IFI16 is required for DNA sensing in human macrophages by promoting production and function of cGAMP.

Innate immune activation by macrophages is an essential part of host defence against infection. Cytosolic recognition of microbial DNA in macrophages leads to induction of interferons and cytokines through activation of cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING). Other host factors, including interferon-gamma inducible factor 16 (IFI16), have been proposed to contribute to immune activation by DNA. However, their relation to the cGAS-STING pathway is not clear. Here, we show that IFI16 functions in the cGAS-STING pathway on two distinct levels. Depletion of IFI16 in macrophages impairs cGAMP production on DNA stimulation, whereas overexpression of IFI16 amplifies the function of cGAS. Furthermore, IFI16 is vital for the downstream signalling stimulated by cGAMP, facilitating recruitment and activation of TANK-binding kinase 1 in STING complex. Collectively, our results suggest that IFI16 is essential for efficient sensing and signalling upon DNA challenge in macrophages to promote interferons and antiviral responses.

[Medicinal treatment of breathing disorders in adenotonsillar hyperplasia]. / Medikamentöse Behandlung von Atmungsstörungen bei adenotonsillärer Hyperplasie.

BACKGROUND: Adenotonsillar hyperplasia (ATH) can lead to severe breathing disorders, such as impaired nasal breathing, mouth breathing, snoring and obstructive sleep apnea. In such cases ATH should be treated mostly by performing adenoidectomy and/or adenotonsillectomy. There is increasing evidence that anti-inflammatory medication (AIM) is effective in treating ATH-related breathing disorders. OBJECTIVES: The aim of this study was to provide evidence and recommendations for the use of AIM in the treatment of ATH-related breathing disorders. METHODS: In this study 12 national pediatric sleep experts were included into a Delphi process and formulated indications and recommendations. RESULTS: The use of AIM in the treatment of ATH-related breathing disorders is sufficiently supported by the results of randomized controlled trials and systematic reviews. Nasal beclometason and nasal mometason have been studied for the treatment of enlarged adenoids and nasal fluticason and oral montelukast for the treatment of obstructive sleep apnea. The use of AIM for first-line treatment should be restricted to selected indications, such as a characteristic patient age and exclusion of an acute upper respiratory tract infection. Evidence-based recommendations are given concerning indications, dosage, treatment duration and correct administration of AIM. CONCLUSIONS: Anti-inflammatory medications are simple and effective alternatives for the treatment of ATH-related breathing disorders. These guidelines are intended to promote the use of AIM by pediatricians in ambulatory care settings.

Determinants of adrenal androgen hypofunction in premenopausal females with rheumatoid arthritis.

The aim of our study was to investigate adrenocortical function in the context of disease activity and inflammatory status in premenopausal RA females. Adrenal glucocorticoid and androgen responses to the 1 microg ACTH 1-24 test were investigated in 23 premenopausal RA and in 15 age- and BMI-matched healthy females. Twelve RA patients were on low-dose prednisone (<8.5 mg/day). Patients with DAS28>3.2 had lower (p<0.05) total plasma cortisol, 17-hydroxyprogesterone, dehydroepiandrosterone and androstenedione responses in the ACTH test compared to healthy controls. Patients with DAS28>3.2 had lower (p<0.05) dehydroepiandrosterone response in the ACTH test compared to patients with DAS28

Microglia: first responders in the central nervous system.

Microglia has emerged not only as an essential inflammatory cell but also as a major player in the development of the adult brain. Microglia phagocytize extra-numerical synapses during postnatal development, maintain and strengthen the remaining subset of synapses, remodel synaptic circuits and clearing apoptotic newborn neurons. Thereby, microglia plays a crucial role for the establishment, plasticity and function of adult neural circuits. In addition to the key role in normal brain function, any imbalance in microglia activity has been associated with neurodegenerative diseases. Microglial cells respond rapidly to smallest pathological changes, this being a vital aspect in many tissue scaring and the local confinement of focal lesions. It is assumed that the high motility of microglial cells represents an important requirement to fulfill the numerous functions. In this review will highlight the role of microglial motility in the healthy and the injured brain, and discuss how impairment of microglia motility can affect normal brain function.

Combined two-photon laser-scanning microscopy and spectral microCT X-ray imaging to characterize the cellular signature and evolution of microstroke foci.

