C5a induction of human interleukin 1. Synergistic effect with endotoxin or interferon-gamma

Interleukin-1 (IL-1) is a potent cytokine which possesses the ability to mediate systemic acute phase responses as well as local tissue inflammation. In these studies, we have examined the ability of C5a and C5a des Arg to induce IL-1 production in vitro. Human C5a and C5a des Arg were purified to homogeneity and were found to stimulate IL-1 release from freshly obtained human mononuclear cells into the extracellular medium. Only 2 hr of exposure to the purified complement components were necessary in order to stimulate IL-1 production. The minimal concentration of C5a required was 25 ng/ml, whereas 125 ng/ml of C5a des Arg induced comparable amounts of IL-1. This dose relationship was maintained at higher concentrations (150 ng/ml vs 750 ng/ml, respectively). That the effect was due to the anaphylatoxins themselves, and not endotoxin contamination, was shown by negative Limulus amebocyte lysate tests and employing preincubation of C5a/C5a des Arg with polymyxin B. The latter blocked a wide dose range of endotoxin-stimulated IL-1 production. However, when endotoxin was added to C5a or C5a des Arg, significant synergism in the stimulation of IL-1 production was observed, occurring at various concentrations of either agent. A similar synergism with C5a/C5a des Arg was seen with interferon-gamma. In these studies, IL-1 production was measured by bioassay employing cloned D . 10 . G4 . 1 murine T cells and by radioimmunoassay for human IL-1 beta; using C5a/C5a des Arg as stimulants, there was a high degree of correlation (r = 0.82) between the two assays. Since traumatic, infectious, and inflammatory diseases may result in the simultaneous appearance of these stimuli, the synergism described herein is likely to be clinically relevant.     

Neuroattenuated bunyavirus variant: derivation, characterization, and revertant clones.

A neuroattenuated variant bunyavirus, designated RFC/25B.5 (B.5), was selected by serial passage of a reassortant clone (RFC virus) of a California serogroup virus in BHK-21 cells, followed by plaque purification of that passaged stock. Based on its virulence index (ratio of PFU/50% lethal dose), clone B5 was over 40,000-fold less virulent than its unpassaged RFC parent after intracerebral (i.c.) inoculation into adult mice. Clone B.5 also exhibited markedly reduced neuroinvasiveness after subcutaneous injection into neonatal mice, although it retained its ability to replicate and kill suckling mice after i.c. injection. A murine neuroblastoma line (NA cells) can be used as an in vitro surrogate for the adult mouse brain, since clone B.5 replicated to at least 1,000-fold-lower titers in NA cells than did several neurovirulent California serogroup viruses. Clone B.5 replicated in BHK-21 cells at 37 degrees C to titers similar to those achieved by other California serogroup viruses but was temperature sensitive (ts) since its replication was markedly restricted at 38.9 degrees C. Ten ts revertant clones of B.5 virus were selected at 38.9 degrees C, and all of them lost their ts phenotype and regained the ability to replicate to high titer in NA cells and to kill adult mice after i.c. injection. Clone B.5 is the first described California serogroup virus which is truly attenuated after i.c. inoculation, and its availability will permit genetic analysis of bunyavirus neurovirulence.     

Tumor necrosis factor-alpha converting enzyme is processed by proprotein-convertases to its mature form which is degraded upon phorbol ester stimulation.

Tumor necrosis factor-alpha converting enzyme (TACE or ADAM17) is a member of the ADAM (a disintegrin and metalloproteinase) family of type I membrane proteins and mediates the ectodomain shedding of various membrane-anchored signaling and adhesion proteins. TACE is synthesized as an inactive zymogen, which is subsequently proteolytically processed to the catalytically active form. We have identified the proprotein-convertases PC7 and furin to be involved in maturation of TACE. This maturation is negatively influenced by the phorbol ester phorbol-12-myristate-13-acetate (PMA), which decreases the cellular amount of the mature form of TACE in PMA-treated HEK293 and SH-SY5Y cells. Furthermore, we found that stimulation of protein kinase C or protein kinase A signaling pathways did not influence long-term degradation of mature TACE. Interestingly, PMA treatment of furin-deficient LoVo cells did not affect the degradation of mature TACE. By examination of furin reconstituted LoVo cells we were able to exclude the possibility that PMA modulates furin activity. Moreover, the PMA dependent decrease of the mature enzyme form is specific for TACE, as the amount of mature ADAM10 was unaffected in PMA-treated HEK293 and SH-SY5Y cells. Our results indicate that the activation of TACE by the proprotein-convertases PC7 and furin is very similar to the maturation of ADAM10 although there is a significant difference in the cellular stability of the mature enzyme forms after phorbol ester treatment.     

