The physiologic increase in expression of some type I IFN-inducible genes during pregnancy is not associated with improved disease activity in pregnant patients with rheumatoid arthritis

During pregnancy, most patients with rheumatoid arthritis (RA) experience a spontaneous improvement in their condition. Since type I interferons (IFN) have immunomodulatory properties, we investigated whether type I IFN-inducible genes are upregulated in pregnant patients with RA. Peripheral blood mononuclear cells were evaluated using quantitative real-time polymerase chain reaction for type I IFN-inducible genes (IFI 35, IFI44, IFI44L, IFIT3, OAS1, and Siglec1) in patients with RA and healthy women during and after pregnancy as well as in nonpregnant controls. IFN-alpha and IFN-beta levels in sera of patients and healthy donors were analyzed by enzyme linked immunosorbent assay. It was found that healthy women did not show a change of gene expression levels from the second trimester until postpartum, yet some type I IFN-inducible genes were significantly upregulated in pregnant and postpartum women compared with nonpregnant individuals. In patients with RA, a pronounced upregulation of IFI35 and IFI44 at the second trimester and a peak expression of Siglec1 at the third trimester were observed. Pregnancy levels of IFI35 and IFI44 in patients with RA were higher than those of nonpregnant patients with RA. No significant association of gene expression levels with disease activity was found. In the sera of patients and healthy women, IFN-beta was undetectable and IFN-alpha levels remained stable throughout pregnancy and postpartum. Thus, pregnancy can give rise to an increased expression of type I IFN-inducible genes, reflecting an upregulation of the innate immune system. However, an association of type I IFN-inducible genes with pregnancy induced disease amelioration seems unlikely.

CopY-like copper inducible repressors are putative 'winged helix' proteins

CopY of Enterococcus hirae is a well characterized copper-responsive repressor involved in copper homeostasis. In the absence of copper, it binds to the promoter. In high copper, the CopZ copper chaperone donates copper to CopY, thereby releasing it from the promoter and allowing transcription of the downstream copper homeostatic genes of the cop operon. We here show that the CopY-like repressors from E. hirae, Lactococcus lactis, and Streptococcus mutans have similar affinities not only for their native promoters, but also for heterologous cop promoters. CopZ of L. lactis accelerated the release of CopY from the promoter, suggesting that CopZ of L. lactis acts as copper chaperone, similar to CopZ in E. hirae. The consensus binding motif of the CopY-like repressors was shown to be TACAxxTGTA. The same binding motif is present in promoters controlled by BlaI of Bacillus licheniformis, MecI of Staphylococcus aureus and related repressors. BlaI and MecI have known structures and belong to the family of 'winged helix' proteins. In the N- terminal domain, they share significant sequence similarity with CopY of E. hirae. Moreover, they bind to the same TACAxxTGTA motif. NMR analysis of the N-terminal DNA binding domain of CopY of L. lactis showed that it contained the same alpha-helical content like the same regions of BlaI and MecI. These findings suggest that the DNA binding domains of CopY-like repressors are also of the 'winged helix' type.

Single-cell analysis divides bovine monocyte-derived dendritic cells into subsets expressing either high or low levels of inducible nitric oxide synthase

Dendritic cells (DC) are important cells at the interface between innate and adaptive immunity. DC have a key role in antigen processing and presentation to T cells. Effector functions of DC related to innate immunity have not been explored extensively. We show that bovine monocyte-derived DC (mDC) express inducible nitric oxide synthase (iNOS) mRNA and protein and produce NO upon triggering with interferon-gamma (IFN-gamma) and heat-killed Listeria monocytogenes (HKLM). An immunocytochemical analysis revealed that a sizeable subset (20-60%) copiously expresses iNOS (iNOShi) upon IFN-gamma/HKLM triggering, whereas the other subset expressed low levels of iNOS (iNOSlo). Monocyte-derived macrophages (mMphi) are more homogeneous with regard to iNOS expression. The number of cells within the iNOSlo mDC subset is considerably larger than the number of dead cells or cells unresponsive to IFN-gamma/HKLM. The large majority of cells translocated p65 to the nucleus upon triggering by IFN-gamma/HKLM. A contamination of mDC with iNOS-expressing mMphi was excluded as follows. (i) Cell surface marker analysis suggested that mDC were relatively homogeneous, and no evidence for a contaminating subset expressing macrophage markers (e.g. high levels of CD14) was obtained. (ii) iNOS expression was stronger in iNOShi mDC than in mMphi. The use of maturation-promoting stimuli revealed only subtle phenotypic differences between immature and mature DC in cattle. Nevertheless, these stimuli promoted development of considerably fewer iNOShi mDC upon triggering with IFN-gamma/HKLM. Immunocytochemical results showed that although a significant proportion of cells expressed iNOS only or TNF only upon triggering with IFN-gamma/HKLM, a significant number of cells expressed both iNOS and TNF, suggesting that TNF and iNOS producing (TIP) DC are present within bovine mDC populations obtained in vitro.

