Cell surface characteristics and DNA content of macrophages in murine bone marrow cultures. A study using simultaneous scanning electron microscopy and fluorescence microscopy

An instrument combining scanning electron microscopy (SEM) and light microscopy (LM) was used to study the cell surface characteristics and DNA content of macrophages in murine bone marrow cultures. After a quantitative Feulgen DNA staining, the DNA content of the individual macrophages was measured and their cell surface morphology was studied immediately thereafter with the SEM part of the instrument. The cells were divided into six groups according to the number of microvilli and/or microridges present on their surface. A proportion of macrophages showed a DNA content more than occurs in diploid cells, which could indicate a future division. No special surface morphology could be detected in this cell type.     

Exposure of bipartite hydrophobic signal triggers nuclear quality control of Ndc10 at the endoplasmic reticulum/nuclear envelope

Proper functioning of the protein-folding quality control network depends on the network's ability to discern diverse structural perturbations to the native states of its protein substrates. Despite the centrality of the detection of misfolded states to cell home-ostasis, very little is known about the exact sequence and structural features that mark a protein as being misfolded. To investigate these features, we studied the requirements for the degradation of the yeast kinetochore protein Ndc10p. Mutant Ndc10p is a substrate of a protein-folding quality control pathway mediated by the E3 ubiquitin (Ub) ligase Doa10p at the endoplasmic reticulum (ER)/nuclear envelope membrane. Analysis of Ndc10p mutant derivatives, employing a reverse genetics approach, identified an autonomous quality control-associated degradation motif near the C-terminus of the protein. This motif is composed of two indispensable hydrophobic elements: a hydrophobic surface of an amphipathic helix and a loosely structured hydrophobic C-terminal tail. Site-specific point mutations expose these elements, triggering ubiquitin-mediated and HSP70 chaperone-dependent degradation of Ndc10p. These findings substantiate the ability of the ER quality control system to recognize subtle perturbation(s) in the native structure of a nuclear protein.     

Modeling tuberculous meningitis in zebrafish using Mycobacterium marinum

Tuberculous meningitis (TBM) is one of the most severe extrapulmonary manifestations of tuberculosis, with a high morbidity and mortality. Characteristic pathological features of TBM are Rich foci, i.e. brain- and spinal-cord-specific granulomas formed after hematogenous spread of pulmonary tuberculosis. Little is known about the early pathogenesis of TBM and the role of Rich foci. We have adapted the zebrafish model of Mycobacterium marinum infection (zebrafish–M. marinum model) to study TBM. First, we analyzed whether TBM occurs in adult zebrafish and showed that intraperitoneal infection resulted in granuloma formation in the meninges in 20% of the cases, with occasional brain parenchyma involvement. In zebrafish embryos, bacterial infiltration and clustering of infected phagocytes was observed after infection at three different inoculation sites: parenchyma, hindbrain ventricle and caudal vein. Infection via the bloodstream resulted in the formation of early granulomas in brain tissue in 70% of the cases. In these zebrafish embryos, infiltrates were located in the proximity of blood vessels. Interestingly, no differences were observed when embryos were infected before or after early formation of the blood-brain barrier (BBB), indicating that bacteria are able to cross this barrier with relatively high efficiency. In agreement with this observation, infected zebrafish larvae also showed infiltration of the brain tissue. Upon infection of embryos with an M. marinum ESX-1 mutant, only small clusters and scattered isolated phagocytes with high bacterial loads were present in the brain tissue. In conclusion, our adapted zebrafish–M. marinum infection model for studying granuloma formation in the brain will allow for the detailed analysis of both bacterial and host factors involved in TBM. It will help solve longstanding questions on the role of Rich foci and potentially contribute to the development of better diagnostic tools and therapeutics.KEY WORDS: Tuberculous meningitis, Tuberculosis, Zebrafish, Mycobacterium marinum, Blood-brain barrier, ESX-1 mutant     

Specificity and multiple forms of beta-galactosidase in the rat.

1. Ten rat tissues and organs have been assayed for beta-galactosidase with phenyl beta-d-galactoside, p-nitrophenyl beta-d-galactoside, p-aminophenyl beta-d-galactoside and 4-methylumbelliferyl beta-d-galactoside as substrates. 2. The relative activities of these tissues are independent of the mode of assay, and maximum rates of hydrolysis are not greatly affected by the nature of the substrate. 3. Inhibition studies suggest the liver enzyme has no associated beta-glucosidase activity. 4. There is no cellular localization of preferential activity towards any of the four substrates in liver, kidney or spleen. 5. Evidence suggesting the non-destructive penetration of liver lysosomal membranes by p-nitrophenyl beta-d-galactoside is presented. 6. Liver lysosomal beta-galactosidase exists in multiple forms that can be separated on DEAE-cellulose, and the enzyme components that are bound to the membrane appear to be similar to those of the lysosome sap. 7. The chromatographic pattern of enzyme excreted in the urine is compared with those from the kidney, intestine, spleen and liver.     

