 20,000 genes tested on a selected human CodeLink UniSet Bioarray, with a prominent modulation of genes involved in transcriptional control, signaling, apoptosis, cell cycle regulation and metabolism. The genetic changes reported here contribute to a better understanding of the role of RA in RCC. They also provide new insights into CRABP-I-mediated signaling and gene expression., (Copyright 2005 S. Karger AG, Basel.)

Cdc42 is a small GTPase involved in the regulation of the cytoskeleton and cell polarity. To test whether Cdc42 has an essential role in the formation of filopodia or directed cell migration, we generated Cdc42-deficient fibroblastoid cells by conditional gene inactivation. We report here that loss of Cdc42 did not affect filopodium or lamellipodium formation and had no significant influence on the speed of directed migration nor on mitosis. Cdc42-deficient cells displayed a more elongated cell shape and had a reduced area. Furthermore, directionality during migration and reorientation of the Golgi apparatus into the direction of migration was decreased. However, expression of dominant negative Cdc42 in Cdc42-null cells resulted in strongly reduced directed migration, severely reduced single cell directionality, and complete loss of Golgi polarization and of directionality of protrusion formation toward the wound, as well as membrane blebbing. Thus, our data show that besides Cdc42 additional GTPases of the Rho-family, which share GEFs with Cdc42, are involved in the establishment and maintenance of cell polarity during directed migration.

The severity of infection with Streptococcus pyogenes is strongly influenced by the host's genetics. This observation extends to the murine model of streptococcal infection, where the background of the mouse strain determines the infection outcome (BALB/c are resistant, whereas C3H/HeN are susceptible). To determine the extent to which the MHC complex (H2) contributed to diseases susceptibility, the response to S. pyogenes of congenic BALB mice from a resistant background (BALB/c), but carrying the H2(k) region of susceptible C3H/HeN mice (BALB/k), was examined. BALB/k were as susceptible as the H2 donor strain (C3H/HeN). Linkage analysis performed in F(2) backcross ([BALB/c x C3H/HeN] x BALB/c) mice confirmed the presence of a susceptibility locus within the H2 region on proximal chromosome 17. The possibility that modulation of T cell responses to streptococcal superantigens (GAS-SAgs) by different H2 haplotypes may influence disease severity was examined. BALB/k exhibited a significantly stronger response at the level of cell proliferation and cytokine production to GAS-SAgs than did BALB/c mice. However, the fact that T cell-deficient SCID-C3H/HeN mice also exhibited a susceptible phenotype suggests a more important contribution of innate effector cells to disease susceptibility. Lower transcriptional levels of certain inflammation-related regulatory genes located on chromosome 17 were detected in macrophages from susceptible than in those from resistant mice in response to infection. These results suggest that susceptibility to S. pyogenes may be associated with an altered transcription of specific genes that may compromise the endogenous regulatory processes controlling the inflammatory cascade and favor the progression to sepsis.

The Liverpool epidemic strain (LES) of Pseudomonas aeruginosa is a transmissible aggressive pathogen of cystic fibrosis (CF) patients. We compared transcriptome profiles of two LES isolates with each other and with a laboratory and genetic reference strain (PAO1) after growth to late exponential phase and following exposure to oxidative stress. Both LES isolates exhibited enhanced antimicrobial resistances linked to specific mutations in efflux pump genes. Although transcription of AmpC beta-lactamase was up-regulated in both, one LES isolate contained a specific mutation rendering the ampC gene untranslatable. The virulence-related quorum-sensing (QS) regulon of LES431, an isolate that caused pneumonia in the non-CF parent of a CF patient, was considerably up-regulated in comparison to either isolate LES400, associated with a chronic CF infection, or strain PAO1. Premature activation of QS genes was detected in isolates from both non-CF parents and the CF patient in a previously reported infection episode. LES isolates lacking the up-regulated QS phenotype contained different frameshift mutations in lasR. When fed to Drosophila melanogaster, isolate LES431 killed the fruit flies more readily than either isolate LES400 or strain PAO1, indicating that virulence varies intraclonally. The LES may represent a clone with enhanced virulence and antimicrobial resistance characteristics that can vary or are lost due to mutations during long-term colonization but have contributed to the successful spread of the lineage throughout the CF population of the United Kingdom.

