The Drosophila neuregulin vein maintains glial survival during axon guidance in the CNS.

Neuron-glia interactions are necessary for the formation of the longitudinal axon trajectories in the Drosophila central nervous system. Longitudinal glial cells are required for axon guidance and fasciculation, and pioneer neurons for trophic support of the glia. Neuregulin is a neuronal molecule that controls glial survival in the vertebrate nervous system. The Drosophila protein Vein has structural similarities with Neuregulin. We show here that Vein functions like a Neuregulin to maintain glial cell survival. We present direct in vivo evidence at single-cell resolution that Vein is produced by pioneer neurons and maintains the survival of neighboring longitudinal glia. This mechanism links axon guidance to control of glial cell number and may contribute to plasticity during the establishment of normal axonal trajectories.

Roundabout signalling, cell contact and trophic support confine longitudinal glia and axons in the Drosophila CNS.

Contrary to our knowledge of the genetic control of midline crossing, the mechanisms that generate and maintain the longitudinal axon pathways of the Drosophila CNS are largely unknown. The longitudinal pathways are formed by ipsilateral pioneer axons and the longitudinal glia. The longitudinal glia dictate these axonal trajectories and provide trophic support to later projecting follower neurons. Follower interneuron axons cross the midline once and join these pathways to form the longitudinal connectives. Once on the contralateral side, longitudinal axons are repelled from recrossing the midline by the midline repulsive signal Slit and its axonal receptor Roundabout. We show that longitudinal glia also transiently express roundabout, which halts their ventral migration short of the midline. Once in contact with axons, glia cease to express roundabout and become dependent on neurons for their survival. Trophic support and cell-cell contact restrict glial movement and axonal trajectories. The significance of this relationship is revealed when neuron-glia interactions are disrupted by neuronal ablation or mutation in the glial cells missing gene, which eliminates glia, when axons and glia cross the midline despite continued midline repellent signalling.

Glia maintain follower neuron survival during Drosophila CNS development.

While survival of CNS neurons appears to depend on multiple neuronal and non-neuronal factors, it remains largely unknown how neuronal survival is controlled during development. Here we show that glia regulate neuronal survival during formation of the Drosophila embryonic CNS. When glial function is impaired either by mutation of the glial cells missing gene, which transforms glia toward a neuronal fate, or by targeted genetic glial ablation, neuronal death is induced non-autonomously. Pioneer neurons, which establish the first longitudinal axon fascicles, are insensitive to glial depletion whereas the later extending follower neurons die. This differential requirement of neurons for glia is instructive in patterning and links control of cell number with axon guidance during CNS development.

Genetic detection of lymph node micrometastases in patients with colorectal cancer.

BACKGROUND: Undetected micrometastases represent the single most important cause of treatment failure in patients undergoing putatively curative resection for colorectal cancer because current staging techniques are unable to identify patients with minimal residual disease. METHODS: A highly sensitive reverse transcription-polymerase chain reaction technique has been used to amplify tissue-specific messenger RNA from lymph nodes classified as tumour-free using both conventional histopathology and immunohistochemistry. RESULTS: Four of 15 patients were restaged after genetic diagnosis of lymph node micrometastases, while in a further two additional positive nodes were detected. CONCLUSION: Sensitive genetic techniques that detect minimal residual disease merit further study, particularly as there is evidence that patients may benefit from adjuvant chemotherapy.

In vitro liposome-mediated DNA transfection of epithelial cell lines using the cationic liposome DC-Chol/DOPE.

Clinical trials using cationic liposome-mediated DNA transfer have now been initiated for several disorders including cystic fibrosis. Previous studies have shown that the level of gene expression achieved may be dependent on the formulation of the DNA-liposome complex and the cell type transfected. We have investigated, in vitro, the effect of parameters such as DNA:liposome ratio, dose and concentration on the level of transgene expression in epithelial cell lines using the cationic liposome DC-Chol/1,2-dioleoyl phosphatidylethanolamine (DOPE). A narrow range of conditions was found to produce maximal level of transgene expression within a particular cell line, as detected using the reporter molecule beta-galactosidase (beta-gal). beta-Gal expression was significantly enhanced by formulation of the DNA-DC-Chol/DOPE complexes in physiological solution at pH 9.0. Under standard in vitro transfection conditions, increased incubation time of the DNA-liposome complexes with cells resulted in increased transgene expression. In contrast, at relatively high DNA and liposome dose and concentrations, beta-gal activity was maximal after only 1 h of incubation, with a subsequent decrease in expression with time. The maximum level of expression that could be produced using fully optimised transfection conditions, however, was still highly dependent on each cell type analysed. Correlation of these findings with similar studies in vivo are now critical to determine the optimal formulation of DNA-liposome complexes for clinical application.

Gene therapy for cystic fibrosis in humans by liposome-mediated DNA transfer: the production of resources and the regulatory process.

