Tetanus toxin fragment C as a vector to enhance delivery of proteins to the CNS.

The non-toxic neuronal binding domain of tetanus toxin (tetanus toxin fragment C, TTC) has been used as a vector to enhance delivery of potentially therapeutic proteins to motor neurons from the periphery following an intramuscular injection. The unique binding and transport properties of this 50-kDa polypeptide suggest that it might also enhance delivery of proteins to neurons after direct injection into the CNS. Using quantitative fluorimetry, we found that labeled TTC showed vastly superior retention within brain tissue after intracerebral injection compared to a control protein (bovine serum album). Fluorescence microscopy revealed that injected TTC was not retained solely in a restricted deposit along the needle track, but was distributed through gray matter in a pattern not previously described. The distribution of injected protein within the extracellular space of the gray matter and neuropil was also seen after injection of a recombinant fusion protein comprised of TTC linked to the enzyme superoxide dismutase (TTC-SOD-1). Injections of native SOD-1 in contrast showed only minimal retention of protein along the injection track. Immunohistochemistry demonstrated that both TTC and TTC-SOD-1 were distributed in a punctate perineuronal and intraneuronal pattern similar to that seen after their retrograde transport, suggesting localization primarily in synaptic boutons. This synaptic distribution was confirmed using HRP-labeled TTC with electron microscopy along with localization within neuronal endosomes. We conclude that TTC may be a useful vector to enhance neuronal delivery of potentially therapeutic enzymes or trophic factors following direct injection into the brain.

Radiosurgery at the Royal Adelaide Hospital: the first 4 1/2 years' clinical experience.

Radiosurgery refers to the treatment of small lesions localized by stereotactic technology using highly focused radiation. This review utilizes prospectively gathered data from the Royal Adelaide Hospital Radiosurgery unit to summarize experience with the first 62 patients (65 lesions) treated between November 1993 and May 1998. This experience included acoustic neuromas (23 patients), arteriovenous malformations (18), brain metastases (12), meningiomas (6), and glomus tumour, subependymoma, dural arteriovenous fistula (1 each). Although follow up is relatively short, the outcome in terms of morbidity and tumour control is thus far comparable with results reported in the literature. Radiosurgery provides a viable alternative to neurosurgery and conventional external beam radiotherapy for several benign and malignant intracranial lesions.

A simple technique for treating age-related macular degeneration with external beam radiotherapy.

PURPOSE: To develop a simple external beam photon radiotherapy technique to treat age-related macular degeneration without the need for simulation, planning computed tomography (CT) or computer dosimetry. METHODS AND MATERIALS: The goal was to enable the treatment to be set up reliably on the treatment machine on Day 1 with the patient supine in a head cast without any prior planning. Using measurements of ocular globe topography from Karlsson et al. (Int J Radiat Oncol Biol Phys 1996; 33: 705-712), we chose a point 1.5 cm behind the anterior surface of the upper eyelid (ASUE) as the isocentre of a half-beam, blocked, 5.0 x 3.0-cm, angled lateral field to treat the involved eye. This would position the isocentre about 0.5 cm behind the posterior surface of the lens, and a little over 1 cm in front of the macula, according to Karlsson et al. The setup requires initial adjustment of the gantry from horizontal (to account for any asymmetry of position of the eyes), then angling 15 degrees posteriorly to avoid the contralateral eye. Finally, the couch is raised to position the isocentre 1.5 cm behind the ASUE. RESULTS: To verify the applicability of the technique, we performed CT and computer dosimetry on the first 11 eyes so treated. Our CT measurements were in good agreement with Karlsson et al. The lens dose was < 5% and the macula was within the 95% isodose curve in each case (6-MV linac). Treatment setup time is approximately 10 min each day. The 11 patients were treated with 5 x 2.00 Gy (2 patients) or 5 x 3.00 Gy (9 patients), and subjective response on follow-up over 1 to 12 months (median 4 months) was comparable to previously reported results, with no significant acute side effects. CONCLUSION: Our technique is easy to set up and reliably treats the macula, with sparing of the lens and contralateral eye. It enables treatment to commence rapidly and cost-effectively without the need for simulation or CT computer planning.