Staphylococcus aureus is a gram-positive bacterium frequently isolated from patients with bloodstream infections. Endothelial cells (EC) play an important role in host defence against bacteria, and recent reports have shown that infection of EC with S. aureus induces expression of cytokines and cell surface receptors involved in activating the innate immune response. The ability of S. aureus to invade nonphagocytic cells, including EC, has been documented. However, the knowledge of the role of EC in pathogenesis of S. aureus infection is still limited. In this study, we investigate the gene-expression program in human EC initiated by internalized S. aureus, using microarray analysis. We found 156 genes that were differentially regulated at least threefold, using arrays representing 14,239 genes. Many of the upregulated genes code for proteins involved in innate immunity, such as cytokines, chemokines and cell adhesion proteins. Other upregulated genes encode proteins involved in antigen presentation, cell signalling and metabolism. Furthermore, intracellular bacteria survived for days without inducing EC death.

The mechanisms controlling the differentiation of dendritic cells (DCs) remain largely unknown. Using a transcriptional profiling approach, we identified Gfi1 as a novel critical transcription factor in DC differentiation. Gfi1-/- mice showed a global reduction of myeloid and lymphoid DCs in all lymphoid organs whereas epidermal Langerhans cells were enhanced in number. In vivo, Gfi1-/- DCs showed striking phenotypic and functional alterations such as defective maturation and increased cytokine production. In vitro, Gfi1-/- hematopoietic progenitor cells were unable to develop into DCs. Instead, they differentiated into macrophages, suggesting that Gfi1 is a critical modulator of DC versus macrophage development. Analysis of hematopoietic chimeras and retrovirus-reconstituted hematopoietic progenitor cells established a cell autonomous and nonredundant role for Gfi1 in DC development. The developmental defect of Gfi1-/- progenitor cells was associated with decreased STAT3 activation. In conclusion, we have identified Gfi1 as a critical transcription factor that controls DC versus macrophage development and dissociates DC maturation and activation.

Background: Several studies have suggested that chronic inflammatory bowel disease may be a consequence of antigen specific recognition by appropriate T cells which expand and induce immunopathology., Aims: We wished to investigate whether autoreactive CD4+ T cells can initiate the disease on recognition of enterocyte specific antigens directly and if induction of mucosal tolerance occurs., Methods: Transgenic mice (VILLIN-HA) were generated that showed specific expression of haemagglutinin from influenza virus A exclusively in enterocytes of the intestinal epithelium. To investigate the impact of enterocyte specific haemagglutinin expression in an autoimmune environment, we mated VILLIN-HA mice with T cell receptor (TCR)-HA mice expressing an alpha/beta-TCR, which recognises an MHC class II restricted epitope of haemagglutinin, and analysed the HA specific T cells for induction of autoimmunity or tolerance., Results: In VILLIN-HAxTCR-HA mice, incomplete central deletion of HA specific lymphocytes occurred. Peripheral HA specific lymphocytes showed an activated phenotype and increased infiltration into the intestinal mucosa, but not into other organs of double transgenic mice. Enterocyte specific lamina propria lymphocytes showed a dose dependent proliferative response on antigen stimulation whereas the proliferative capacity of intraepithelial lymphocytes was reduced. Mucosal lymphocytes from VILLIN-HAxTCR-HA mice secreted lower amounts of interferon gamma and interleukin (IL)-2 but higher levels of tumour necrosis factor alpha, monocyte chemoattractant protein 1, and IL-6. Mucosal immune reactions were accompanied by broad changes in the gene expression profile with expression of proinflammatory genes, but strikingly also a remarkable set of genes discussed in the context of peripheral induction of regulatory T cells, including IL-10, Nrp-1, and Foxp3., Conclusions: Enterocyte specific antigen expression is sufficient to trigger a specific CD4+ T cell response leading to mucosal infiltration. In our model, progression to overt clinical disease was counteracted most likely by induction of regulatory T cells.

