c-Jun N-terminal kinase (JNK)-dependent internalization and Rab5-dependent endocytic sorting mediate long-distance retrograde neuronal death induced by axonal BDNF-p75 signaling.

During the development of the sympathetic nervous system, signals from tropomyosin-related kinase receptors (Trks) and p75 neurotrophin receptors (p75) compete to regulate survival and connectivity. During this process, nerve growth factor (NGF)- TrkA signaling in axons communicates NGF-mediated trophic responses in signaling endosomes. Whether axonal p75 signaling contributes to neuronal death and how signaling endosomes contribute to p75 signaling has not been established. Using compartmentalized sympathetic neuronal cultures (CSCGs) as a model, we observed that the addition of BDNF to axons increased the transport of p75 and induced death of sympathetic neurons in a dynein-dependent manner. In cell bodies, internalization of p75 required the activity of JNK, a downstream kinase mediating p75 death signaling in neurons. Additionally, the activity of Rab5, the key GTPase regulating early endosomes, was required for p75 death signaling. In axons, JNK and Rab5 were required for retrograde transport and death signaling mediated by axonal BDNF-p75 in CSCGs. JNK was also required for the proper axonal transport of p75-positive endosomes. Thus, our findings provide evidence that the activation of JNK by p75 in cell bodies and axons is required for internalization to a Rab5-positive signaling endosome and the further propagation of p75-dependent neuronal death signals.

Investigation of the peak action wavelength of light-activated gene transduction.

Light-activated gene transduction (LAGT) is an approach to localize gene therapy via preactivation of cells with UV light, which facilitates transduction by recombinant adeno-associated virus vectors. Previous studies demonstrated that UVC induces LAGT secondary to pyrimidine dimer formation, whereas UVA induces LAGT secondary to reactive-oxygen species (ROS) generation. However, the empirical UVB boundary of these UV effects is unknown. Thus, we aimed to define the action spectra for UV-induced LAGT independent of DNA damage and determine an optimal wavelength to maximize safety and efficacy. UV at 288, 311 and 320 nm produced significant dose-dependent LAGT effects, of which the maximum (800-fold) was observed with 4 kJ m⁻² at 311 nm. Consistent with its robust cytotoxicity, 288 nm produced significantly high levels of DNA damage at all doses tested, whereas 311, 320 and 330 nm did not generate pyrimidine dimers and produced low levels of DNA damage detected by comet assay. Although 288 nm failed to induce ROS, the other wavelengths were effective, with the maximum (10-fold) effect observed with 30 kJ m⁻² at 311 nm. An in vivo pilot study assessing 311 nm-induced LAGT of rabbit articular chondrocytes demonstrated a significant 6.6-fold (P<0.05) increase in transduction with insignificant cytotoxicity. In conclusion, 311 nm was found to be the optimal wavelength for LAGT on the basis of its superior efficacy at the peak dose and its broad safety range that is remarkably wider than the other UV wavelengths tested.

ADF/Cofilin-actin rods in neurodegenerative diseases.

Dephosphorylation (activation) of cofilin, an actin binding protein, is stimulated by initiators of neuronal dysfunction and degeneration including oxidative stress, excitotoxic glutamate, ischemia, and soluble forms of beta-amyloid peptide (Abeta). Hyperactive cofilin forms rod-shaped cofilin-saturated actin filament bundles (rods). Other proteins are recruited to rods but are not necessary for rod formation. Neuronal cytoplasmic rods accumulate within neurites where they disrupt synaptic function and are a likely cause of synaptic loss without neuronal loss, as occurs early in dementias. Different rod-inducing stimuli target distinct neuronal populations within the hippocampus. Rods form rapidly, often in tandem arrays, in response to stress. They accumulate phosphorylated tau that immunostains for epitopes present in "striated neuropil threads," characteristic of tau pathology in Alzheimer disease (AD) brain. Thus, rods might aid in further tau modifications or assembly into paired helical filaments, the major component of neurofibrillary tangles (NFTs). Rods can occlude neurites and block vesicle transport. Some rod-inducing treatments cause an increase in secreted Abeta. Thus rods may mediate the loss of synapses, production of excess Abeta, and formation of NFTs, all of the pathological hallmarks of AD. Cofilin-actin rods also form within the nucleus of heat-shocked neurons and are cleared from cells expressing wild type huntingtin protein but not in cells expressing mutant or silenced huntingtin, suggesting a role for nuclear rods in Huntington disease (HD). As an early event in the neurodegenerative cascade, rod formation is an ideal target for therapeutic intervention that might be useful in treatment of many different neurological diseases.

