Fc receptor-mediated phagocytosis in tissues as a potent mechanism for preventive and therapeutic HIV vaccine strategies.

Although the development of a fully protective HIV vaccine is the ultimate goal of HIV research, to date only one HIV vaccine trial, the RV144, has successfully induced a weakly protective response. The 31% protection from infection achieved in the RV144 trial was linked to the induction of nonneutralizing antibodies, able to mediate antibody-dependent cell-mediated cytotoxicity (ADCC), suggestive of an important role of Fc-mediated functions in protection. Similarly, Fc-mediated antiviral activity was recently shown to play a critical role in actively suppressing the viral reservoir, but the Fc effector mechanisms within tissues that provide protection from or after infection are largely unknown. Here we aimed to define the landscape of effector cells and Fc receptors present within vulnerable tissues. We found negligible Fc receptor-expressing natural killer cells in the female reproductive and gastrointestinal mucosa. Conversely, Fc receptor-expressing macrophages were highly enriched in most tissues, but neutrophils mediated superior antibody-mediated phagocytosis. Modifications in Fc domain of VRC01 antibody increased phagocytic responses in both phagocytes. These data suggest that non-ADCC-mediated mechanisms, such as phagocytosis and neutrophil activation, are more likely to play a role in preventative vaccine or reservoir-eliminating therapeutic approaches.

The TSC1 tumour suppressor hamartin regulates cell adhesion through ERM proteins and the GTPase Rho.

Loss of the tumour-suppressor gene TSC1 is responsible for hamartoma development in tuberous sclerosis complex (TSC), which renders several organs susceptible to benign tumours. Hamartin, the protein encoded by TSC1, contains a coiled-coil domain and is expressed in most adult tissues, although its function is unknown. Here we show that hamartin interacts with the ezrin-radixin-moesin (ERM) family of actin-binding proteins. Inhibition of hamartin function in cells containing focal adhesions results in loss of adhesion to the cell substrate, whereas overexpression of hamartin in cells lacking focal adhesions results in activation of the small GTP-binding protein Rho, assembly of actin stress fibres and formation of focal adhesions. Interaction of endogenous hamartin with ERM-family proteins is required for activation of Rho by serum or by lysophosphatidic acid (LPA). Our data indicate that disruption of adhesion to the cell matrix through loss of hamartin may initiate the development of TSC hamartomas and that a Rho-mediated signalling pathway regulating cell adhesion may constitute a rate-limiting step in tumour formation.

Stimulus history alters behavioral responses of neuronal growth cones.

Generally, it is assumed that growth cones respond to a specific guidance cue with a single, specific, and stereotyped behavior. However, there is evidence to suggest that previous exposure to a given cue might alter subsequent responses to that cue (Snow and Letourneau, 1992; Shirasaki et al., 1998). We therefore tested the hypothesis that growth cone responses to stimuli are dependent on the history of previous stimulation. Growth cones of chick dorsal root ganglion neurons were exposed to well characterized stimuli: (1) contact with a laminin-coated bead, which causes growth cone turning, or (2) electrical stimulation, which causes growth cone collapse. Although the expected behavioral responses were observed after the initial stimulation, strikingly different responses to a subsequent stimulation were observed. Growth cones that had recovered from electrical stimulation-induced collapse rapidly developed insensitivity to a second identical electrical stimulation. Growth cones that previously turned in response to contact with a laminin-coated bead responded to a second bead with a "stall" or cessation in outgrowth. This stimulus history dependence of growth cone behavior could be generalized across dissimilar stimuli: after contact with a laminin-coated bead, growth cones failed to collapse in response to electrical stimulation. The calcium/calmodulin-dependent protein kinase II (CaMKII) was implicated in this history dependence by pharmacological experiments. Together, these results demonstrate that growth cones can alter their behavioral response rapidly to a given stimulus in a manner dependent on previous history and that knowledge of past events in growth cone navigation may be required to predict future growth cone behavior.

Membrane recycling in the neuronal growth cone revealed by FM1-43 labeling.

