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.

Glutamate-pyruvate transaminase protects against glutamate toxicity in hippocampal slices.

Elimination of glutamate through enzymatic degradation is an alternative to glutamate receptor blockade in preventing excitotoxic neuronal injury. Glutamate pyruvate transaminase (GPT) is a highly active glutamate degrading enzyme that requires pyruvate as a co-substrate. This study examined the ability of GPT to protect neurons of the hippocampal slice preparation against glutamate toxicity. Two methods were used to elevate the concentration of glutamate in the peri-neuronal space. In an endogenous release paradigm, slices were incubated with 100-500 microM L-trans-pyrrolidine-2,4-dicarboxylate (PDC), an inhibitor of glutamate re-uptake. One hour of exposure to PDC in normal, pyruvate-free slice maintenance medium caused a dose dependent increase in neuronal death assessed 24 h later by propidium iodide uptake in dead cell nuclei. GPT (10 U/ml) decreased neuronal death caused by exposure to PDC at all PDC concentrations tested. Neuroprotection in this model was not dependent on added or non-physiologic levels of pyruvate. In a different paradigm, glutamate was added directly to the normal, pyruvate-free slice maintenance medium and not rinsed away, exposing the slices to a range of 1-5 mM glutamate for an extended period. Twenty-four hours later, neuronal death was again assessed by propidium iodide uptake. GPT was again neuroprotective, decreasing neuronal death in the range from 3 to 5 mM glutamate. In the setting of incubation with this large load of glutamate, neuroprotection by GPT was enhanced by adding pyruvate to the medium. GPT is an effective neuroprotectant against glutamate excitotoxicity. When exposure is limited to endogenously released glutamate, neuroprotection by GPT is not dependent on added pyruvate.

Interaction of tetanus toxin derived hybrid proteins with neuronal cells.

The non-toxic ganglioside binding domain of tetanus toxin (Hc fragment C or TTC) has been studied as a vector for delivering therapeutic proteins to neurons. There is little information on the cellular processing of proteins delivered by linkage to TTC. We have evaluated the cellular handling of a multi-domain hybrid protein containing TTC and both the human enzyme superoxide dismutase and the maltose binding protein from E. coli. Binding, internalization, and cleavage of this protein during prolonged incubation with fetal cortical neurons or cells of the N18-RE-105 line was evaluated by immunoblot analysis, ELISA, and immunocytochemistry. Hybrid proteins were bound and internalized in a manner very similar to TTC. Internalized proteins showed long-term stability within cells, and were degraded into predictable large protein fragments in both cell types. Fragments that were cleaved away from the TTC domain were released into extracellular fluid after internalization. Proteins coupled to TTC share its long-term stability after cellular internalization. After internalization, dissociation of proteins linked to TTC facilitates their release from the cell, but not into other cellular compartments such as the cytosol. TTC linked proteins are probably enclosed within a stable endosomal compartment throughout their cellular lifetime.

Protective effect of supplemental superoxide dismutase on survival of neuronal cells during starvation. Requirement for cytosolic distribution.

There is evidence that raising cellular levels of Cu2+/Zn2+ superoxide dismutase (SOD1) can protect neurons from oxidative injury. We compared a novel method of elevating neuronal SOD activity using a recombinant hybrid protein composed of the atoxic neuronal binding domain of tetanus toxin (C fragment or TTC) and human SOD1 (hSOD1) with increasing cellular SOD levels through overexpression. Fetal murine cortical neurons or N18-RE-105 cells were incubated with the TTC-hSOD1 hybrid protein and compared to cells constitutively expressing hSOD1 for level of SOD activity, cellular localization of hSOD1, and capacity to survive glucose and pyruvate starvation. Cells incubated with TTC-hSOD1 showed a threefold increase in cellular SOD activity over control cells. This level of increase was comparable to fetal cortical neurons from transgenic mice constitutively expressing hSOD1 and transfected N18-RE-105 cells expressing a green fluorescent protein-hSOD1 fusion protein (GFP-hSOD1). Human SOD1 was distributed diffusely throughout the cytoplasm of the transgenic murine neurons and transfected N18-RE-105 cells. In contrast, cells incubated with TTC-hSOD1 showed hSOD1 localized to the cell surface and intra-cytoplasmic vesicles. The cells expressing hSOD1 showed enhanced survival in glucose- and pyruvate-free medium. Neither cortical neurons nor N18-RE-105 cells incubated in TTC-hSOD1 showed increased survival during starvation. Access to the site where toxic superoxides are generated or their targets may be necessary for the protective function of SOD1.

