Biomedical subjects

Hermann J Schluesener

Publications and source records attributed to Hermann J Schluesener.

At least 19 recordsLinked to original sources

PubMed2006

Polyguanosine motif increases cellular oligonucleotide uptake in the brain cortex.

Cellular uptake and localization of fluorescein isothiocyanate-labelled or biotin-labelled CpG-oligonucleotides (oligonucleotides containing unmethylated CpG dinucleotides within specific flanking bases) with or without 3'-end continuous guanosines (polyG motif) were studied in the rat cortex. Twenty minutes after intracerebral microinjection, labelled CpG-oligonucleotide accumulated in both nuclear and cytoplasmic compartments of neuronal and nonneuronal cells localized near the injection site. In the brain, polyG motif significantly increased the cellular uptake of phosphodiester CpG-oligonucleotide (P < 0.05) but not phosphothioate CpG-oligonucleotide (P > 0.05). These data not only provide in-vivo evidence for the local uptake and distribution of oligonucleotides in the brain cortex but also indicate that phosphodiester CpG-oligonucleotides containing a 3'-end polyG motif show an increased cellular uptake and thus might be promising alternate analogues for application to the brain.

Amino Acid Motifs↗

PubMed2006

Acute but not chronic stimulation of glial cells in rat spinal cord by systemic injection of lipopolysaccharide is associated with hyperalgesia.

We have analyzed development of mechanical hyperalgesia after repeated systemic lipopolysaccharide (LPS) injections and correlated these findings with stimulation of astrocytes and microglia in spinal cord. Male Lewis rats received a single or seven intraperitoneal injections of LPS. Mechanical hyperalgesia was measured as rat hindpaw withdrawal thresholds (PWTs). We observed that a single LPS injection elicited a specific change of PWTs while stimulated spinal glial activation was identified by immunoreactivities of specific markers, ED1, P2X4 receptor, endothelial monocyte activating polypeptide II (EMAP II) and glial fibrillary acidic protein (GFAP), respectively; multiple LPS treatments induced tolerance to mechanical hyperalgesia, whereas expression of ED1 and GFAP were further increased. In conclusion, we have demonstrated that the number of activated spinal glial cells was increased as an acute effect of LPS correlating with increased sensitivity to mechanical stimulation. However chronic exposure to LPS can develop a tolerance to mechanical hyperalgesia despite ongoing signs of CNS glial activation.

Animals↗

PubMed2006

Combinations of TLR and NOD2 ligands stimulate rat microglial P2X4R expression.

As ATP-gated ion channels, P2X4 receptors (P2X4R) of microglial cells play a crucial role in central nervous system (CNS) inflammation. In this study, we used rat microglial cell cultures to examine P2X4R expression in response to stimulation by combination of toll-like receptors (TLRs) and nucleotide-binding oligomerization domain 2 (NOD2) receptors. Various TLR1-9 ligands and NOD2 agonist muramyldipeptide (MDP) were investigated. Our results showed that certain combination of ligands had additive effects on upregulating microglial P2X4R at both mRNA and protein levels, and induced nitric oxide increase and tumor necrosis factor-alpha production. Thus TLRs and NOD2 combinations are contributors to the signaling cascades resulting in purinergic microglial activation.

Animals↗

PubMed2006

Molecular addresses of tumors: selection by in vivo phage display.

In vivo phage display has been used extensively to screen for novel targets of tumor therapy. Phage display peptide libraries can express random peptides or protein fragments and the aim of phage display is to identify peptide molecules that bind stably to a given target. Angiogenesis is essential to tumor development. Both blood and lymphatic vessels of tumors are different from those of normal tissues. Phage display has been used to analyze the structure and molecular diversity of tumor vasculature and to select tumor-specific antigens which have revealed stage- and type-specific markers of tumor blood vessels. Furthermore, peptides identified by in vivo phage display also work as vehicles to transport cargo therapeutic reagents to tumors. These peptides and their corresponding cellular proteins and ligands may provide molecular tools to selectively target the addresses of tumors and their pathological blood vessels and might increase the efficacy of therapy while decreasing side effects.

Amino Acid Sequence↗

PubMed2006

siRNA binding proteins of microglial cells: PKR is an unanticipated ligand.

