Experimental therapies for repair of the central nervous system: stem cells and tissue engineering.

Several stem cell-based therapeutic tools are currently being investigated for the regeneration of central nervous system (CNS) injuries. This review focuses on innovative approaches for CNS tissue repair via the use of implantable cellular devices. These devices are supported by biopharmaceuticals and conventional physiotherapy for the restoration of lost neuronal circuits and CNS function. This paper further reviews new and promising tools currently in pre-clinical and clinical tests for the treatment of CNS diseases where substantial loss of cellular and extracellular components of neural tissue has occurred such as stroke, encephalopathy and traumatic neural injuries. We also discuss selected 3D bioscaffolds co-cultured with clinically applicable human mesenchymal stem cells. Recent advances in neural tissue engineering and stem cell differentiation methods have shown promise for their clinical application in treating yet incurable CNS deficits.

Biosignature detection at an Arctic analog to Europa.

The compelling evidence for an ocean beneath the ice shell of Europa makes it a high priority for astrobiological investigations. Future missions to the icy surface of this moon will query the plausibly sulfur-rich materials for potential indications of the presence of life carried to the surface by mobile ice or partial melt. However, the potential for generation and preservation of biosignatures under cold, sulfur-rich conditions has not previously been investigated, as there have not been suitable environments on Earth to study. Here, we describe the characterization of a range of biosignatures within potentially analogous sulfur deposits from the surface of an Arctic glacier at Borup Fiord Pass to evaluate whether evidence for microbial activities is produced and preserved within these deposits. Optical and electron microscopy revealed microorganisms and extracellular materials. Elemental sulfur (S°), the dominant mineralogy within field samples, is present as rhombic and needle-shaped mineral grains and spherical mineral aggregates, commonly observed in association with extracellular polymeric substances. Orthorhombic α-sulfur represents the stable form of S°, whereas the monoclinic (needle-shaped) γ-sulfur form rosickyite is metastable and has previously been associated with sulfide-oxidizing microbial communities. Scanning transmission electron microscopy showed mineral deposition on cellular and extracellular materials in the form of submicron-sized, needle-shaped crystals. X-ray diffraction measurements supply supporting evidence for the presence of a minor component of rosickyite. Infrared spectroscopy revealed parts-per-million level organics in the Borup sulfur deposits and organic functional groups diagnostic of biomolecules such as proteins and fatty acids. Organic components are below the detection limit for Raman spectra, which were dominated by sulfur peaks. These combined investigations indicate that sulfur mineral deposits may contain identifiable biosignatures that can be stabilized and preserved under low-temperature conditions. Borup Fiord Pass represents a useful testing ground for instruments and techniques relevant to future astrobiological exploration at Europa.

Peripheral neural cell sensitivity to mTHPC-mediated photodynamic therapy in a 3D in vitro model.

BACKGROUND: The effect of photodynamic therapy (PDT) on neural cells is important when tumours are within or adjacent to the nervous system. The purpose of this study was to investigate PDT using the photosensitiser, meta-tetrahydroxyphenyl chlorin (mTHPC), on rat neurons and satellite glia, compared with human adenocarcinoma cells (MCF-7). METHODS: Fluorescence microscopy confirmed that mTHPC was incorporated into all three cell types. Sensitivity of cells exposed to mTHPC-PDT (0-10 microg ml(-1)) was determined in a novel 3-dimensional collagen gel culture system. Cell death was quantified using propidium iodide and cell types were distinguished using immunocytochemistry. In some cases, neuron survival was confirmed by measuring subsequent neurite growth in monolayer culture. RESULTS: MCF-7s and satellite glia were significantly more sensitive to PDT than neurons. Importantly, 4 microg ml(-1) mTHPC-PDT caused no significant neuron death compared with untreated controls but was sufficient to elicit substantial cell death in the other cell types. Initially, treatment reduced neurite length; neurons then extended neurites equivalent to those of untreated controls. The protocol was validated using hypericin (0-3 microg ml(-1)), which caused neuron death equivalent to other cell types. CONCLUSION: Neurons in culture can survive mTHPC-PDT under conditions sufficient to kill tumour cells and other nervous system cells.