Occlusive brain ischemia and micro-strokes are the most frequent brain pathologies, particularly in older patients and a major cause of dementia. Currently, we are missing appropriate methodology to study micro-strokes in experimental animals. In vivo two-photon laser-scanning microscopy (2P-LSM) and transgenic mouse models expressing cell type specific reporters have been used to examine ischemia-related insults, e.g. perturbations of neuronal process morphology and local blood flow in the MCAO - middle cerebral artery occlusion-model. Glia and pericytes can be visualized by selective fluorescent protein expression, e.g. astrocytes by their cyan-fluorescent ECFP, pericytes by red-fluorescent tdtomato and microglia by green fluorescent EGFP expression. In these mice, the breakdown of the blood brain barrier and the immediate as well as long-term cellular responses can be monitored. A new prototype of microCT incorporating a fast X-ray XPAD3 camera has been recently set up to allow cerebral angiography at high sampling rate. Preliminary data indicate that it is useful to monitor blood perfusion disturbance (i.e. lateralization) in the brain of tumor-bearing mice following retro-orbital injection of iodinated contrast agent. We expect this technology to be adequate to assess in real time the impact of acute stroke models on brain blood perfusion. By localizing perfusion anomalies, we will evaluate the extent of non-perfused areas and correlate these observations with subsequent behavioral deficits, and with local changes in myelin content in white matter tracks. The spectral properties of the XPAD3 detector moreover allow for the simultaneous identification and localization of several contrast agents opening the way to whole body multicolor imaging of vessels and inflammatory cells in the context of microstrokes.

Constitutive Notch2 signaling in neural stem cells promotes tumorigenic features and astroglial lineage entry.

Recent studies identified a highly tumorigenic subpopulation of glioma stem cells (GSCs) within malignant gliomas. GSCs are proposed to originate from transformed neural stem cells (NSCs). Several pathways active in NSCs, including the Notch pathway, were shown to promote proliferation and tumorigenesis in GSCs. Notch2 is highly expressed in glioblastoma multiforme (GBM), a highly malignant astrocytoma. It is therefore conceivable that increased Notch2 signaling in NSCs contributes to the formation of GBM. Here, we demonstrate that mice constitutively expressing the activated intracellular domain of Notch2 in NSCs display a hyperplasia of the neurogenic niche and reduced neuronal lineage entry. Neurospheres derived from these mice show increased proliferation, survival and resistance to apoptosis. Moreover, they preferentially differentiate into astrocytes, which are the characteristic cellular population of astrocytoma. Likewise, we show that Notch2 signaling increases proliferation and resistance to apoptosis in human GBM cell lines. Gene expression profiling of GBM patient tumor samples reveals a positive correlation of Notch2 transcripts with gene transcripts controlling anti-apoptotic processes, stemness and astrocyte fate, and a negative correlation with gene transcripts controlling proapoptotic processes and oligodendrocyte fate. Our data show that Notch2 signaling in NSCs produces features of GSCs and induces astrocytic lineage entry, consistent with a possible role in astrocytoma formation.

Role of Nef in primate lentiviral immunopathogenesis.

More than a decade ago it was established that intact nef genes are critical for efficient viral persistence and greatly accelerate disease progression in SIVmac-infected rhesus macaques and in HIV-1-infected humans. Subsequent studies established a striking number of Nef functions that evidently contribute to the maintenance of high viral loads associated with the development of immunodeficiency in the 'evolutionary-recent' human and the experimental macaque hosts. Recent data show that many Nef activities are conserved across different lineages of HIV and SIV. However, some differences also exist. For example, Nef alleles from most SIVs that do not cause disease in their natural monkey hosts, but not those of HIV-1 and its simian precursors, down-modulate TCR-CD3 to suppress T cell activation and programmed death. This evolutionary loss of a specific Nef function may contribute to the high virulence of HIV-1 in humans.

Rapid development of severe end-organ damage in C57BL/6 mice by combining DOCA salt and angiotensin II.