Small interfering RNA (siRNA), double-stranded RNA (dsRNA) 21-23 nucleotides (nt) long with two nt 3' overhangs, has been shown to mediate powerful sequence-specific gene silence in mammalian cells through RNA interference (RNAi). Due to its high efficiency and high specificity siRNA has been used as a powerful post genomic tool and a potent therapeutic candidate. However, there is still a lot to learn about the mobility of siRNA inside cells and the cellular factors that might interfere with the specificity and activity of siRNA. Microglia are the brain's effector cells of the innate immune system and suitable targets in the development of novel therapeutic strategies. Here, we show the cellular uptake and intracellular distribution of siRNA in murine microglial N9 cells. siRNA was internalized by microglial N9 cells without transfection reagent and mainly localized to the endosomes However, no significant gene silencing effects were observed. Its cellular uptake and cellular distribution pattern were similar with that of a same length single stranded DNA (ssDNA). Further, cellular binding proteins of siRNA were purified and identified by mass spectrometry. Negative control siRNA and siRNA targeted to beta-actin were used in this part of experiment. Most of the siRNA binding proteins for negative control siRNA and siRNA targeted to beta-actin were dsRNA-binding proteins, such as dsRNA-dependent protein kinase R (PKR). Furthermore, both control siRNA and siRNA targeted to beta-actin activated PKR in N9 cells, which suggest that siRNA might cause off-target effects through activation of PKR.

Animals↗

PubMed2006

Experimental strategies to promote spinal cord regeneration--an integrative perspective.

Detailed pathophysiological findings of secondary damage phenomena after spinal cord injury (SCI) as well as the identification of inhibitory and neurotrophic proteins have yielded a plethora of experimental therapeutic approaches. Main targets are (i) to minimize secondary damage progression (neuroprotection), (ii) to foster axon conduction (neurorestoration) and (iii) to supply a permissive environment to promote axonal sprouting (neuroregenerative therapies). Pre-clinical studies have raised hope in functional recovery through the antagonism of growth inhibitors, application of growth factors, cell transplantation, and vaccination strategies. To date, even though based on successful pre-clinical animal studies, results of clinical trials are characterized by dampened effects attributable to difficulties in the study design (patient heterogeneity) and species differences. A combination of complementary therapeutic strategies might be considered pre-requisite for future synergistic approaches. Here, we line out pre-clinical interventions resulting in improved functional neurological outcome after spinal cord injury and track them on their intended way to bedside.

Animals↗

PubMed2006

Therapeutic effects of cisplatin on rat experimental autoimmune encephalomyelitis.

INTRODUCTION: Experimental autoimmune encephalomyelitis (EAE) is a prototypic Th1-mediated autoimmune inflammatory disease of the central nervous system (CNS), and serves as a model for the human demyelinating disease, multiple sclerosis. Cisplatin is a drug widely used in the treatment of a variety of human neoplasias, such as advanced bladder carcinoma, adrenal cortex carcinoma, breast cancer, head and neck or lung carcinoma. Cisplatin binds to DNA and interferes with cellular repair and other mechanism, which eventually result into cell death. It is known that cisplatin can induce immunosuppressive effects through inhibition of T cell activity. Therefore we analyzed the anti-inflammatory effects of cisplatin in a rat EAE model. MATERIALS AND METHODS: EAE was induced in male LEW rats by immunizing with a synthetic peptide of guinea pig myelin basic protein. The development of EAE and neurological signs were evaluated by a standard protocol. Immunohistochemistry was applied to show immune cell infiltration into the CNS. RESULTS: Early treatment of EAE rats with cisplatin effectively ameliorated the development of disease and provided a significant protective effect compared to control rats. Further, histological analysis demonstrated that the formation of the typical perivascular cuffs and brain infiltration of monocytes and lymphocytes were complete absent in cisplatin treated rats, while abundant T cell infiltration was seen in the CNS of EAE rats. CONCLUSIONS: Our data show that cisplatin has protective effects in EAE, indicating that cisplatin could be a candidate in the treatment of human CNS autoimmunity.

Animals↗

PubMed2006

Endostatin/collagen XVIII accumulates in patients with traumatic brain injury.

In patients with traumatic brain injury (TBI), hypoperfusion contributes to ongoing and expanding areas of neuronal damage long after the initial trauma has ceased. In order to evaluate whether the antiangiogenic protein endostatin may play a role in this process, we analyzed its spatial distribution in brains of 18 patients with TBI. We observed an increase of endostatin/collagen XVIII(+) macrophages/microglial cells but not astrocytes up to day 14 and a consequent decrease to day 16 post-TBI. In addition, paracellular endostatin/collagen XVIII deposits were detected. In vitro experiments revealed that microglial endostatin release is induced predominantly by hypoxia and, to a lesser extent, by reactive oxygen intermediates. Common NO synthase inhibitor pharmacotherapy with aminoguanidine and L-NAME completely abolished endostatin release from microglial cells, raising hopes of altering endostatin release in vivo.