Enzymatic degradation protects neurons from glutamate excitotoxicity.

Several enzymes with the capacity to degrade glutamate have been suggested as possible neuroprotectants. We initially evaluated the kinetic properties of glutamate pyruvate transaminase (GPT; also known as alanine aminotransferase), glutamine synthetase, and glutamate dehydrogenase under physiologic conditions to degrade neurotoxic concentrations of glutamate. Although all three enzymes initially degraded glutamate rapidly, only GPT was able to reduce toxic (500 microM) levels of glutamate into the physiologic (<20 microM) range. Primary cultures of fetal murine cortical neurons were subjected to paradigms of either exogenous or endogenous glutamate toxicity to evaluate the neuroprotective value of GPT. Neuronal survival after exposure to added glutamate ranging from 100 to 500 microM was improved significantly in the presence of GPT (> or =1 U/ml). Cultures were also exposed to the glutamate transporter inhibitor L-trans-pyrrolidine-2,4-dicarboxylate (PDC), which produces neuronal injury by elevating extracellular glutamate. GPT significantly reduced the toxicity of PDC. This reduction was associated with a reduction in the PDC-dependent rise in the medium concentration of glutamate. These results suggest that enzymatic degradation of glutamate by GPT can be an alternative to glutamate receptor blockade as a strategy to protect neurons from excitotoxic injury.

Micronutrients and host resistance to viral infection.

Previous work in our laboratory demonstrated that a virus could undergo rapid mutation in a host deficient in Se, leading to a normally avirulent virus acquiring virulence due to genome changes. Once these mutations occur, even a host with adequate Se-nutriture is susceptible to the newly virulent virus. What influence does the deficiency in Se have on the immune response of the host? Infection with myocarditic strains of coxsackievirus induces an inflammatory response in the cardiac tissue. It is this immune response that induces the heart damage, rather than direct viral effects on the heart tissue. Chemokines are chemo-attractant molecules that are secreted during an infection in order to attract immune cells to the site of the injury, and have been found to be important for the development of coxsackievirus-induced myocarditis. We found that a deficiency in Se influences the expression of mRNA for the chemokine monocyte chemo-attractant protein-1, which may have implications for the development of myocarditis in the Se-deficient host. Expression of mRNA for interferon-gamma was also greatly decreased in the Se-deficient animal. Thus, a deficiency in Se can have profound effects on the host as well as on the virus itself. How the alteration of the immune response of the Se-deficient animal affects the development of the virulent genotype remains to be answered.

Neuronal binding of tetanus toxin compared to its ganglioside binding fragment (H(c)).

The non-toxin 50 kD C-terminus peptide of the heavy chain of tetanus H(c) contains the ganglioside binding domain of tetanus toxin (TTX). H(c) retains much of the capacity of tetanus toxin for binding internalization and transport by neurons. For this reason tetanus H(c) has been studied as a vector for delivery of therapeutic proteins to neurons. We directly compared H(c) and TTX in the capacity to bind and be internalized by neurons by ELISA. Primary cultures of dissociated fetal cortical neurons were incubated with equimolar amounts of TTX or H(c). Neuronal associated tetanus protein was 4-8 fold greater on a molar basis with tetanus toxin compared to H(c) (1 h incubation). This increase in neuronal tetanus protein was evident with incubation in concentrations from 0.1 microM to 2 microM. There were greater amounts of TTX delivered to the cultured cells at both 0 degrees C (representing membrane bound tetanus protein) and 37 degrees C (bound and internalized tetanus protein). Unlike H(c), TTX showed significant continued accumulation of protein with increasing incubation durations. Neuronal associated TTX increased 2-3 fold over incubation times ranging from 1 to 8 h. Tetanus toxin appears to be clearly superior to the ganglioside binding fragment (H(c)) in the capacity for neuronal binding and internalization. Atoxic tetanus proteins containing additional molecular domains as well as H(c) may be more suitable vectors for linkage with therapeutic proteins and delivery to neurons.

