In vitro comparison of different carrier materials with rat bone marrow MSCs.

OBJECTIVES: Injectable or implantable scaffolds seeded with autologous chondrogenic cells may represent a promising option for treatment of cartilage defects in the future. Current problems with the autologous chondrocyte implantation including dedifferentiation and the development of fibrocartilage suggest the use of alternative chondrogenic cell sources such as mesenchymal stromal cells (MSCs). The aim of this study was to compare the early effects of different scaffolds on the proliferation and metabolic activity of chondrogenic MSCs in vitro. MATERIALS AND METHODS: Multipotent stromal cells were isolated from rat bone marrow, phenotyped by flow cytometry, and differentiated into distinct lineages proved by lineage-specific staining and gene expression (RT-PCR) pattern. Cell proliferation on Tutodent® Membrane, Bio-Gide®, TissuFleece E, and Belotero® Soft was quantified by the MTT and WST-1 assay and direct determination of total cell numbers. Potential cytotoxic effects of eluates obtained from the materials were quantified by lactate dehydrogenase (LDH) and 5-bromo-2-deoxyuridine (BrdU) assay. RESULTS: TissuFleece E displayed the best results regarding cell proliferation on the biomaterials and metabolic activity (MTT, WST-1) (p < 0.001). Yet, the eluates of TissuFleece E caused an increased LDH release and lower values in the BrdU test. Cell proliferations on Bio-Gide®, Tutodent® Membrane, and Belotero® Soft were similar to the control. The eluates of Belotero® Soft exhibited the highest LDH release and lowest values in the BrdU assay (p < 0.05). CONCLUSIONS: Our results support the use of Tissufleece E as scaffold for chondrogenic rat MSCs. However, it should be prewashed with culture medium before seeding of the cells. CLINICAL RELEVANCE: Tissufleece E may serve as a promising carrier material for chondrogenic MSCs for cartilage tissue engineering attempts.

A low level of extragalactic background light as revealed by gamma-rays from blazars.

The diffuse extragalactic background light consists of the sum of the starlight emitted by galaxies through the history of the Universe, and it could also have an important contribution from the 'first stars', which may have formed before galaxy formation began. Direct measurements are difficult and not yet conclusive, owing to the large uncertainties caused by the bright foreground emission associated with zodiacal light. An alternative approach is to study the absorption features imprinted on the gamma-ray spectra of distant extragalactic objects by interactions of those photons with the background light photons. Here we report the discovery of gamma-ray emission from the blazars H 2356 - 309 and 1ES 1101 - 232, at redshifts z = 0.165 and z = 0.186, respectively. Their unexpectedly hard spectra provide an upper limit on the background light at optical/near-infrared wavelengths that appears to be very close to the lower limit given by the integrated light of resolved galaxies. The background flux at these wavelengths accordingly seems to be strongly dominated by the direct starlight from galaxies, thus excluding a large contribution from other sources-in particular from the first stars formed. This result also indicates that intergalactic space is more transparent to gamma-rays than previously thought.

Discovery of very-high-energy gamma-rays from the Galactic Centre ridge.

The source of Galactic cosmic rays (with energies up to 10(15) eV) remains unclear, although it is widely believed that they originate in the shock waves of expanding supernova remnants. At present the best way to investigate their acceleration and propagation is by observing the gamma-rays produced when cosmic rays interact with interstellar gas. Here we report observations of an extended region of very-high-energy (> 10(11) eV) gamma-ray emission correlated spatially with a complex of giant molecular clouds in the central 200 parsecs of the Milky Way. The hardness of the gamma-ray spectrum and the conditions in those molecular clouds indicate that the cosmic rays giving rise to the gamma-rays are likely to be protons and nuclei rather than electrons. The energy associated with the cosmic rays could have come from a single supernova explosion around 10(4) years ago.

Pre-activated antiviral innate immunity in the upper airways controls early SARS-CoV-2 infection in children.

Children have reduced severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection rates and a substantially lower risk for developing severe coronavirus disease 2019 compared with adults. However, the molecular mechanisms underlying protection in younger age groups remain unknown. Here we characterize the single-cell transcriptional landscape in the upper airways of SARS-CoV-2-negative (n = 18) and age-matched SARS-CoV-2-positive (n = 24) children and corresponding samples from adults (n = 44), covering an age range of 4 weeks to 77 years. Children displayed higher basal expression of relevant pattern recognition receptors such as MDA5 (IFIH1) and RIG-I (DDX58) in upper airway epithelial cells, macrophages and dendritic cells, resulting in stronger innate antiviral responses upon SARS-CoV-2 infection than in adults. We further detected distinct immune cell subpopulations including KLRC1 (NKG2A)+ cytotoxic T cells and a CD8+ T cell population with a memory phenotype occurring predominantly in children. Our study provides evidence that the airway immune cells of children are primed for virus sensing, resulting in a stronger early innate antiviral response to SARS-CoV-2 infection than in adults.

