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Specifically tailored amino acid–based formulations were previously shown to have a high potential to avoid stress-mediated degradation of complex molecules such as monoclonal antibodies and viral vectors. By using adenovirus 5 (Ad5) as a model, we studied whether such formulations may also efficiently protect viral vectors in thermal stress experiments and during long-term liquid storage. Algorithm-based amino acid preselection using an excipient database and subsequent application of design of experiments (DoE) in combination with a 37°C challenging model enabled the prediction of long-term storage stability of Ad5. By statistical analysis of the Ad5 infectivity, amino acids with significant influence on Ad5 stability were detected after 2 and 3 weeks of liquid storage at 37°C. Ad5 formulations comprising positively selected amino acids did not reveal any loss of infectivity after 24 months in liquid storage at 5°C. By contrast, a 2 log reduction after 3 months and complete loss of infectivity after 18 months was observed with a standard viral vector formulation. By an optimization round, we designed a simple and well-balanced formulation avoiding MgCl2, previously considered essential in Ad5 formulations. This work demonstrates the efficacy of an algorithm-based development approach in the formulation development for viral vectors.

Calcium (Ca2+) and redox signaling enable cells to quickly adapt to changing environments. The signaling protein calredoxin (CRX) from the green alga Chlamydomonas reinhardtii is a chloroplast-resident thioredoxin having Ca2+-dependent activity and harboring a unique combination of an EF-hand domain connected to a typical thioredoxin-fold. Using small-angle X-ray scattering (SAXS), FRET, and NMR techniques, we found that Ca2+-binding not only induces a conformational change in the EF-hand domain, but also in the thioredoxin domain, translating into the onset of thioredoxin redox activity. Functional analyses of CRX with genetically altered EF-hands revealed that EF-hand 4 is important for mediating the communication between the two domains. Moreover, we crystallized a variant (C174S) of the CRX target protein peroxiredoxin 1 (PRX1) at 2.4 A resolution, modeled the interaction complex of the two proteins, and analyzed it by cross-linking and MS analyses, revealing that the interaction interface is located close to the active sites of both proteins. Our findings shed light on the Ca2+ binding-induced changes in CRX structure in solution at the level of the overall protein and individual domains and residues.

Objective There is a number of different methods to localize a glioma intraoperatively. Neuronavigation, intraoperative MRI, 5-aminolevulinic acid, as well as intraoperative sonography. Every method has its advantages and disadvantages. Low grade gliomas do not show a specific signal with 5-aminolevulinic acid and are difficult to distinguish macroscopically from normal tissue. In the present study we stress out the importance of intraoperative diagnostic ultrasound for localization of low grade gliomas. Methods We retrospectively evaluated the charts and MRIs of 34 patients with low grade gliomas operated in our department from 2011 until December 2014. The efficacy of ultrasound as an intraoperative navigational tool was assessed. In 15 patients ultrasound was used and in 19 not. Only histologically proven low grades gliomas (astrocytomas grade II) were evaluated. Results In none of the patients where ultrasound (combined with neuronavigation) was used (N = 15) to find the tumors, the target was missed, whereas the exclusive use of neuronavigation missed the target in 5 of 19 cases of small subcortical low grade gliomas. Conclusions Intraoperative ultrasound is an excellent tool in localizing low grade gliomas intraoperatively. It is an inexpensive, real time neuronavigational tool, which overcomes brain shift. Even when identifying the tumors with ultrasound is very reliable, the extend of resection and the decision to remove any residual tumor with the help of ultrasound is at the moment unreliable.

