Regulation of membrane trafficking by a novel Cdc42-related protein in Caenorhabditis elegans epithelial cells.

Rho GTPases are mainly known for their implication in cytoskeleton remodeling. They have also been recently shown to regulate various aspects of membrane trafficking. Here, we report the identification and the characterization of a novel Caenorhabditis elegans Cdc42-related protein, CRP-1, that shows atypical enzymatic characteristics in vitro. Expression in mouse fibroblasts revealed that, in contrast with CDC-42, CRP-1 was unable to reorganize the actin cytoskeleton and mainly localized to trans-Golgi network and recycling endosomes. This subcellular localization, as well as its expression profile restricted to a subset of epithelial-like cells in C. elegans, suggested a potential function for this protein in polarized membrane trafficking. Consistent with this hypothesis, alteration of CRP-1 expression affected the apical trafficking of CHE-14 in vulval and rectal epithelial cells and sphingolipids (C(6)-NBD-ceramide) uptake and/or trafficking in intestinal cells. However, it did not affect basolateral trafficking of myotactin in the pharynx and the targeting of IFB-2 and AJM-1, two cytosolic apical markers of intestine epithelial cells. Hence, our data demonstrate a function for CRP-1 in the regulation of membrane trafficking in a subset of cells with epithelial characteristics.

Does the source of support matter for different health outcomes? Findings from the Normative Aging Study.

OBJECTIVES: This study investigated the differential relationships between different types and sources of social support and physical and mental health. METHODS: Using data from the Normative Aging Study, 1,386 older men (median age = 62.7 years) were categorized into four groups separately for frequency of interaction with networks and perceived support. RESULTS: More than half the sample reported high levels of support from both sources. One-way ANOVAs revealed that those with high perceived support from both sources reported better physical health and fewer depressive symptoms than those with low support from both sources or high support from family alone. Similarly, those with high perceived support from both sources had lower levels of depressive symptoms than those with low support from both sources, but frequency of contact was unrelated to physical health. DISCUSSION: In general, those with high support from both family and friends reported the highest level of well-being.

Relations between health-related quality of life and well-being: the gerontologist's new clothes?

Is the recent construct of health-related quality of life (HQL) distinct from what gerontologists have long referred to as "well-being" or "life satisfaction?" We addressed this question using data from men in the VA Normative Aging Study to examine relations among twelve scales assessing HQL and seven scales of well-being (WB). We hypothesized that these two constructs would be distinct factorially, and that the derived factors would have different correlates. Correlations between scales of HQL and WB were moderate. When the nineteen scales were factored, four factors were extracted with HQL and WB scales generally loading on separate factors. The factors had distinct patterns of relations with general quality of life, personality, and the presence of a health problem, controlling for sociodemographics. These results suggest that HQL is distinct from the older construct of well-being. Although the two constructs are conceptually related, there is only a moderate amount of statistical overlap between them. Gerontologists should readily adopt health-related quality of life, which maintains continuity with such classics as well-being. This new construct, although needing slight alterations to broaden its assessment of well-being and life satisfaction, holds promise as more than an accessory in the study of health and well-being among older persons.

Production of stem-cell transplants according to good manufacturing practice.

Peripheral blood stem cells (PBSCs) are used for transplantation to reconstitute the hematopoietic system after high-dose chemotherapy. They are harvested from peripheral blood after mobilization by cytokines and/or chemotherapy. Further ex vivo manipulation steps (e.g., selection of CD34+ PBSCs, purging, expansion, and differentiation or gene transfer) can be performed. In 1997, more than 12,000 PBSC preparations were transplanted in Europe and the total number is steadily increasing [1]. To ensure quality and safety of the final cell products intended for clinical use, national and international guidelines and regulations have been issued. The implementation of a quality assurance (QA) program including the principles of good manufacturing practice (GMP) and a quality control system is a major requirement. GMP regulations apply to all phases of cell collection, processing, and storage, and to documentation, training of personnel, and equipment of the cell processing laboratory. They have to be followed by pharmaceutical companies and medical doctors who are involved in PBSC processing at academic institutions. The complicated regulatory network for the manufacturing of cell products will help to standardize these procedures and ensure consistent quality and safety in the long term. This will be in the interest of patients and reduce risks of application of individual cell preparations.