Membrane dynamics within the chick ciliary neuronal growth cone were investigated by using the membrane-impermeant dye FM1-43. A depolarization-evoked endocytosis was observed that shared many properties with the synaptic vesicle recycling previously described at the presynaptic terminal. In addition, in the absence of depolarization a basal level of constitutive endocytotic activity was observed at approximately 30% of the rate of evoked endocytosis. This constitutive endocytosis accounted for large amounts of membrane retrieval: the equivalent of the entire growth cone surface area could be internalized within a 30 min period. Endosomes generated via constitutive and evoked processes were highly mobile and could move considerable distances both within the growth cone and out to the neurite. In addition to their different requirements for formation, evoked and constitutive endosomes displayed a significant difference in release properties. After a subsequent depolarization of labeled growth cones, evoked endosomes were released although constitutive endosomes were not released. Furthermore, treatment with latrotoxin released evoked endosomes, but not constitutive endosomes. Although the properties of evoked endosomes are highly reminiscent of synaptic vesicles, constitutive endosomes appear to be a separate pool resulting from a distinct and highly active process within the neuronal growth cone.

Early development of an identified serotonergic neuron in Helisoma trivolvis embryos: serotonin expression, de-expression, and uptake.

In early-stage embryos of Helisoma trivolvis, a bilateral pair of identified neurons (ENC1) express serotonin and project primary descending neurites that ramify in the pedal region of the embryo prior to the formation of central ganglia. Pharmacological studies suggest that serotonin released from ENC1 acts in an autoregulatory pathway to regulate its own neurite branching and in a paracrine or synaptic pathway to regulate the activity of pedal ciliary cells. In the present study, several key features of early ENC1 development were characterized as a necessary foundation for further experimental studies on the mechanisms underlying ENC1 development and its physiological role during embryogenesis. ENC1 morphology was determined by confocal microscopy of serotonin-immunostained embryos and by differential-interference contrast (DIC) microscopy of live embryos. The soma was located at an anteriolateral superficial position and contained several distinguishing features, including a large spherical nucleus with prominent central nucleolus, large granules in the apical cytoplasm, a broad apical dendrite ending in a sensory-like structure at the embryonic surface, and a ventral neurite. ENC1 first expressed serotonin immunoreactivity around stage E13, followed immediately by the appearance of an immunoreactive neurite (stage E14). Both the intensity of immunoreactivity and primary neurite length were consistently greater in the right ENC1 at early stages. Serotonin uptake, as indicated by 5,7-dihydroxytryptamine-induced fluorescence, first occurred between stages E18 and E25. At later stages of embryogenesis (after stage E65), serotonin immunoreactivity disappeared, whereas serotonin uptake and normal cell morphology were retained.

Neurite branch development of an identified serotonergic neuron from embryonic Helisoma: evidence for autoregulation by serotonin.

Previous studies have shown that in select neurons, neurite outgrowth can be regulated by the same neurotransmitter that is synthesized and released by those neurons. However, it is not known whether such an autoregulatory mechanism is utilized during the normal course of nervous system development in either invertebrates or vertebrates. In the present study, we tested this hypothesis on the first pair of identified serotonergic neurons to be expressed in embryos of the pulmonate gastropod, Helisoma trivolvis. Embryonic neurons C1 (ENC1) elaborate a stereotyped pattern of neurite outgrowth prior to the differentiation of subsequent serotonergic neurons. Embryos were treated with either p-chlorophenylalanine (pCPA) or 5-hydroxytryptophan (5-HTP) to lower or raise embryonic serotonin content, respectively. High-performance liquid chromatography with electrochemical detection was used to measure the effects of these treatments on serotonin content, and serotonin immunohistochemistry was carried out to quantify the extent of neurite outgrowth of ENC1. Embryonic serotonin content was significantly reduced at both 24 and 48 hr after treatment with 0.02% pCPA, whereas dopamine levels were unchanged. Although the proximal neurite outgrowth of ENC1 appeared unaffected by the pCPA treatment at both of these time points, the distal outgrowth in the target cell region appeared more profuse. This effect on outgrowth was quantified by counting the number of neurite branch points, which was significantly increased both 24 and 48 hr after pCPA treatment. In contrast, 5-HTP treatment resulted in an increase in embryonic serotonin content and a significant decrease in the number of ENC1 branch points. Treatment with dopamine had no effect on the pattern of ENC1 neurite outgrowth. Together, these results support the hypothesis that a neuron may utilize its own transmitter in an autoregulatory fashion to regulate neurite formation during embryonic development.

Pharmacological characterization of a serotonin receptor involved in an early embryonic behavior of Helisoma trivolvis.