Glycerol-induced unraveling of the tight helical conformation of Escherichia coli type 1 fimbriae.

Glycerol was found to unravel the helical conformation of Escherichia coli type 1 fimbriae without appreciable depolymerization. The linearized fimbrial polymers have a diameter of 2 nm, react strongly with a monoclonal antibody directed at an inaccessible epitope on native fimbriae, and display greater mannose-binding activity and trypsin sensitivity than native fimbriae. Removal of glycerol by dialysis results in spontaneous reassembly of the linear polymers into structures morphologically, antigenically, and functionally indistinguishable from native fimbriae.     

Ectogalactosyltransferase studies in fibroblasts and concanavalin A- stimulated lymphocytes

In this communication, we have demonstrated that hydrolysis of the nucleotide sugar can cause errors in the detection of an ectoglycosyltransferase. Spleen cell suspensions can incorporate radioactivity when incubated with labeled UDP-galactose, but all the activity is due to decomposition of the nucleotide sugar and uptake of the free sugar. The fibroblast cell lines can incroporate carbohydrate directly from UDP-galactose. Several criteria are presented with can be used to demonstrate that a nucleotide sugar is the direct carbohydrate donor.     

Crystal structure of Nsp15 endoribonuclease NendoU from SARS‐CoV‐2

Severe Acute Respiratory Syndrome coronavirus 2 (SARS‐CoV‐2) is rapidly spreading around the world. There is no existing vaccine or proven drug to prevent infections and stop virus proliferation. Although this virus is similar to human and animal SARS‐CoVs and Middle East Respiratory Syndrome coronavirus (MERS‐CoVs), the detailed information about SARS‐CoV‐2 proteins structures and functions is urgently needed to rapidly develop effective vaccines, antibodies, and antivirals. We applied high‐throughput protein production and structure determination pipeline at the Center for Structural Genomics of Infectious Diseases to produce SARS‐CoV‐2 proteins and structures. Here we report two high‐resolution crystal structures of endoribonuclease Nsp15/NendoU. We compare these structures with previously reported homologs from SARS and MERS coronaviruses.     

A novel in vitro bovine cartilage punch model for assessing the regeneration of focal cartilage defects with biocompatible bacterial nanocellulose

Introduction

Current therapies for articular cartilage defects fail to achieve qualitatively sufficient tissue regeneration, possibly because of a mismatch between the speed of cartilage rebuilding and the resorption of degradable implant polymers. The present study focused on the self-healing capacity of resident cartilage cells in conjunction with cell-free and biocompatible (but non-resorbable) bacterial nanocellulose (BNC). This was tested in a novel in vitro bovine cartilage punch model.

Methods

Standardized bovine cartilage discs with a central defect filled with BNC were cultured for up to eight weeks with/without stimulation with transforming growth factor-β1 (TGF-β1. Cartilage formation and integrity were analyzed by histology, immunohistochemistry and electron microscopy. Content, release and neosynthesis of the matrix molecules proteoglycan/aggrecan, collagen II and collagen I were also quantified. Finally, gene expression of these molecules was profiled in resident chondrocytes and chondrocytes migrated onto the cartilage surface or the implant material.