The number of clinical trials using gene transfer technology, either active or under discussion, is increasing rapidly. However, little information is available describing the regulatory procedures or safety specifications that must be considered before initiation of such trials in Europe. We describe the procedure used by our group to produce resources for the first stage of a phase I trial of liposome-mediated gene therapy for cystic fibrosis. The current lack of written and co-ordinated guidance from the numerous interested regulatory agencies within the UK and Europe makes determination of the appropriate safety specifications and procedures for these novel trials difficult, as does the fact that some new agencies (such as the Genetic Therapy Advisory Committee in the UK) and some which are unfamiliar with clinical trials (such as the Department of the Environment) are involved as well as the Medicines Control Agency. In addition, we estimate that the realistic cost of these trials, which in many cases will have to be covered from research budgets provided by government agencies or medical charities, could lead to delays in the clinical application of this important new therapeutic strategy.

Distribution of herpes simplex virus DNA in the brains of human long-term survivors of encephalitis.

We have examined the distribution of type 1 herpes simplex virus (HSV-1) DNA in the brains of 8 humans surviving for between 4 months and 17 years after acute encephalitis. Histological examination showed neuronal loss and gliosis largely confined to the temporal and frontal lobes, typical of HSV-1 encephalitis. There was a widespread persistent inflammatory infiltrate, present in both the cerebrum and brainstem. Viral DNA was detected in paraffin sections of formalin-fixed paraffin-embedded autopsy brain by use of the polymerase chain reaction to amplify a fragment of the HSV-1 thymidine kinase gene. HSV-1 DNA was amplified from the cerebrum in 6/8 cases and from the brainstem in 4/8 cases. These findings suggest the possibility that HSV-1 may persist within the human central nervous system after acute herpes simplex encephalitis. The distribution of the viral DNA correlates better with that of the persistent inflammatory infiltrate than with the destructive lesions of the acute encephalitis.

Sequencing and quantitative assessment of mutant and wild-type mitochondrial DNA in paraffin sections from cases of MELAS.

MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes) is a clinically devastating disease of children and young adults. The cause of the stroke-like episodes is not known. We have sequenced the mitochondrial DNA (mtDNA) in archival paraffin-embedded material from two cases. In only one of these did the mitochondrial tRNA(Leu(UUR) gene contain the nucleotide 3243 A-to-G mutation that is most commonly responsible for MELAS. In this case, we determined the relative proportion of mutant:wild-type mtDNA in sections of the central nervous system and other tissues by PCR amplification, PalI digestion, DNA electrophoresis, and scanning densitometry of the ethidium bromide-stained gels. The technique allowed the proportion of mitochondria that contain the mutant genome to be compared with the histological findings in immediately adjacent sections of tissue. The mutant mtDNA was detectable in most tissues, the percentage of mtDNA ranging from barely detectable levels to 78 per cent. The relative amount of mutant mtDNA correlated poorly with the distribution of histological lesions, both within the central nervous system and in other tissues examined. The proportion was high in tissues such as liver, kidney, adrenal, and pancreas that appeared histologically normal. Relatively low levels were present in some regions of the central nervous system, such as the occipital lobe, which contained many of the characteristic infarct-like lesions. These observations do not support previous speculation that the distribution of these lesions reflects that of the defective mitochondria. The results emphasize the usefulness of the polymerase chain reaction in correlative histogenetic studies.

PCR-mediated search for herpes simplex virus DNA in sections of brain from patients with multiple sclerosis and other neurological disorders.

Herpes simplex virus (HSV) has been shown to cause central nervous system demyelination in experimental animals and several studies have implicated HSV in the aetiology of multiple sclerosis (MS). We have used the polymerase chain reaction to look for DNA of both type 1 HSV (HSV-1) and type 2 HSV (HSV-2) in formalin-fixed paraffin-embedded brain tissues from patients with MS and other neurological diseases. Primers which amplify a fragment of the normal cellular gene c-myc were included in the reactions to assess the preservation of DNA in the tissue samples. 77 plaques of demyelination from 23 patients with MS were examined. HSV-1 DNA was amplified from only one plaque. This plaque involved the trigeminal root entry zone in the pons and it is suggested that the presence of viral DNA was related to the site examined rather than to the demyelination per se. HSV-2 DNA was amplified from none of the plaques. As expected, HSV-1 DNA was detected in the brains of 6 patients who died of HSV-1 encephalitis and HSV-2 DNA was amplified from the brain of a neonate with congenital HSV-2 infection. In sections of brain from 39 patients with a wide range of other neurological diseases HSV-1 DNA was detected in the pons of only 1 patient, who had AIDS associated with cytomegalovirus ventriculitis; subsequent investigation confirmed the presence of concomitant HSV-1 brain stem infection.(ABSTRACT TRUNCATED AT 250 WORDS)