International audience; Chlamydomonas reinhardtii is a green unicellular eukaryotic model organism for studying relevant biological and biotechnological questions. The availability of genomic resources and the growing interest in C. reinhardtii as an emerging cell factory for the industrial production of biopharmaceuticals require an in-depth analysis of protein N-glycosylation in this organism. Accordingly, we used a comprehensive approach including genomic, glycomic, and glycoproteomic techniques to unravel the N-glycosylation pathway of C. reinhardtii. Using mass-spectrometry-based approaches, we found that both endogenous soluble and membrane-bound proteins carry predominantly oligomannosides ranging from Man-2 to Man-5. In addition, minor complex N-linked glycans were identified as being composed of partially 6-O-methylated Man-3 to Man-5 carrying one or two xylose residues. These findings were supported by results from a glycoproteomic approach that led to the identification of 86 glycoproteins. Here, a combination of in-source collision-induced dissodiation (CID) for glycan fragmentation followed by mass tag-triggered CID for peptide sequencing and PNGase F treatment of glycopeptides in the presence of (18)O-labeled water in conjunction with CID mass spectrometric analyses were employed. In conclusion, our data support the notion that the biosynthesis and maturation of N-linked glycans in the endoplasmic reticulum and Golgi apparatus occur via a GnT I-independent pathway yielding novel complex N-linked glycans that maturate differently from their counterparts in land plants.

The demand to develop convergent technology platforms, such as bio-functionalized medical devices, is rapidly increasing. However, the loss of biological function of the effector molecules during sterilization represents a significant and general problem. Therefore, we have developed and characterized a nano-coating (NC) formulation capable of maintaining the functionality of proteins on biological-device combination products. As a proof of concept, the NC preserved the structural and functional integrity of an otherwise highly fragile antibody immobilized on polyurethane during deleterious sterilizing irradiation (≥ 25 kGy). The NC procedure enables straight-forward terminal sterilization of bio-functionalized materials while preserving optimal conditioning of the bioactive surface.

Posttrauma apoptosis resistance of neutrophils (PMN) is related to overshooting immune responses, systemic inflammatory response syndrome (SIRS) and multiple organ failure (MOF). Recently, we have shown that the apoptosis resistance in circulating PMN from severely injured patients which is known to be mediated by high serum levels of pro-inflammatory cytokines can be overcome by the activation of Fas death receptor. Here, we aimed to study whether stimulation of surface Fas leads to the inactivation of hyperactivated PMN from critically ill patients with SIRS. PMN from 23 multiple trauma patients (mean injury severity score (ISS) 34±1.9) were isolated at day 1 after admission to the trauma center. PMN from 17 volunteer blood donors served as controls. Neutrophil activity has been determined after ex vivo short (1 h) and long-term (4 h) stimulation of freshly isolated PMN with immobilized agonistic anti-Fas antibodies. We found neutrophil chemotactic migration in response to IL-8, phagocytosis and oxidative burst to be significantly inhibited in control cells already after short-term (1 h) Fas stimulation. In contrast, inactivation of trauma PMN by agonistic anti-Fas antibodies was found to be efficient only after long-term (4 h) incubation of cells with agonistic antibodies. Thus, in trauma PMN down-regulation of neutrophil activity seems to be delayed when compared to cells isolated from healthy controls, suggesting impaired susceptibility for Fas stimulation in these cells. Interestingly, whereas Fas-mediated inhibition of phagocytosis and oxidative burst could be prevented by the broad range caspase inhibitor t-butoxycarbonyl-aspartyl(O-methyl)-fluoromethyl ketone (BocD-fmk), the chemotactic activity in response to IL-8 was unaffected. In conclusion, we demonstrate that stimulation of neutrophil Fas does not only initiate apoptosis but also induces inhibition of neutrophil functions, partially by non-apoptotic signaling.

The aim of this study was to adapt the ultrasonographical techniques developed for brain perfusion imaging to an intraoperative setting for topographic diagnosis of cerebral tumours. During surgery, the patients underwent contrast-enhanced ultrasonography (phase inversion harmonic imaging, bolus kinetic, fitted model function). Endocavity curved array (6.5EC10, 6.5 MHz) was used intraoperatively. The ultrasound contrast agent SonoVue (Bracco) was administered IV as a bolus injection. Off-line, time-intensity curves as well as perfusion maps were calculated and parameters such as peak intensity were locally extracted to characterise perfusion. Seven patients with brain tumours of different histologic types were subjected to contrast-enhanced ultrasonography during surgery. Tissue differentiation with contrast agent was superior to conventional B-mode ultrasound imaging. Intraoperative contrast-enhanced ultrasonography enabled visualisation of cerebral tumours in high spatial resolution.