Results

Non-stimulated and especially TGF-β1-stimulated cartilage discs displayed a preserved structural and functional integrity of the chondrocytes and surrounding matrix, remained vital in long-term culture (eight weeks) without signs of degeneration and showed substantial synthesis of cartilage-specific molecules at the protein and mRNA level. Whereas mobilization of chondrocytes from the matrix onto the surface of cartilage and implant was pivotal for successful seeding of cell-free BNC, chondrocytes did not immigrate into the central BNC area, possibly due to the relatively small diameter of its pores (2 to 5 μm). Chondrocytes on the BNC surface showed signs of successful redifferentiation over time, including increase of aggrecan/collagen type II mRNA, decrease of collagen type I mRNA and initial deposition of proteoglycan and collagen type II in long-term high-density pellet cultures. Although TGF-β1 stimulation showed protective effects on matrix integrity, effects on other parameters were limited.

Conclusions

The present bovine cartilage punch model represents a robust, reproducible and highly suitable tool for the long-term culture of cartilage, maintaining matrix integrity and homoeostasis. As an alternative to animal studies, this model may closely reflect early stages of cartilage regeneration, allowing the evaluation of promising biomaterials with/without chondrogenic factors.     

Prehospital Thrombolysis: A Manual from Berlin

In acute ischemic stroke, time from symptom onset to intervention is a decisive prognostic factor. In order to reduce this time, prehospital thrombolysis at the emergency site would be preferable. However, apart from neurological expertise and laboratory investigations a computed tomography (CT) scan is necessary to exclude hemorrhagic stroke prior to thrombolysis. Therefore, a specialized ambulance equipped with a CT scanner and point-of-care laboratory was designed and constructed. Further, a new stroke identifying interview algorithm was developed and implemented in the Berlin emergency medical services. Since February 2011 the identification of suspected stroke in the dispatch center of the Berlin Fire Brigade prompts the deployment of this ambulance, a stroke emergency mobile (STEMO). On arrival, a neurologist, experienced in stroke care and with additional training in emergency medicine, takes a neurological examination. If stroke is suspected a CT scan excludes intracranial hemorrhage. The CT-scans are telemetrically transmitted to the neuroradiologist on-call. If coagulation status of the patient is normal and patient''s medical history reveals no contraindication, prehospital thrombolysis is applied according to current guidelines (intravenous recombinant tissue plasminogen activator, iv rtPA, alteplase, Actilyse).Thereafter patients are transported to the nearest hospital with a certified stroke unit for further treatment and assessment of strokeaetiology. After a pilot-phase, weeks were randomized into blocks either with or without STEMO care. Primary end-point of this study is time from alarm to the initiation of thrombolysis. We hypothesized that alarm-to-treatment time can be reduced by at least 20 min compared to regular care.     

Accelerating all-atom protein folding simulations through reduced dihedral barriers

Protein folding requires extensive changes of backbone and sidechain dihedral angles, whose energy barriers constitute obstacles for the folding kinetics. Folding of small proteins is furthermore thought to be path-independent. Here, we propose that time-consuming all-atom protein folding simulations may be accelerated through a reduction of the dihedral barriers of the force field. In order to investigate this hypothesis, we performed various folding simulations of two small proteins. We report an acceleration towards smaller root-mean-square deviations from the native protein structure using our proposed method.     

High cytokine levels at admission are associated with fatal outcome in patients with necrotizing fasciitis

We evaluated in a blinded fashion the cytokine profiles of patients with suspected necrotizing fasciitis. In 15 out of 20 patients, the diagnosis of necrotizing fasciitis was established; five patients had cellulitis. Eighteen of the 20 patients were i.v. drug users. Five of the 15 patients with necrotizing fasciitis died (33%). On admission, serum levels for interleukin-1beta (IL-1beta), IL-1-receptor antagonist (IL-1Ra), IL-18 and interferon-gamma (IFNgamma) as well as white blood cells (WBC) were significantly elevated in patients with fatal outcome compared to survivors with necrotizing fasciitis. IL-1Ra and WBC levels were also higher than in patients with cellulitis. No differences were observed between patients groups for IL-6 and IL-8. In summary, significantly elevated levels of proinflammatory cytokines and particularly IL-1Ra are associated with fatal outcome in patients with necrotizing fasciitis. The measurement of proinflammatory cytokines and IL-1Ra may help to establish early diagnosis of life-threatening necrotizing fasciitis and thus to initiate aggressive treatment.