Employment and health among older bereaved men in the normative aging study: one year and three years following a bereavement event.

Research has indicated that the negative effects of bereavement on health among elderly men occur within the first six to twelve months following a bereavement event while other studies indicate that the death of a loved one can have long-term effects on social functioning and mental health (Arbuckle & DeVries, 1995; Vinick, 1983a). However, employment has been found to buffer the strain produced by stressful life events. The purpose of this study was to examine the differential effects of employment on physical and mental health between elderly men bereaved for one year and elderly men bereaved for two to three years. We selected two groups of men from the Normative Aging Study: those bereaved within the past year (N = 248) and those bereaved from two to three years (N = 262). Ordinary least squares multiple regression analyses examined the direct effect of employment, controlling for age, education, income, marital status, and stress, on physical and mental health among the two groups of men. Although there were no significant differences between the two groups of men, the results from the separate analyses indicated that employment had a direct positive effect on physical health among those bereaved for one year and those bereaved from two to three years, but no significant effects were observed on mental health. The results suggest that employment can benefit men soon after a bereavement event and also over a longer period of time, especially on physical health. Implications for clinical practice and future research are discussed.

Gonadal hormones modulate 5-hydroxytryptamine2A receptors: emphasis on the rat frontal cortex.

The effect of short-term (Sprague-Dawley rats, two weeks) and long-term ovariectomy (Sprague-Dawley and Fischer rats, three months) on serotonin 5-hydroxytryptamine2A receptors in different regions of the brain and its possible correction with an 17 beta-estradiol treatment (10 micrograms, b.i.d., two weeks) were studied in comparison to intact rats. Saturation binding assays were performed using [3H]ketanserin to estimate 5-hydroxytryptamine2A receptor density and affinity in tissue homogenates of frontal cortex of Fischer rats and quantitative autoradiography was performed to evaluate receptor specific binding in frontoparietal cortex, nucleus accumbens, striatum and dorsal raphe nucleus of Fischer rats, and in frontal cortex of the two strains of rats. Messenger RNA levels of 5-hydroxytryptamine2A receptors were measured by in situ hybridization in frontal cortex of the two strains of rats. An overall decrease of 5-hydroxytryptamine2A receptor densities was found in all the brain regions of ovariectomized Fischer rats assayed, and this could be restored towards control levels by estradiol treatment. No change in the 5-hydroxytryptamine2A receptor affinity was measured in the frontal cortex. A similar pattern of changes was observed for the messenger RNA levels encoding the 5-hydroxytryptamine2A receptors and receptor density, suggesting the implication of a genomic mechanism. Experiments in Sprague-Dawley rats confirmed and extended the results obtained with Fischer rats. By analogy, in humans, this 5-hydroxytryptamine2A receptor modulation may underlie the mood and movement disorders associated with menopause.

Anterior pituitary hypoplasia and dwarfism in mice lacking the dopamine transporter.

Deletion of the dopamine transporter (DAT) results in increased dopaminergic tone, anterior pituitary hypoplasia, dwarfism, and an inability to lactate. DAT elimination alters the spatial distribution and dramatically reduces the numbers of lactotrophs and somatotrophs in the pituitary. Despite having normal circulating levels of growth hormone and prolactin in blood, hypoplastic glands from DAT-/- mice fail to respond to secretagog stimulation. The effects of DAT deletion on pituitary function result from elevated DA levels that down-regulate the lactotroph D2 DA receptors and depress hypothalamic growth hormone-releasing hormone content. These results reveal an unexpected and important role or DA in the control of developmental events in the pituitary gland and assign a critical role for hypothalamic DA reuptake in regulating these events.

Ovariectomy and estradiol treatment affect the dopamine transporter and its gene expression in the rat brain.