Overexpression of acid ceramidase protects from tumor necrosis factor-induced cell death.

Tumor necrosis factor (TNF) signals cell death and simultaneously induces generation of ceramide. To evaluate the contribution of ceramide to TNF-dependent cell death, we generated clones of the TNF-sensitive cell line L929 that constitutively overexpress human acid ceramidase (AC). Ceramidase, in concert with sphingosine kinase, metabolizes ceramide to sphingosine-1-phosphate (SPP), an inducer of proliferation. In response to TNF, parental L929 cells display a significant increase in intracellular ceramide correlated with an "atypical apoptosis" characterized by membrane blebbing, DNA fragmentation and degradation of poly(ADP-ribose) polymerase despite a lack of caspase activity. These features are strongly reduced or absent in AC-overexpressing cells. Pharmacological suppression of AC with N-oleoylethanolamine restored the accumulation of intracellular ceramide as well as the sensitivity of the transfectants to TNF, implying that an enhanced metabolization of intracellular ceramide by AC shifts the balance between intracellular ceramide and SPP levels towards cell survival. Correspondingly, inhibition of ceramide production by acid sphingomyelinase also increased survival of TNF-treated L929 cells.

Human NK cells require caspases for activation-induced proliferation and cytokine release but not for cytotoxicity.

Natural killer (NK) cells are innate immune cells involved in antiviral defence and tumour surveillance. To fulfil these tasks, NK cells make use of two major effector functions, cytokine and chemokine release and cytotoxicity. In addition, NK cells proliferate in response to cytokines such as IL-2. NK cells possess a large array of activating and inhibitory receptors and their activation demands a complex crosstalk between those receptors. The signalling pathways leading to NK-cell activation are a field of intensive research. The first clue for signal specificity was provided by studies showing that a pathway leading to NF-κB activation selectively induces cytokine release, but is dispensable for cytotoxicity. Here, we demonstrate that in human NK cells caspase activity is required for the upregulation of select activation markers and IFN-γ and TNF production, but not for cytotoxicity. Interestingly, caspases have previously been linked in T cells to the same mechanism of NF-κB induction that is active in NK cells. Moreover, we provide evidence that caspases are involved in IL-2-induced proliferation. Thus, our data provide the basis for a novel approach using caspase inhibitors to generate cytotoxic NK cells, while simultaneously suppressing cytokine release.

Involvement of FAN in TNF-induced apoptosis.

TNF-alpha is a pleiotropic cytokine activating several signaling pathways initiated at distinct intracellular domains of the TNF receptors. Although the C-terminal region is believed to be responsible for apoptosis induction, the functions of more membrane-proximal domains, including the domain that couples to neutral sphingomyelinase activation, are not yet fully elucidated. The roles of this region and of the associated adapter protein FAN (factor associated with neutral SMase activation) in the cytotoxic response to TNF have been investigated. We have now shown that stable expression in human fibroblasts of a dominant negative form of FAN abrogates TNF-induced ceramide generation from sphingomyelin hydrolysis and reduces caspase processing, thus markedly inhibiting TNF-triggered apoptosis. However, the cytotoxic responses to daunorubicin and exogenous ceramide remain unaltered, as do the TNF-induced p42/p44 MAPK activation and CD54 expression. Fibroblasts from FAN-knockout mice also proved to be resistant to TNF toxicity. These findings highlight the previously unrecognized role of the adapter protein FAN in signaling cell death induction by TNF.

Molecular cloning and analysis of cDNA encoding the murine c-yes tyrosine protein kinase.

The cellular yes (c-yes) gene is a member of the class of proto-oncogenes that encode non-receptor tyrosine protein kinases. In this report we describe the isolation of cDNAs that encode the murine c-yes gene product and analysis of the nucleotide sequence of the murine c-yes cDNA clones. The reading frame encodes a protein of 541 amino acids with a calculated molecular mass of 60.63 kDa that is reactive with anti-Yes antisera and possesses protein kinase activity.

Distinct adapter proteins mediate acid versus neutral sphingomyelinase activation through the p55 receptor for tumor necrosis factor.

