Targeting gene-modified hematopoietic cells to the central nervous system: use of green fluorescent protein uncovers microglial engraftment.

Gene therapy in the central nervous system (CNS) is hindered by the presence of the blood-brain barrier, which restricts access of serum constituents and peripheral cells to the brain parenchyma. Expression of exogenously administered genes in the CNS has been achieved in vivo using highly invasive routes, or ex vivo relying on the direct implantation of genetically modified cells into the brain. Here we provide evidence for a novel, noninvasive approach for targeting potential therapeutic factors to the CNS. Genetically-modified hematopoietic cells enter the CNS and differentiate into microglia after bone-marrow transplantation. Up to a quarter of the regional microglial population is donor-derived by four months after transplantation. Microglial engraftment is enhanced by neuropathology, and gene-modified myeloid cells are specifically attracted to the sites of neuronal damage. Thus, microglia may serve as vehicles for gene delivery to the nervous system.

Blood-borne soluble protein antigen intensifies T cell activation in autoimmune CNS lesions and exacerbates clinical disease.

We explored the effect of i.v. soluble antigen on autoaggressive, myelin basic protein-specific effector T cells within their target organ during acute experimental autoimmune encephalomyelitis (EAE). Intravital two-photon imaging revealed that i.v. autoantigen reached the CNS and was taken up and processed by antigen-presenting cells within 30 min after injection. The exogenous autoantigen dramatically changed the motility and function of autoreactive effector T cells within the EAE lesions: T cells that had been cruising through the tissue slowed down and became tethered to local antigen-presenting cells within 1 h. One hour later, the effector T cells massively produced proinflammatory cytokines and up-regulated membranous activation markers. This strong activation of the T cells boosted CNS inflammation and aggravated clinical disease. Postactivated effector and resting memory T cells specific for a non-CNS antigen (ovalbumin) were recruited to EAE lesions and moved there without contacting antigen-presenting cells. These cells were similarly arrested and activated after i.v. infusion of ovalbumin, and they also exacerbated clinical disease. Our data are relevant for autoantigen-based therapies of autoimmune disorders. Further, the study indicates how brain unrelated antigens (microbial components) leaking into the chronically inflamed CNS through the bloodstream might trigger relapses in multiple sclerosis.

Cellular effects and metabolic stability of N1-cyclic inosine diphosphoribose and its derivatives.

BACKGROUND AND PURPOSE: Recently, a number of mimics of the second messenger cyclic ADP-ribose (cADPR) with replacement of adenosine by inosine were introduced. In addition, various alterations in the molecule ranging from substitutions at C8 of the base up to full replacement of the ribose moieties still retained biological activity. However, nothing is known about the metabolic stability and cellular effects of these novel analogues. EXPERIMENTAL APPROACH: cADPR and the inosine-based analogues were incubated with CD38, ADP-ribosyl cyclase and NAD-glycohydrolase and metabolism was analysed by RP-HPLC. Furthermore, the effect of the analogues on cytokine expression and proliferation was investigated in primary T-lymphocytes and T-lymphoma cells. KEY RESULTS: Incubation of cADPR with CD38 resulted in degradation to adenosine diphosphoribose. ADP-ribosyl cyclase weakly catabolised cADPR whereas NAD-glycohydrolase showed no such activity. In contrast, N1-cyclic inosine 5'-diphosphoribose (N1-cIDPR) was not hydrolyzed by CD38. Three additional N1-cIDPR analogues showed a similar stability. Proliferation of Jurkat T-lymphoma cells was inhibited by N1-cIDPR, N1-[(phosphoryl-O-ethoxy)-methyl]-N9-[(phosphoryl-O-ethoxy)-methyl]-hypoxanthine-cyclic pyrophosphate (N1-cIDP-DE) and N1-ethoxymethyl-cIDPR (N1-cIDPRE). In contrast, in primary T cells neither proliferation nor cytokine expression was affected by these compounds. CONCLUSIONS AND IMPLICATIONS: The metabolic stability of N1-cIDPR and its analogues provides an advantage for the development of novel pharmaceutical compounds interfering with cADPR mediated Ca2+ signalling pathways. The differential effects of N1-cIDPR and N1-cIDPRE on proliferation and cytokine expression in primary T cells versus T-lymphoma cells may constitute a starting point for novel anti-tumor drugs.

[The balloon dissector in expander treatment: A ten-year experience in plastic and reconstructive surgery]. / Der Einsatz des Ballondissektors in der Expanderchirurgie: Zehn Jahre Erfahrung in der plastischen und rekonstruktiven Chirurgie.