Nerve-dependent factors regulating transcript levels of glycogen phosphorylase in skeletal muscle.

1. Muscle glycogen phosphorylase (MGP), the rate-limiting enzyme for glycogen metabolism in skeletal muscle, is neurally regulated. Steady-state transcript levels of the skeletal muscle isozyme of MGP decrease significantly following muscle denervation and after prolonged muscle inactivity with an intact motor nerve. These data suggest that muscle activity has an important influence on MGP gene expression. The evidence to this point, however, does not preclude the possibility that MGP is also regulated by motor neuron-derived trophic factors. This study attempts to distinguish between regulation provided by nerve-evoked muscle contractile activity and that provided by the delivery of neurotrophic factors. 2. Steady-state MGP transcript levels were determined in rat tibialis anterior (TA) muscles following controlled interventions aimed at separating the contributions of contractile activity from axonally transported trophic factors. The innervated TA was rendered inactive by daily epineural injections of tetrodotoxin (TTX) into the sciatic nerve. Sustained inhibition of axonal transport was accomplished by applying one of three different concentrations of the antimicrotubule agent, vinblastine (VIN), to the proximal sciatic nerve for 1 hr. The axonal transport of acetylcholinesterase (AChE) was assessed 7, 14, and 28 days after the single application of VIN. 3. MGP transcript levels normalized to total RNA were reduced by 67% in rat TA, 7 days after nerve section. Daily injection of 2 microg TTX into the sciatic nerve for 7 days eliminated muscle contractile activity and reduced MGP transcript levels by 60%. 4. A single, 1-hr application of 0.10% (w/v) VIN to the sciatic nerve reduced axonal transport but did not alter MGP transcript levels in the associated TA, 7 days after treatment. Application of 0.10% VIN to the sciatic nerve also did not affect IA sensory or motor nerve conduction velocities or TA contractile function. 5. Treatment of the sciatic nerve with 0.40% (w/v) VIN for 1 hr reduced axonal transport and decreased MGP transcript levels by 50% within 7 days, but also reduced sensory and motor nerve conduction velocities and depressed TA contractile function. 6. Myogenin, a member of a family of regulatory factors shown to influence the transcription of many muscle genes, including MGP, was used as a molecular marker for muscle inactivity. Myogenin transcript levels were increased following denervation and after treatment with TTX or 0.40% VIN but not after treatment with 0.10% VIN. 7. The results suggest that MGP transcript levels in TA are regulated predominantly by muscle activity, rather than by the delivery of neurotrophic factors. Intrinsic myogenic factors, however, also play a role in MGP expression, since denervation did not reduce MGP transcript levels below 30% of control TA. The dominant influence of activity in the regulation of MGP contrasts with the proposed regulation of oxidative enzyme expression, which appears to depend on both activity and trophic factor influences.

Insulin-like growth factor-I is an osmoprotectant in human neuroblastoma cells.