This study of 169 consecutive individually prefabricated CAD/CAM titanium implants for cranioplasty between 1993 and 2000 yields comprehensive data of geometric, surgical, and medical aspects, and answers various questions of quality of life. The scheme for the classification of size and location of the defects is based on the precise CAD data of each implant. CAD/CAM-prefabricated titanium implants are of increased benefit in the neurocranium compared to the craniofacial area, including the forehead and the periorbital rims, which are more problematic with regard to primary fit and cosmetic result. Neurocranial protection and the consecutive improvement of the quality of life are more distinct in this group and come along with the highest benefit for patients with very large defects (> 100 cm2 defect area). For the first time, the defect area could be determined so precisely with the CAD implant data and such a high number of extremely large consecutive cranioplasties could be evaluated.

Recently, the term oncomodulation has been proposed to express the ability of human cytomegalovirus (HCMV) to modify tumor cell biology, a phenomenon that is independent from transformation. Because past studies have failed to show that HCMV can transform normal human cells, HCMV has not been regarded as an oncogenic tumor virus. However, recent investigations have revealed a high frequency of HCMV in tumor cells of malignancies such as colon cancer, malignant glioma, prostatic intraepithelial neoplasia, and carcinoma. Data from experiments with HCMV-infected tumor cell lines have highlighted the oncomodulatory potential of HCMV and provided important insights into the patho-mechanisms associated with aberrant signaling pathways and transcription factor and/or tumor suppressor function of the host cell.

Selection of an optimal treatment plan requires the comparison of dose distributions and dose-volume histograms (DVH) of all plan variants calculated for the patient. Each treatment plan consists generally of 30 to 40 CT slices, making the comparison difficult and time consuming. The present study proposes an objective index that takes into account both physical and biological criteria for the evaluation of the dose distribution. The DHV-based evaluation index can be calculated according to the following four criteria: ICRU conformity (review of the differences between the dose in the planning target volume and the ICRU recommendations); mean dose and dose homogeneity of the planning target volume; the product of tumour complication probability (TCP) and normal tissue complication probability (NTCP); and finally a criterion that takes into account the dose load of non-segmented tissue portions within the CT slice. The application of the objective index is demonstrated for two different clinical cases (esophagus and breast carcinoma). During the evaluation period, the objective index showed a good correlation between the doctor's decision and the proposed objective index. Thus, the objective index is suitable for a computer-based evaluation of treatment plans.

Thrombospondin-1 (TSP-1) is a potent inhibitor of angiogenesis. It has been shown that promoter sequences of the TSP-1 gene can be transactivated by the wild-type tumor suppressor protein p53. As human cytomegalovirus (HCMV) infection inactivates wild-type p53 of various cell types, we investigated whether HCMV infection is associated with reduced TSP-1 production. We found, in conjunction with accumulated p53, that TSP-1 mRNA and protein expression was significantly reduced in HCMV-infected cultured human fibroblasts. To determine whether the observed TSP-1 suppression depends on p53 inactivation, the p53-defective astrocytoma cell line U373MG was infected with HCMV. In these cells TSP-1 expression was also significantly reduced by HCMV infection whereas expression of the p53 mutant variant remained unaltered. In both cell lines the decreased expression of TSP-1 mRNA occurred early after infection (4 hours), indicating that HCMV inhibits TSP-1 transcription during the immediate-early phase of infection before HCMV DNA replication. Inhibition of HCMV DNA synthesis by ganciclovir did not influence TSP-1 reduction whereas the antisense oligonucleotide ISIS 2922, complementary to HCMV immediate-early mRNA, completely prevented the HCMV-mediated TSP-1 suppression. These findings strongly suggest a novel role for HCMV in the modulation of angiogenesis due to p53-independent down-regulation of TSP-1 expression.

Despite the increased bandwidth and the Quality of Service (QoS) capabilities of ATM networks, measurements show that only a fraction of the available bandwidth can be used by applications with current approaches for realizing data communications over ATM employing the TCP/IP protocol suite [1, 2]. This is largely due to the unsuited protocol mechanisms and the lack of QoS support on transport level. Native Mode ATM offers full access to ATM's Quality of Service capabilities in homogenous ATM networks and provides support for a wide range of information streams with different properties and requirements [6]. In order to exploit the capabilities of ATM networks, and to optimally use the available resources, in the network and end system, new transport protocols are inevitable. In this paper we propose the transport protocols ADTS (ATM Data Transfer Service), which uses efficient protocol mechanisms suitable for ATM systems with a minimum of protocol processing overhead. We discuss implementation issues and present performance results obtained with a reference implementation of the ADTS protocol.