BACKGROUND: The use of the balloon dissector ("space maker") for the implantation of soft tissue expanders is illustrated and technical aspects and advantages are compared to the conventional method. PATIENTS AND METHODS: Over a 10-year period a group of 90 patients with evaluation records (m = 34, f = 56) was analysed retrospectively. The mean age was 23 years (range: 5 to 62 years). Overall, 164 expanders were implanted and in 73 cases (44.5%) a balloon dissector was used. RESULTS: The mean intraoperative expander filling was increased up to 27% of the volume of the tissue expander after using the balloon dissector; in contrast it was 15% after conventional dissection. The mean duration of expansion was decreased by 9.8% after space maker dissection. CONCLUSION: The use of the space maker is a scar-sparing technique. Time of operation and overall duration of expansion are reduced. Patient comfort is clearly improved. The negligible costs of a space maker are compensated by the cost reduction due to shorter operating time, fewer outpatient contacts and a low complication rate. The indications for the use of balloon dissectors are the expansion of skin (scar correction after burns, trauma, tumour), breast reconstruction and augmentation, and the prefabrication and rapid intraoperative expansion of musculocutaneous flaps.

[Defect coverage of the hand with the free serratus fascial flap]. / Defektdeckung an der Hand mit dem freien Serratus-Faszien-Lappen.

PURPOSE: Coverage of exposed functional structures such as tendons, bones, vessels or nerves at the dorsal and palmar surface of the hand requires thin, supple tissue to provide adequate range of motion and a satisfying aesthetic result. Free fascial flaps are possible alternatives to cutaneous, fasciocutaneous and muscle flaps. The purpose of this retrospective study was to evaluate the functional and aesthetic results after coverage of the hand with free serratus fascial flaps in our department. METHOD AND CLINICAL MATERIAL: From 1994 to 2002, ten patients underwent free fascial flap coverage of the hand with eleven serratus fascial flaps. Six patients could be re-examined and answered a questionnaire about their satisfaction with the functional and aesthetic results. The mean follow-up was after 34 months. RESULTS: Average active range of motion of the hand, functional improvement and the aesthetic result were satisfying in all follow-up patients. No secondary debulking or other contouring procedures were required. CONCLUSION: We recommend the use of free serratus fascial flaps as a valuable alternative to fasciocutaneous or muscle flaps since the functional results are excellent; no additional procedures were necessary and the aesthetical results are appealing.

Potential of allospecific gene-engineered T cells in transplantation gene therapy: specific T cell activation determines transgene expression in vitro and in vivo.

T lymphocytes, regardless of their specificity, are considered key targets for genetic modification in the treatment of inherited or acquired human diseases. In this study, we generated Lewis T cell lines specific for Dark Agouti rat alloantigens and tested the potential of allospecific T lymphocytes as carriers of genes encoding therapeutic proteins in transplantation gene therapy. These allospecific T lymphocytes were successfully, stably transduced with enhanced green fluorescent protein (EGFP) by an Mo-MuLV-based retrovirus vector. A novel gene delivery protocol was utilized, resulting in nearly 100% EGFP-expressing T cells. This approach enabled tracking of allospecific transduced T cells in vivo and illustrates their transgene production by fluorometric determination after ex vivo isolation. Quantitation of EGFP transgene expression was used to determine the influence of T cell receptor-specific activation on transgene regulation. A strict positive correlation between activation state and expression level was detected in vitro and in vivo. The activation-induced increase in transgene expression could be blocked by interference with T cell activation signaling pathways by cyclosporin A, anti-CD4 MAb, or CTLA4-Ig. These data provide strong evidence that direct or indirect effects caused by activation-induced transcription factors are crucial in transgene upregulation. Allospecific activation in spleens, lymph nodes, and transplanted grafts can be considered as antigen-specific targeting strategy. This activation might be useful in expressing therapeutic proteins such as TGF-beta or IL-10 specific to these sites. T lymphocyte priming and activation might be prevented or altered by modification of the local microenvironments, thereby exerting a therapeutic influence on acute and chronic graft rejection processes.

Neuronal FasL induces cell death of encephalitogenic T lymphocytes.

Apoptosis of inflammatory cells plays a crucial role in the recovery from autoimmune CNS disease. However, the underlying mechanisms of apoptosis induction are as yet ill-defined. Here we report on the neuronal expression of FasL and its potential function in inducing T-cell apoptosis. Using a combination of facial nerve axotomy and passive transfer encephalomyelitis, the fate of CD4+ encephalitogenic T cells engineered to express the gene for green fluorescent protein was followed. FasL gene transcripts and FasL protein were detected in neurons by in sit-hybridization and immunohistochemistry. T cells infiltrating preferentially the injured brain parenchyma were found in the immediate vicinity of FasL expressing neurons and even inside their perikarya. In contrast to neurons, T cells rapidly underwent apoptosis. In co-cultures of hippocampal nerve cells and CD4 T lymphocytes, we confirmed expression of FasL in neurons and concomitant induction of T-cell death. Antibodies blocking neuronal FasL were shown to have a protective effect on T-cell survival. Thus, FasL expression by neurons in neuroinflammatory diseases may constitute a pivotal mechanism underlying apoptosis of encephalitogenic T cells.