Although considerable evidence indicates a role for CD4(+) T lymphocytes (T cells) in airway inflammation, little data exist regarding the mechanisms underlying the induction and regulation of CD4(+) T cell reactivity to lung-specific antigens. To dissect the immunologic and molecular mechanisms of CD4(+) T cell dysregulation, reactivity to a self-antigen expressed in the lung of mice bearing a major histocompatibility complex class-II-restricted T cell receptor specific for this antigen was studied. Transgenic mice developed a progressive interstitial pneumonitis characterized by massive lymphocytic and plasmacytic infiltration of interalveolar septa, a clinical picture closely resembling some of the interstitial lung diseases. Pulmonary inflammation reached a plateau state in older mice with prominent formation of lymphoid follicles but reduced interstitial infiltration. Extensive immunologic characterization of self-reactive CD4(+) T cells isolated from the inflamed lung suggested the induction of regulatory T cells in the site of inflammation. Moreover, inflammation was accompanied by broad changes in the gene expression pattern toward a profile partially resembling that of activated, but strikingly, also that of regulatory CD4(+) T cells. Together our data provide important insights into functional and molecular alterations being associated with the induction and/or regulation of T cell-mediated pulmonary inflammation.

Liver is a quiescent organ with >90% of the cells present in the G(0) stage of the cell cycle. However, adult hepatocytes have enormous ability to proliferate in response to liver injury. After 70% liver resection hepatocytes enter the cell cycle in a highly synchronized manner and undergo 1 to 2 rounds of cell division to restore the lost organ mass, thus, representing one of the most reliable model systems to study cell cycle progression in vivo. Using high density oligonucleotide micro-array we analyzed the expression patterns of genes at the G(1)/S transition of liver regeneration in comparison to quiescent livers. The G(1)/S boundary was identified by in vivo BrdU pulse labeling and we observed 199 genes/ESTs which were either up/down regulated at this time point. These differentially regulated genes have a wide range of functions including transcriptional regulation, signal transduction, cell cycle regulation, chromatin reorganization, protein targeting, metabolism, transport, surface receptors, circadian rhythms, xenobiotic metabolism, inflammation and acute phase response. The functions of most of the genes identified in this screen are not known in the process of liver regeneration and cell cycle control at G(1)/S transition.

In this paper, we report the synthesis of Pseudomonas aeruginosa cDNA in the presence of oligo(dT) primers. Hybridization of oligonucleotide DNA microarrays indicates that under the experimental conditions used, at least 43.7% of the expressed genes from P. aeruginosa PAO1, representing many different functional classes, can be detected by using oligo(dT)-primed cDNAs.

CD4+CD25+ regulatory T cells (Treg cells) control immune responsiveness to a large variety of antigens. The isolation and therapeutic manipulation of Treg cells requires the use of reliable surface receptors that are selectively up-regulated in Treg cells. On the basis of global gene expression studies, we identified neuropilin-1 (Nrp1) as a specific surface marker for CD4+CD25+ Treg cells. Nrp1, a receptor involved in axon guidance, angiogenesis, and the activation of T cells, is constitutively expressed on the surface of CD4+CD25+ Treg cells independently of their activation status. In contrast, Nrp1 expression is down-regulated in naive CD4+CD25- T cells after TCR stimulation. Furthermore, CD4+Nrp1(high) T cells express high levels of Foxp3 and suppress CD4+CD25- T cells. Thus, Nrp1 constitutes a useful surface marker to distinguish Treg cells from both naive and recently activated CD4+CD25+ non-regulatory T cells.

Epithelial cells express genes whose products signal the presence of pathogenic microorganisms to the immune system. Pathogenicity factors of enteric bacteria modulate host cell gene expression. Using microarray technology we have profiled epithelial cell gene expression upon interaction with Yersinia enterocolitica. Yersinia enterocolitica wild-type and isogenic mutant strains were used to identify host genes modulated by invasin protein (Inv), which is involved in enteroinvasion, and Yersinia outer protein P (YopP) which inhibits innate immune responses. Among 22 283 probesets (14,239 unique genes), we found 193 probesets (165 genes) to be regulated by Yersinia infection. The majority of these genes were induced by Inv, whose recognition leads to expression of NF-kappa B-regulated factors such as cytokines and adhesion molecules. Yersinia virulence plasmid (pYV)-encoded factors counter regulated Inv-induced gene expression. Thus, YopP repressed Inv-induced NF-kappa B regulated genes at 2 h post infection whereas other pYV-encoded factors repressed host cell genes at 4 and 8 h post infection. Chromosomally encoded factors of Yersinia, other than Inv, induced expression of genes known to be induced by TGF-beta receptor signalling. These genes were also repressed by pYV-encoded factors. Only a few host genes were exclusively induced by pYV-encoded factors. We hypothesize that some of these genes may contribute to pYV-mediated silencing of host cells. In conclusion, the data demonstrates that epithelial cells express a limited number of genes upon interaction with enteric Yersinia. Both Inv and YopP appear to modulate gene expression in order to subvert epithelial cell functions involved in innate immunity.