Ceramide, generated by the enzymatic function of sphingomyelinases (SMases) has emerged as an important signaling pathway transducing diverse biological effects of various cytokine receptors. The 55-kDa receptor for tumor necrosis factor (TNF-R55) activates two types of SMases through distinct cytoplasmic domains. The death domain that is responsible for the initiation of the apoptotic pathway also signals for the activation of an acid SMase (A-SMase). The adapter protein TRADD binds to TNF-R55 in a ligand-dependent manner and serves as anchor for the subsequent recruitment of other proteins into the signaling complex that directly lead to cell death or nuclear factor-kappaB (NF-kappaB) induction. Notably, the two pro-apoptotic adapter proteins TRADD and FADD are also involved in the activation of A-SMase. In contrast, the NF-kappaB-inducing adapters TRAF2 and RIP do not signal for A-SMase. Thus, activation of A-SMase appears to belong to signals leading to TNF-induced cell death. A second signaling domain (NSD) is located upstream of the death domain and directly links the TNF-R55 to the activation of a neutral SMase (N-SMase). A novel adapter protein, FAN, has been identified that specifically binds to the NSD. FAN contains five WD repeats at its carboxy terminus, while it shows significant sequence homology with the mouse beige protein and its human homolog, the CHS protein, in the center portion of the protein. Overexpression of full-length FAN enhanced N-SMase activity in TNF-treated cells, whereas truncated mutants of FAN produced dominant negative effects. FAN, however, did not interfere with any of the TNF responses signaled for by the death domain. Taken together, our data suggest that distinct cytoplasmic domains of TNF-R55 initiate independent signaling pathways by binding different adapter proteins.

[Methods of resolution for haptic assistance during catheterization]. / Lösungsansätze für haptische Assistenz bei Katheterisierungen.

During catheterization navigation within the patient is mainly dependent on a live x-ray image on the screen. Although methods for 3D visualisation and remote navigation of the catheter are discussed and tested still precise positioning is merely the result of intense training and a high skill and level of training of the performing surgeon. This article refers to a system which can be considered as an add-on for existing procedures of catheterization. It compromises of a miniaturised force sensor located at the tip of guide-wires whose prototype is shown here. The measured forces will be presented to the surgeon amplified by an external actuator described in this article. As a result a haptic perception of the forces between the tip of the guide-wire and the vessels walls will be available and enable the surgeon to gain an impression which is comparable to palpation of living vessels from the inside

Multiplicity dependent expression of the predominant phosphoprotein pp65 of human cytomegalovirus.

Recent clinical isolates of human cytomegalovirus (HCMV) display considerable quantitative differences in the expression of the structural phosphoprotein pp65. This study shows that a reduced production of pp65 correlates with reduced amounts of its coding transcript in wild-type strains compared to the high level expression in the laboratory strain AD169. The cleavage pattern of the encoding DNAs including the promoter region did not show major differences among the isolates. Therefore the variable expression of pp65 is supposed to be dependent on cell culture conditions. Increasing multiplicities of infection resulted in overproduction of pp65 and this may lead to the increased formation of dense body particles.

Identification of p55 tumor necrosis factor receptor-associated proteins that couple to signaling pathways not initiated by the death domain.

The 55 kDa receptor for tumor necrosis factor (TNF-R55) has become a paradigm for membrane receptors that are devoid of intrinsic enzymatic activity. The initiation of intracellular signaling events observed in TNF-stimulated cells appears to depend on protein intermediates that interact with specific cytoplasmic domains of TNF-R55. By use of TNF-R55 deletion mutants, we have defined a novel domain of TNF-R55 (NSD) distinct from the death domain which is specifically required for activation of neutral sphingomyelinase (N-SMase). In addition, using the yeast interaction trap system, we have identified one protein (FAN) that binds to the NSD and mediates activation of N-SMase as well as seventeen other potential interaction partners of TNF-R55. These candidate interactors include the adaptor protein Grb2-linking TNF-R55 to the Ras pathway-as well as the enzyme phosphoglycerate mutase, suggesting a role of TNF-R55 in glycolysis/ energy metabolism of cells.

Second-line drug susceptibility breakpoints for Mycobacterium tuberculosis using the MODS assay.

