Identification and characterization of 13 new mutations in mucopolysaccharidosis type I patients

In this study we have investigated a group of 29 Brazilian patients, who had been diagnosed with the lysosomal storage disorder, Mucopolysaccharidosis type I (MPS-I). MPS I is caused by a deficiency in the lysosomal hydrolase, alpha-L-iduronidase. Ninety percent of the MPS I patients in this study were genotyped and revealed 10 recurrent and thirteen novel IDUA gene mutations. Eight of these new mutations and three common mutations W402X, P533R, and R383H were individually expressed in CHO-K1 cells and analyzed for alpha-L-iduronidase protein and enzyme activity. A correlation was observed between the MPS I patient clinical phenotype and the associated mutant alpha-L-iduronidase protein/enzyme activity expressed in CHO-K1 cells. This was the first time that Brazilian MPS I patients had been thoroughly analyzed and highlighted the difficulties of mutation screening and clinical phenotype assessment in populations with high numbers of unique mutations.     

Impermeability to quinolones in gram-positive and gram-negative bacteria

The initial step in the accumulation of fluoroquinolone antimicrobial agents is binding to cell surface components reduced by lowered pH and divalent cations. Uptake into gram-negative and gram-positive bacteria is by simple diffusion. Entry through the outer membrane occurs preferentially for most agents by the porin route but a second process using the self-promoted uptake pathway is active especially for more hydrophobic agents. Fluoroquinolones bind to vesicles of phospholipid which may be the initiating step in cross-cytoplasmic membrane diffusion. An active efflux system has been described inEscherichia coli with evidence supporting its presence in several other bacteria. Total uptake is not altered by a resistant gyrase. Resistant isolates associated with reduced total quinolone accumulation due to lowered uptake have been described for laboratory mutants and clinical isolates. Most but not all of these have had alterations in outer membrane proteins. A functionally dominant resistance gene has been cloned from resistantStaphylococcus aureus and codes for a highly hydrophobic protein most likely membrane associated. This gene is expressed inEscherichia coli and specifies resistance especially to hydrophilic quinolones, possibly by altered accumulation.     

Intralesion transplantation of serotonergic precursors enhances locomotor recovery but has no effect on development of chronic central pain following hemisection injury in rats

The effects of intralesion grafts of serotonergic precursors on locomotor recovery and development of chronic pain were assessed after chronic spinal cord hemisection injury (SCI) in rats. Serotonin- and brain-derived neurotrophic factor-secreting (RN46A-B14) and RN46A-vector-only cells were transplanted into the site of T13 lateral hemisection 10 days following injury in immunosuppressed animals, and locomotor and pain related behaviors were assessed weekly for 28 days. There were significant improvements in the degree of spontaneous locomotor recovery, but no significant difference was found in the magnitude of development of mechanical allodynia or thermal hyperalgesia in any transplant group. From these results, we conclude that intraparenchymal engraftment of RN46A-B14 cells is largely ineffective in influencing somatosensory outcomes after SCI, in contrast with the efficacy of dorsal intrathecal placement.     

Influence of glial growth factor and Schwann cells in a bioresorbable guidance channel on peripheral nerve regeneration

Using an established rat peripheral nerve regeneration model, we investigated the role of glial growth factor (GGF) in nerve regeneration in combination with a novel bioresorbable poly(lactic-co-glycolic) acid (PLGA) guide in vivo. Schwann cells, established from a 1-cm segment of excised rat sciatic nerve, were isolated and seeded onto nerve guides with or without GGF (n = 24/group). Living nerve guides were re-established in these animals, and nerve regeneration was assessed over a period of 12 weeks. Histological studies revealed a reduction in the total axon count and the number of myelinated axons in the presence of exogenously added Schwann cells compared to saline controls. In contrast, the addition of GGF alone enhanced the total number of axons and significantly increased the number of blood vessels. Although combining GGF with Schwann cells negated the enhanced numbers of axons and blood vessels seen with GGF alone, this combination resulted in the highest myelination index and the fastest conduction velocities recorded. The PLGA guide material did not trigger any histologically detectable host response and was permissive for nerve regeneration in this animal model. The results from this study demonstrate the potential utility of this guide in vivo and establish a promotional role for GGF in nerve regeneration.     

