Efficient gene transfer in mouse neural precursors with a bicistronic retroviral vector.

Gene transfer into neural precursors is a powerful approach to study the function of specific gene products during nervous system development. Here we describe a retrovirus-based methodology to transduce foreign genes into mouse neural precursors. We used a high-titer bicistronic retroviral vector that encodes a marker gene, placental alkaline phosphatase (plap), and a selection gene, neomycin phosphotransferase II (neoR), under the translational control of two retroviral internal ribosome entry segments. Transduction efficiency even without selection was up to 95% for multipotential neurospheres derived from embryonic striata and grown with basic fibroblast growth factor 2. Expression of plap and neoR was sustained with time in culture and upon differentiation into neurons, astrocytes, and oligodendrocytes, as shown by double immunofluorescence labeling with cell type-specific markers, Western blotting, and neomycin resistance. However, levels of plap were decreased in differentiated oligodendrocytes. Transduction with the same vector of neonatal oligodendrocyte precursors grown in oligospheres consistently resulted in a lower proportion of plap-immunoreactive cells and enhanced cell death in the absence of neomycin. However, plap expression was maintained in some differentiated oligodendrocytes expressing galactocerebroside or myelin basic protein. In that neurospheres can be easily expanded in vitro and factors enabling their differentiation into the three main central nervous system cell types are being elucidated, this methodology could be used in the future to produce large number of transduced, differentiated neural cells.

Developmental pattern of expression of the alpha chemokine stromal cell-derived factor 1 in the rat central nervous system.

Stromal cell-derived factor 1 (SDF-1) is an alpha-chemokine that stimulates migration of haematopoietic progenitor cells and development of the immune system. SDF-1 is also abundantly and selectively expressed in the developing and mature CNS, as we show here. At embryonic day 15, SDF-1 transcripts were detected in the germinal periventricular zone and in the deep layer of the forming cerebral cortex. At birth, granule cells in the cerebellum and glial cells of the olfactory bulb outer layer showed an SDF-1 in situ hybridization signal that decreased progressively within the next 2 weeks. In other regions such as cortex, thalamus and hippocampus, SDF-1 transcripts detected at birth progressively increased in abundance during the postnatal period. SDF-1 protein was identified by immunoblot and/or immunocytochemistry in most brain regions where these transcripts were detected. SDF-1 was selectively localized in some thalamic nuclei and neurons of the fifth cortical layer as well as in pontine and brainstem nuclei which relay the nociceptive response. The presence of SDF-1 transcripts in cerebellar granule cells was correlated with their migration from the external to the inner granular layers with disappearance of the signal when migration was completed. In contrast, SDF1 mRNA signal increased during formation of the hippocampal dentate gyrus and stayed high in this region throughout life. The selective and regulated expression of SDF-1 in these regions suggests a role in precursor migration, neurogenesis and, possibly, synaptogenesis. Thus this alpha chemokine may be as essential to nervous system function as it is to the immune system.

Neuregulin is a mitogen and survival factor for olfactory bulb ensheathing cells and an isoform is produced by astrocytes.

The rat olfactory bulb is an exceptional CNS tissue. Unlike other areas of the brain, growing axons are able to enter the olfactory bulb and extend within this CNS environment throughout adult life. It appears that the glial cells of the olfactory system, known as olfactory bulb ensheathing cells (OBECs), may have an important role in this remarkable process of CNS neural regeneration. OBECs are unusual glial cells, possessing properties of both astrocytes and Schwann cells. In this study we show that astrocytes (in the form of astrocyte-conditioned medium; ACM) produce two critical regulatory functions for OBECs: mitogenic activity and a survival factor. Interestingly, the ACM-derived activity for OBECs appears to reside in a signalling protein(s) belonging to the neuregulin (NRG) family of growth factors, and specifically appears to coincide with one or more products of the nrg-1 gene. Our observations provide evidence for the following: recombinant human neu differentiation factors (NDFbeta1, -2 and -3) are mitogenic to OBECs; the activity in ACM can be neutralized by NDF antibodies; these same antibodies detect a 50-kDa, non-heparin binding protein in concentrated ACM; astrocytes express detectable nrg-1 transcripts; and OBECs express functional NRG receptors erbB2 and erbB4.

Adhesion molecule expression and phenotype of glial cells in the olfactory tract.

These data illustrate that OBECs have a highly plastic nature in keeping with their need to respond rapidly to changing environmental cues. This relates to their required function in supporting axonal extension throughout life. Future studies using antibodies to PSA-NCAM and L-NGFr together with FACS sorting to purify the two types of OBEC should give us a clearer understanding of the lineage relationship of the two phenotypes. With purified populations of the astrocyte-like and Schwann cell-like OBEC we should be able to determine if these cells have different functions in vivo, using several approaches namely: i) identifying the growth factors that regulate their growth and differentiation, ii) measuring the ability of the purified cells to remyelinate the experimentally-created CNS lesions and iii) carry out more detailed cellular and molecular comparisons of the two phenotypes.