Heterogeneity of subcellular localization and electrophoretic mobility of survival motor neuron (SMN) protein in mammalian neural cells and tissues.

Spinal muscular atrophy is caused by defects in the survival motor neuron (SMN) gene. To better understand the patterns of expression of SMN in neuronal cells and tissues, we raised a polyclonal antibody (abSMN) against a synthetic oligopeptide from SMN exon 2. AbSMN immunostaining in neuroblastoma cells and mouse and human central nervous system (CNS) showed intense labeling of nuclear "gems," along with prominent nucleolar immunoreactivity in mouse and human CNS tissues. Strong cytoplasmic labeling was observed in the perikarya and proximal dendrites of human spinal motor neurons but not in their axons. Immunoblot analysis revealed a 34-kDa species in the insoluble protein fractions from human SY5Y neuroblastoma cells, embryonic mouse spinal cord cultures, and human CNS tissue. By contrast, a 38-kDa species was detected in the cytosolic fraction of SY5Y cells. We conclude that SMN protein is expressed prominently in both the cytoplasm and nucleus in multiple types of neurons in brain and spinal cord, a finding consistent with a role for SMN as a determinant of neuronal viability.

Interleukin-1-beta and tumor necrosis factor-alpha increase peripheral-type benzodiazepine binding sites in cultured polygonal astrocytes.

Peripheral-type benzodiazepine binding sites (PTBBS) are markedly increased in the injured CNS. Astrocytes appear to be the primary cell type which express increased PTBBS. Because certain cytokines within the injured CNS are potent mitogens for astrocytes, we examined the effects of two such cytokines, interleukin (IL)-1 beta and tumor necrosis factor (TNF), on PTBBS in cultured astrocytes using [3H]Ro 5-4864 as the specific ligand. Purified cultures of either polygonal or process-bearing astrocytes were prepared from neonatal rat cerebral hemispheres. At a concentration of 1.8 nM, specific binding of the radioactive ligand to polygonal astrocytes reached equilibrium within 60 min and was half-maximal by 5-10 min. By contrast, specific binding to process-bearing astrocytes barely exceeded background levels. IL-1 and TNF increased PTBBS within polygonal astrocytes in both dose- and time-dependent manners. At 10-50 ng/ml, IL-1 beta and TNF-alpha elevated [3H]Ro 5-4864 binding in polygonal astrocyte cultures 65 and 87%, respectively, above the level in control cultures. However, no changes in PTBBS were seen within polygonal astrocytes after IL-2 treatment. Scatchard analysis of saturation binding experiments suggested that the increase in PTBBS promoted by TNF was due to an increased number of binding sites present in polygonal astrocytes and not due to an increase in receptor affinity. Binding data suggested that PTBBS within cultures of process-bearing astrocytes were virtually absent irrespective of the treatment. These in vitro data suggest that certain cytokines found in the injured brain may be involved in up-regulating PTBBS within a particular subtype of astrocyte.

Film dosimetry for the junction region of four compensated photon beams.

A set of four 4 MV photon beam compensators were produced, one for each field of a Hodgkins Disease treatment plan. The resultant dose profiles at various depths were measured by an ion chamber in water and by Kodak X-Omat V film in a Solid Water phantom, and compared to the doses calculated by a GE RT/Plan treatment planning computer. After normalisation and correction for the film's non-linear dose response, the film and ion chamber results compared well with each other. They both showed cold spots of 80% in the junction region of the four fields which were not shown on the computed isodose plan. Film dosimetry is faster than ion chamber dosimetry and is shown to be accurate enough to use for measuring the dose uniformity of compensated beams.