OBJECTIVE: To establish breakpoint concentrations for the fluoroquinolones (moxifloxacin [MFX] and ofloxacin [OFX]) and injectable second-line drugs (amikacin [AMK], kanamycin [KM] and capreomycin [CPM]) using the microscopic observation drug susceptibility (MODS) assay. SETTING: A multinational study conducted between February 2011 and August 2012 in Peru, India, Moldova and South Africa. DESIGN: In the first phase, breakpoints for the fluoroquinolones and injectable second-line drugs (n = 58) were determined. In the second phase, MODS second-line drug susceptibility testing (DST) as an indirect test was compared to MGIT™ DST (n = 89). In the third (n = 30) and fourth (n = 156) phases, we determined the reproducibility and concordance of MODS second-line DST directly from sputum. RESULTS: Breakpoints for MFX (0.5 µg/ml), OFX (1 µg/ml), AMK (2 µg/ml), KM (5 µg/ml) and CPM (2.5 µg/ml) were determined. In all phases, MODS results were highly concordant with MGIT DST. The few discrepancies suggest that the MODS breakpoint concentrations for some drugs may be too low. CONCLUSION: MODS second-line DST yielded comparable results to MGIT second-line DST, and is thus a promising alternative. Further studies are needed to confirm the accuracy of the drug breakpoints and the reliability of MODS second-line DST as a direct test.

Modulation of CD4+ T-cell activation by CD95 co-stimulation.

CD95 is a dual-function receptor that exerts pro- or antiapoptotic effects depending on the cellular context, the state of activation, the signal threshold and the mode of ligation. In this study, we report that CD95 engagement modulates TCR/CD3-driven signaling pathways in resting T lymphocytes in a dose-dependent manner. While high doses of immobilized CD95 agonists silence T cells, lower concentrations augment activation and proliferation. We analyzed the co-stimulatory capacity of CD95 in detail in resting human CD4(+) T cells, and demonstrate that low-dose ligand-induced co-internalization of CD95 and TCR/CD3 complexes enables non-apoptotic caspase activation, the prolonged activation of MAP kinases, the upregulation of antiapoptotic proteins associated with apoptosis resistance, and the activation of transcription factors and cell-cycle regulators for the induction of proliferation and cytokine production. We propose that the levels of CD95L on antigen-presenting cells (APCs), neighboring T cells or epithelial cells regulate inhibitory or co-stimulatory CD95 signaling, which in turn is crucial for fine-tuning of primary T-cell activation.

The involvement of demethylation in the myeloid-specific function of the mouse M lysozyme gene downstream enhancer.

Lysozyme gene expression is a specific marker for the macrophage/granulocyte lineage of hematopoietic differentiation in mammals, its expression being gradually increased during maturation. Analysis of the mechanisms regulating mouse M lysozyme gene expression during myeloid differentiation revealed a complicated pattern of DNase I hypersensitive sites (HS sites) within the flanking regions of the gene. The HS-3 site, located in the 3'-flanking region of the gene, overlapped with an enhancer element, which is the only strong enhancer identified in the vicinity of the gene. We demonstrate a positive correlation between undermethylation of the entire 3'-flanking region, the appearance of the HS-3 site, and M lysozyme gene expression during in vitro differentiation of hematopoietic stem cells. We furthermore show that methylation of a single CpG site within the enhancer core element, only observed in immature macrophage cells in vivo, is sufficient to inhibit nuclear factor binding to this element in vitro and to inhibit its transactivation potential in DNA transfection experiments.

Late expiratory inhibition of stage 2 expiratory neurons in the cat--a correlate of expiratory termination.

1. Intracellular recordings were made from stage 2 expiratory bulbospinal neurons (E2Ns) in the caudal part of the ventral respiratory group in pentobarbitone-anesthetized cats, to characterize changes in neuronal input resistance (Rn) and synaptic inhibition occurring at the time of the expiratory-inspiratory phase transition of the respiratory cycle. 2. Rn was maximal between 30-90% of stage 2 expiration, but decreased significantly during the last 10% of stage 2 expiration. Mean normalized Rn for 60-90% of stage 2 expiration was 0.9 +/- 0.02, while mean Rn during the last 10% of stage 2 expiration was 0.68 +/- 0.09 (n = 8). This decrease in Rn began 200-300 ms before rapid hyperpolarization of E2N membrane potential and onset of phrenic nerve activity. 3. Under conditions of strong central respiratory drive, constant injection of positive current into E2Ns sometimes revealed a transient membrane hyperpolarization that straddled the expiratory-inspiratory phase transition. During this transient event, Rn was markedly reduced. 4. Intracellular injection of Cl- or NO3- ions into E2Ns produced reversal of chloride-dependent inhibitory synaptic potentials (IPSPs). Comparison of averages of membrane potential pattern over the whole respiratory cycle during control conditions and IPSP reversal revealed several periods of synaptic inhibition: 1) weak but progressively increasing synaptic inhibition during the second half of stage 2 expiration, 2) strong transient synaptic inhibition beginning 200-300 ms before the onset of phrenic nerve activity and ending shortly after the onset of phrenic nerve activity, and 3) strong but progressively decreasing synaptic inhibition throughout inspiration.(ABSTRACT TRUNCATED AT 250 WORDS)

CMER, an RNA encoded by human cytomegalovirus is most likely transcribed by RNA polymerase III.

