Indirect haemagglutination reaction (LH)-the method of choice for the detection of anamnestic antibodies to varicella - zoster (VZ) virus.

The method of indirect haemagglutination is a sensitive quantitative serological reaction for the detection of anamnestic titres of VZ antibodies. The curve obtained by the examination of a representative population sample has shown that the occurrence of VZ antibodies increases with the age so that 60% of children of under-school age and 90% of 15-year-old children have antibodies to varicella-zoster virus. In adult age groups, seropositivity ranges between 90--100%. The incidence and level of antibodies are identical in both sexes and do not decrease in old age groups. The mean titre of indirect haemagglutinating antibodies was 1 : 128.     

Testing the properties of an experimental batch of zoster immune gammaglobulin (ZIG).

The methods of indirct haemagglutination (IH) and precipitation in gel (ID) were employed to test the level of varicella-zoster (VZ) antibodies in an experimental batch of zoster gammaglobulin (ZIG). The titre of indirect haemagglutinating antibodies in ZIG was about 64 times higher than in the ordinary batches of normal immunoglobulin and about 8 times higher in comparison with the level of the initial plasma pool. In the reaction of precipitation in gel, ZIG produced 5 to 6 zones. In comparison with the initial pool of convalescent plasma, ZIG also showed an 8-fold concentration of precipitating antibodies. ZIG was administered preventively to 6 children with risk diagnoses. None of the children fell ill with varicella. According to the results of subsequent serological examination in the reactions of indirect haemagglutination and radioimmunologic analysis, only 3 children were definitely susceptible to VZ infection. In two other children (very low antibody titres) the risk could not be excluded. No substantial increase in the levels of IH and RIA antibodies was observed in the 4 children under serological observation in a period of 6 months following the administration of ZIG. ZIG was administered therapeutically to four children with varicella. The effect of ZIG therapy was very suggestive, especially in two newborn infants lacking maternal antibodies, where the dose of ZIG per 1 kg body weigt was unusually high.     

Development of three-dimensional architecture of the neuroepithelium: Role of pseudostratification and cellular 'community'

This review discusses the development of the neuroepithelium (NE) and its derivative ventricular zone (VZ), from which the central nervous system (CNS) is formed. First, the histological features of the NE and VZ are summarized, highlighting the phenomenon of pseudostratification, which is achieved by polarization and interkinetic nuclear migration (INM) of neural progenitor cells. Next, our current understanding of the cellular and molecular mechanisms and biological significance of INM and pseudostratification are outlined. The recent three-dimensional time-lapse observations revealing heterogeneity in cell lineages within the NE and VZ are also described, focusing on the neuronal lineage. Finally, the necessity of comprehensive studies on cell-cell interactions in the NE/VZ is discussed, as well as the importance of electrophysiological and biomechanical approaches. In particular, we suggest that a systems biology approach to the NE/VZ as a cellular 'community' may be fruitful.     

Development of Cerebellar Neurons and Glias Revealed by in Utero Electroporation: Golgi-Like Labeling of Cerebellar Neurons and Glias

Cerebellar cortical functions rely on precisely arranged cytoarchitectures composed of several distinct types of neurons and glias. Studies have indicated that cerebellar excitatory and inhibitory neurons have distinct spatial origins, the upper rhombic lip (uRL) and ventricular zone (VZ), respectively, and that different types of neurons have different birthdates. However, the spatiotemporal relationship between uRL/VZ progenitors and their final phenotype remains poorly understood due to technical limitations. To address this issue, we performed in utero electroporation (IUE) of fluorescent protein plasmids using mouse embryos to label uRL/VZ progenitors at specific developmental stages, and observed labeled cells at maturity. To overcome any potential dilution of the plasmids caused by progenitor division, we also utilized constructs that enable permanent labeling of cells. Cerebellar neurons and glias were labeled in a Golgi-like manner enabling ready identification of labeled cells. Five types of cerebellar neurons, namely Purkinje, Golgi, Lugaro and unipolar brush cells, large-diameter deep nuclei (DN) neurons, and DN astrocytes were labeled by conventional plasmids, whereas plasmids that enable permanent labeling additionally labeled stellate, basket, and granule cells as well as three types of glias. IUE allows us to label uRL/VZ progenitors at different developmental stages. We found that the five types of neurons and DN astrocytes were labeled in an IUE stage-dependent manner, while stellate, basket, granule cells and three types of glias were labeled regardless of the IUE stage. Thus, the results indicate the IUE is an efficient method to track the development of cerebellar cells from uRL/VZ progenitors facing the ventricular lumen. They also indicate that while the generation of the five types of neurons by uRL/VZ progenitors is regulated in a time-dependent manner, the progenitor pool retains multipotency throughout embryonic development.     