Adolescent↗

PubMed2006

Spinal cord injury-induced expression of the immune-regulatory chemokine interleukin-16 caused by activated microglia/macrophages and CD8+ cells.

OBJECT: Spinal cord injury (SCI) elicits a strong inflammatory response that readily participates in lipid oxygenation, edema formation, apoptotic cell death, and tissue remodeling. Because cytokines determine the postinjury inflammatory milieu, the authors analyzed the expression of the immunomodulatory chemokine interleukin- 16 (IL- 16) after SCI. METHODS: The authors detected a highly significant, persistent, lesional accumulation of parenchymal IL-16+ microglia/macrophages, which reached a maximal level 3 days postinjury compared with control rats. The majority of cells that demonstrated positive labeling for IL-16 also had positive labeling for ED1 (> 70%) and OX-8/CD8; these cells exhibited the morphological hallmarks of activated microglia/macrophages and pronounced MHC Class II expression. In contrast to IL-16+ED1+ cells, IL-16+ microglia/macrophages that coexpressed OX-8 were exclusively seen in the pannecrotic lesion core. In addition, clustering of IL-16+ cells was observed in perivascular Virchow-Robin-like spaces in areas of the primary injury (lesion core) and in immediately adjacent areas of secondary injury. Furthermore, on Day 3 postinjury, IL-16+ microglia/macrophages were frequently observed in a perineuronal position. CONCLUSIONS: The early lesional accumulation of IL-16+ microglia/macrophages suggests a role for IL-16 in the early postinjury immune response such as recruitment and activation of immune cells, leading to microvessel clustering and secondary damage progression.

Animals↗

PubMed2005

Lesional accumulation of P2X4 receptor+ monocytes following experimental traumatic brain injury.

P2X4 receptor (P2X4R) is an ATP-gated ion channel. ATP is an important messenger in traumatic brain injury. Here, we report expression of P2X4R in rat traumatic brain injury with focus on the early phase, most amenable to therapy. Accumulation of P2X4R+ cells was observed as early as 6 h after injury and continued to increase 4 days post-injury at the lesion and remote areas. Double staining revealed that most P2X4R+ cells co-expressed ED-1, a marker for reactive microglia/macrophages, but not nestin or W3/13. Our data suggest that P2X4R expression defines a subtype of activated microglia/macrophages involved in the early processes following traumatic brain injury.

Animals↗

PubMed2005

Spinal cord glia activation following peripheral polyinosine-polycytidylic acid administration.

Effects of repeated intraperitoneal injections of polyinosine-polycytidylic acid on spinal cord cells were analysed. After each injection, the number of ED-1 microglia significantly increased in rat spinal cords. Expression of endothelial monocyte-activating polypeptide II, however, was not observed. The morphology of microglia indicated an incomplete activation state even after three repeated polyinosine-polycytidylic acid injections. Astrocyte activation was observed after the first injection using glial fibrillary acidic protein staining. Simultaneously with glia activation, hyperalgesia was observed, but the expression of P2X4 receptor, which is considered to be closely associated with hyperalgesia, on microglia was not detected. In sum, our data suggest that repeated peripheral injections of polyinosine-polycytidylic acid might alert the central nervous system through limited activation of microglia and astrocytes.

Animals↗

PubMed2005

Prolonged lesional expression of RhoA and RhoB following spinal cord injury.

Inhibition of the small GTPase ras homology protein (Rho) or its downstream target, the Rho-associated kinase (ROCK), has been shown to promote axon regeneration and to improve functional recovery following spinal cord injury (SCI) in the adult rat. Here, we have analyzed the expression of RhoA and RhoB following spinal cord injury in order to assess whether Rho is a possible target for late pharmacological intervention. In control spinal cords, RhoA(+) cells were almost absent, whereas RhoB was localized to some ependymal cells, a few microglia, and some dissociated neurons. In injured spinal cords, RhoA(+) and RhoB(+)cells accumulated at perilesional areas and in the developing necrotic core early after injury at day 1. After reaching their maximum levels (RhoA at day 3; RhoB at day 1), RhoA(+) and RhoB(+) cell numbers remained significantly elevated until day 28. In areas remote from the lesion (> or =0.75 mm), a more discrete accumulation of RhoA(+) and RhoB(+) cells was observed, primarily in areas of ongoing Wallerian degeneration. RhoA and RhoB were predominantly expressed by polymorphonuclear granulocytes, ED1(+) microglia/macrophages, oligodendrocytes, some neurons, and swollen axons/neurites. Furthermore, expression was located to lesional, reactive astrocytes and fibroblastoid cells confined to areas of scar formation. Our experiments have determined that most RhoA(+) and RhoB(+) cells (>70%) are of mononuclear origin. The persistent presence of lesional RhoA(+) and RhoB(+) axon/neurite fibers over a period of 4 weeks after injury suggests that Rho inhibition is a putative therapeutic concept also for delayed intervention after SCI.

