Fibrin matrices with affinity‐based delivery systems and neurotrophic factors promote functional nerve regeneration

Glial‐derived neurotrophic factor (GDNF) and nerve growth factor (NGF) have both been shown to enhance peripheral nerve regeneration following injury and target different neuronal populations. The delivery of either growth factor at the site of injury may, therefore, result in quantitative differences in motor nerve regeneration and functional recovery. In this study we evaluated the effect of affinity‐based delivery of GDNF or NGF from fibrin‐filled nerve guidance conduits (NGCs) on motor nerve regeneration and functional recovery in a 13 mm rat sciatic nerve defect. Seven experimental groups were evaluated consisting of GDNF or NGF and the affinity‐based delivery system (DS) within NGCs, control groups excluding the DS and/or growth factor, and nerve isografts. Groups with growth factor in the conduit demonstrated equivalent or superior performance in behavioral tests and relative muscle mass measurements compared to isografts at 12 weeks. Additionally, groups with GDNF demonstrated greater specific twitch and tetanic force production in extensor digitorum longus (EDL) muscle than the isograft control, while groups with NGF produced demonstrated similar force production compared to the isograft control. Assessment of motor axon regeneration by retrograde labeling further revealed that the number of ventral horn neurons regenerating across NGCs containing GDNF and NGF DS was similar to the isograft group and these counts were greater than the groups without growth factor. Overall, the GDNF DS group demonstrated superior functional recovery and equivalent motor nerve regeneration compared to the isograft control, suggesting it has potential as a treatment for motor nerve injury. Biotechnol. Bioeng. 2010;106: 970–979. © 2010 Wiley Periodicals, Inc.     

Revascularization of peripheral nerve autografts and allografts

The timing and mechanisms of peripheral nerve revascularization were investigated using a 2-cm sciatic nerve graft model in 58 rats. Epineurial perfusion was consistently established by 48 hours and endoneurial perfusion by 72 hours. The pattern of endoneurial perfusion was "all-or-none"--either all or none of the vessels in a fascicle exhibited blood flow. Conventional allografts exhibited similar revascularization dynamics and patterns. Capping the ends of the autograft with Silastic significantly delayed revascularization; no flow was observed at 4 days, and only a peripheral rim of perfused fascicular vessels was observed at 7 days. These patterns suggested that the primary method of revascularization in the conventional graft was longitudinal inosculation; no evidence of peripheral neovascularization or dependence on the graft bed as a source of revascularization was observed. The introduction of a major histocompatibility complex barrier between the grafted tissue and the recipient animal did not alter the timing or the mechanics of blood flow reestablishment.     

Dose-dependent effects of FK506 on neuroregeneration in a rat model

This study explored the effects of different doses of FK506 on peripheral nerve regeneration, to determine whether neuroregeneration could be enhanced without the toxicity of systemic immunosuppression. In the first part of the study, subimmunosuppressive doses of FK506 were determined by examining skin allograft survival in a rat model. Full-thickness skin grafts (2 cm2) from Wistar rats were grafted to recipient Lewis rats. The procedure was performed for six groups (n = 6). The control group received no FK506, and the other five groups received daily doses of FK506 of 0.125, 0.25, 0.5, 1.0, or 2.0 mg/kg. Animals that received 2.0 mg/kg FK506 per day exhibited complete skin graft take, whereas all other groups demonstrated complete rejection. After determination of the immunosuppressive dose of FK506, the neuroregenerative effects of different doses of FK506 were explored by assessing nerve regeneration in 80 rats after tibial nerve transection and repair. The control group received no FK506, whereas the other four groups were given daily doses of FK506 of 0.25, 0.5, 1.0, or 2.0 mg/kg. Rats were euthanized at three time points (25, 30, and 35 days), to fully investigate the effects of different FK506 dosing regimens on neuroregeneration. Histomorphometric analyses performed on postoperative days 30 and 35 demonstrated statistically significant improvements in neuroregeneration with subimmunosuppressive FK506 doses of 0.5 and 1.0 mg/kg per day. Therefore, the study demonstrated that neuroregeneration was enhanced at low doses of FK506 that were not sufficient to prevent skin allograft rejection.     