β1 Integrin Maintains Integrity of the Embryonic Neocortical Stem Cell Niche

During embryogenesis, the neural stem cells (NSC) of the developing cerebral cortex are located in the ventricular zone (VZ) lining the cerebral ventricles. They exhibit apical and basal processes that contact the ventricular surface and the pial basement membrane, respectively. This unique architecture is important for VZ physical integrity and fate determination of NSC daughter cells. In addition, the shorter apical process is critical for interkinetic nuclear migration (INM), which enables VZ cell mitoses at the ventricular surface. Despite their importance, the mechanisms required for NSC adhesion to the ventricle are poorly understood. We have shown previously that one class of candidate adhesion molecules, laminins, are present in the ventricular region and that their integrin receptors are expressed by NSC. However, prior studies only demonstrate a role for their interaction in the attachment of the basal process to the overlying pial basement membrane. Here we use antibody-blocking and genetic experiments to reveal an additional and novel requirement for laminin/integrin interactions in apical process adhesion and NSC regulation. Transient abrogation of integrin binding and signalling using blocking antibodies to specifically target the ventricular region in utero results in abnormal INM and alterations in the orientation of NSC divisions. We found that these defects were also observed in laminin α2 deficient mice. More detailed analyses using a multidisciplinary approach to analyse stem cell behaviour by expression of fluorescent transgenes and multiphoton time-lapse imaging revealed that the transient embryonic disruption of laminin/integrin signalling at the VZ surface resulted in apical process detachment from the ventricular surface, dystrophic radial glia fibers, and substantial layering defects in the postnatal neocortex. Collectively, these data reveal novel roles for the laminin/integrin interaction in anchoring embryonic NSCs to the ventricular surface and maintaining the physical integrity of the neocortical niche, with even transient perturbations resulting in long-lasting cortical defects.     

Application of fluorescent probes to study structural changes in Aspergillus fumigatus exposed to amphotericin B, itraconazole, and voriconazole

The broad objective of this study was to document patterns of structural changes following antifungal treatment, and to determine any relationship with minimum inhibitory concentration (MIC) of an antifungal. Three clinical isolates of Aspergillus fumigatus, with high, intermediate, and low amphotericin B (AB), itraconazole (IZ), and voriconazole (VZ) MICs were studied in 24-well plates with cover slips. The fluorescent probes used were Calcofluor White (cell wall), propidium iodide (nucleus), and MitoTracker Green FM (mitochondria). Fluorescent microscopy as early as 3-h after exposure revealed that AB treated hyphae had intact cell wall with deformed mitochondria and nuclei while IZ and VZ treated hyphae revealed no intact cell wall, and deformation of mitochondria and nuclei. At 48 h, AB treated cells revealed rupture of hyphae and disintegration of mitochondria, and nuclei, IZ treated hyphae were swollen with disintegration of mitochondria, and nuclei while VZ treated hyphae showed rupture and disintegration of mitochondria and nuclei. The structural changes for the three strains studied were similar in fluorescent microscopy as long as the incubation time and their respective MICs were used. Thus, AB, IZ, and VZ induced gross organelle defects in A. fumigatus nuclei, mitochondria, and cell wall, which were consistent with respective MICs of antifungals used.     

Enzymatic basis for the selective inhibition of varicella-zoster virus by 5-halogenated analogues of deoxycytidine.