Assessment of the activity of a novel nociceptin/orphanin FQ analogue at recombinant human nociceptin/orphanin FQ receptors expressed in Chinese hamster ovary cells.

The neuropeptide nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand for the nociceptin receptor (NOP). In an attempt to identify high potency NOP agonists for use in the brain we have compared the activity of a novel N/OFQ analogue [Phe(1)Psi(CH(2)-O)Gly(2)]N/OFQ(1-13)NH(2) ([F/G-O]) with the existing [Phe(1)Psi(CH(2)-NH)Gly(2)]N/OFQ(1-13)NH(2) ([F/G]). Both peptides are modified between the first two N-terminal amino acids and are further compared with the agonist template N/OFQ(1-13)NH(2) in [(3)H]N/OFQ binding, GTPgamma[(35)S] binding and cAMP inhibition studies using Chinese hamster ovary cells expressing the recombinant human NOP. All peptides displaced [(3)H]N/OFQ, stimulated GTPgamma[(35)S] binding and inhibited cAMP formation. In [(3)H]N/OFQ binding and GTPgamma[(35)S] binding the rank order affinity and potency was N/OFQ(1-13)NH(2)>[F/G-O]>[F/G]. In GTPgamma[(35)S] binding [F/G] was a clear partial agonist with intrinsic activity (E(max) stimulation factor, mean+/-SEM, n=4) of 7.75+/-1.02 compared with N/OFQ(1-13)NH(2) of 11.13+/-1.76. The efficacy of [F/G-O] (10.17+/-1.88) approached that of the full agonist N/OFQ(1-13)NH(2). Downstream, at the level of cAMP formation, all peptides were full agonists with the following rank order potency: N/OFQ(1-13)NH(2)>[F/G-O]=[F/G]. The enhanced potency and intrinsic activity of the novel [F/G-O] modification makes this an interesting peptide for further in vivo analysis.

Sorting of glycoprotein B from human cytomegalovirus to protein storage vesicles in seeds of transgenic tobacco.

As part of ongoing studies into the use of plant expression systems for making human therapeutic proteins, we have successfully expressed the major glycoprotein, gB, of human cytomegalovirus (HCMV) in transgenic tobacco plants. Viral glycoprotein was detectable in the protein extracts of mature tobacco seeds using neutralizing and non-neutralizing monoclonal antibodies specific for gB. Although several mammalian proteins have been expressed in tobacco, localization of these proteins in transgenic tobacco tissue has not been extensively examined. The objective of this study was to identify the site(s) of recombinant gB deposition in mature tobacco seeds. Using immunogold labelling and electron microscopy, we found specific labelling for gB in the endosperm of transgenic seeds, with gB localized almost exclusively in protein storage vesicles (PSV). This occurred in seeds that were freshly harvested and in seeds that had been stored for several months. These data indicate that gB behaves like a plant storage protein when expressed in tobacco seeds, and provide further support for the suitability of plants for producing recombinant proteins of potential clinical relevance.

Thymocyte-targeted overexpression of xiap transgene disrupts T lymphoid apoptosis and maturation.

The X-linked inhibitor of apoptosis (XIAP) and other members of the inhibitor of apoptosis (IAP) family can suppress apoptosis induced by a diverse variety of triggers. Functional studies done to date have focused on tissue culture models and adenovirus overexpression of XIAP and other IAP proteins. Here we report the phenotype of an engineered transgenic mouse overexpressing a human IAP, as well as assessing the long-term consequence of IAP overexpression. We document the relative protein expression levels of the endogenous mouse homologue to XIAP, mouse inhibitor of apoptosis (MIAP 3), within thymocyte and T cell subpopulations. The consequence of lymphoid-targeted overexpression of XIAP in transgenic mice suggests a physiological role for the endogenous protein, MIAP3. Xiap-transgenic mice accumulated thymocytes and/or T cells in primary and secondary lymphoid tissue, T cell maturation was perturbed, and transgenic thymocytes resisted a variety of apoptotic triggers both in vitro and in vivo. These observations imply a possible key function for the intrinsic cellular inhibitor XIAP in maintaining the homeostasis of the immune system.