Amyloid beta-Protein Precursor↗

PubMed2005

Spinal cord injury induces early and persistent lesional P2X4 receptor expression.

Following spinal cord injury (SCI), neuropathic, chronic pain is a major cause of disability. Recently, glial P2X4 receptor (P2X4R) has been identified as a major contributor to the development of neuropathic pain after peripheral nerve injury. Here we report analysis of P2X4R expression following rat SCI. Significant lesional accumulation of P2X4R+ cells was detected as early as 24 h after SCI, reaching maximum cell numbers on Day 7. Thereafter cell numbers declined but persisted at significantly elevated, sub-maximal levels (>70%) until 1 month post injury. Double-immunolabeling identified the majority of lesional P2X4R+ cells as activated microglia/macrophages and surviving neurons/neurites. Increase of P2X4R+, beta-APP+ hypertrophic neurites correlated with proximity to the lesion. Further, P2X4R+ cells coexpressed the intracellular regulators of signalling cascades, COX-1 (>20%), COX-2 (>5%), RhoA (>60%) and RhoB (>10%).

Amino Acid Sequence↗

PubMed2005

Expression of P2X4 receptor by lesional activated microglia during formalin-induced inflammatory pain.

P2X4 receptor (P2X4R) is an ion channel gated by adenosine 5'-triphosphate. Here we report the presence and the distribution of P2X4R in rat spinal cord by immunohistochemical analysis in an inflammatory pain model. Peripheral inflammation was induced by subcutaneous injection of 4% formalin into the rat hindpaw. Morphology, spatial localization, and activation state of P2X4R+ cells were described at 1, 5, 7, 14, and 28 days after injury. In normal and saline treated control rats, P2X4R was rarely seen. After formalin administration, an increase of P2X4R+ microglia were observed in the spinal cord dorsal horn on the side ipsilateral to the injection, reaching maximal levels by day 7, and then decreasing to normal levels by day 14. This implicates a role of P2X4R in the spinal inflammatory pain process. Furthermore, formalin-induced region-specific increase in activated microglia was confirmed by ED1 and endothelial monocytes activating polypeptide II (EMAP-II) expression. In conclusion, this is the first demonstration that P2X4R is expressed by microglia in the inflammatory pain.

Animals↗

PubMed2005

Uptake, intracellular distribution, and novel binding proteins of immunostimulatory CpG oligodeoxynucleotides in microglial cells.

Microglial cells are central components of the innate immune system of the brain and contribute to inflammatory and degenerative processes. DNA with unmethylated CpG dinucleotides is a potent stimulant of microglial cells. We have analyzed uptake, intracellular distribution, and cellular binding proteins of CpG oligdeoxynucleotides (ODNs) by the microglial cell line N9. The uptake of CpG-ODN is concentration-, time-, and temperature-dependent, but, interestingly, independent of the CpG dinucleotides. After internalisation, CpG-ODN localized to the cytoplasm and showed a typical speckled distribution pattern. We further purified the cellular binding proteins of CpG-ODN and identified several binding proteins by tryptic digestion and mass spectrometry. Most of the CpG-ODN binding proteins are RNA processing enzymes, which are important for RNA splicing, export, and stability. Further, we identified a protein, pigpen, which has not been observed in microglial cells, so far. These proteins apparently bind CpG-ODN with low selectivity, as binding is independent of CpG dinucleotides. Interference of immunostimulatory and therapeutic oligonucleotides with proteins and enzymes of RNA transport and processing has not been described so far and might affect the physiological functions of these proteins and also might influence cellular localization of therapeutic ODN. These findings are helpful in understanding the cellular fate of ODN and the nonsequence-specific effects of ODN and for rational design and evaluation of ODN-based therapeutic strategies.

ADP Ribose Transferases↗

PubMed2005

Central nervous system injury-induced repulsive guidance molecule expression in the adult human brain.