Positive reinforcement training as enrichment for singly housed rhesus macaques (Macaca mulatta).

Positive reinforcement training is one component of behavioural management employed to improve psychological well-being. There has been regulatory promotion to compensate for restricted social housing in part by providing human interaction to singly caged primates, implying an efficacy standard for evaluating human interaction. The effect of positive reinforcement training on the behaviour of 61 singly housed laboratory rhesus macaques (Macaca mulatta) was evaluated at two large primate facilities. Training involved body part presentation and basic control behaviours. Baseline data were compared to two treatment phases presented in varying order across individuals, six minutes per week of positive reinforcement training and six minutes per week of unstructured human interaction. While a MANOVA involving behavioural categories and study conditions across study subjects was significant, univariate ANOVAs found no effect of phase within any behavioural category. Categorising subjects according to rearing, housing facility, or baseline levels of abnormal behaviour did not reveal changes in behaviour with positive reinforcement training or human interaction. This study failed to detect, to any degree, the types of behavioural changes documented in the scientific literature to result from pairing singly housed monkeys. Implementing short durations of positive reinforcement training across large numbers of singly housed animals may not be the most effective manner for incorporating positive reinforcement training in the behavioural management of laboratory macaques. Rather, directing efforts toward individuals with specific behavioural, management, clinical, research or therapeutic needs may represent a more fruitful approach to improving psychological well-being with this technique.

Production and use of replication-deficient adenovirus for transgene expression in neurons.

Adenoviruses infect a wide range of cell types, do not require integration into the host cell genome, and can be produced as replication-deficient viruses capable of expressing transgenes behind any desired promoter. Thus, they are ideal for use in expressing transgenes in the postmitotic neuron. This chapter describes simplifications in the protocols for making recombinant adenoviruses and their use in expressing transgenes in primary neurons of several different types.

Synergistic effect of verotoxin and interferon-alpha on erythropoiesis.

Previous studies have shown that the verotoxin receptor globotriaosyl ceramide is involved in interferon-alpha (IFN-alpha) signaling pathways. The results of the present study indicate that verotoxin used in combination with IFN-alpha had a direct cytotoxic effect on erythrocyte development by targeting nucleated erythrocyte precursors. Toxin treatment alone had no significant effect on erythropoiesis. However, treatment with 100 ng/ml of verotoxin (VT) in combination with 100 U/ml of IFN-alpha was highly cytotoxic (>90%) to erythroid cells in human cord blood cultures by day 14 post-treatment. The lack of effect on other hematopoietic cells, and the relatively modest decrease in the number of erythroid colonies formed (28%) as a result of IFN-alpha/VT treatment, indicate that the cytotoxicity was targeted specifically toward cells committed to the erythrocyte lineage. IFN-alpha treatment alone did not result in cytotoxicity or a significant reduction in the number of erythroid colonies formed. However, IFN-alpha treatment did result in an increase in the surface expression of verotoxin receptors as determined by flow cytometry following labeling of cells with VT-FITC. Abnormalities in erythrocyte morphology and anemia are associated with infection by verotoxin-producing Escherichia coli such as serotype O157:H7. These symptoms are frequently attributed to passage of erythrocytes through partially occluded blood vessels following toxin-induced damage to endothelial cells. The present results document a synergistic cytotoxic effect of IFN-alpha and verotoxin on erythropoiesis, which could have relevance to clinical infection with verotoxin-producing bacteria.

Effect of novel filler particles on the mechanical and wear properties of dental composites.