A role in neuronal homeostasis is suggested by the persistent expression of the insulin-like growth factors in the adult nervous system. SH-SY5Y human neuroblastoma cells, a well-characterized in vitro model of human neurons, were used to investigate the effects of hyperosmotic stress on neurons. Neuronal DNA fragmentation was detected within 1 h and pyknotic nuclei were apparent in attached cells after 12 h of hyperosmotic stress. In parallel, flow cytometry measurements revealed a sudden increase in the rate of cells irreversibly undergoing programmed cell death after 12 h of hyperosmotic exposure. Insulin-like growth factor-I delayed the onset of a laddered DNA fragmentation pattern for 24 h and provided continuing protection against hyperosmotic exposure for 72 h. Amino acid uptake was decreased in hyperosmotic medium even in the presence of insulin-like growth factor-I; the protein synthesis inhibitor cycloheximide neither prevented the induction of programmed cell death nor interfered with the ability of insulin-like growth factor-I to act as an osmoprotectant in hyperosmotic medium. Cysteine and serine protease inhibitors each prevented DNA fragmentation under hyperosmotic conditions, suggesting that the osmoprotectant activity of insulin-like growth factor-I involves the suppression of protease activity. Collectively, these results indicate that insulin-like growth factor-I limits the death of neurons under stressful environmental conditions, suggesting that it may provide a candidate therapy in the treatment of hyperosmolar coupled neurological injury.

Insulin-like growth factor I rescues SH-SY5Y human neuroblastoma cells from hyperosmotic induced programmed cell death.

Insulin-like growth factor I (IGF-I) and the type I IGF receptor are widely distributed in developing and adult mammalian nervous systems. In vitro, IGF-I is a mitogen for primary neurons and also for cells from the SH-SY5Y human neuroblastoma cell line, a well-characterized model system of neuronal growth. In the current study, we examined the effects of osmotic stress on SH-SY5Y cell viability and the mechanism by which IGF-I serves as a neuronal osmoprotectant. Within 24 hr, exposure of SH-SY5Y cells to hyperosmotic serum-free media decreased (1) the number of viable cells, (2) the rate of 3H-thymidine incorporation, and (3) cell cycle progression. The inclusion of 10 nM IGF-I with hyperosmotic media prevented the loss of cell viability. The osmoprotective effects of IGF-I were inhibited by alpha-IR3, a blocking antibody of the type I IGF receptor. The observed loss of SH-SY5Y cell viability following hyperosmotic shock was due to an induction of programmed cell death as determined by flow cytometry and gel electrophoresis. Our results suggest that IGF-I can protect SH-SY5Y cells from hyperosmotic induced programmed cell death.

Portal vein air embolization after blunt abdominal trauma.

Gas in the portal veins is a rare, and usually fatal, condition. This case report describes a patient with air embolization of the portal veins secondary to blunt trauma. The condition was clinically benign and resolved spontaneously. Computed tomography documented the findings.

Recovery of free muscle grafts in rat: improvement is associated with an increase in cyclic adenosine monophosphate concentration or use of the condition/test paradigm.

The muscle fibers in freely grafted skeletal muscles degenerate and are replaced by new fibers which develop within the graft. Myogenesis in regenerating muscle recapitulates, to a large extent, developmental myogenesis and may depend on similar modulating influences. In addition to the generation of new fibers, functional recovery of free muscle grafts also requires reinnervation and revascularization of the new fibers. Recovery of function should be improved by enhancing either myogenesis or reinnervation and revascularization. We have used two procedures, shown previously to stimulate peripheral nerve regeneration, to improve the morphologic and functional recovery of free, orthotopic grafts of rat extensor digitorum longus muscle. Each of the procedures was effective, but had potentially different sites of action. The first procedure, the condition/test paradigm, presumably increases the rate and extent of graft reinnervation. The second procedure, continuous infusion of the adenylate cyclase activator forskolin during the first 21 days after grafting, may influence both myogenesis and nerve regeneration. Each procedure increased regenerating muscle fiber size and functional capacity, and forskolin also significantly increased capillary density and fatigue resistance.

Digital imaging of the chest.