The C57BL/6 mouse strain serves as the genetic background of many transgenic and gene knockout models; however, this strain appears to be resistant to hypertension-induced renal injury. We developed a new model of hypertensive end-organ damage in C57BL/6 mice by combining deoxycorticosterone acetate (DOCA) and salt with angiotensin II infusion. The systolic blood pressure (SBP) was significantly elevated in DOCA salt-angiotensin II mice compared to control mice or mice treated individually with DOCA salt or angiotensin II. Hypertensive glomerular damage, increased expression of profibrotic and inflammatory genes, albuminuria, tubular casts, increased plasma cholesterol, cardiac hypertrophy, and fibrosis were found in mice treated with DOCA salt-angiotensin II. The SBP in the angiotensin II-infused group was further increased by increasing the infusion rate; only mild injury was observed in these mice, suggesting that blood pressure was not a causal factor. Removal of DOCA and the angiotensin pump lowered blood pressure to normal; however, albuminuria along with the glomerular and cardiac damage did not completely resolve. Our study describes a new model of hypertensive end-organ damage and repair in C57BL/6 mice.

Glia: the fulcrum of brain diseases.

Neuroglia represented by astrocytes, oligodendrocytes and microglial cells provide for numerous vital functions. Glial cells shape the micro-architecture of the brain matter; they are involved in information transfer by virtue of numerous plasmalemmal receptors and channels; they receive synaptic inputs; they are able to release 'glio'transmitters and produce long-range information exchange; finally they act as pluripotent neural precursors and some of them can even act as stem cells, which provide for adult neurogenesis. Recent advances in gliology emphasised the role of glia in the progression and handling of the insults to the nervous system. The brain pathology, is, to a very great extent, a pathology of glia, which, when falling to function properly, determines the degree of neuronal death, the outcome and the scale of neurological deficit. Glial cells are central in providing for brain homeostasis. As a result glia appears as a brain warden, and as such it is intrinsically endowed with two opposite features: it protects the nervous tissue as long as it can, but it also can rapidly assume the guise of a natural killer, trying to eliminate and seal the damaged area, to save the whole at the expense of the part.

Exosomes are released by cultured cortical neurones.

Accumulating evidence shows that several cell types have the capacity to secrete membrane proteins by incorporating them into exosomes, which are small lipid vesicles derived from the intralumenal membranes of multivesicular bodies (MVBs) of the endocytic pathway. Exosomes are expelled in the extracellular space upon fusion of the MVB with the plasma membrane. Exosomal release is a way of secreting membrane proteins meant to be discarded, or to be passed on to other cells. Here, we demonstrate, using primary cortical cultures, that neurones and astrocytes can secrete exosomes. We find that exosomes released by cortical neurones contain the L1 cell adhesion molecule, the GPI-anchored prion protein, and the GluR2/3 but not the NR1 subunits of glutamate receptors. We also show that exosomal release is regulated by depolarisation. Our observation suggests that exosomes may have a regulatory function at synapses and could also allow intercellular exchange of membrane proteins within the brain.

GABA(A)-receptor expression in glioma cells is triggered by contact with neuronal cells.

The expression of functional GABA(A)-receptors in glioma cells correlates with low malignancy of tumours and cell lines from glioma lack these receptors. Here we show that contact with neurons induces the expression of functional GABA(A)-receptors. C6 and F98 glioma cell lines were labelled by recombinant expression of enhanced green fluorescent protein injected into rat brain and studied in acute slices after two to three weeks of tumour growth. The cells responded to GABA or the specific agonist, muscimol with a current typical for GABA(A)-receptors, as studied with the patch-clamp technique. To get insight into the mechanism of GABA(A) receptor induction, the C6 or F98 cells were co-cultured with neurons, astrocytes, oligodendrocytes and microglia. Glioma cells expressed functional GABA(A) receptors within 24 h only in cultures where physical contact to neurons occurred. Activation of GABA(A)-receptors in the co-cultures attenuated glioma cell metabolism while blockade of the receptors increased metabolism. We conclude that with this form of interaction, neurons can influence tumour behaviour in the brain.

Pathways of neuron-astrocyte interactions and their possible role in neuroprotection.

Astrocytes are the most numerous cell type within the central nervous system. Early during development they act as guiding structures for migratory neurons; later they are not only the main source for nutrients and growth factors in the brain, but they are also communication partners of neighboring neurons. For this purpose astrocytes are equipped with several types of transmitter receptors and the capacity to release neuroactive substances. In addition, they form an extended syncytium via gap junction channels which allows fast intercellular signaling pathways. The pivotal involvement of astrocytes in brain function during disease situations is the topic of many studies. Here, we will review the role of astrocytic gap junctions, astroglial metabolism and neuron-astrocyte signaling. Identification of the molecular mechanisms of these three functions will improve our understanding of neuroprotection.