Interferon-inducible expression of APOBEC3 editing enzymes in human hepatocytes and inhibition of hepatitis B virus replication

Hypermutations in hepatitis B virus (HBV) DNA by APOBEC3 cytidine deaminases have been detected in vitro and in vivo, and APOBEC3G (A3G) and APOBEC3F (A3F) have been shown to inhibit the replication of HBV in vitro, but the presumably low or even absent hepatic expression of these enzymes has raised the question as to their physiological impact on HBV replication. We show that normal human liver expresses the mRNAs of APOBEC3B (A3B), APOBEC3C (A3C), A3F, and A3G. In primary human hepatocytes, interferon alpha (IFN-alpha) stimulated the expression of these cytidine deaminases up to 14-fold, and the mRNAs of A3G, A3F, and A3B reached expression levels of 10%, 3%, and 3%, respectively, relative to GAPDH mRNA abundance. On transfection, the full-length protein A3B(L) inhibited HBV replication in vitro as efficiently as A3G or A3F, whereas the truncated splice variant A3B(S) and A3C had no effect. A3B(L) and A3B(S) were detected predominantly in the nucleus of uninfected cells; however, in HBV-expressing cells both proteins were found also in the cytoplasm and were associated with HBV viral particles, similarly to A3G and A3F. Moreover, A3G, A3F, and A3B(L), but not A3B(S), induced extensive G-to-A hypermutations in a fraction of the replicated HBV genomes. In conclusion, the editing enzymes A3B(L), A3F, and most markedly A3G, which are expressed in liver and up-regulated by IFN-alpha in hepatocytes, are candidates to contribute to the noncytolytic clearance of HBV.

Cellular differentiation determines the expression of the hypoxia-inducible protein NDRG1 in pancreatic cancer

N-myc downstream-regulated gene-1 (NDRG1) is a recently described hypoxia-inducible protein that is upregulated in various human cancers. Pancreatic ductal adenocarcinoma, called pancreatic cancer, is a highly aggressive cancer that is characterised by its avascular structure, which results in a severe hypoxic environment. In this study, we investigated whether NDRG1 is upregulated in these tumours, thus providing a novel marker for malignant cells in the pancreas. By immunohistochemistry, we observed that NDRG1 was highly expressed in well-differentiated cells of pancreatic cancer, whereas the poorly differentiated tumour cells were negative. In addition, hyperplastic islets and ducts of nonquiescent pancreatic tissue were positive. To further explore its selective expression in tumours, two well-established pancreatic cancer cell lines of unequal differentiation status were exposed to 2% oxygen. NDRG1 mRNA and protein were upregulated by hypoxia in the moderately differentiated Capan-1 cells; however, its levels remained unchanged in the poorly differentiated Panc-1 cell line. Taken together, our data suggest that NDRG1 will not serve as a reliable marker of tumour cells in the pancreas, but may serve as a marker of differentiation. Furthermore, we present the novel finding that cellular differentiation may be an important factor that determines the hypoxia-induced regulation of NDRG1.

Activation of non-ischemic, hypoxia-inducible signalling pathways up-regulate cytoprotective genes in the murine liver