The development of effective methods for producing digital chest radiographs is essential if the totally digital radiology department of the future is to evolve. Digital radiography of the chest is now possible with a variety of techniques. The most promising approach currently in clinical use involves the use of large-area detectors composed of photostimulable phosphors read with a laser to produce high-quality digital chest images. Image processing to modify contrast and latitude of the image display as well as enhancement of selected spatial frequencies and energy subtraction is possible with digital chest radiography. The wide exposure latitude of digital image receptors may allow useful images to be obtained with significantly smaller exposure doses than those required in conventional techniques and may also be used to compensate for inadvertent overexposure, eliminating the need for repeat examinations because of errors in exposure technique. Limitations of many current systems result from inadequate resolution provided by CRT display monitors. The production of hard copy images by laser printer on small format film overcomes this problem, allowing application of digital techniques for chest radiology to be incorporated into clinical practice with images comparable in spatial resolution to those of film-screen systems.

Incidental lesions discovered on intravenous digital subtraction angiography.

Recognition of the clinically significant incidental lesion has always been a challenge to the diagnostic radiologist. We present a series of such lesions discovered in patients undergoing intravenous digital subtraction angiography (DSA). The increasingly widespread use of DSA and the subsequent large numbers of patients involved will no doubt result in a substantial number of cases of this type.

Computerized tomography in acute and chronic pancreatitis.

Modern imaging techniques have revolutionized the diagnostic evaluation of pancreatitis, primarily demonstrating its complications. Computerized tomography (CT) is a more sensitive method than ultrasonography and pancreatic ductography. A chart review revealed 214 patients at our hospital with a discharge diagnosis of pancreatitis. Sixty patients had CT for evaluation of possible complications. Only five scans were normal. Of 37 cases of acute pancreatitis, 92% demonstrated localized or diffuse enlargement, and 65% showed loss of pancreatic outline. Other frequent findings included thickening of perirenal fascia (49%), ileus (43%), edema of mesentery (35%), and inflammatory exudate (32%). Abscess and pseudocyst were each detected in 8% of cases. In chronic pancreatitis 65% of patients showed localized or diffuse pancreatic enlargement. Atrophy of the gland (30%), calcification (30%), pseudocyst (26%), and dilated pancreatic ducts (17%) were also seen. CT is effective in evaluating pancreatitis and its complications.

CT demonstration of cystadenocarcinoma of the pancreas with calcified lymphadenopathy.

We believe our patient's pancreatic lesion represents a primary macrocystic adenocarcinoma with calcified retroperitoneal lymphadenopathy. CT examination was useful in characterizing the primary tumor and evaluating the extent of disease. The presence of calcified metastatic lesions was an additional key point in the differential diagnosis.

Modification of pulmonary vascular responses to arachidonic acid by alterations in physiologic state.

The effects of bolus injections of arachidonic acid and prostaglandins (PG) E2 and F2a on the pulmonary vascular bed were compared under resting conditions and after alteration in the physiologic state of the lung. Studies were carried out in the vascularly isolated lung lobe of the intact, anesthetized dog under conditions of controlled blood flow. Arachidonic acid, PGE2 and PGF2a increased pulmonary vascular resistance by constricting intrapulmonary veins and arteries in a dose-related manner, as did an analog of the endoperoxide, PGH2, whereas PGI2 dilated the pulmonary vascular bed. The response to arachidonate was associated with a 2- to 3-fold increase in levels of PGE- and PGF-like substances in pulmonary venous blood and was blocked by indomethacin. The effects of arachidonic acid, but not the PGs, were greatly enhanced during perfusion with either dextran or saline and the enhanced response in saline was associated with a 15 to 20-fold increase in levels of PG-like substances in the pulmonary effluent. Responses to arachidonate were not dependent upon the presence of formed elements in blood but were related to perfusate protein concentration. Alveolar hypoxia decreased responses to the precursor while those to PGE2 and PGF2a were enhanced. Responses to the PGs, but not those to arachidonate, were affected by changes in blood pH. Sublethal doses of Escherichic coli endotoxin increased the response to arachidonic acid, but not those to PGE2 and PGF2a. Results of the present study indicate that the effects of bolus injection of arachidonic acid on the pulmonary vascular bed are due mainly to formation of constrictor metabolites which may overshadow the actions of any dilator (PGI2) formed and suggest that metabolism of the precursor is altered by changes in physiologic state.