5-Bromodeoxycytidine (BrdC) and 5-iododeoxycytidine, at a concentration of 100 mug/ml, effectively inhibit the replication of varicella-zoster (VZ) virus in tissue culture. No toxicity could be demonstrated in uninfected cells under the same conditions. Studies on the enzymatic basis for this selective inhibition were undertaken. Infection of human embryonic lung cell monolayers with VZ virus-infected cells results in the induction of thymidine (dT), deoxycytidine (dC), and BrdC kinase activities (which are increased 10-, 40-, and 60-fold, respectively) and in a 70-fold stimulation in the incorporation of 3H nucleotide (5-bromodeoxyuridylate) derived from BrdC into DNA. The thermal stability of the VZ virus-induced activities differs significantly from the activities induced by herpes simplex virus type 1 and herpes simplex virus type 2 and those present in uninfected human embryonic lung cells. The VZ virus-induced dT, dC, and BrdC kinase are similarly affected by temperature and cofractionate upon Sephadex gel filtration, findings consistent with the hypothesis that these activities are the function of a single enzyme: a pyrimidine deoxyribonucleoside kinase. The molecular weight, calculated on the basis of the elution pattern on Sephadex G-150, is 70,000. Kinetic studies, demonstrating that dT and dC competively inhibit the phosphorylation of BrdC, are consistent with the phosphorylation of these substrates at a common active site. Kinetic parameters include: KidT = 0.6 MUM; KidC = 60 muM; KmBrdC = 8.5 muM. In contrast to its relatively high affinity for the VZ virus-induced kinase, BrdC is a relatively poor substrate for the host kinases. Therefore, the basis for the selective inhibition of VZ virus by 5-halogenated analogues of dC is reflected in the induction of a pyrimidine deoxyribonucleoside kinase with a high affinity for BrdC.     

CNTF/LIF/gp130 receptor complex signaling maintains a VZ precursor differentiation gradient in the developing ventral forebrain

The extrinsic signaling pathways responsible for the formation and maintenance of the unique laminar organization of the forebrain germinal zones are largely unknown. In the present study, we asked whether ciliary neurotrophic factor (CNTF)/leukemia inhibitory factor (LIF)/gp130 signaling plays a role in the development of the germinal layers in the lateral ganglionic eminence. We found that CNTF/LIF/gp130 receptor signaling promotes the self-renewal/expansion of a subpopulation of fibroblast growth factor-responsive ventricular zone (VZ) precursors in the ventral forebrain. Analysis of Lifr-/- mice suggests that CNTF/LIF/gp130 signaling maintains a subpopulation of GSH2+ VZ precursors, which are necessary for normal growth of the early ventral forebrain and for maintaining a gradient of VZ precursor differentiation in the lateral ganglionic eminence, as defined by GSH2, MASH1 and DLX2 expression. Furthermore, addition of exogenous CNTF to embryonic forebrain explant cultures deprived of choroid plexus-derived CNTF, was sufficient to promote a VZ differentiation gradient. In contrast to the forebrain, CNTF/LIF/gp130 signaling reduced, rather than enhanced, precursor self-renewal/expansion in the spinal cord. These results demonstrate a novel region-specific role for CNTF/LIF/gp130 signaling in the development of the germinal layers of the embryonic telencephalon.     

Proliferation "hot spots" in adult avian ventricular zone reveal radial cell division

Neurogenesis in the adult avian brain is restricted to the telencephalon. New neurons originate in the ventricular zone (VZ) from cells that have not been identified. We mapped the position of [3H]thymidine-labeled cells in the walls of the ventricles of the adult canary brain. Labeled VZ cells were restricted to the telencephalon (lateral ventricles) and concentrated in "hot spots". The coincidence of these hot spots with regions rich in radial cells suggested that radial cells may be the cells undergoing mitosis. We used smears prepared from fragments of the VZ containing the hot spots to show directly that radial cells accumulate [3H]thymidine. In addition, grain counts at different survival times demonstrated that these cells divide. Hot spots of VZ cell division also coincided with sites of neuronal origin. We suggest that radial cell division may give rise to new neurons.     

Enhancement of varicella-zoster virus replication in cultured human embryonic lung cells treated with the pesticide carbaryl

In studies designed to determine the factors responsible for control of herpesvirus replicaton in an infected cell, we examined the interaction of varicella-zoster (VZ) virus-infected human embryonic lung cells with the pesticide carbaryl. The replication of the cell-associated VZ virus was enhanced 2- to 13-fold as compared to control cultures in Sevin 4 Oil-treated cultures and in cultures treated with the pesticide's active ingredient, carbaryl. The replication of VZ virus in cultures teated with the base oil plus inert ingredients found in the pesticide formulation was not enhanced. Possible differences in cytotoxicity induced by Seven 4 Oil, pure carbaryl, or the base oil preparation were ruled out since treated and control cultures were shown to have similar numbers of viable cells when measured by trypan blue exclusion tests or by the ability of treated cells to form foci. A dose response study showed a decrease in viral enhancement in cells treated with decreasing carbaryl concentrations.     