Evaluation of a neonatal rat model for prediction of mumps virus neurovirulence in humans.

Neurovirulence of several mumps virus strains was assessed in a prototype rat neurovirulence test and compared to results obtained in the monkey neurovirulence test. The relative human neurovirulence of these strains was proportional to the severity of hydrocephalus in rats but not to lesion scores in the monkeys.

Biological characteristics of genetic variants of Urabe AM9 mumps vaccine virus.

The Urabe AM9 mumps vaccine is composed of a mixture of variants distinguishable by a difference at nucleotide (nt) 1081 of the hemagglutinin-neuraminidase (HN) gene (Brown, E.G., Dimock, K., Wright, K.E., 1996. The Urabe AM9 mumps vaccine is a mixture of viruses differing at amino acid (aa) 335 of the hemagglutinin-neuraminidase gene with one form associated with disease. J. Infect. Dis. 174, 619-622.). Further genetic and biological variation was detected in plaque purified viruses from the Urabe AM9 vaccine by examining the HN gene sequence, plaque morphology, cytopathic effects and growth in Vero cells, and temperature sensitivity (ts). Infection of Vero cells with plaque purified viruses with a G at nt 1081 of the HN gene produced large, clear plaques, caused significant CPE early after infection but yielded lower titres of virus than other purified viruses. None of these viruses were ts. In contrast, half of the plaque purified viruses with an A at nt 1081 were sensitive to a temperature of 39.5 degrees C. These viruses produced small plaques, caused significant CPE and grew to low titres. Two ts viruses possessed a unique aa substitution at aa 468 of HN. The remaining A(1081) viruses were not ts, produced large plaques but little CPE, and grew to titres 10-fold higher than the G(1081) viruses. Isolates of Urabe AM9 associated with post-vaccination illness were similar to these non-ts A(1081) viruses, but could be further sub-divided into two groups on the basis of a difference at aa 464 of HN. The post-vaccination isolates may represent insufficiently attenuated components of the vaccine, while the G(1081) and ts subset of A(1081) viruses may be more fully attenuated.

Iatrogenic spondylolysis leading to contralateral pedicular stress fracture and unstable spondylolisthesis: a case report.

STUDY DESIGN: A case report of iatrogenic spondylolysis as a complication of microdiscectomy leading to contralateral pedicular stress fracture and unstable spondylolisthesis. OBJECTIVE: To improve understanding of this condition by presenting a case history and roentgenographic findings of a patient that differ from those already reported and to propose an effective method of surgical management. METHODS: A 67-year-old woman with no history of spondylolysis or spondylolisthesis underwent an L4-L5 microdiscectomy for a left herniated nucleus pulposus 1 year before the current consultation. For the preceding 8 months, she had been experiencing low back and bilateral leg pain. Imaging studies revealed a left L4 spondylolytic defect and a right L4 pedicular stress fracture with an unstable Grade I spondylolisthesis. RESULTS: The patient was treated with posterior spinal fusion, which resulted in complete resolution of her clinical and neurologic symptoms. CONCLUSIONS: Iatrogenic spondylolysis after microdiscectomy is an uncommon entity. However, it can lead to contralateral pedicular stress fracture and spondylolisthesis, and thus can be a source of persistent back pain after disc surgery. Surgeons caring for these patients should be aware of this potential complication.

Viscosupplementation for osteoarthritis.