Primary cell cultures, such as isolated epithelial cells, neuronal cells, or hepatocytes are characterized by a very low mitotic activity. Monitoring of small changes in cell numbers requires staining with a DNA-specific dye with an extremely high sensitivity and a low inter- and intraassay variability. For this purpose, an ultrasensitive in vitro assay has been developed based on the fluorescent nucleic acid stain PicoGreen. PicoGreen has been shown to detect as little as 0.5 ng pure DNA or 10(2) cells (interassay SD < 10%, intraassay SD < 5%). This is far above the limit of sensitivity of conventional fluorochromes, such as Hoechst 33342 or propidium iodide. To obtain optimum efficacy of PicoGreen, cells were digested with papain for 20 h at 60 degrees C prior to staining. Under these conditions, the slope factor was calculated to be 0.105 relative fluorescence units (RFU)/cell, which is far superior to the slope factor of Hoechst 33342 (0.0137 RFU/cell) or propidium iodide (0.0077 RFU/cell). Analysis of the blank values revealed a very low autofluorescence of PicoGreen, which is only 1/50th of the autofluorescence of Hoechst 33342 and 1/5th of the autofluorescence of propidium iodide. Additional coating of the culture plates with extracellular matrix proteins to prevent cellular dedifferentiation did not influence the high sensitivity of PicoGreen. In conclusion, the PicoGreen-assay seems to be the method of choice when the growth capacity of primary cell cultures needs to be analyzed with high accuracy.

The biliary epithelium is a major target for allograft-directed immune responses during rejection crises after liver transplantation. This paper deals with in vitro studies on the immunogenetic potential of cultured biliary epithelial cells (BECs) to elicit an allogeneic cellular immune response. Therefore, BECs were cocultured with syngeneic and allogeneic lymphocytes in order to study lymphocyte activation. The respective lymphocytes [3H]thymidine incorporation was a measure for the proliferative activity. While syngeneic peripheral blood lymphocytes (PBL) never exhibited BEC-induced proliferation allogeneic PBL were significantly (P0.05) activated in all experiments (n = 6). In experiments with purified subpopulations CD8+ cells but not CD4+ cells proved to be activated by BECs. In time kinetics (n = 5) the maximum of the BEC-induced proliferation was on day 9 while the endothelial cell-induced proliferation was found to be 2 days earlier on day 7 after onset of the experiments (P0.05). BEC-induced proliferation was accompanied by induced IL-2 secretion (300 pg/ml) by activated lymphocytes as determined by ELISA. Stimulation of BECs with 500 U/ml interferon-gamma, 1000 U/ml interferon-alpha or blocking expression of HLA molecules on the surface membrane of BECs by monoclonal antibodies did not alter BEC-induced allogeneic lymphocyte proliferation. Monoclonal antibodies against CD8+ but not CD4+ suppressed proliferative activity of PBL and CD8+ cells by 40 and 45%, respectively. Overall, these results provide evidence that BECs may induce CD8+ lymphocyte activation in vivo and therefore might play a crucial role in triggering immune responses related to liver transplant rejection episodes.

Desferrioxamine (DFO), commonly used in therapy as a chelator of ferric ion in disorders of iron overload, is a potent inhibitor of human cytomegalovirus (HCMV) replication in cultured fibroblast cells. Moreover, DFO has immunomodulatory activity both in vitro and in vivo. We studied DFO effects on HCMV replication in cultured human endothelial cells and on the expression of several cell surface molecules, which mediate interactions of endothelial cells with other cell types in the immune system. The concentrations of DFO required for 50% reduction in the number of endothelial cells expressing HCMV late antigen, ranged for several HCMV strains from 5.2 to 8.8 microM. DFO concentrations ranging from 5 to 40 microM inhibited cellular DNA synthesis in a dose-dependent manner without any significant effects on the cell viability. DFO at 10 microM concentration suppressed expression of intercellular adhesion molecule-1 (ICAM-1) and endothelial leucocyte adhesion molecule-1 (ELAM-1), while it had no significant effect on the expression of vascular cell adhesion molecule-1 (VCAM-1). Expression of HLA class I and class II was not influenced by DFO treatment. The results showed that DFO is both effective in inhibition of HCMV replication and expression of ICAM-1 and ELAM-1 in endothelial cells, a combination that warrants attention to its potential use to prevent HCMV-induced allograft rejection in transplant recipients.