Enhancement of varicella-zoster virus replication in cultured human embryonic lung cells treated with the pesticide carbaryl.

In studies designed to determine the factors responsible for control of herpesvirus replicaton in an infected cell, we examined the interaction of varicella-zoster (VZ) virus-infected human embryonic lung cells with the pesticide carbaryl. The replication of the cell-associated VZ virus was enhanced 2- to 13-fold as compared to control cultures in Sevin 4 Oil-treated cultures and in cultures treated with the pesticide's active ingredient, carbaryl. The replication of VZ virus in cultures teated with the base oil plus inert ingredients found in the pesticide formulation was not enhanced. Possible differences in cytotoxicity induced by Seven 4 Oil, pure carbaryl, or the base oil preparation were ruled out since treated and control cultures were shown to have similar numbers of viable cells when measured by trypan blue exclusion tests or by the ability of treated cells to form foci. A dose response study showed a decrease in viral enhancement in cells treated with decreasing carbaryl concentrations.     

Isolation of varicella-zoster virus from pharyngeal and nasal swabs in varicella patients

Thirteen children aged 5 months to 4 years were observed during a varicella epidemic in an Infants' Hospital; except for two normal individuals, the children had various forms of congenital defects. Eleven of the children developed varicella. During the first 3 days of exanthem, a total of 17 VZ virus strains were isolated: 12 from vesicular fluid, 3 from 23 nasal and 2 from 22 pharyngeal swabs. No strain was isolated during the incubation period despite 57 and 56 swabs having been collected from the throat and nose, respectively; nor was VZ virus isolated from 6 pharyngeal and 7 nasal swabs taken on the first day of exanthem. Isolation attempts performed from vesicular fluid to control quality of the isolation conditions gave a positivity rate of 100%. Under these optimal isolation conditions VZ virus was found in the nose or throat alongside skin vesicles in four of the 11 ill children. Besides VZ virus, the pharyngeal and nasal swabs yielded, respectively, four and four cytomegalovirus strains. The cytomegalovirus infections were inapparent.     

Progressive restriction of cell fates in relation to neuroepithelial cell mingling in the mouse cerebellum

Neurogenesis in the cerebellum proceeds through a temporal series of cell production from two separate epithelia, the ventricular zone (VZ) and the external granule cell layer (EGL). Using the laacZ cell lineage tracer in transgenic mice, we describe cellular clones whose dates of birth span the entire period of cerebellar development and deduce a sequence of cell dispersion leading to the final allocation of cells in the cerebellum. Clones probably labeled early during neural tube formation show that individual progenitors can give rise to all cerebellar cell types. The distribution of clonally related granule cells in these clones indicates a mediolateral organization of EGL progenitors already established before the allocation of the EGL progenitors to the cerebellum. Clones restricted to the cerebellar VZ show that the VZ derives progenitors for deep nuclei and multipotent cortical progenitors, which lose their systematic lineage relationship when longitudinal cell intermingling in the cerebellar VZ becomes more limited. The small clones also show that cell dispersion is radial in the internal granule layer and tangential in the molecular layer. Together, the data demonstrate the broad maintenance of the relative order of cells from neural tube stages to the adult cerebellum.     

Lysophosphatidic acid (LPA) is a novel extracellular regulator of cortical neuroblast morphology

During cerebral cortical neurogenesis, neuroblasts in the ventricular zone (VZ) undergo a shape change termed "interkinetic nuclear migration" whereby cells alternate between fusiform and rounded morphologies. We previously identified lp(A1), the first receptor gene for a signaling phospholipid called lysophosphatidic acid (LPA) and showed its enriched expression in the VZ. Here we report that LPA induces changes in neuroblast morphology from fusiform to round in primary culture, accompanied by nuclear movements, and formation of f-actin retraction fibers. These changes are mediated by the activation of the small GTPase, Rho. In explant cultures, where the cerebral cortical architecture remains intact, LPA not only induces cellular and nuclear rounding in the VZ, but also produces an accumulation of rounded nuclei at the ventricular surface. Consistent with a biological role for these responses, utilization of a sensitive and specific bioassay indicates that postmitotic neurons can produce extracellular LPA. These results implicate LPA as a novel factor in cortical neurogenesis and further implicate LPA as an extracellular signal from postmitotic neurons to proliferating neuroblasts.