OBJECTIVES: The purpose of this study was to investigate the method of producing pre-polymerized fused-fiber filler modified composite (PP-FFMC) particles and the effectiveness of incorporating these novel filler particles into dental composites. METHODS: Fused-fiber filler (FFF) blocks were impregnated with composite by two different methods. Three-point flexure tests were utilized to determine which was more effective. In order to assess the effect of the addition of PP-FFMC particles, two Bis-GMA/TEGDMA based conventional composite compositions were utilized as baselines, to which the novel particles were added. Mechanical and wear tests were performed to determine the fracture toughness, biaxial flexure strength, and in vitro wear of the materials. RESULTS: Mechanical testing showed that the addition of PP-FFMC particles decreased the strength and toughness of the conventional composites. Wear tests indicated that addition of the same particles improved the wear behavior of the conventional composites. SEM analysis of the fracture surfaces indicated that the PP-FFMC particles were incorporated without creating porosity, and that fracture was transgranular through the reinforcing particles. Microscopic flaws observed in the novel particles are the likely explanation for the observed strength and toughness values. SIGNIFICANCE: The results indicate that PP-FFMC particles have the potential to improve the wear properties of dental composites, however, they adversely affect the fracture behavior. Existing processing techniques for these particles, which introduce imperfections, limit their current usefulness.

Effects of high copper supplements on performance, health, plasma copper and enzymes in goats.

Six growing female Nubian goats (average BW=34.8+/-0.55kg, 7-8 months of age) were randomly assigned to either a basal diet (BD, 10-15ppm Cu/DM), or to medium Cu (MC, BD+50mgCu), or to high Cu (HC, BD+100mgCu) diets for 9 weeks. This level would cause Cu toxicity in sheep, but none occurred in the goats. Therefore, Cu supplementation was then increased to 150 and 300mg per head per day, for the following 14 weeks; to 300 and 600mg per head per day, for the next 8 weeks; and to 600 and 1200mg per head per day, for an additional 4 weeks, in the MC and HC group, respectively. Body weight and vital signs were recorded and blood samples collected at different time intervals. Hematological parameters, plasma Cu, sorbitol dehydrogenase (SDH), glutamic oxaloacetic transaminase (GOT), and gamma-glutamyl transferase (GGT) were determined. At the termination of the study, tissue Cu concentration in different organs was also determined. During first 23 weeks (<300mgCu per day) of the study there were no apparent signs of Cu toxicity. Cu supplementation at 600mg per head per day in young Nubian does, had no effect on respiration rate (RR), heart rate (HR), and decreased (P<0.05) rectal temperature (RT) in the HC group only. Leukocyte counts were positively correlated with Cu supplementation (r=+0.296, P<0.02) and negatively correlated (r=-0.254, P<0.05) with RT in the HC group. Plasma SDH increased (P<0.05) when Cu supplementation was >/=300mg per head per day, thus, SDH may serve as an early indicator of Cu toxicosis in goats. Increases (P<0.05) in GOT were noted when Cu intake was >/=600mg per head per day. Contrary to the results observed for SDH and GOT, feeding goats 50mgCu per day or more, resulted in an increased plasma GGT as compared to BD goats. Levels of SDH, GOT and GGT of the BD goats were within normal range. Plasma Cu was not indicative of Cu status of animals. Copper improved ADG by 28% at the 100-150ppm level in diet. No relationship between Cu intake and hair Cu was found in the present study. Highest concentration of Cu was found in liver, followed by duodenum, rumen and brain. Results of this study indicate that goats are more resistant to Cu toxicity than sheep. This is one of the first reports documenting significant differences in Cu requirements and tolerance between goats and sheep.

Dense inflammation does not mask residual primary basal cell carcinoma during Mohs micrographic surgery.

BACKGROUND: Areas of dense inflammation are commonly removed during Mohs micrographic surgery for basal cell carcinoma because of the concern that they may mask areas of tumor. OBJECTIVE: Our purpose was to determine whether inflammation masks tumor during Mohs surgery for primary basal cell carcinoma. METHODS: Twenty-five consecutive cases of primary basal cell carcinoma with areas of dense inflammation encountered during Mohs surgery were sectioned and stained with hematoxylin and eosin and Ber-EP4. RESULTS: In no cases did the dense inflammation mask residual tumor. CONCLUSION: Dense inflammation does not mask primary basal cell carcinoma during Mohs surgery and should be carefully evaluated before additional surgery is performed.

Equipotent generation of protective antitumor immunity by various methods of dendritic cell loading with whole cell tumor antigens.