Deficiency in immune complex uptake by chronic granulomatous disease neutrophils.

Receptor-mediated binding and endocytosis by normal and chronic granulomatous disease (CGD) polymorphonuclear (PMN) leucocytes have been investigated. Our results suggest that activation of the respiratory burst is a requisite for normal immune complex internalization. Cell-associated immune complexes were detected by fluorescence microscopy, electron microscopy and flow cytometric techniques. These measurements indicate that immune complex uptake is defective in CGD patients, but not in normal subjects. Antibody-dependent binding and complement-dependent binding and endocytosis were not affected. The functional association of the respiratory burst and antibody-dependent endocytosis was further explored using rhodamine-labelled glucose oxidase:anti-glucose oxidase immune complexes. These complexes generate H2O2 in the presence of glucose. When treated with these complexes, CGD PMNs showed similar glucose-dose-dependent increases in both H2O2 production and cell-associated fluorescence. Therefore: (1) respiratory burst and immune complex uptake are coexisting deficiencies that may be a manifestation of a single defect; and (2) the respiratory burst may be one participant in immune complex endocytic triggering.

Optical microscopy of antibody-dependent phagocytosis and lysis of erythrocytes by living normal and chronic granulomatous disease neutrophils: a role of superoxide anions in extra- and intra-cellular lysis.

Novel optical microscopic techniques have been developed to observe neutrophil-mediated effector functions at the level of individual cells. Conventional absorption spectrophotometry has shown that exposure of hemoglobin to superoxide anions decreases the intensity of the Soret band and shifts it to lower wavelengths. This oxidative event can be visualized within intact erythrocytes using bright-field microscopy in conjunction with violet illumination at 430 nm. The sequential oxidation of IgG-opsonized sheep erythrocytes bound to normal human neutrophils can be observed. Chronic granulomatous disease (CGD) neutrophils which do not generate superoxide anions were not capable of influencing target absorption at 430 nm. Cytolytic events were visualized by fluorescence microscopy. Cytosolic or membrane compartments of sheep erythrocytes were labeled with eosin Y or fluorescein isothiocyanate, respectively. Time-dependent studies of erythrolysis show that targets are lysed extra- and intra-cellularly. The fluorescent diffusion gradient generated at the site of membrane rupture suggests that a pore of approximately 30 nm in diameter is formed in the target membrane. The site of pore formation is not found at the target-effector cell interface. CGD neutrophils did not display these cytolytic phenomena. Furthermore, the cytosolic label eosin Y could be followed into an associated granule compartment; we suggest that the phenomenon of piranhalysis may participate in antibody-dependent effector mechanisms. Phagocytosis can also be observed using fluorescently-labeled erythrocytes. Determinations of phagocytic index are more reliable with this approach. These microscopical methods are both simple and efficient. To our knowledge, these are the first direct microscopic studies of effector cell-mediated target cell oxidation and cytolysis. These experiments provide a fresh approach to the study of phagocyte effector functions at the cellular level and illuminate the importance of superoxide anions in antibody-dependent erythrolysis.

Influence of immune complexes on macrophage membrane fluidity: a nanosecond fluorescence anisotropy study.