Through computer analysis of a human cytomegalovirus (HCMV) genomic region, previously identified to be homologous to human genomic DNA, an element showing significant similarity to the 3'-internal control region (3'-ICR or B-block) of a eukaryotic RNA polymerase III promoter could be detected. This region-located on the EcoRI b fragment within the UL segment of the viral genome of HCMV strain AD 169-cannot be transcribed in vitro in an RNA polymerase III specific transcription system. However, this part of the viral genome is able to compete for components of the RNA polymerase III transcription complex as shown in template exclusion experiments and by gel retardation assays. Two different synthetic oligonucleotides complementary to the 3'-ICR and to nucleotides located immediately downstream of this promoter element can anneal specifically to a HCMV-encoded ribonucleic acid (termed CMER) synthesized in human foreskin fibroblasts (HFF) late in virus replication. As a consequence of identifying the transcription initiation point by primer extension analyses the position of the 5'-internal control region (5'-ICR or A-block) of the CMER gene could be uncovered. Both identified control regions (the A-block as well as the B-block) of the transcription unit exhibit significant similarities to corresponding regulatory elements of other class III genes, including virus encoded class III genes. Initiation of in vivo transcription occurs 15 nucleotides upstream of the 5'-border of the 5'-ICR and the two non-contiguous gene internal promoter elements are separated by 79 nucleotides.

Peptide design aided by neural networks: biological activity of artificial signal peptidase I cleavage sites.

De novo designed signal peptidase I cleavage sites were tested for their biological activity in vivo in an Escherichia coli expression and secretion system. The artificial cleavage site sequences were generated by two different computer-based design techniques, a simple statistical method, and a neural network approach. In previous experiments, a neural network was used for feature extraction from a set of known signal peptidase I cleavage sites and served as the fitness function in an evolutionary design cycle leading to idealized cleavage site sequences. The cleavage sites proposed by the two algorithms were active in vivo as predicted. There seems to be an interdependence between several cleavage site features for the constitution of sequences recognized by signal peptidase. It is concluded that neural networks are useful tools for sequence-oriented peptide design.

Procaryotic expression of phosphorylated tegument protein pp65 of human cytomegalovirus and application of recombinant peptides for immunoblot analyses.

The tegument of human cytomegalovirus (HCMV) contains a phosphorylated protein of 65 kilodaltons, termed pp65, which was reported to carry significant epitopes for the stimulation of the humoral immune response during natural infection. A monoclonal antibody directed against this protein was used to screen a lambda gt11 cDNA library for recombinant polypeptides. Two DNA fragments from purified lambda clones and one fragment from genomic DNA were used for cloning in a bacterial high-level expression vector. The resulting fusion proteins were tested for their reactivity with a panel of monoclonal antibodies directed against pp65 and with polyspecific anti-HCMV rabbit antisera. The binding site for all the monoclonal antibodies tested was found to be contained in one of the recombinant proteins with a viral portion of 26 amino acids. Immunoblot analyses with HCMV-positive human sera revealed that pp65 alone is not a reliable antigen for serodiagnosis but may be very useful in combination with other HCMV proteins.

Primary structure and transcription of the genes coding for the two virion phosphoproteins pp65 and pp71 of human cytomegalovirus.

Human cytomegalovirus contains a phosphorylated matrix protein of 65,000 apparent molecular weight (65K phosphoprotein; pp65) and a related phosphoprotein of 71,000 molecular weight (pp71). The 65K phosphoprotein is usually by far the most abundant structural component found in culture-grown purified virus particles. This study describes the precise mapping of the genes for both polypeptides, giving the entire nucleotide sequences and the exact positions of the respective transcripts. The 65K phosphoprotein is coded for by the 5'-terminal part of an abundant 4-kilobase (kb) mRNA. The 71K phosphoprotein corresponds to the single translational reading frame of a rare nonspliced 1.9-kb mRNA that is coterminal with the 4-kb transcript. The promoter for 4-kb mRNA appears to be unusual in structure; it does not contain a characteristic TATA sequence. The expression of antigenic epitopes from pp65 may allow improved serodiagnosis of human cytomegalovirus infections.