CD4 down-modulation by human immunodeficiency virus type 1 Nef correlates with the efficiency of viral replication and with CD4(+) T-cell depletion in human lymphoid tissue ex vivo.

The human immunodeficiency virus type 1 (HIV-1) Nef protein is an important virulence factor. Nef has several functions, including down-modulation of CD4 and class I major histocompatibility complex cell surface expression, enhancement of virion infectivity, and stimulation of viral replication in peripheral blood mononuclear cells. Nef also increases HIV-1 replication in human lymphoid tissue (HLT) ex vivo. We analyzed recombinant and primary nef alleles with highly divergent activity in different in vitro assays to clarify which of these Nef activities are functionally linked. Our results demonstrate that Nef activity in CD4 down-regulation correlates significantly with the efficiency of HIV-1 replication and with the severity of CD4(+) T-cell depletion in HLT. In conclusion, HIV-1 Nef variants with increased activity in CD4 down-modulation would cause severe depletion of CD4(+) T cells in lymphoid tissues and accelerate AIDS progression.

DC-SIGN interactions with human immunodeficiency virus: virus binding and transfer are dissociable functions.

The C-type lectins DC-SIGN and DC-SIGNR capture and transfer human immunodeficiency virus (HIV) to susceptible cells, although the underlying mechanism is unclear. Here we show that DC-SIGN/DC-SIGNR-mediated HIV transmission involves dissociable binding and transfer steps, indicating that efficient virus transmission is not simply due to tethering of virus to the cell surface.

Efficient class I major histocompatibility complex down-regulation by simian immunodeficiency virus Nef is associated with a strong selective advantage in infected rhesus macaques.

Substitution of Y223F disrupts the ability of simian immunodeficiency virus (SIV) Nef to down-modulate major histocompatibility complex (MHC) class I from the cell surface but has no effect on other Nef functions, such as down-regulation of CD4, CD28, and CD3 cell surface expression or stimulation of viral replication and enhancement of virion infectivity. Inoculation of three rhesus macaques with the SIVmac239 Y223F-Nef variant revealed that this point mutation consistently reverts and that Nef activity in MHC class I down-modulation is fully restored within 4 weeks after infection. Our results demonstrate a strong selective pressure for a tyrosine at amino acid position 223 in SIV Nef, and they constitute evidence that Nef-mediated MHC class I down-regulation provides a selective advantage for viral replication in vivo.

Effects of lectins on CCK-8-stimulated enzyme secretion and differentiation of the rat pancreatic cell line AR42J.

INTRODUCTION: The peptide hormone cholecystokinin (CCK) plays an important role in the gastrointestinal tract. The rat pancreatic CCK receptor is a highly glycosylated membrane receptor that is able to bind to plant lectins such as wheat germ agglutinin (WGA) and Ulex europaeus agglutinin (UEA-I). AIMS AND METHODOLOGY: We used both lectins to block this receptor for studying the pathophysiologic relevance of its oligosaccharide side chains. In the present study we investigated the influence of WGA and UEA-I on CCK-8-induced alpha-amylase secretion of the rat pancreatic tumor cell line AR42J, which expresses both CCK-A and CCK-B receptors. RESULTS: Under the influence of WGA (25 microg/mL), the alpha-amylase release was reduced by 25% after 30 minutes compared with the hormone-stimulated controls. UEA-I (25 microg/mL) caused a reduction of 20%. The simultaneous application of the lectins with CCK antagonists L 364,718 or L 365,260 led to a reduction of secretion, but the assignment to CCK-A or CCK-B receptors was not possible. CONCLUSION: In long-term studies, both lectins revealed no toxic or apoptosis-inducing effects. On the contrary, WGA showed an inhibitory effect on cell proliferation and led to improved differentiation of cells.

Simian immunodeficiency virus in which nef and U3 sequences do not overlap replicates efficiently in vitro and in vivo in rhesus macaques.