Time-resolved fluorescence anisotropy (TRFA) and steady-state anisotropy measurements and fluorescence intensification microscopic observations were made on RAW264 macrophages labeled with 1,6-diphenyl-1,3,5-hexatriene (DPH) or 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH). Microscopic analysis revealed that the fluorescent probe DPH was found in association with plasma membranes and small vesicles. Macrophages treated with immune complexes could not be distinguished from untreated cells, indicating that the same membrane compartments were labeled. The probe TMA-DPH was exclusively localized to the plasma membrane. Steady-state anisotropy measurements indicated that in vitro culture conditions did not significantly affect membrane fluidity. TRFA measurements were conducted to determine the physical properties of macrophage membranes during immune recognition and endocytosis. Data were analyzed by iterative deconvolution to yield phi, the rotational correlation time, and r infinity, the limiting anisotropy. These parameters may be interpreted as the "fluidity" and order parameter of the membrane environment, respectively. Typical values for untreated macrophages were phi = 7.8 ns and r infinity = 0.12. Binding and endocytosis of immune complexes prepared in 4-fold antigen excess increase these values to phi = 22.1 ns and r infinity = 0.15. However, receptor-independent phagocytosis of latex beads decreases these values to phi = 2.2 ns and r infinity = 0.10. Addition of catalase before, but not after, immune complex incubation with cells diminishes the effect upon membrane structure, suggesting that H2O2 participates in fluidity changes. Pretreatment of macrophages with the membrane-impermeable sulfhydryl blocker p-(chloromercuri)benzenesulfonic acid also diminished these effects.(ABSTRACT TRUNCATED AT 250 WORDS)

A monoclonal antibody (BMA-1) reactive with murine B cells as well as resident and elicited but not activated macrophages.

Hybridoma technology has been employed to prepare a monoclonal antibody that recognizes a subpopulation of mononuclear leukocytes. Enzyme-linked immune assay revealed a cell clone producing a monoclonal antibody reactive with elicited but not activated C57Bi/6 peritoneal macrophages. Detailed analyses using fluorescence flow cytometry demonstrated that this monoclonal antibody binds to B cells, B cell blasts, as well as to the resident and elicited macrophages, but not to activated macrophages, T cells, red blood cells, or syngeneic fibroblasts. This antigen has been designated BMA-1. Antigenic expression is greatest upon resident macrophages. A bimodal level of expression is found on elicited macrophages while activated macrophages possess low levels of expression. The unique cellular distribution of this antigen indicates that it is lost during macrophage differentiation to the activated state. Immunoprecipitation studies indicate that this antigen is composed of multiple subunits; the primary subunit possesses a molecular weight of 38,000. This new tool should be valuable in the analysis of heterogeneous macrophage populations and in defining molecular differentiation pathways.

Novel fluorescence method to visualize antibody-dependent hydrogen peroxide-associated "killing" of liposomes by phagocytes.

We have developed a new methodology to examine effector-cell-mediated immune attack using liposomes as targets. Hydrogen peroxide-associated killing of liposomes was observed with fluorescence intensification microscopy. Liposomes were composed of 98-99 mol % egg phosphatidylcholine and 1-2 mol % dinitrophenyl lipid hapten. Anti-dinitrophenyl IgG antibody was used to opsonize liposomes. Liposomes were loaded with dihydroxymandelic acid (DHMA) and peroxidase. Macrophage- or neutrophil-mediated recognition of liposomes triggers the release of H2O2 and other oxidative products. Upon interaction of H2O2 or OH radical with liposome contents, DHMA dimerizes forming a fluorescent derivative. Our studies indicate that individual living neutrophils and macrophages deposit oxidative products in a heterogenous fashion among bound targets.

Novel fluorescence method to visualize antibody-dependent hydrogen peroxide-associated "killing" of liposomes by phagocytes.

We have developed a new methodology to examine effector-cell-mediated immune attack using liposomes as targets. Hydrogen-peroxide-associated killing of liposomes was observed with fluorescence intensification microscopy. Liposomes were composed of 98-99 mol % egg phosphatidylcholine and 1-2 mol % dinitrophenyl lipid hapten. Anti-dinitrophenyl IgG antibody was used to opsonize liposomes. Liposomes were loaded with dihydroxymandelic acid (DHMA) and peroxidase. Macrophage- or neutrophil-mediated recognition of liposomes triggers the release of H2O2 and other oxidative products. Upon interaction of H2O2 or OH radical with liposome contents, DHMA dimerizes forming a fluorescent derivative. Our studies indicate that individual living neutrophils and macrophages deposit oxidative products in a heterogeneous fashion among bound targets.