BACKGROUND: The repulsive guidance molecule (RGM) is involved in formation of the central nervous system during development by moderating the repulsion of growing axons. However, the role of RGM in adult central nervous system lesions remains to be clarified. OBJECTIVE: To identify and determine RGM expression in adult brains with focal cerebral ischemia or traumatic brain injury and in neuropathologically unaffected control brains. Patients Twenty-one brains of patients with focal cerebral ischemia, 25 brains after traumatic brain injury, and 4 control brains. Main Outcome Measure Expression of RGM as assessed by immunohistochemical analysis. RESULTS: In normal brains, RGM expression was detected on the perikarya of some neurons, choroid plexus, smooth muscle and endothelial cells, oligodendrocytes, and myelinated white matter fibers. After focal cerebral ischemia and traumatic brain injury, RGM-immunopositive cells accumulated in lesional and perilesional areas. In hemorrhagic lesions, a massive accumulation of RGM-immunopositive cells was observed. During the first week after insult, RGM expression remained confined to neurons, smooth muscle and endothelial cells, and leukocytes infiltrating the lesion. Thereafter, with maturation of the lesion, we observed RGM expression by components of the developing scar tissue, such as fibroblastoid cells, reactive astrocytes, and a pronounced extracellular RGM deposition resembling neo-laminae. CONCLUSIONS: This is the first study of RGM in the human central nervous system. Following central nervous system injury, RGM, a novel, potent axonal growth inhibitor, is present in axonal growth impediments: the mature myelin, choroid plexus, and components of the developing scar.

Adult↗

PubMed2005

Uptake, cellular distribution and novel cellular binding proteins of immunostimulatory CpG oligodeoxynucleotides in glioblastoma cells.

Glioblastomas are the most malignant and most frequent brain tumors and exciting targets of gene and immunotherapy. Despite rapid development of experimental therapy little is known about the cellular behaviour of therapeutic oligodeoxynucleotides (ODNs). Here we designed uptake, cellular distribution and cellular binding proteins of immunostimulatory CpG-ODNs in glioblastoma cells by flow cytometry, fluorescence microscopy and mass spectrometry. Our data show that the phosphorothioate (PS) CpG-ODNs uptake in T98G and C6 cells is dose-, time-, temperature-dependent and independent of the CpG dinucleotides. Uptake can be inhibited by sodium azide, polyanions but not by chloroquine. After internalisation FITC labelled CpG-ODNs showed a spotted distribution in cytoplasm. Dozens of cellular binding proteins were identified using mass spectrometry. The binding of ODNs to proteins is dependent on modification and sequence but independent on CpG motif. ODNs bind to cellular proteins that are important for RNA processing and transport. Furthermore, three novel membrane proteins were identified, which might contribute to uptake of ODNs. ODNs binding to these proteins might interfere with the physiological function and thus might cause unwanted effects. Such binding also might influence the uptake efficiency or cellular distribution of therapeutic ODNs.

Adjuvants, Immunologic↗

PubMed2005

The immunostimulatory activity of CpG oligonucleotides on microglial N9 cells is affected by a polyguanosine motif.

Oligonucleotides (ODN) with hexameric motifs containing central unmethylated CpG dinucleotides are immunostimulatory. Also ODN with continuous guanosines (polyG motif) show a wide range of immunological activity. Depending on the position, the chemical property of the ODN backbone and the cell type, polyG motifs have either an enhancing or a suppressing effect on the immunostimulatory activity of the CpG-ODN. Microglial cells are central components of the innate immune system of the brain and are activated by CpG-ODN in vitro and in vivo. Here we present the analysis of the immunomodulatory effects of CpG-ODN carrying a polyG motif on the microglial cell line N9. Our data show that N9 cells express Toll-like receptor 9 (TLR9) and are activated by CpG-ODN, which leads to expression of interleukin-12p40 (IL12p40), tumor necrosis factor-alpha (TNF-alpha) and inducible nitric oxide synthase (iNOS). A 3'-end polyG motif inhibits phosphothioate (PS) CpG-ODN immunostimulatory activity but enhances the immunostimulatory activity of phosphodiester (PE) CpG-ODN. Correspondingly, a 3'-end polyG motif improves the cellular uptake of PE CpG-ODN but does not change their cellular distribution pattern. Furthermore, PE CpG-ODN with a 3'-end polyG motif interact with a much higher number of cellular proteins than PE CpG-ODN. These data indicate that the 3'-end polyG motif could enhance the immunostimulatory activity of PE CpG-ODN in microglial N9 cells through increasing interaction with cellular proteins. Therefore PE CpG-ODN containing a 3'-end polyG motif resulting in increased immunostimulatory activity might be promising alternate analogues for studies in the central nervous system.

Animals↗