BACKGROUND/AIMS: We investigated the molecular response of a non-ischemic hypoxic stress in the liver, in particular, to distinguish its hepatoprotective potential. METHODS: The livers of mice were subjected to non-ischemic hypoxia by clamping the hepatic-artery (HA) for 2h while maintaining portal circulation. Hypoxia was defined by a decrease in oxygen saturation, the activation of hypoxia-inducible factor (HIF)-1 and the mRNA up-regulation of responsive genes. To demonstrate that the molecular response to hypoxia may in part be hepatoprotective, pre-conditioned animals were injected with an antibody against Fas (Jo2) to induce acute liver failure. Hepatocyte apoptosis was monitored by caspase-3 activity, cleavage of lamin A and animal survival. RESULTS: Clamping the HA induced a hypoxic stress in the liver in the absence of severe metabolic distress or tissue damage. The hypoxic stimulus was sufficient to activate the HIF-1 signalling pathway and up-regulate hepatoprotective genes. Pre-conditioning the liver with hypoxia was able to delay the onset of Fas-mediated apoptosis and prolong animal survival. CONCLUSIONS: Our data reveal that hepatic cells can sense and respond to a decrease in tissue oxygenation, and furthermore, that activation of hypoxia-inducible signalling pathways function in part to promote liver cell survival.

Inducible endothelial cell-specific gene expression in transgenic mouse embryos and adult mice

Utilizing both the TET-OFF and TET-ON systems in combination with transcriptional control elements of the Tie-2 gene, we have established a series of transgenic activator and responder mice for TET-regulated endothelial cell-specific transgene expression in double transgenic mouse embryos and in adult mice. TET-regulated expression of LacZ reporter genes could be achieved in virtually all endothelia in mid gestation stage mouse embryos. In contrast in adult mice, using the very same Tie-2 tTA activator mouse strain, we observed striking differences of TET-induced gene expression from various inducible expression constructs in different vascular beds. Non-endothelial expression was never detected. The prominent differences in completeness of TET-induced endothelial expression highlight the still underestimated critical role of the responder mouse lines for uniform TET-induced gene expression in heterogeneous cell populations such as endothelial cells. Interestingly, in double transgenic mice inducibly expressing several different adhesion molecules, no adverse effects were observed even though these proteins were robustly expressed on endothelial cells in adult tissues. These transgenic model systems provide versatile tools for the TET-regulated manipulation of endothelial cell-specific gene expression in the entire embryonic vasculature and distinct vascular beds in adult mice.

Inducible nitric oxide synthase and nitrotyrosine in listeric encephalitis: a cross-species study in ruminants

Listeria monocytogenes (LM) is a Gram-positive facultative intracellular bacterium that causes fatal meningoencephalitis in humans and ruminants. A current paradigm predicts that intracellular bacteria are controlled by nitric oxide (NO) whose synthesis is catalyzed by inducible nitric oxide synthase (iNOS). The ability of macrophages (Mphi) to express iNOS shows extreme interspecies variability. Here the expression of iNOS and synthesis of NO was studied in listeric encephalitis of cattle, sheep, and goats. iNOS was expressed by a subset of Mphi in cerebral microabscesses in all three species. The level of iNOS expression and the density of cells per lesion expressing iNOS was highest in cattle, intermediate in sheep, and lowest in goats. The accumulation of nitrotyrosine (NT), an indicator of local NO synthesis, was observed in lesions of cattle but not in those of small ruminants. The density of iNOS-expressing cells in lesions was inversely correlated with the number of bacteria. No species differences were observed in regard to reactive oxygen intermediate (ROI) production by stimulated granulocytes, using the flow cytometric dihydrorhodamine-123 (DHR) method indicating ROI generation. Thus, the marked species differences in iNOS expression, NT accumulation, and LM content in lesions of ruminants with listeric encephalitis are explained by different amounts of ROI produced. It suggests that variations in the ability of Mphi to synthesize NO are of pathophysiological significance in listeriosis.

Inducible nitric oxide synthase and the effect of aminoguanidine in experimental neonatal meningitis

This study explored the role of inducible nitric oxide (NO) synthase (iNOS) in an infant rat model of group B streptococcal meningitis. Brain iNOS activity increased during meningitis (P < .001), and iNOS was detected by immunocytochemistry in the walls of meningeal vessels and cells of the cerebrospinal fluid (CSF) inflammation. Animals treated with iNOS inhibitor aminoguanidine (AG; 130 mg/kg every 8 h) had reduced NO production (P < .05), higher CSF bacterial titers (P < .05), and increased incidence of seizures (P < .01) compared with untreated infected animals. AG also increased areas of severe hypoperfusion in the cortex (31% +/- 14% in controls vs. 56% +/- 16% in AG; P < .01) and the extent of cortical neuronal injury, both when administered at the time of infection (P < .05) and in established meningitis (P < .02). Thus, NO produced by iNOS may be beneficial in this model of experimental meningitis by reducing cerebral ischemia.