The nef genes of human immunodeficiency virus and simian immunodeficiency virus (SIV) overlap about 80% of the U3 region of the 3' long terminal repeat (LTR) and contain several essential cis-acting elements (here referred to as the TPI region): a T-rich region, the polypurine tract, and attachment (att) sequences required for integration. We inactivated the TPI region in the nef reading frame of the pathogenic SIVmac239 clone (239wt) by 13 silent point mutations. To restore viral infectivity, intact cis-regulatory elements were inserted just downstream of the mutated nef gene. The resulting SIV genome contains U3 regions that are 384 bp shorter than the 517-bp 239wt U3 region. Overall, elimination of the duplicated Nef coding sequences truncates the proviral genome by 350 bp. Nonetheless, it contains all known coding sequences and cis-acting elements. The TPI mutant virus expressed functional Nef and replicated like 239wt in all cell culture assays and in vivo in rhesus macaques. Notably, these SIVmac constructs allow us to study Nef function in the context of replication-competent viruses without the restrictions of overlapping LTR sequences and important cis-acting elements. The genomes of all known primate lentiviruses contain a large overlap between nef and the U3 region. We demonstrate that this conserved genomic organization is not obligatory for efficient viral replication and pathogenicity.

Basic amino acid residues in the V3 loop of simian immunodeficiency virus envelope alter viral coreceptor tropism and infectivity but do not allow efficient utilization of CXCR4 as entry cofactor.

In contrast to human immunodeficiency viruses type 1 and type 2 (HIV-1 and HIV-2, respectively), simian immunodeficiency virus (SIVmac) rarely uses CXCR4 (X4) for efficient entry into target cells. Basic amino acid residues in the V3 loop of HIV Env allow efficient coreceptor utilization of X4. Therefore, we investigated if similar changes in the SIVmac Env protein also mediate a coreceptor switch from CCR5 (R5) to X4. Functional analysis revealed that none of eight SIVmac variants, containing V3 regions with an overall charge between +4 and +10, efficiently utilized X4 as entry cofactor. Nonetheless, these alterations had differential effects on SIV coreceptor tropism and on Env expression levels. A single amino acid substitution of L328R, located near the tip of the V3 loop, resulted in grossly reduced Env expression levels and impaired viral infectivity. Notably, additional basic residues restored efficient Env expression and virion incorporation but not infectivity. In comparison to the L328R mutation, changes of P334K and D337K had little disruptive effects on SIVmac entry and replication. Interestingly, mutation of L320K and P321R disrupted coreceptor usage of GPR15 but not R5. These changes also impaired SIVmac replication in peripheral blood mononuclear cells (PBMC) derived from a Delta32/Delta32 donor but not in R5-expressing human or simian PBMC. Our results show that positively charged amino acid residues in the V3 loop affect SIVmac coreceptor tropism and infectivity but do not allow efficient utilization of X4.

AN2/NG2 protein-expressing glial progenitor cells in the murine CNS: isolation, differentiation, and association with radial glia.

During early neural development, the lineage specification of initially pluripotent progenitor cells is associated with proliferation, differentiation, and migration. Oligodendroglial progenitor cells migrate from their sites of origin to reach the axons that they will myelinate. We have described a cell-surface protein, AN2, expressed by oligodendroglial progenitor cells in vitro and showed that antibodies against AN2 inhibited the migration of cultured primary oligodendroglial progenitor cells, suggesting that the AN2 antigen plays a role in their migration. Recently, results from MALDI mass spectroscopy showed that AN2 is the mouse homologue of the rat NG2 protein. In this study, we have analyzed cells staining with AN2 antibodies during development and in the adult murine central nervous system (CNS), carried out double stainings with antibodies against NG2, and investigated the differentiation potential of cells in vitro after isolation from early postnatal brain using AN2 antibodies. AN2 and NG2 antibodies stained totally overlapping populations of cells in the CNS. AN2/NG2 expressing cells in embryonic and postnatal brain expressed the PDGF-alpha-receptor and in postnatal brain exhibited electrophysiological properties typical of glial progenitor cells. Cells isolated from early postnatal brain using AN2 monoclonal antibody developed into oligodendrocytes in low serum medium or into astrocytes in the presence of fetal calf serum. In the embryonic spinal cord, cells staining with AN2 antibodies were found closely apposed to radial glial cells, suggesting that glial precursors, like neurons, may use radial glia as scaffolds for migration.