Direct estimation of the frequency of human cytotoxic T lymphocytes and their precursors following in vitro allosensitization

Cell mediated lympholysis (CML) has been proposed as an in vitro model of the rejection process that results from transplantation of allogeneic tissue. To date, the absolute frequencies of cytotoxic T lymphocytes (CTL) and their precursors (CTL.P) have not been directly estimated in man because of technical difficulties. Through optimizing the conditions for radiometric detection of 51Cr release and the attendant improvement in CML sensitivity, direct CTL frequency estimates have been determined in peripheral blood (PBL), spleen (SPL), and lymph nodes (LNC) after in vitro allostimulation using unrelated human cells and limiting dilution assays. The mean frequency of CTL generated from PBL is 1 in 826 cells (0.121% +/- 0.101%) which, from preliminary experiments, is significantly greater than that generated from either LNC or SPL (p less than 0.05). With restimulation of primed cells on day 10, the frequency of CTL generated from PBL was increased 400%. The CTL.P frequency (0.0064% +/- 0.0050%) was approximately 5% of the corresponding CTL frequency. The CTL.P frequencies were found to be minimal estimates as both accessory "filler" cells and T cell growth factors increased the level of detection of CTL.P an average of threefold. The limiting cell dilution assay as detailed in this report should be a powerful tool for defining the cellular requirements and related factors necessary for optimal induction of a CTL response and should provide the means for determination of the immunogenetic requirements and the allospecificity of human cytotoxic lymphocytes.     

Evidence for the synthesis of defective interfering particles by Aedes albopictus cells persistently infected with sindbis virus

The species of double- and single-stranded viral RNA in Aedes albopictus cells persistently infected with Sindbis virus have been analyzed. In addition to small amounts of 42-S and 26-S single-stranded and 23-S double-stranded viral RNA, persistently infected mosquito cells contain several new species of single-stranded RNA with molecular weights ranging from 0.65 x 108 to 1.0 x 108. New double-stranded RNA forms with sedimentation coefficients in the 12- to 15-S range are also detected. Similar double- and single-stranded RNA forms are synthesized in BHK cells after infection with virus released from persistently infected cells. Treatment of the virus from persistently infected cells with anti-Sindbis antiserum inhibits the synthesis of all the viral RNA species in BHK cells. These results suggest that DI particles of Sindbis virus may be synthesized and released from persistently infected A. albopictus cells.     

A canine model for reaginic hypersensitivity and allergic bronchoconstriction

Immunization of neonatal dogs with a conjugate of 2,4-dinitrobenzene and ovalbumin (DNP₂-OA), using aluminum hydroxide as the adjuvant, elicited long-lasting (over 30 wk) anti-DNP and anti-OA IgE antibody responses of high titers as determined by homologous passive cutaneous anaphylaxis. Low antigen doses of 10 or 50 μg were more effective than the higher doses of 250 or 1,250 μg in inducing high IgE antibody levels. However, this method of immunization failed to elicit any detectable IgE antibody response in adult dogs. Bronchoprovocation with antigen of sensitized animals having IgE antibody titers in excess of 64 resulted in a marked increase in airflow resistance, which could be corrected by the administration of nebulized isoproterenol. On the other hand, sensitized animals with IgE antibody titers in the order of 64 did not manifest significant bronchoconstriction on inhalation challenge but developed anaphylaxis following intravenous injection of the antigen.     

Differential distribution of spinal cord field potentials

Summary Electrical stimulation of the sural, superficial peroneal and plantar nerves in anesthetized cats produces a sequence of potentials in the spinal cord lumbosacral enlargement. The distributions of the spinal cord dorsum negative intermediary potential (N1 wave) and of the associated field potential recorded in depth from the spinal gray matter were mapped. The N1 wave produced by the sural nerve was largest at the junction of the S1 and L7 segments, whereas that evoked by the other two nerves was maximum in L6 and L7. The field potentials recorded in depth also showed a differential distribution. The maximum negativity during phase 2, corresponding to the N1 cord dorsum potential, was found to lie laterally in the dorsal horn when the sural nerve was stimulated, but medially when the plantar nerve was activated. The superficial peroneal nerve produced its largest negative field potential in the central region of the dorsal horn. The negative field potentials from the sural and superficial peroneal nerves were not as well separated spatially from each other as they were from the potential evoked by the plantar nerve.     

Effect of interferon alpha,interferon beta,and interferon gamma on the in vitro growth of human renal adenocarcinoma cells

Interferon-, interferon-, and interferon- differ in their antiproliferative effects for several cell lines. Interferons were thus assessed for their activity in inhibiting proliferation of three renal cell carcinoma cell lines. The malignant epithelial phenotype of each of these cell lines was confirmed by electron microscopy, histology, karyotype and tumorigenicity. When compared on an anti-viral unit basis, naturally produced interferon- was more effective than natural interferon- for all cell lines and clones. Proliferation of each of the cell lines was inhibited by interferon-. In all cases, removal of interferons from culture media resulted in resumption of the rate of cell growth after a variable delay of 6–10 days. If the antiproliferative effects of interferons predominate in mediating tumor regression, clinical response may depend upon the type of interferon to which the tumor is exposed.