Antibacterial activity of beta-lactam antibiotics in experimental meningitis due to Streptococcus pneumoniae

In order to define the characteristics of the antibacterial activity of beta-lactam antibiotics in the treatment of bacterial meningitis, the relationship between cerebrospinal fluid (CSF) drug concentrations and the rate of bacterial killing was investigated for penicillin G and four new cephalosporins in an animal model of meningitis due to Streptococcus pneumoniae. All five drugs showed a significant correlation between increasing drug concentrations in CSF and increasing bactericidal rates. Minimal activity was observed in CSF at drug concentrations of approximately the broth minimal bactericidal concentration (MBC). Maximal activity occurred with CSF concentrations 10-30 times higher. In vitro tests did not reproduce the unique correlation of increasing drug concentrations and killing activity found in vivo. When evaluating new beta-lactam antibiotics for the treatment of bacterial meningitis, it is reasonable to establish a minimum standard of CSF drug concentrations of greater than or equal to 30 times the MBC against the infecting organism.

closo-borane conjugated regulatory peptides retain high biological affinity: synthesis of closo-borane conjugated Tyr(3)-octreotate derivatives for BNCT

Despite the improvements in cancer therapy during the past years, high-grade gliomas and many other types of cancer are still extremely resistant to current forms of therapy. Boron neutron capture therapy (BNCT) provides a promising way to destroy cancer cells without damaging healthy tissue. However, BNCT in practice is still limited due to the lack of boron-containing compounds that selectively deliver boron to cancer cells. Since many neuroendocrine tumors show an overexpression of the somatostatin receptor, it was our aim to synthesize compounds that contain a large number of boron atoms and still show high affinity toward this transmembrane receptor. The synthetic peptide Tyr (3)-octreotate (TATE) was chosen as a high-affinity and internalizing tumor targeting vector (TTV). Novel boron cluster compounds, containing 10 or 20 boron atoms, were coupled to the N-terminus of TATE. The obtained affinity data demonstrate that the use of a spacer between TATE and the closo-borane moiety is the option to avoid a loss of biological affinity of closo-borane conjugated TATE. For the first time, it was shown that closo-borane conjugated regulatory peptides retain high biological affinity and selectivity toward their transmembrane tumor receptors. The results obtained and the improvement of spacer and boron building block chemistry may stimulate new directions for BNCT.

Catapult-like release of mitochondrial DNA by eosinophils contributes to antibacterial defense

Although eosinophils are considered useful in defense mechanisms against parasites, their exact function in innate immunity remains unclear. The aim of this study is to better understand the role of eosinophils within the gastrointestinal immune system. We show here that lipopolysaccharide from Gram-negative bacteria activates interleukin-5 (IL-5)- or interferon-gamma-primed eosinophils to release mitochondrial DNA in a reactive oxygen species-dependent manner, but independent of eosinophil death. Notably, the process of DNA release occurs rapidly in a catapult-like manner--in less than one second. In the extracellular space, the mitochondrial DNA and the granule proteins form extracellular structures able to bind and kill bacteria both in vitro and under inflammatory conditions in vivo. Moreover, after cecal ligation and puncture, Il5-transgenic but not wild-type mice show intestinal eosinophil infiltration and extracellular DNA deposition in association with protection against microbial sepsis. These data suggest a previously undescribed mechanism of eosinophil-mediated innate immune responses that might be crucial for maintaining the intestinal barrier function after inflammation-associated epithelial cell damage, preventing the host from uncontrolled invasion of bacteria.

Regulation of mitochondrial function by hypoxia and inflammation in sepsis: A putative role for hypoxia inducible factor

Sepsis-related organ failure is the leading cause of mortality in European intensive care units (ICU). Although the inflammatory cascade of mediators in response to infection is well known, the relationships between regional inflammation, microvascular heterogeneity, hypoxia and hypoxia-inducible gene expression, and finally, organ dysfunction, are unknown. Growing evidence suggests that not only low oxygen supply to the tissues secondary to macrovascular and microvascular alterations, but also altered cellular oxygen utilization is involved in the development of multiorgan dysfunction [1]–[3]. Microbial products and innate and adaptive dysregulated immune response to infection directly affect parenchymal cells of organs and may contribute to multiorgan dysfunction.