The impact of gonadal hormone withdrawal and estrogen therapy was investigated on the rat dopamine transporter (DAT). Short-term ovariectomized (ST-OVX, 2 weeks) and long-term ovariectomized (LT-OVX, 3 months) rats were treated or not with 17beta-estradiol (E2) for 2 weeks. DAT mRNA expression was measured by in situ hybridization in the substantia nigra pars compacta (SNc) for the nigrostriatal pathway and the ventral tegmental area (VTA) for the mesolimbic pathway whereas DAT levels were assessed by [3H]GBR-12935 autoradiography, respectively, in the striatum and the nucleus accumbens. Ovariectomy produced a time-dependent decrease of the DAT density in the striatum and the nucleus accumbens and the E2 treatment did not significantly restore these DAT levels. Neither ST-OVX nor E2 treatment of the ST-OVX animals altered the DAT mRNA expression in the SNc and the VTA. However, LT-OVX animals showed increased DAT mRNA levels in these regions. E2 treatment of LT-OVX animals partially restored DAT mRNA levels in the SNc and left these levels unchanged in the VTA. These opposite variations induced by OVX on the DAT density and their mRNA levels suggest the involvement of non-genomic mechanisms, such as post-transcriptional events and/or membrane effects. Altered neurotransmission following gonadal hormone withdrawal may contribute to CNS disorders occurring at menopause in predisposed women. Ovariectomized rats constitute a useful model to study the changes in neurotransmitters balance occurring after menopause.

Differential effect of E-selectin antibodies on neutrophil rolling and recruitment to inflammatory sites.

The selectins are inducible adhesion molecules critically important for the inflammatory response. We investigate here the functional effects of three monoclonal antibodies (MoAbs) raised against murine E-selectin (9A9, 10E6, and 10E9.6) on neutrophil recruitment in vivo, leukocyte rolling and circulating leukocyte concentrations in vivo, and adhesion of myeloid cells to E-selectin transfectants and recombinant E-selectin-IgG fusion protein in vitro. MoAbs 9A9 and 10E6 map to the lectin and epidermal growth factor (EGF)-like domains of murine E-selectin, whereas 10E9.6 binds to the consensus repeat region. 10E9.6 blocked neutrophil recruitment in a model of thioglycollate-induced peritonitis in Balb/c mice by more than 90% but had no effect in C57BL/6 mice. 9A9 and 10E6 blocked neutrophil recruitment in this assay only when combined with a P-selectin antibody, 5H1. Neither 9A9 nor 10E9.6 alone blocked leukocyte rolling in tumor necrosis factor-alpha-treated venules of Balb/c mice, but 9A9 almost completely inhibited leukocyte rolling when combined with the function-blocking murine P-selectin MoAb, RB40.34. In contrast, 10E9.6 had no effect on leukocyte rolling in RB40.34-treated Balb/c or C57BL/6 mice. 10E9.6 did not affect adhesion of myeloid cells to E-selectin transfectants or attachment, rolling, and detachment of myeloid cells to murine E-selectin-IgG fusion protein. However, adhesion was completely blocked in the same assays by 9A9. Taken together, these results indicate that E-selectin serves a function, other than rolling, that appears to be critically important for neutrophil recruitment to inflammatory sites in Balb/c mice.

VE-cadherin antibody accelerates neutrophil recruitment in vivo.

Neutrophils enter sites of inflammation by crossing the endothelial lining of the blood vessel wall. VE-cadherin is an endothelial specific, homophilic adhesion molecule located at the lateral cell surface. We have generated a monoclonal antibody against mouse VE-cadherin which inhibits electrical resistance of endothelial cell monolayers in vitro as well as aggregation of VE-cadherin transfected cells. In vivo, this antibody was found to increase vascular permeability and to accelerate the entry of neutrophils into chemically inflamed mouse peritoneum. Thus, VE-cadherin is essential for the integrity of the endothelial barrier in vivo. Our data suggest that opening of VE-cadherin mediated endothelial cell contacts may be a relevant step during neutrophil extravasation.

Good manufacturing practice production of human stem cells for somatic cell and gene therapy.