In contrast to the abundance of information on the many physiological and developmental actions of serotonin in molluscan nervous systems, comparatively little is known about the serotonin receptors involved in these responses. Embryos of the pulmonate gastropod, Helisoma trivolvis, display a cilia-driven rotational behavior that is regulated by endogenous serotonin. In the present study, two functional assays were used to determine some of the pharmacological properties of the receptors that mediate the cilio-excitatory action of serotonin. Time-lapse video microscopy was used to measure whole embryo rotation rate and cilia beat frequency in isolated cells. In dose-response experiments, serotonin was approximately 10 times more potent in stimulating cilia beat frequency over embryo rotation. In rotation experiments, 5-carboxyamidotryptamine and methysergide had effective agonist activity in dose ranges similar to that of serotonin (1 to 100 microM). In contrast, 8-hydroxydipropylaminotetralin HBr (8-OH-DPAT) displayed agonist activity of lower potency and effectiveness. Several compounds displayed antagonist activity in the 1 to 100 microM dose range, including mianserin, spiperone, ritanserin, 1-(1-naphthyl)piperazine, and propranolol. alpha-Methylserotonin had mixed agonist-antagonist activity, and metoclopramide, MDL-72222, and ketanserin were inactive. Experiments on isolated cells suggested that the extremely effective antagonism displayed by mianserin in the embryo rotation assay was due to its specific activity at ciliary serotonin receptors. These results implicate the presence of a novel serotonin receptor on embryonic ciliated cells that is pharmacologically distinct from those previously characterized in vertebrate or invertebrate systems.

Characterization and development of rotational behavior in Helisoma embryos: role of endogenous serotonin.

Cilia-driven rotational behavior displayed by embryos of the pond snail Helisoma trivolvis was characterized in terms of its behavioral subcomponents, developmental changes, and response to exogenous serotonin. Rotation was found to be a complex behavior characterized by four parameters; rotational direction, rotation rate, rotational surges, and periods of inactivity. These parameters all exhibited characteristic developmental changes from embryonic stage E15 through stage E30. Notably, both rotation rate and frequency of rotational surges increased from stage E15 to E25 and declined to an intermediate level by stage E30. It appeared that the developmental increase in overall rotation rate was caused primarily by an increase in surge frequency, rather than an increase in the rate of nonsurge rotation. Immersion of embryos inserotonin-containing pond water resulted in a dose-dependent, reversible increase in rotation rate as well as a dose-dependent, reversible decrease in surge frequency. The serotonin antagonist, mianserin, abolished the excitatory effect of exogenous serotonin. Furthermore, application of mianserin alone reduced rotation rate and virtually abolished rotational surges. Taken together, these pharmacological results suggest that endogenous serotonin is responsible for generating rotational surges. Given that early embryos contain only a single pair of serotonergic neurons (Goldberg and Kater, 1989) during the stages when rotational surges are expressed, these results also prompt the hypothesis that these neurons, embryonic neurons C1, act as cilioexcitatory motor neurons during embryonic development.

Glial fibrillary acidic protein, J1-31 antigen and vimentin in adult hamster brain: an immunohistochemical study.

Different regions of the prosencephalon and mesencephalon of the adult hamster brain displayed differences in the immunofluorescence expression of astrocytic proteins, namely glial fibrillary acidic protein and J1-31 antigen (30 kD protein). Neither of these proteins could be detected in layers II-VI of the cerebral cortex. However, varying degrees of immunostaining were detectable in perivascular glia, stria medullaris thalamus, the basal cerebral peduncle and the dentate molecular layer of the hippocampus. Vimentin was conspicuous in neurons, particularly in the cerebral cortex and hippocampus, and in glial fibrillary acidic protein-positive astrocytes in major fibre tracts. These observations are discussed in relation to interspecies differences in the expression of intermediate filament proteins.

Characterization of intermediate filament proteins in astroglia of hamster cerebellum.

This investigation was initiated to determine whether the organization of intermediate filament (IF) proteins is affected in the central nervous system of polymyopathic hamsters, as the Duchenne muscular dystrophy gene is normally expressed in the nervous system, in addition to cardiac and skeletal muscle, and changes in cell shape and cytoplasmic organization can serve as the regulators of growth, gene expression and cellular differentiation. The cerebellum of dystrophic hamster (CHF 146) was selected as the model system with the CHF 148 hamster providing the control for normal cerebellum. Using immunofluorescence microscopy for IF proteins, no difference could be detected in the cerebellum of dystrophic and normal hamsters. However, the glial fibrillary acidic protein and an IF-associated protein (J1-31 antigen), are lacking in Bergmann glia of the molecular layer. Moreover, vimentin persists in the cerebellum, its presence being most pronounced in a subset of Purkinje neurons in the adult hamster. These results are in contrast to those obtained in the rat cerebellum which has been extensively studied in respect to the organization of IFs.