Identification of a gene (lpt-3) required for the addition of phosphoethanolamine to the lipopolysaccharide inner core of Neisseria meningitidis and its role in mediating susceptibility to bactericidal killing and opsonophagocytosis

We have identified a gene, lpt-3, that is required for the addition of phosphoethanolamine to the 3-position (PEtn-3) on the beta-chain heptose (HepII) of the inner core lipopolysaccharide (LPS) of Neisseria meningitidis (Nm). The presence of this PEtn-3 substituent is characteristic of the LPS of a majority ( approximately 70%) of hypervirulent Nm strains, irrespective of capsular serogroup, and is required for the binding of a previously described monoclonal antibody (mAb B5) to a surface-accessible epitope. All strains of Nm that have PEtn-3 possess the lpt-3 gene. In some lpt-3-containing strains, the 3-position on HepII is preferentially substituted by glucose instead of PEtn, the result of lgtG phase variation mediated by slippage of a homopolymeric tract of cytidines. Inactivation of lpt-3 resulted in loss of PEtn-3, lack of reactivity with mAb B5 and conferred relative resistance to bactericidal killing and opsonophagocytosis by mAb B5 in vitro. Thus, the identification of lpt-3 has facilitated rigorous genetic, structural and immunobiological definition of an immunodominant epitope that is a candidate immunogen for inclusion in an LPS-based vaccine to protect against invasive meningococcal disease.     

Cross-site differences in foraging behavior of white-faced capuchins (Cebus capucinus)

Researchers have identified a variety of cross-site differences in the foraging behavior of free-ranging great apes, most notably among chimpanzees (Pan troglodytes) and more recently orangutans (Pongo pygmaeus), that are not due to obvious genetic or ecological differences. These differences are often referred to as "traditions." What is not known is whether this high level of interpopulation variation in behavior is limited to hominoids. In this study, we use long-term data from three Costa Rican field sites that are geographically close and similar ecologically to identify potential foraging traditions in white-faced capuchins (Cebus capucinus). Foraging traditions are predicted in Cebus because of many behavioral and morphological convergences between this genus and the great apes. The processing techniques used for the same food species were compared across sites, and all differences found were classified as present, habitual, or customary. Proximity data were also analyzed to determine if social learning mechanisms could explain variation in foraging behavior. Of the 61 foods compared, we found that 20 of them are processed differently by capuchins across sites. The differences involve pound, rub, tap, "fulcrum," "leaf-wrap," and "army ant following." For most of the differences with enough data to analyze, the average proximity score of the "matched" dyads (two individuals within a group who shared a "different" processing technique) was statistically higher than the average proximity score of the remaining "unmatched" dyads.     

No association between the serotonin transporter-linked promoter region polymorphism and either schizophrenia or density of the serotonin transporter in human hippocampus.

BACKGROUND: Allelic association case-control analysis of a deletion/insertion polymorphism in the serotonin transporter-linked polymorphic region (5-HTTLPR) has suggested associations with unipolar disorder, bipolar disorder, depression, and Alzheimer's disease. Moreover, the heterozygous long form of the 5-HTTLPR has been associated with increased levels of mRNA for the serotonin transporter (5-HTT) and increased serotonin uptake in lymphoblastic cell lines. This study attempts to determine whether there is an association between 5-HTTLPR genotype and schizophrenia or the binding of [3H]paroxetine to the human hippocampal 5-HTT. MATERIALS AND METHODS: DNA from the cerebellum from 58 schizophrenic and 62 control subjects was used to genotype the 5-HTTLPR. In addition, the relationship between 5-HTTLPR genotype and the affinity and density of [3H]paroxetine binding to the hippocampal 5-HTT was assessed. RESULTS: There were no associations between 5-HTTLPR allotype or genotype and/or the parameters of [3H]paroxetine binding to the hippocampal 5-HTT. CONCLUSIONS: Our data suggest that 5-HTTLPR genotype neither confers an increased susceptibility for schizophrenia nor dictates the expression of the 5-HTT in the human hippocampus.     