Peripheral blood stem cells (PBSC) are used for transplantation to reconstitute the hematopoietic system after high-dose chemotherapy. PBSC are harvested from peripheral blood upon successful mobilization by cytokines and/or chemotherapy. Further in vitro manipulation steps like enrichment of CD34+ PBSC or gene transfer can be performed. To ensure the quality and safety of the final cell preparations intended for transplantation, national and international guidelines and regulations have been issued. Herein the implementation of a quality assurance program including the principles of good manufacturing practice (GMP) and a quality control (QC) system is one major concern. GMP regulations apply to all phases of cell collection, processing and storage as well as documentation, training of personnel, and the laboratory facility. QC measures have to be taken to ensure consistent quality and safety with an emphasis on preventing any deficiencies.

Activated T cells induce expression of E-selectin in vitro and in an antigen-dependent manner in vivo.

E-selectin is an endothelial adhesion molecule for polymorphonuclear cells, monocytes and skin-homing T cells. We have analyzed whether murine T cells are able to induce expression of E-selectin in vitro and in vivo. Using models of inflammation in which T cells play either a significant or only a minor role, we compared induction of E-selectin between normal mice and mice lacking functional T cells (athymic nude mice). In irritant contact dermatitis, a model without a major role for T cells, E-selectin was transiently expressed within the first 24 h in both normal and nude mice. In experimental leishmaniasis (where specific T cells play an important role), a high expression of E-selectin was maintained for 48 h in normal mice, whereas in nude mice expression was only transient. However, reconstitution of nude mice with 10(8) T cells from draining lymph nodes (LN) of Leishmania-infected normal mice could restore sustained expression of E-selectin. Transfer of T lymphocytes from normal LN or from LN of mice sensitized to the contact allergen trinitrochlorobenzene (TNCB) did not have this effect. T cells from TNCB-sensitized mice, however, did induce sustained expression of E-selectin in nude mice when TNCB was applied locally; here, reconstitution with Leishmania-specific T cells had no effect. In vitro, T cells from infected or TNCB-sensitized normal mice increased expression of E-selectin on microvascular endothelial cells after 4 h of co-culture. T cells from untreated mice were less effective. Induction was dependent on direct cell-cell contact, but not on the action of interleukin-1 alpha, interleukin-1 beta, tumor necrosis factor-alpha or interferon-gamma. We conclude that sensitized T cells induce sustained expression of E-selectin in vivo in an antigen-dependent manner. This novel way of regulation could be relevant for cell-mediated immunity and chronic disease. The mechanisms are unknown, but, as in vitro, might require direct cell-cell contact.

Assessing oral health-related quality of life: findings from the normative aging study.

The contribution of oral health to health-related quality of life (HQOL) has seldom been examined. This study was designed to develop and validate a measure of oral health-related quality of life (OHQOL), examine relations between OHQOL and HQOL, and explore OHQOL's relation to problem-based dental care utilization in a sample of 1,242 older men, using data gathered by a mail survey. Factor analysis of eight oral health items revealed that three items related to the impact of oral conditions on daily functioning defined a factor labeled OHQOL. Factor analysis of the HQOL items and these three OHQOL items showed that OHQOL represents a separate and independent factor. Correlational analyses supported the construct validity of the OHQOL measure: Men with better OHQOL scores reported less dental pain or discomfort, fewer eating problems, and less problem-based dental care utilization. Logistic regression analysis showed that the measures of dental pain and oral discomfort were related positively to utilization, whereas OHQOL was related negatively. These results suggest that OHQOL represents a separate and distinct facet of HQOL that is associated with dental care utilization. Thus they support the validity of the OHQOL construct and suggest its use in future studies of HQOL.

The modulation of brain dopamine and GABAA receptors by estradiol: a clue for CNS changes occurring at menopause.