The TRANSFAC database on eukaryotic transcriptional regulation, comprising data on transcription factors, their target genes and regulatory binding sites, has been extended and further developed, both in number of entries and in the scope and structure of the collected data. Structured fields for expression patterns have been introduced for transcription factors from human and mouse, using the CYTOMER database on anatomical structures and developmental stages. The functionality of Match, a tool for matrix-based search of transcription factor binding sites, has been enhanced. For instance, the program now comes along with a number of tissue-(or state-)specific profiles and new profiles can be created and modified with Match Profiler. The GENE table was extended and gained in importance, containing amongst others links to LocusLink, RefSeq and OMIM now. Further, (direct) links between factor and target gene on one hand and between gene and encoded factor on the other hand were introduced. The TRANSFAC public release is available at http://www.gene-regulation.com. For yeast an additional release including the latest data was made available separately as TRANSFAC Saccharomyces Module (TSM) at http://transfac.gbf.de. For CYTOMER free download versions are available at http://www.biobase.de:8080/index.html.

The maize GapC4 promoter harbours a complex arrangement of cis-sequences involved in activation of anaerobic gene expression in tobacco. As shown by transient expression assays, four copies of a 50 bp anaerobic response element (ARE) increase anaerobic gene expression compared to the ARE alone. Expression strength is similar to a 190 bp fragment that contains most sequences required for anaerobic expression, including the 50 bp ARE. This supports the notion that redundancy of cis-acting sequences contribute to the anaerobic expression strength of the promoter. Mutation analysis of the 50 bp ARE revealed that cis-regulatory sequences are located within 30 bp at the 5' end of the ARE. Of these 30 bp a putative binding site for a Myb transcription factor is essential for anaerobic induction. The TATA box of the GapC4 promoter is also required for anaerobic gene expression and is bound specifically by a recombinant TATA box binding protein (TBP) from tobacco. A model for anaerobic induction of the GapC4 minimal promoter in tobacco that summarizes the presented data is discussed.

The TRANSFAC database on transcription factors and their DNA-binding sites and profiles (http://www.gene-regulation.de/) has been quantitatively extended and supplemented by a number of modules. These modules give information about pathologically relevant mutations in regulatory regions and transcription factor genes (PathoDB), scaffold/matrix attached regions (S/MARt DB), signal transduction (TRANSPATH) and gene expression sources (CYTOMER). Altogether, these distinct database modules constitute the TRANSFAC system. They are accompanied by a number of program routines for identifying potential transcription factor binding sites or for localizing individual components in the regulatory network of a cell.

The promoter of the maize glyceraldehyde-3-phosphate dehydrogenase 4 gene (GapC4) confers strong, specific and ubiquitous anaerobic reporter gene expression in tobacco. To identify factors required for heterologous anaerobic gene expression, 19 progressive 5' and 3' promoter deletions were linked to a chimeric GapC4 TATA box-beta-glucuronidase (GUS) reporter gene construct and transformed into tobacco. In all transgenic lines aerobic expression values were in the range obtained for negative controls while histochemical GUS assays reveal some weak expression in roots only. Anaerobic induction of about 100-fold to more than 1000-fold above unspecific background is mediated by a region of about 190 bp of the GapC4 promoter. Anaerobic reporter gene induction strongly decreases upon deletion of a 20 bp fragment from -286 to -266 relative to the transcription start point. This fragment harbours putative cis-acting sequences. Electrophoretic mobility shift assays with a 50 bp fragment harbouring these cis sequences reveal a high-mobility complex that is formed with nuclear extracts from aerobic and anaerobic leaf tissue while an additional low-mobility complex is anaerobiosis-specific. The formation of the high-mobility complex requires the sequence GTGGGCCCG. The 50 bp fragment alone confers weak and orientation-dependent anaerobic induction to a GapC4 TATA box-beta-glucuronidase (GUS) reporter gene.