VIRULENCE OF MIXED-CLONE AND SINGLE-CLONE INFECTIONS OF THE RODENT MALARIA PLASMODIUM CHABAUDI

Most evolutionary models treat virulence as an unavoidable consequence of microparasite replication and have predicted that in mixed-genotype infections, natural selection should favor higher levels of virulence than is optimal in genetically uniform infections. Increased virulence may evolve as a genetically fixed strategy, appropriate for the frequency of mixed infections in the population, or may occur as a conditional response to mixed infection, that is, a facultative strategy. Here we test whether facultative alterations in replication rates in the presence of competing genotypes occur and generate greater virulence. An important alternative, not currently incorporated in models of the evolution of virulence, is that host responses mounted against genetically diverse parasites may be more costly or less effective than those against genetically uniform parasites. If so, mixed clone infections will be more virulent for a given parasite replication rate. Two groups of mice were infected with one of two clones of Plasmodium chabaudi parasites, and three groups of mice were infected with 1:9, 5:5, or 9:1 mixtures of the same two clones. Virulence was assessed by monitoring mouse body weight and red blood cell density. Transmission stage densities were significantly higher in mixed- than in single-clone infections. Within treatment groups, transmission stage production increased with the virulence of the infection, a phenotypic correlation consistent with the genetic correlation assumed by much of the theoretical work on the evolution of virulence. Consistent with theoretical predictions of facultative alterations in virulence, we found that mice infected with both parasite clones lost more weight and had on average lower blood counts than those infected with single-clone infections. However, there was no consistent evidence of the mechanism invoked by evolutionary models that predict this effect. Replication rates and parasite densities were not always higher in ???mixed-clone infections, and for a given replication rate or parasite density, mixed-clone infections were still more virulent. Instead, prolonged anemia and increased transmission may have occured because genetically diverse infections are less rapidly cleared by hosts. Differences in maximum weight loss occured even when there were comparable parasite densities in mixed- and single-clone infections. We suggest that mounting an immune response against more that one parasite genotype is more costly for hosts, which therefore suffer higher virulence.     

Induction of sister chromatid exchanges and chromosome aberrations in cho cells arrested in the cell cycle by arginine deprivation

Summary Sister chromatid exchanges (SCE) and chromosome aberrations were induced in nondividing CHO cells that had been arrested in their cell cycle by deprivation of the essential amino acid arginine. Cells arrested in arginine-deficient medium (ADM) were treated with one of the mutagenic agents N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) or mitomycin C (MMC) and refed with complete medium; the recovering cell population was sampled at various intervals thereafter and mitotic cells analyzed for the presence of chromosome aberrations and SCE. Both chemicals were observed to cause delays in the cell cycle of recovering cells and to induce, chromosome aberrations and SCE at low doses. We have described the variation in the incidence of chromosome aberrations and SCE with respect to sampling time and the number of cell cycles traversed. When ADM-arrested CHO cells were treated with three mutagens at various intervals either before or after release from ADM, it was observed that: (a) UV light induced the greatest number of SCE when applied to cells undergoing DNA synthesis, and SCE yeilds induced by this agent could be reduced by postirradiation incubation in ADM; (b) MNNG induced fewer SCE when applied to cells undergoing DNA synthesis, and SCE yields induced by this agent could not be reduced by posttreatment incubation in ADM for 24 hr. (c) MMC induced the same level irrespective of the time of exposure, and SCE yields induced by this agent could not be reduced by posttreatment incubation in ADM for 24 hr. This work was supported by grants from the British Columbia Foundation for Non-Animal Research (to W. D. M.), and the National Cancer Institute of Canada and the National Research Council of Canada (to H. F. S.). Professor H. F. Stich is a Research Associate of the NCI.     

A modular treatment of molecular traffic through the active site of cholinesterase

We present a model for the molecular traffic of ligands, substrates, and products through the active site of cholinesterases (ChEs). First, we describe a common treatment of the diffusion to a buried active site of cationic and neutral species. We then explain the specificity of ChEs for cationic ligands and substrates by introducing two additional components to this common treatment. The first module is a surface trap for cationic species at the entrance to the active-site gorge that operates through local, short-range electrostatic interactions and is independent of ionic strength. The second module is an ionic-strength-dependent steering mechanism generated by long-range electrostatic interactions arising from the overall distribution of charges in ChEs. Our calculations show that diffusion of charged ligands relative to neutral isosteric analogs is enhanced approximately 10-fold by the surface trap, while electrostatic steering contributes only a 1.5- to 2-fold rate enhancement at physiological salt concentration. We model clearance of cationic products from the active-site gorge as analogous to the escape of a particle from a one-dimensional well in the presence of a linear electrostatic potential. We evaluate the potential inside the gorge and provide evidence that while contributing to the steering of cationic species toward the active site, it does not appreciably retard their clearance. This optimal fine-tuning of global and local electrostatic interactions endows ChEs with maximum catalytic efficiency and specificity for a positively charged substrate, while at the same time not hindering clearance of the positively charged products.