The German trial on Aciclovir and Corticosteroids in Herpes-simplex-virus-Encephalitis (GACHE): a multicenter, randomized, double-blind, placebo-controlled trial.

INTRODUCTION: Comprehensive treatment of Herpes-simplex-virus-encephalitis (HSVE) remains a major clinical challenge. The current therapy gold standard is aciclovir, a drug that inhibits viral replication. Despite antiviral treatment, mortality remains around 20% and a majority of survivors suffer from severe disability. Experimental research and recent retrospective clinical observations suggest a favourable therapy response to adjuvant dexamethasone. Currently there is no randomized clinical trial evidence, however, to support the routine use of adjuvant corticosteroid treatment in HSVE. METHODS: The German trial of Aciclovir and Corticosteroids in Herpes-simplex-virus-Encephalitis (GACHE) studied the effect of adjuvant dexamethasone versus placebo on top of standard aciclovir treatment in adult patients aged 18 up to 85 years with proven HSVE in German academic centers of Neurology in a randomized and double blind fashion. The trial was open from November 2007 to December 2012. The initially planned sample size was 372 patients with the option to increase to up to 450 patients after the second interim analysis. The primary endpoint was a binary functional outcome after 6 months assessed using the modified Rankin scale (mRS 0-2 vs. 3-6). Secondary endpoints included mortality after 6 and 12 months, functional outcome after 6 months measured with the Glasgow outcome scale (GOS), functional outcome after 12 months measured with mRS and GOS, quality of life as measured with the EuroQol 5D instrument after 6 and 12 months, neuropsychological testing after 6 months, cranial magnetic resonance imaging findings after 6 months, seizures up to day of discharge or at the latest at day 30, and after 6 and 12 months. RESULTS: The trial was stopped prematurely for slow recruitment after 41 patients had been randomized, 21 of them treated with dexamethasone and 20 with placebo. No difference was observed in the primary endpoint. In the full analysis set (n = 19 in each group), 12 patients in each treatment arm achieved a mRS of 0-2. Similarly, we did not observe significant differences in the secondary endpoints (GOS, mRS, quality of life, neuropsychological testing). CONCLUSION: GACHE being prematurely terminated demonstrated challenges encountered performing randomized, placebo-controlled trials in rare life threatening neurological diseases. Based upon our trial results the use of adjuvant steroids in addition to antiviral treatment remains experimental and is at the decision of the individual treating physician. Unfortunately, the small number of study participants does not allow firm conclusions. TRIAL REGISTRATION: EudraCT-Nr. 2005-003201-81.

Renal salt wasting as part of dysautonomia in Guillain-Barre syndrome.

Cerebral salt-wasting syndrome and the syndrome of inappropriate antidiuresis (SIAD) are the most important causes of non-iatrogenic hyponatraemia that can significantly complicate various brain diseases. Salt wasting without an underlying CNS disease may have been disregarded so far by clinicians and has been described as renal salt-wasting (RSW) in patients as drug side effect (eg, cisplatin), in older people with various common diseases (eg, hip fracture, pulmonary infections) and other sporadic conditions. In Guillain-Barré Syndrome (GBS), however, hyponatraemia has been described mainly as SIAD. However, symptoms of hyponatraemia rarely develop in GBS. Here, we report on a woman with GBS with dominant symptoms of dysautonomia and moderate severe hyponatraemia. We could identify RSW as part of the autonomic dysfunction that significantly contributed to disease worsening.

Quick extended x-ray absorption fine structure instrument with millisecond time scale, optimized for in situ applications.

In order to learn about in situ structural changes in materials at subseconds time scale, we have further refined the techniques of quick extended x-ray absorption fine structure (QEXAFS) and quick x-ray absorption near edge structure (XANES) spectroscopies at beamline X18B at the National Synchrotron Light Source. The channel cut Si (111) monochromator oscillation is driven through a tangential arm at 5 Hz, using a cam, dc motor, pulley, and belt system. The rubber belt between the motor and the cam damps the mechanical noise. EXAFS scan taken in 100 ms is comparable to standard data. The angle and the angular range of the monochromator can be changed to collect a full EXAFS or XANES spectrum in the energy range 4.7-40.0 KeV. The data are recorded in ascending and descending order of energy, on the fly, without any loss of beam time. The QEXAFS mechanical system is outside the vacuum system, and therefore changing the mode of operation from conventional to QEXAFS takes only a few minutes. This instrument allows the acquisition of time resolved data in a variety of systems relevant to electrochemical, photochemical, catalytic, materials, and environmental sciences.

[Acute viral and emerging viral CNS infections]. / Akute virale und importierte virale Entzündungen des ZNS.