Viscosupplementation therapy can restore the elastic and viscous properties of synovial fluid and thus recreate the intra-articular joint homeostasis that is disrupted in the degenerative joint. Hyaluronan (hyaluronic acid) products have been developed and used for viscosupplementation therapy in osteoarthritis. Viscosupplementation treatments using these products are well tolerated. Because viscosupplementation therapy is based on the concept of replenishing a normal physiological component of synovial fluid and cartilaginous tissue, exogenous administration of hyaluronic acid has the potential to have few side effects or local or systemic reactions. Viscosupplementation represents an alternative treatment for patients with osteoarthritis in which oral medications and/or surgery are not options or are ineffective.

The mumps virus neurovirulence safety test in Rhesus monkeys: a comparison of mumps virus strains.

Wild type mumps viruses are highly neurotropic and a frequent cause of aseptic meningitis in unvaccinated humans. To test whether attenuated mumps viruses used in the manufacture of mumps vaccines have neurovirulent properties, a monkey neurovirulence safety test (MNVT) is performed. However, results with several mumps virus MNVTs have raised questions as to whether the test can reliably discriminate neurovirulent from nonneurovirulent mumps virus strains. Here, various mumps virus strains representing a wide range of neuropathogenicity were tested in a standardized MNVT. A trend of higher neurovirulence scores was observed in monkeys inoculated with wild type mumps virus versus vaccine strains, although differences were not statistically significant. Results indicated the need for further examination and refinement of the MNVT or for development of alternative MNVTs.

An assessment of learning styles among undergraduate athletic training students.

OBJECTIVE: Increased attention has been directed toward assessing and improving academic quality in athletic training education. The educational process has been assessed from a global level, but little is known about how athletic training students learn. The purpose of this investigation was to assess the learning styles of undergraduate athletic training students. DESIGN AND SETTING: Undergraduate students enrolled in a Committee on Accreditation of Allied Health Education Programs (CAAHEP)-accredited athletic training education program completed a learning styles inventory during a regularly scheduled athletic training class at the start of the spring semester. SUBJECTS: Twenty-seven student athletic trainers (age range, 19-30 yrs, mean age = 20.5 yrs) served as subjects. Sixteen subjects (7 male, 9 female) were in the first year of this 3-year program. Eleven subjects (7 male, 4 female) were second-year students. MEASUREMENTS: Learning style was assessed using the Productivity Environmental Preference Survey. RESULTS: Parametric and nonparametric one-way analyses of variance for each learning subscale by sex and by year in program revealed significant differences (P < .05) in light preferences for male and female students. There were also significant differences (P < .05) between first-and second-year students in preferences for afternoon learning activities. CONCLUSIONS: These findings suggest that undergraduate athletic training students function best as leamers in a well-lit leaming environment. The significance of aftemoon as the preferred time for learning reinforces the importance of the clinical setting in the introduction and mastery of skills. Athletic training educators and clinical instructors can use these results as they examine their teaching strategies and educational environments.

Specificity analysis of murine monoclonal antibodies reactive with Tn, sialylated Tn, T, and monosialylated (2-->6) T antigens.