Neuronal MCP-1 expression in response to remote nerve injury.

Direct injury of the brain is followed by inflammatory responses regulated by cytokines and chemoattractants secreted from resident glia and invading cells of the peripheral immune system. In contrast, after remote lesion of the central nervous system, exemplified here by peripheral transection or crush of the facial and hypoglossal nerve, the locally observed inflammatory activation is most likely triggered by the damaged cells themselves, that is, the injured neurons. The authors investigated the expression of the chemoattractants monocyte chemoattractant protein MCP-1, regulation on activation normal T-cell expressed and secreted (RANTES), and interferon-gamma inducible protein IP10 after peripheral nerve lesion of the facial and hypoglossal nuclei. In situ hybridization and immunohistochemistry revealed an induction of neuronal MCP-1 expression within 6 hours postoperation, reaching a peak at 3 days and remaining up-regulated for up to 6 weeks. MCP-1 expression was almost exclusively confined to neurons but was also present on a few scattered glial cells. The authors found no alterations in the level of expression and cellular distribution of RANTES or IP10, which were both confined to neurons. Protein expression of the MCP-1 receptor CCR2 did not change. MCP-1, expressed by astrocytes and activated microglia, has been shown to be crucial for monocytic, or T-cell chemoattraction, or both. Accordingly, expression of MCP-1 by neurons and its corresponding receptor in microglia suggests that this chemokine is involved in neuron and microglia interaction.

Microglia only weakly present glioma antigen to cytotoxic T cells.

Microglia and brain macrophages represent a substantial fraction of the cells present in astrocytic gliomas. Yet, the functional role of microglia in these tumors has remained enigmatic. We have compared rat microglial cells and thymocytes with regard to their ability to present purified CNS proteins, MBP and S100beta, as well as C6 glioma cells to specific T lymphocytes. In addition, a new cytotoxicity assay based on fluorescence activated cell sorting of tumor cells carrying the green fluorescent protein was established. This assay was used to determine the influence of microglial population density and activational state on C6 glioma cell survival in vitro. Microglia were consistently found to present MBP and S100beta less efficiently than thymocytes and appeared to be unable to present C6 glioma cells to cytotoxic T lymphocytes. In addition, high concentrations of microglial cells attenuated the cytotoxic effects of these T cells on C6 glioma cells whereas thymocytes significantly supported their specific killing. It is suggested that defense functions of microglial cells against C6 glioma are severely compromised and that the observed deficiency in antigen presentation may play an important role for astrocytoma growth in vivo.

[Principles and possibilities of reconstruction with microsurgical flaps]. / Prinzipien und Möglichkeiten der Rekonstruktion mit mikrochirurgischen Lappenplastiken.

Microsurgical reconstructions are considered an integral art of modern reconstructive concepts, especially in the fields of trauma, tumor reconstruction, and correction of congenital deformities. Patient expectations regarding function and aesthetics of plastic surgical reconstructions are satisfied with permanently improved flap designs that also lead to a significant reduction in donor site morbidity. Together with steadily decreasing complication rates, these options have made microsurgical reconstructions a prime choice in plastic surgery, in contrast to the past, where they were considered the "last line of defense."

The potential for genetically altered microglia to influence glioma treatment.

Diffuse and unstoppable infiltration of brain and spinal cord tissue by neoplastic glial cells is the single most important therapeutic problem posed by the common glioma group of tumors: astrocytoma, oligoastrocytoma, oligodendroglioma, their malignant variants and glioblastoma. These neoplasms account for more than two thirds of all malignant central nervous system tumors. However, most glioma research focuses on an examination of the tumor cells rather than on host-specific, tumor micro-environmental cells and factors. This can explain why existing diffuse glioma therapies fail and why these tumors have remained incurable. Thus, there is a great need for innovation. We describe a novel strategy for the development of a more effective treatment of diffuse glioma. Our approach centers on gaining control over the behavior of the microglia, the defense cells of the CNS, which are manipulated by malignant glioma and support its growth. Armoring microglia against the influences from glioma is one of our research goals. We further discuss how microglia precursors may be genetically enhanced to track down infiltrating glioma cells.