Multiple clinically applicable methods have been used to induce dendritic cells (DCs) to express whole cell tumor antigens, including pulsing DCs with tumor lysate, and mixing DCs with apoptotic or live tumor cells. Herein we demonstrate, using two different tumor systems, that these methods are equipotent inducers of systemic antitumor immunity. Furthermore, tumor lysate pulsed DC vaccines generate more potent antitumor immunity than immunization with irradiated tumor cells plus the classic adjuvant, Corynebacterium parvum.

Acrochordons as a presenting sign of nevoid basal cell carcinoma syndrome.

BACKGROUND: Nevoid basal cell carcinoma syndrome (NBCCS) is a genodermatosis with autosomal dominant inheritance. In identified kindreds the diagnosis is relatively easy, but for the patients without family history of this syndrome a high clinical suspicion is necessary for diagnosis. OBJECTIVE: Acrochordons are distinctly uncommon in childhood. Our purpose was to evaluate skin tags that develop at an early age. METHODS: This is a retrospective series evaluation of 7 children who presented with pedunculated papules (acrochordon-like growths). A full history was then correlated with biopsy results in each patient. RESULTS: Clinically, lesions consisted of flesh-colored and pigmented pedunculated papules. Histopathologic examination of these papules showed basal cell carcinomas in each biopsy specimen. CONCLUSION: We consider that "skin tag"-like basal cell carcinomas in childhood may represent a marker for NBCCS. Early diagnosis of this syndrome and early sun protection of the affected children could help decrease the number of lifetime tumors. Biopsy should be performed on acrochordons in children because they may be the presenting sign of NBCCS. Because these tags may precede other stigmata of the NBCCS, recognition may facilitate early diagnosis and allow early treatment and sun protection.

Indirect recognition of allopeptides promotes the development of cardiac allograft vasculopathy.

Graft loss from chronic rejection has become the major obstacle to the long-term success of whole organ transplantation. In cardiac allografts, chronic rejection is manifested as a diffuse and accelerated form of arteriosclerosis, termed cardiac allograft vasculopathy. It has been suggested that T-cell recognition of processed alloantigens (allopeptides) presented by recipient antigen-presenting cells through the indirect pathway of allorecognition plays a critical role in the development and progression of chronic rejection. However, definitive preclinical evidence to support this hypothesis is lacking. To examine the role of indirect allorecognition in a clinically relevant large animal model of cardiac allograft vasculopathy, we immunized MHC inbred miniature swine with synthetic polymorphic peptides spanning the alpha(1) domain of an allogeneic donor-derived swine leukocyte antigen class I gene. Pigs immunized with swine leukocyte antigen class I allopeptides showed in vitro proliferative responses and in vivo delayed-type hypersensitivity responses to the allogeneic peptides. Donor MHC class I disparate hearts transplanted into peptide-immunized cyclosporine-treated pigs not only rejected faster than unimmunized cyclosporine-treated controls (mean survival time = 5.5 +/-1.7 vs. 54.7 +/-3.8 days, P < 0.001), but they also developed obstructive fibroproliferative coronary artery lesions much earlier than unimmunized controls (<9 vs. >30 days). These results definitively link indirect allorecognition and cardiac allograft vasculopathy.

Intranodal immunization with tumor lysate-pulsed dendritic cells enhances protective antitumor immunity.

We developed a technique for direct inguinal lymph node injection in mice to compare various routes of immunization with tumor lysate-pulsed dendritic cell (DC) vaccines. Syngeneic, bone marrow-derived, tumor lysate-pulsed DCs administered intranodally generated more potent protective antitumor immunity than s.c. or i.v. DC immunizations. Intranodal immunization with ovalbumin peptide-pulsed DCs induced significantly greater antigen-specific T-lymphocyte expansion in the spleen than either s.c. or i.v. immunization. Furthermore, a significantly more potent, antigen-specific TH1-type response to the ovalbumin peptide was induced by intranodal, compared with s.c. or i.v., immunization. Intranodal immunization, designed to enhance DC-T cell interaction in a lymphoid environment, optimizes induction of T lymphocyte-mediated protective antitumor immunity. These results support the use of intranodal immunization as a feasible and effective route of DC vaccine administration.