Schwann cell-like myelination following transplantation of an olfactory bulb-ensheathing cell line into areas of demyelination in the adult CNS.

In this study we have transplanted a clonal olfactory bulb-ensheathing cell line into focal areas of the rat spinal cord which contain demyelinated axons but neither oligodendrocytes nor astrocytes. The cell line was created by retroviral incorporation of the temperature-sensitive Tag gene into FACS-sorted 04+ cells from 7-day-old rat pup olfactory bulb. The spinal cord lesions were obtained by injecting small volumes of ethidium bromide into the dorsal white matter of spinal cord previously exposed to 40 Grays of X-irradiation. Many of the axons were remyelinated by PO+ myelin sheaths 21 days after transplantation. Light and electron microscopy revealed cells engaging and myelinating axons in a manner highly reminiscent of Schwann cells within similar lesions. GFAP+ cells were also present within the lesion. This study provides the first in vivo evidence that olfactory bulb-ensheathing cells are able to produce peripheral-type myelin sheaths around axons of the appropriate diameter.

Low-affinity NGF-receptor and E-N-CAM expression define two types of olfactory nerve ensheathing cells that share a common lineage.

Previously, we have shown that the O4 antibody can be used to define and purify olfactory nerve ensheathing cells (ONECs) from the rat olfactory bulb by fluorescence-activated cell sorting. In this study, using a larger panel of neural markers, we demonstrate that this apparently homogeneous population of ONECs possess a heterogeneous antigenic profile both in vivo and in vitro. The antigenic profile of the sorted cells initially correlated with their antigenic profile in vivo, although expression of some of the markers was either lost or gained during time in culture. These changes were influenced by the culture conditions, with a greater loss of "typical" ONEC markers in serum-containing medium. In serum-free medium, which maintains the cells in a phenotype that closely resembles their in vivo counterparts, we were able to reclassify the ONECs into two cell types based on morphology and antigenic phenotype by using antibodies to polysialic acid (correlating with the embryonic form of N-CAM expression) and the low-affinity nerve growth factor receptor. A detailed immunocytochemical study of the developing olfactory system showed that these two cell types could also be detected along the entire length of the olfactory nerve and the outer layer of the olfactory bulb from Embryonic Day 14 to adulthood, suggesting they were not an in vitro artefact. To address the relationship between the two cell types we constructed a clonal ONEC cell line by retroviral infection with the temperature-sensitive mutant gene of the large T antigen. This clonal cell line contained cells that expressed antigenic phenotypes of both classes of ONECs, suggesting that both cell types are related and share a common lineage.

Tratamento cirúrgico da comunicaçäo inter-auricular (CIA) / Surgical treatment of the atrial septal defect (ASD)

A CIA é uma cardiopatia congênita frequente que pode ocasionar manifestaçöes clínicas graves quando näo tratada. A correçäo cirúrgica é feita com baixa mortalidade hospitalar e significa, na maioria dos casos, cura total da lesäo, por isto os pediatras e cardiologistas necessitam ficar atentos para näo deixar a lesÝo passar desapercebida, como frquentemente ocorre. Os autores analisam um grupo de 104 pacientes consecutivos portadores da cardiopatia e que foram operados com baixa mortalidade hospitalar (1,9 por cento) e chamam a atençäo pela frequente presença de lesöes associadas (30 por cento) que também devem ser corrigidas em procedimento cirúrgico único

Válvula aórtica de "Epoxy": experiência clínica de 9 anos / Epoxy aortic valve: clinical experience of 9 years

De marco de 1973 a marco de 1982, 60 pacientes foram submetidos a troca de valva aórtica por prótese de "epoxy". Oito pacientes estavam na classe II e os demais nas classes III e IV. Houve 6 óbitos hospitalares, sendo um relacionado com a prótese. A curva atuarial evidenciou proporção de sobreviventes de 72% em 9 anos, com 18 pacientes seguidos por mais de 6 anos. Dos 8 óbitos tardios, nenhum se deveu a tromboembolismo ou disfunção da prótese. Dos 54 pacientes com seguimento de 6 a 108 meses (226 pacientes-ano), houve um acidente microembolico transitório, perfazendo 0,4 eventos por 10 pacientes-ano. Não usam anticoagulantes 74% dos pacientes. Somente os pacientes com fibrilação atrial, prótese mitral concomitante ou acidente vascular cerebral prévio receberam anticoagulantes. Em nossa experiência, o "epoxy" apresenta baixo potencial trombogênico e alta resistência ao desgaste quando usado na confecção de próteses cardíacas