[Ulnar nerve lesions after osteosynthesis of a supercondylar humerus fracture during childhood. Indications for revision]. / Ulnarisläsion nach Osteosynthese der suprakondylären Humerusfraktur im Kindesalter. Indikationen zur Revision.

Since 1948, closed reduction and osteosynthesis for supracondylar humeral fractures using two K-wires from the medial and lateral side has been performed on a regular basis. Although this procedure is used routinely, many authors have described paralysis of the ulnar nerve after blindly inserting the medial K-wire. Only very few publications describe the treatment options after iatrogenic paralysis of this nerve. The patients described showed progressive paralysis of the ulnar nerve after K-wire osteosynthesis. Intraoperatively, all patients showed scarring but intact continuity. After surgical revision and neurolysis, all four patients showed complete restitution after 1 year. If patients show progressive paralysis of the ulnar nerve early operative revision after 3 months should be performed.

Coverage of soft-tissue defects of the hand with free fascial flaps.

Coverage of exposed functional structures such as tendons, bones, vessels, or nerves at the dorsal and palmar surface of the hand requires thin, supple tissue to provide adequate range of motion and a satisfying aesthetic result. The purpose of this retrospective study was to evaluate the functional and aesthetic results after coverage of the hand with free fascial flaps. From 1994-2002, 14 patients underwent free fascial flap coverage of the hand with 4 tempo-parietal fascia flaps and 11 serratus fascia flaps. Eight patients could be reexamined and answered a questionnaire about their satisfaction with the functional and aesthetic results. The mean follow-up was 41.7 months. Average active range of motion of the hand, functional improvement, and the aesthetic result were satisfying in all follow-up patients. No secondary debulking or other contouring procedures were required. We recommend the use of free fascial flaps as a valuable alternative to fasciocutaneous or muscle flaps, since the functional results are excellent, no additional procedures were necessary, and the aesthetic results are appealing.

New tools to trace populations of inflammatory cells in the CNS.

Cells of the central nervous system (CNS) and immune system communicate regularly. There is a constant surveillance of the intact, healthy CNS by activated T-cells, and massive infiltration of the CNS by immune cells under pathological conditions such as neurodegeneration or neuroinflammation. Labeling CNS-infiltrating T-cells is an essential tool to identify the signals and mechanisms, which mediate the interaction between immune cells and cells of the CNS. In this article, we will present an overview describing currently used cellular markers and demonstrate how these markers have contributed to our current knowledge of CNS inflammation and immune surveillance.

Release of prostaglandin D2 by murine mast cells: importance of metabolite formation for antiproliferative activity.

Prostaglandin (PG) D2, PGJ2 and delta12-PGJ2 are antiproliferative eicosanoids. We investigated the production of PGD2 by murine bone marrow-derived mast cells (BMMC) taking into consideration metabolism of PGD2 to PGD2 and delta12-PGJ2. PG-metabolites were quantified by high performance liquid chromatography (HPLC) combined with radioimmunoassay (RIA). Stimulated with calcium ionophore A23187 BMMC released eight-fold more PGJ2 and delta12-PGJ2 than PGD2. Conversion of endogenously produced PGD2 to PGJ2 and delta12-PGJ2 proceeded rapidly in contrast to metabolism of exogenously added PGD2. The antiproliferative potency of these prostaglandins is demonstrated in vitro. We conclude that determination of PGD2 production by mast cells must take into consideration rapid conversion to active derivatives, which may play a significant role in growth regulation.

Substitution of two histidine residues in YadA protein of Yersinia enterocolitica abrogates collagen binding, cell adherence and mouse virulence.

The plasmid-encoded surface protein YadA of Yersinia enterocolitica mediates binding to diverse extracellular matrix (ECM) proteins, adherence to epithelial cell lines, resistance to complement lysis, autoagglutination, and is required for mouse virulence. Using site-directed mutagenesis we attempted to analyse the relationship between structural domains and functions of YadA. In a first approach we could abrogate collagen binding by chemical modification of histidyl residues of YadA protein. This result prompted us to substitute histidyl residues (His) of conserved regions of YadA protein of Y. enterocolitica O8 by tyrosine residues using site-directed mutagenesis. Substitution of His-156 and His-159 (YadA-2 mutant) resulted in abrogation of binding to ECM proteins, of cell adherence, and in reduction of mouse virulence, whereas autoagglutination, serum complement resistance and oligomer formation remained unaffected. A striking result was obtained from the orogastric mouse-infection model: the YadA-2 mutant retained the ability to colonize the small intestine and to invade and multiply within the Peyer's patches but was impaired in colonizing mesenteric lymph nodes and spleen in comparison to the wild-type strain.