T, Tn, and sialyated Tn (sTn) are pancarcinoma antigens, and increased expression of these carbohydrate epitopes has been correlated with a poor prognosis in several epithelial malignancies. Ten murine monoclonal antibodies have been generated to these antigens, and compared by ELISA and immunohistochemistry to established mAbs reactive with these antigens. Nine mAbs (3 IgM and 6 IgG) reactive with synthetic T-human serum albumin (T-HSA) were produced after immunizing BALB/c mice with a synthetic T-keyhole limpet hemocyanin glycoconjugate (T-KLH). An additional IgM mAb (145.22) was produced in mice immunized with erythrocytes isolated from a patient with Tn syndrome. Three IgM and six IgG1 mAbs reactive with T-HSA did not react with natural T antigen present on desialyated glycophorin. All three IgM and several IgG1 mAbs, however, did react with LS-174T, a mucinous colon carcinoma cell line, 647V, a human bladder carcinoma cell line, and TA3Ha, a murine mammary carcinoma cell line as well as fresh frozen colon carcinomas. MAb 145.22 reacted with both natural and synthetic sources of sTn and Tn, as well as with LS-174T cells and mucin deposits in 10/11 colon carcinomas on fresh-frozen sections. MAb B72.3 reacted strongly with ovine submaxillary mucin (OSM) and sTn-HSA, while mAb CC49, a second-generation mAb to TAG-72 carcinoma mucin, reacted strongly with OSM, less strongly with desialyated OSM, and only weakly with sTn-HSA, suggesting that the epitope specificity for mAb CC49 is distinct from that of B72.3.

Mutations in the hemagglutinin and matrix genes of a virulent influenza virus variant, A/FM/1/47-MA, control different stages in pathogenesis.

The mouse adapted strain of influenza A/FM/1/47 virus, FM-MA, has increased virulence due to mutations in HA, M1 and at least one other, unmapped, genome segment. Genetic reassortants that differ due to the HA or M1 mutations were used to define the role of these mutations in pathogenesis. Pathological changes in lungs of infected mice were assessed by hematoxylin phloxine saffron (HPS) staining, and viral infection was measured by fluorescent antibody staining of thin sections and flow cytometry of lung parenchymal cells. HA played a role in bronchiolar pathology by increasing necrosis of bronchiolar epithelium, peribronchiolar lymphocytes, and airway obstruction. The HA mutation was shown to be responsible for a 0.2 unit decreased in the pH optimum of fusion and controlled resistance to alpha and beta inhibitors of hemagglutination. Both these changes in biology may confer a replicative advantage in bronchioles seen in the first day of infection. Thus the HA mutation may have conferred a survival advantage in the extracellular lung environment. The M1 mutation resulted in improved growth in the lung and cultured cells and was associated with increases in recruitment of macrophages, spread of infection into the alveoli of the lung and interstitial pneumonia. Sequence analysis indicated that the unmapped mutation in the control of FM-MA virulence is either the K482-->R substitution in the PB2 protein or the D538-->G substitution in the PB1 protein. One or other of these mutations results in a growth advantage in infected lung but not in cultured cells as well as a further increased recruitment and infection of macrophages in the lung. Infection with virulent strains of influenza that induced increases in macrophage recruitment caused hypothermia in the mouse.

The Urabe AM9 mumps vaccine is a mixture of viruses differing at amino acid 335 of the hemagglutinin-neuraminidase gene with one form associated with disease.

The reason for the high incidence of vaccine-associated meningitis due to the Urabe AM9 vaccine was assessed by comparing the nucleotide (nt) sequence of the hemagglutinin-neuraminidase (HN) gene from vaccine virus to those of viruses isolated from persons with postvaccination meningitis. A G1081--> A nt substitution that was predicted to result in a Glu335--> Lys reversion in the HN protein was detected between Urabe AM9 (G) and postvaccine meningitis mumps virus isolates (A). Further analysis showed that the Urabe AM9 vaccine was a mixture of viruses with wild type (A) and variant (G) nt at position 1081. Urabe AM9 vaccinees who developed meningitis or parotitis possessed predominantly A (98%-100%) at nt 1081, indicating strong selection of the wild type (A) form relative to the variant (G) form. Mumps virus homogeneous for the variant Glu335 form of the HN gene may be safer than the original Urabe AM9 vaccine.

Effect of incremental time experience on the results of in vitro fertilization with intracytoplasmic sperm injection (ICSI).