The morbidity and mortality of viral CNS infections can be reduced through early initiation of therapy, which could be specific or in most cases symptomatic. This in turn requires the physician to consider meningoencephalitis as a differential diagnosis.Increasing climate changes, global air travel and changing immune responses contribute to the emergence of rather rare viral pathogens on our continent. Thus, neurologists in Europe are facing an enormous challenge due to lacking knowledge and experience of these new germs, which needs to be taken care of.Novel therapeutic approaches with e.g. monoclonal antibodies awaken the hope of mitigating the partially lethal courses of these diseases.

[Vasculitis of the nervous system in infectious diseases]. / Vaskulitis des Nervensystems bei Infektionserkrankungen.

Vasculitis and vasculopathies of the central and peripheral nervous system can be caused by infectious diseases. Vasculitis can lead to stenosis, occlusion and aneurysm formation of blood vessels which may result in stroke or cerebral haemorrhage. In cases of peripheral nervous system involvement mononeuritis multiplex and symmetric peripheral neuropathy are possible. The diagnosis is based on clinical presentation, serology, cerebrospinal fluid analysis and neuroradiologic examinations. In cases of peripheral neuropathy neurophysiologic examinations and biopsy of the sural nerve can lead to the diagnosis. A fast and efficient antimicrobial therapy is the most important treatment option. In cases of peripheral neuropathies short-term treatment with corticosteroids and plasma exchange may be helpful.

Generation and degradation of human endostatin proteins by various proteinases.

The angiogenesis inhibitor endostatin is a fragment of the NC1 domain of collagen XVIII. The generation of endostatin has been investigated only in murine hemangioendothelioma cell cultures and was ascribed to cathepsin L. Distinct endostatin-like fragments were detected in human tissues and serum. To identify proteinases able to generate such fragments, we incubated human NC1 with proteinases of all classes, including cathepsin L. Eleven out of 12 generate fragments with an N-terminus within the same 15 residue stretch as those occurring physiologically, indicating that this region is sensitive to many proteinases. None correspond to mouse endostatin. However, the efficiencies of these proteinases differed markedly. Some proteinases also proved to degrade endostatin, pointing to another regulatory loop of angiogenesis.

Matrix metalloproteinase 9 and vascular endothelial growth factor are essential for osteoclast recruitment into developing long bones.

Bone development requires the recruitment of osteoclast precursors from surrounding mesenchyme, thereby allowing the key events of bone growth such as marrow cavity formation, capillary invasion, and matrix remodeling. We demonstrate that mice deficient in gelatinase B/matrix metalloproteinase (MMP)-9 exhibit a delay in osteoclast recruitment. Histological analysis and specialized invasion and bone resorption models show that MMP-9 is specifically required for the invasion of osteoclasts and endothelial cells into the discontinuously mineralized hypertrophic cartilage that fills the core of the diaphysis. However, MMPs other than MMP-9 are required for the passage of the cells through unmineralized type I collagen of the nascent bone collar, and play a role in resorption of mineralized matrix. MMP-9 stimulates the solubilization of unmineralized cartilage by MMP-13, a collagenase highly expressed in hypertrophic cartilage before osteoclast invasion. Hypertrophic cartilage also expresses vascular endothelial growth factor (VEGF), which binds to extracellular matrix and is made bioavailable by MMP-9 (Bergers, G., R. Brekken, G. McMahon, T.H. Vu, T. Itoh, K. Tamaki, K. Tanzawa, P. Thorpe, S. Itohara, Z. Werb, and D. Hanahan. 2000. Nat. Cell Biol. 2:737-744). We show that VEGF is a chemoattractant for osteoclasts. Moreover, invasion of osteoclasts into the hypertrophic cartilage requires VEGF because it is inhibited by blocking VEGF function. These observations identify specific actions of MMP-9 and VEGF that are critical for early bone development.

Proteinases in bone resorption: obvious and less obvious roles.

Bone resorption is critical for the development and the maintenance of the skeleton, and improper regulation of bone resorption leads to pathological situations. Proteinases are necessary for this process. In this review, we show that this need of proteinases is not only because they are required for the solubilization of bone matrix, but also because they are key components of the mechanism that determines where and when bone resorption will be initiated. Moreover, there are indications that proteinases may also determine whether resorption will be followed by bone formation. Some of the proteinases involved in these different steps of the resorption processes were recently identified, as for instance cathepsin K, MMP-9 (gelatinase B), and interstitial collagenase. However, there is also increasing evidence showing that the critical proteinase(s) may vary depending on the bone type or on other factors.

Developmental expression of GDNF, neurturin and their receptors in rat hippocampus.