Activated N-ras controls the transformed phenotype of HT1080 human fibrosarcoma cells

To investigate whether the activated N-ras oncogene of HT1080 human fibrosarcoma cells contributes to the expression of the transformed phenotype, we have isolated flat revertants. In two independent revertant lines, an increase in chromosomal ploidy occurred without a concomitant increase in the number of copies of the N-ras transforming allele. Immunoprecipitation confirms that the level of the mutant N-ras p21 gene product in the revertants is correspondingly lower than in HT1080. Analysis of sporadic tumors derived from the revertant cells reveals an increased dosage of the transforming allele. The revertants also retransform after transfection of cloned activated ras oncogenes. These results imply direct participation of an N-ras oncogene in maintaining the transformed phenotype of a human tumor cell line.     

Mycobacteria employ two different mechanisms to cross the blood–brain barrier

Central nervous system (CNS) infection by Mycobacterium tuberculosis is one of the most devastating complications of tuberculosis, in particular in early childhood. In order to induce CNS infection, M. tuberculosis needs to cross specialised barriers protecting the brain. How M. tuberculosis crosses the blood–brain barrier (BBB) and enters the CNS is not well understood. Here, we use transparent zebrafish larvae and the closely related pathogen Mycobacterium marinum to answer this question. We show that in the early stages of development, mycobacteria rapidly infect brain tissue, either as free mycobacteria or within circulating macrophages. After the formation of a functionally intact BBB, the infiltration of brain tissue by infected macrophages is delayed, but not blocked, suggesting that crossing the BBB via phagocytic cells is one of the mechanisms used by mycobacteria to invade the CNS. Interestingly, depletion of phagocytic cells did not prevent M. marinum from infecting the brain tissue, indicating that free mycobacteria can independently cause brain infection. Detailed analysis showed that mycobacteria are able to cause vasculitis by extracellular outgrowth in the smaller blood vessels and by infecting endothelial cells. Importantly, we could show that this second mechanism is an active process that depends on an intact ESX‐1 secretion system, which extends the role of ESX‐1 secretion beyond the macrophage infection cycle.     

The neurotrophins and CNTF: specificity of action towards PNS and CNS neurons.

The availability of relatively large amounts of nerve growth factor (NGF) has allowed extensive in vitro and in vivo characterization of the neuronal specificity of this neurotrophic factor. The restricted neuronal specificity of NGF (sympathetic neurons, neural crest-derived sensory neurons, basal forebrain cholinergic neurons) has long predicted the existence of other neurotrophic factors possessing different neuronal specificities. Whereas there have been many reports of "activities" distinct from NGF, full characterization of such molecules has been hampered by their extremely low abundance. The recent molecular cloning of brain-derived neurotrophic factor (BDNF) revealed that this protein is closely related to NGF and suggested that these two factors might be members of an even larger gene family. A PCR cloning strategy based on homologies between NGF and BDNF has allowed us to identify and clone a third member of the NGF family which we have termed neurotrophin-3 (NT-3). The establishment of suitable expression systems has now made available sufficient quantities of these proteins to allow us to begin to establish the neuronal specificity of each member of the neurotrophin family, and the role of each in development, maintenance and repair of the PNS and CNS. Using primary cultures of various PNS and CNS regions of the developing chick and rat, and Northern blot analysis, we describe novel neuronal specificities of BDNF, NT-3 and an unrelated neurotrophic factor-ciliary neurotrophic factor (CNTF).     

Meningeal and perivascular macrophages of the central nervous system play a protective role during bacterial meningitis

Meningeal (MM) and perivascular macrophages (PVM) constitute major populations of resident macrophages in the CNS that can be distinguished from microglial cells. So far, there is no direct evidence that demonstrates a possible role of MM and PVM in the CNS during normal or pathologic conditions. To elucidate the role of the MM and PVM during CNS inflammation, we have developed a strategy using a single intraventricular injection of mannosylated clodronate liposomes, which results in a complete and selective depletion of the PVM and MM from the CNS. Depletion of the MM and PVM during experimental pneumococcal meningitis resulted in increased illness, which correlated with higher bacteria counts in the cerebrospinal fluid and blood. This was associated with a decreased influx of leukocytes into the cerebrospinal fluid, which occurred despite an elevated production of relevant chemokines (e.g., macrophage-inflammatory protein-2) and a higher expression of vascular adhesion molecules (e.g., VCAM-1). In contrast, the higher bacterial counts correlated with elevated production of local and systemic inflammatory mediators (e.g., IL-6) indicating enhanced local leukocyte and systemic immune activation, and this may explain the worsening of the clinical signs. These findings show that the PVM and MM play a protective role during bacterial meningitis and suggest that a primary action of these macrophages is to facilitate the influx of leukocytes at the blood-brain barrier. More in general, we demonstrate for the first time that the PVM and MM play a crucial role during inflammation in the CNS.