PURPOSE: Our objective was to determine the effect of experience on the results with intracytoplasmic sperm injection. METHODS: The quarterly outcome with both ICSI and traditional in vitro fertilization (IVF) in 1994 was analyzed in 475 patients under age 40 undergoing 595 oocyte retrievals. The data represent 307 patients undergoing 379 retrievals for IVF and 165 patients undergoing 216 retrievals for ICSI. RESULTS: Fertilization rates with ICSI improved significantly each quarter (52.96, 62.17, 70.17, and 74.87% in Q-I, Q-II, Q-III, and Q-IV, respectively), while the rate with IVF improved significantly between Q-I (69.9%) and Q-II (80.10%) and slightly but significantly between Q-II and Q-IV (82.88%). The implantation rate per embryo after ICSI improved significantly after Q-I (6.17%) compared to Q-II (10.70%) and Q-IV (12.14%). The pregnancy rate per transfer with ICSI increased steadily after Q-I (13.79, 21.88, 23.53, and 25.00% in Q-I through Q-IV), reaching statistical significance between Q-I and Q-III and between Q-I and Q-IV. CONCLUSIONS: Although acceptable results can be obtained with ICSI after a relatively short period of time, optimum results require substantial experience.

Stimulation of megakaryocytopoiesis in mice by human modified C-reactive protein (mCRP)

The prototypic human acute phase reactant, C-reactive protein (CRP), and a structurally modified form of CRP (mCRP) were studied as agents which could stimulate thrombopoiesis in both in vitro and in vivo mouse models. mCRP, but not the widely studied (native) pentameric form of CRP, demonstrated significant megakaryocyte colony-stimulating activity. This activity was measured in plasma clot cultures incubated with pokeweed mitogen-stimulated spleen cell conditioned medium (PWM-SCM). mCRP increased the number of mouse megakaryocyte colonies in a dose-dependent manner. While significantly more colonies were observed in mCRP-treated cultures compared to controls, the kinetics of megakaryocyte growth and maturation were similar to those measured in cultures stimulated with PWM-SCM lacking mCRP. A low level of megakaryocyte growth-promoting activity was noted when mCRP was added to plasma clot cultures not incubated with spleen cell conditioned medium. However, the most striking activity of mCRP was in potentiating stimulated megakaryocyte colony formation (i.e., as a Meg-POT factor). In in vivo experiments, mCRP injected subcutaneously into normal mice resulted in significant increases in blood platelet numbers compared to control mice receiving sham injections. These results suggest that a modified form of CRP has thrombopoietic activity in both in vitro and in vivo mouse models, Therefore, one important biological role for CRP during an acute-phase response might be to contribute, after a structural modification, to the hematopoietic regulation of blood platelets.

Use of computer-based instruction in athletic training education.

Computer-based instruction is being widely used in the education programs of many allied health professions. However, there has been little, if any, documentation of computer-based instruction use in athletic training education. The primary purpose of this study was to determine what percentage of undergraduate and graduate NATA-approved athletic training education programs are using some form of computer-based instruction (ie, computer-assisted instruction or interactive video). We also addressed the following research questions: 1) What athletic training educational software is currently being used by athletic training students and educators? 2) What factors currently impede the use of computer-based instruction in athletic training education? 3) What instructional methods are commonly used to incorporate computer-based instruction into the athletic training curricula? and 4) What are the attitudes of athletic training program directors toward the use of computer-based instruction in athletic training education? Surveys were mailed to the program directors (n = 97) of all graduate and undergraduate NATA-approved athletic training education programs. Eighty-six (87.7%) usable surveys were returned. Forty-eight (55.8%) of the respondents reported using some form of computer-based instruction in their athletic training education program; 47 (54.7%) used computer-assisted instruction and 9 (10.6%) used interactive video. Respondents also identified the educational software they use and their method for implementing this software. Software was used most often to supplement traditional instructional methods. A lack of funds was reported to be the primary impeding factor for those programs not using computer-based instruction. Respondents reported an overall positive attitude toward computer-based instruction use in athletic training education and indicated the need for increased development of athletic training/sports medicine software.