Cultured human umbilical vein endothelial cells (HUVEs) were infected with human cytomegalovirus (HCMV) strain AD169. Up to 50% HUVEs proved to be positive for HCMV early nuclear antigens 24 hours after inoculation with virus. Following infection kinetics of surface expression of HLA class I and II, intercellular adhesion molecule (ICAM-1) and endothelial lymphocyte adhesion molecule (ELAM-1) on HUVEs were investigated by means of flow cytometry. A slight increase in HLA class I expression was observed, whereas expression of HLA class II (DR, DP, DQ) antigens was not induced by infection with HCMV. Furthermore, when compared with uninfected cells treated with interferon-gamma (IFN-gamma), reduced enhancement of HLA-DR expression was conspicuous in HCMV-infected cells treated with IFN-gamma. There is evidence that only a portion of HUVE is affected in its ability to upregulate HLA class II antigens. While expression of ICAM-1 was found to be enhanced between 8 and 20 hours after infection with a maximum at 12 hours after infection, no modulation of ELAM-1 was seen.

A 46-year-old man with diabetes insipidus, hypothyroidism and hypocortisolism was found to have a tumor of the pituitary stalk. After transsphenoidal resection of the tumor substitution of cortisol and thyroxin was necessary. Pathological examination of the lesion revealed an extremely rare granulomatous infiltration classified as Gagel's granuloma (Langerhans' cell histiocytosis). Pre- and postoperative magnetic resonance imaging (MRI) studies, differential diagnosis and treatment of such lesions are presented.

Object Accidental dural tears during craniotomy constitute a possible source of CSF leakage and wound infection. This can turn an elective procedure into a complicated and cost-intensive problem. Only a few studies have addressed the incidence of dural tears, but there have been many studies dealing with various techniques that can be employed to repair dural tears. The present study was carried out to analyze predisposing factors for dural tears during trepanation in order to optimize the design of a robot-assisted trepanation system. Patients 100 patients were analyzed prospectively. An evaluation sheet was designed to document size and location of the lesion and the craniotomy, the geometry and number of burr holes, and the auxiliary tools used during bone flap removal. Furthermore, the suspected histology was noted and anatomical facts, including cranial vault thickness and the presence of hyperostosis frontalis interna, were documented. Results In 100 craniotomies performed, in the majority of cases (64%), in order to gain access to intracerebral lesions, 30 dural tears were seen, involving both dural layers in 26 cases. There were 26 tears located under the margins of the craniotomy; the length was 0-3 cm in 18 patients (69%). Significant predisposing factors were the thickness of the cranial vault and the presence of a hyperostosis frontalis. Furthermore, the location (frontal) and the diagnosis of an extracerebral pathology, including meningiomas, were significant factors for dural tears. Elderly patients and the use of the drill to complete the trepanation were also significant predisposing factors. Dural repair was done using suturing, in most of the cases combined with a free periostal flap. Central dural tears were integrated into the planned dural opening. A vascularized flap or muscle was used in the minority of cases. Postoperative cerebral fluid leakage was seen in two patients, wound infections in three. Conclusions Dural tears occurring during craniotomy cannot be prevented, when predisposing factors are taken into account. The absence of brain damage may due to two factors: 1) in elderly patients with hyperostosis, an additional atrophy of the brain is present; 2) extracerebral tumors, with their space-occupying growth, shift the underlying brain away from the calvaria. Considering the design of a robot-assisted trepanation system, the following conclusions seem possible: Dural tears cannot be avoided because predisposing factors are overriding. For improved safety, additional, specialized instrumentation is required.