GLIAL cell-line derived neurotrophic factor (GDNF) and neurturin (NTN) are members of a new family of factors within the transforming growth factor-beta (TGFbeta) super-family. Signaling by GDNF and NTN depends on the tyrosine kinase receptor Ret and on GFRalpha-1 and -2. We have investigated by competitive RT-PCR the developmental expression of these molecules within the rat hippocampus. All transcripts reach high expression levels between postnatal day 0 and 14 (P0-P14), a critical period of hippocampal differentiation, suggesting roles for these molecules in this process. All mRNAs (with the exception of Ret) can be detected as early as embryonic day 14 (E14). The absence of Ret expression at E14 suggests that both GDNF and NTN may employ an additional, unknown receptor. NTN expression shows a biphasic expression pattern, with maximum expression levels at E14 and P5.

Characterization of the human gonadotropin-releasing hormone receptor heterologously produced using the baculovirus/insect cell and the Semliki Forest virus systems.

1. Two eukaryotic viral systems, the baculovirus/insect cell and the Semliki Forest virus systems, were tested for heterologous expression of human gonadotropin-releasing hormone receptor (GnRHR) cDNA. 2. An unmodified as well as a c-myc epitope-tagged human GnRH receptor was produced in two insect cell lines (Spodoptera frugiperda, Trichoplusia ni) after infection with the respective recombinant baculoviruses. In both insect cell lines, the receptor was identified by immunoblot analysis as a triplet of bands between 35 and 40 kDa. After deglycosylation of the receptor the molecular mass decreased to 35 kDa. The GnRH receptor was localized in membrane compartments within the infected insect cells. However, only in membranes of infected Trichoplusia ni insect cells could approximately 2000 receptors per cell be detected. 3. Production of the GnRH receptor in BHK cells using the Semliki Forest virus system resulted in approximately 50,000 receptors per cell. A maximal yield of 0.42 pmol/mg membrane protein was obtained 24 hr after electroporation of BHK cells with in vitro synthesized RNA. Binding of the antagonist [125I]Cetrorelix was saturable with a KD of 1.3 nM. The receptor produced in the BHK cells was further characterized by ligand displacement studies. The rank order of agonist and antagonist affinities was Cetrorelix > Triptorelin > Antide > GnRH.

Characterization of the human dopamine transporter heterologously expressed in BHK-21 cells.

1. cDNA of the human dopamine transporter (hDAT) was cloned into a cloning vector based on the Semliki Forest virus. Electroporation of in vitro transcribed mRNA from this plasmid into BHK-21 cells resulted in production of the transporter as measured by [3H]dopamine uptake (Km = 2.0 +/- 0.4 microM), which was specifically inhibited in the presence of cocaine. 2. The recombinant transporter protein exhibited an apparent molecular mass of 56 kDa, which was reduced to 50 kDa after tunicamycin treatment of the producing BHK-21 cells. Tunicamycin treatment of the electroporated cells also resulted in a decrease in transport activity with no change in the Km value (2.1 +/- 0.4 microM). 3. The localization of the heterologously produced transporter in the BHK cells either with or without tunicamycin treatment was studied by electron microscopic immunogold staining. The glycosylated transporter was found to be localized at the plasma membrane, whereas in the case of the unglycosylated transporter, transport to the plasma membrane was blocked.

Engineering the folding pathway of insect cells: generation of a stably transformed insect cell line showing improved folding of a recombinant membrane protein.

The baculovirus-insect cell expression system has proven to be a valuable tool for the high level production of a multitude of recombinant proteins. However, production of membrane proteins in infected insect cells is often hampered by incorrect folding and processing which results in the accumulation of non-functional protein. Here, we report the construction of a Sf9 insect cell line stably transformed with the ninaA gene from D. melanogaster (Sfn cell line). The ninaA protein is a membrane bound cyclophilin which acts as a peptidyl-prolyl cis/trans isomerase during the folding process of rhodopsin 1 in D. melanogaster rhabdomere. Engineered Sfn insect cells infected with a recombinant baculovirus bearing the human dopamine transporter gene under the control of the polyhedrin promoter showed a > or = 5 times enhanced uptake of [3H]dopamine in comparison to similarly infected Sf9 cells. This increase in specific transport activity was not due to an altered Km value in the Sfn cell line. The uptake in infected Sfn cells was blocked by the peptidyl-prolyl cis/trans isomerase inhibitor cyclosporin A which had no effect on infected Sf9 cells. From these results we conclude that the prolyl-cis/trans isomerase activity of the ninaA in the stably transformed Sfn cell line was responsible, directly or indirectly, for the improved folding of the heterologously produced human dopamine transporter.

A new set of versatile vectors for the heterologous expression of foreign genes using the baculovirus system.

A new set of versatile advanced baculovirus (BV) vectors for the production of fused proteins in insect cells, under the control of the strong polyhedrin promoter of the Autographa california nuclear polyhedrosis virus (AcMNPV), has been constructed. The vectors contain peptide tags which allow immunological detection, as well as purification of recombinant protein produced via the BV expression system.