Investigating microglia-brain tumor cell interactions in vivo in the larval zebrafish brain.

Glioblastoma is the most frequent and aggressive primary malignant brain tumor. Gliomas exhibit high genetic diversity in addition to complex and variable clinical features. Glioblastoma tumors are highly resistant to multimodal therapies and there is significant patient mortality within the first two years after prognosis. At present clinical treatments are palliative, not curative. Glioblastomas contain a high number of microglia and infiltrating macrophages, which are positively correlated with glioma grade and invasiveness. Microglia are the resident macrophages of the central nervous system. These cells constantly scan the brain and react promptly to any abnormality, removing detrimental factors and safeguarding the central nervous system against further damage. Microglia and macrophages that have colonized the glioblastoma display protumoral functions and promote tumor growth. The optically transparent zebrafish larva facilitates imaging of fluorescently labeled cells at high spatial and temporal resolution in vivo. It is therefore an excellent model to investigate microglia-glioma cell interactions at the early stages of tumor development. Here we provide several methods that can be used to study the early stages of microglia-glioma cell interactions in the zebrafish. We present a technique for the xenotransplantation of mammalian oncogenic cells into the zebrafish brain and provide advice for image capture and analysis.

Surfactant protein A and lipid are internalized via the coated-pit pathway by type II pneumocytes.

Surfactant protein (SP) A and SP-A-mediated lipid uptake by isolated type II cells were investigated with biochemical and morphological methods. Inhibition of coated-pit function by potassium depletion severely reduced both SP-A and SP-A-mediated lipid internalization. Lipid uptake in the absence of SP-A was not affected. With confocal laser scanning microscopy and immunoelectron microscopy, SP-A and lipid predominantly (60%) colocalized in intracellular vesicles carrying early endosomal markers (EEA1) 5 min after endocytosis but were negative for the late endosomal or lysosomal marker LAMP-1. As estimated by subcellular fractionation, at this time point, 23% of the internalized SP-A and 45% of internalized lipid were localized within light (<0.38 M sucrose) fractions, which contain lamellar bodies and are positive for EEA1. The remaining label was predominantly found within EEA1-positive and plasma membrane-containing subfractions (> or = 1 M sucrose). We suggest that in isolated type II cells in vitro, SP-A and lipid are taken up together via the coated-pit pathway and that at early time points, both components reside in the same early endosomal compartment.

Identification of a new surfactant protein A binding protein at the cell membrane of rat type II pneumocytes.

Antibodies against a type II pneumocyte component were developed by an auto-anti-idiotypic approach using surfactant-associated protein A (SP-A) as the immunogen. The antibodies recognize an SP-A-binding protein (approximately 170-200 kDa under non-reducing conditions, 55 kDa under reducing conditions) on the cell membrane of rat type II pneumocytes. One of the antibodies competes with SP-A for binding to type II cells. In immunization assays in vitro, with this antibody as the antigen, anti-SP-A antibodies have been generated. The SP-A-binding cell membrane protein recognized by this auto-anti-idiotypic antibody may be involved in the interaction of extracellular SP-A with the type II pneumocyte and may play a role in the regulation of alveolar surfactant metabolism.

Peroxisomal dysfunction in chondrodysplasia punctata, rhizomelic type.

The rhizomelic type of chondrodysplasia punctata (RCDP) is recognizable at birth because of the typical phenotype and radiological features. Most patients die young, some survive until their teens but all are severely retarded. Recent studies showed RCDP to be a peroxisomal disorder. Peroxisomal investigations may be important in defining the prognosis for an individual patient, and are definitely of use in antenatal diagnosis.

Serology in ocular toxoplasmosis.

The diagnostic value of toxoplasma serology in ocular disease was evaluated in the following groups of patients: (I) uveitis cases of various causes (n = 291); (II) consecutive posterior and panuveitis patients (n = 60); (III) patients with definite congenital and ocular toxoplasmosis (n = 8); (IV) cases of clinical ocular toxoplasmosis (n = 25); and control patients with uveitis of non-toxoplasma origin (n = 12). No relation was observed between the level of the dye test titres and the diagnosis of ocular toxoplasmosis (groups I and II). During the active stages of the disease no typical change of the titres occurred in several longitudinally studied patients with toxoplasmosis. In group III one case was discovered to be negative by the dye test despite active ocular disease; however, IgG antibodies against toxoplasma were detected by the ELISA technique. In group IV, which was investigated by the ELISA technique, 100% of the toxoplasmosis patients were positive for IgG versus 58% of the control patients. Circulating immune complexes containing IgG and toxoplasma antigen were detected in seven of 25 toxoplasmosis patients (28%) and in two of 12 control patients (16%). Our study shows that the definite diagnosis of ocular toxoplasmosis or its exclusion by serological means only is not yet feasible. The possible superiority of the ELISA test to the dye test warrants further investigation.

Results of 20-year follow-up of congenital toxoplasmosis.

In patients with congenital toxoplasmosis new lesions continue to appear well after the age of 5 years, and the impairments can be severe. Screening of women for toxoplasmosis before pregnancy thus seems advisable.

[Early diagnosis of vision disorders]. / Vroegtijdig onderkennen van visuele stoornissen (VOV).

In recent years some pilot-studies were performed at health centres in Amsterdam, on infants of 8-9 months of age, concerning early detection of visual disorders. The method consisted of a programme of corneal light reflexes, cover test, monocular following movements and inspection of the cornea, the pupil and the pupillary reaction. The aim of the studies was: 1. Is this programme efficient for early detection of organic eye disorders and strabismus at this age? 2. Is the method reliable, if performed by doctors of the health centres? The results of the studies were positive. In at least 1,9% of the 1200 children of the pilot-study a visual disorder was detected. The method can be incorporated in the regular working scheme of the health centres and can be performed on infants from the age of 6 1/2 months.

Oculocutaneous albinism and anterior chambre cleavage malformations. Not a coincidence.

We report on 6 patients out of a group of 86 albinos, with an additional eye defect: an anterior chamber cleavage disorder, i.e. a frequency of 7%. Given this high percentage of the coexistence of these two, we believe that this is not a coincidence.

The olivocerebellar projection to the uvula in the mouse.

The organization of the olivocerebellar projection in the mouse was studied with the use of microinjections of horseradish peroxidase (HRP) or HRP conjugated to wheat germ agglutinin (WGA-HRP). Injections were made in medial, intermediate, and lateral sites along the width of the uvula. The purpose of this investigation was to determine the subnuclear origin of olivary afferents to different mediolateral regions of the uvula. Injections made in or adjacent to the midline of the uvula resulted in the retrograde labeling of cells, bilaterally in the caudal portion of the medial accessory olive (MAO). These labeled cells were located primarily in subnucleus C and nucleus beta of the MAO. Injections into the intermediate part of the uvula resulted in the labeling of cells in the caudal MAO (primarily nucleus beta), the dorsomedial cell column (dmcc), and a few cells in the ventral lamella of the principal olive (vPO). Laterally placed injections produced labeling of cells in dmcc and the vPO. These results are discussed in reference to the parasagittal organization of olivary afferents to the cerebellar cortex in the mouse (Beyerl et al., '82) and the organization of afferents to the involved regions of the inferior olivary (IO) complex. It is suggested that these parasagittal zones in the uvula may play different roles in the control of eye movements.