1. Tardive dyskinesia is more important in postmenopausal women than men of comparable age and a peak of first episodes of schizophrenia is observed in postmenopausal women. The effect of ovariectomy (2 weeks or 3 months) in rats was investigated as a model of decreased gonadal function associated with menopause. 2. Frontal cortex D1 receptor density and affinity were similar in intact male compared to intact female rats and progressively decreased in density with time after ovariectomy, with no change of affinity. Striatal D1 and D2 receptors also decreased in density after ovariectomy for both receptor subtypes, with no change of affinity. Striatal D1 receptor density and affinity were similar in intact male and female rats, whereas the density of D2 receptors was higher in females. Treatment with estradiol for 2 weeks restored the D2 but not the D1 receptor changes. 3. In the substantia nigra pars reticulata, striatum, nucleus accumbens, and entopeduncular nucleus, a progressive increase in [3H]flunitrazepam specific binding associated with GABAA receptors was observed as a function of time following ovariectomy; this was corrected with estradiol treatment. In contrast, the opposite was observed for [3H] flunitrazepam binding in the globus pallidus, where ovariectomy decreased binding, which was corrected with estradiol replacement therapy. 4. Low prefrontal cortex dopamine activity with implications of D1 receptors in negative symptoms of schizophrenia is hypothesized. Furthermore, GABAergic overactivity in the internal globus pallidus-substantia nigra pars reticulata complex is hypothesized in tardive dyskinesia. 5. The present data suggest that gonadal hormone withdrawal by reducing brain dopamine receptors and producing an imbalance of GABAA receptors in the output pathways of the striatum may predispose to schizophrenia and dyskinesia.

Dopamine and GABAA receptor imbalance after ovariectomy in rats: model of menopause.

A peak of first episodes of schizophrenia can occur in postmenopausal women. Furthermore, tardive dyskinesia is more common in postmenopausal women than in men of comparable age. This study investigated the effect of ovariectomy (2 weeks or 3 months) in rats as a model of decreased gonadal function associated with menopause. After ovariectomy, frontal cortex D1 receptors progressively decreased in density with no change of affinity over time. Striatal D1 and D2 receptors also had decreased density after ovariectomy with no change of affinity. In the substantia nigra pars reticulata, a progressive increase in [3H]flunitrazepam-specific binding associated with GABAA receptors was observed as a function of time following ovariectomy. It is hypothesized that low prefrontal cortex dopamine activity has implications in negative symptoms of schizophrenia and, furthermore, that GABAergic overactivity in the internal globus pallidus-substantia nigra pars reticulata complex plays a role in tardive dyskinesia. The present results suggest that, by reducing brain dopamine receptors and increasing GABAA receptors, gonadal hormone withdrawal may predispose to schizophrenia and dyskinesia.

P-selectin/ICAM-1 double mutant mice: acute emigration of neutrophils into the peritoneum is completely absent but is normal into pulmonary alveoli.

Neutrophil emigration during an inflammatory response is mediated through interactions between adhesion molecules on endothelial cells and neutrophils. P-Selectin mediates rolling or slowing of neutrophils, while intercellular adhesion molecule-1 (ICAM-1) contributes to the firm adhesion and emigration of neutrophils. Removing the function of either molecule partially prevents neutrophil emigration. To analyze further the role of P-selectin and ICAM-1, we have generated a line of mice with mutations in both of these molecules. While mice with either mutation alone show a 60-70% reduction in acute neutrophil emigration into the peritoneum during Streptococcus pneumoniae-induced peritonitis, double mutant mice show a complete loss of neutrophil emigration. In contrast, neutrophil emigration into the alveolar spaces during acute S. pneumoniae-induced pneumonia is normal in double mutant mice. These data demonstrate organ-specific differences, since emigration into the peritoneum requires both adhesion molecules while emigration into the lung requires neither. In the peritoneum, P-selectin-independent and ICAM-1-independent adhesive mechanisms permit reduced emigration when one of these molecules is deficient, but P-selectin-independent mechanisms cannot lead to ICAM-1-independent firm adhesion and emigration.

Sequential contribution of L- and P-selectin to leukocyte rolling in vivo.

Leukocyte recruitment into inflammatory sites is initiated by a reversible transient adhesive contact with the endothelium called leukocyte rolling, which is thought to be mediated by the selectin family of adhesion molecules. Selectin-mediated rolling precedes inflammatory cell emigration, which is significantly impaired in both P- and L-selectin gene-deficient mice. We report here that approximately 13% of all leukocytes passing venules of the cremaster muscle of wild-type mice roll along the endothelium at < 20 min after surgical dissection. Rolling leukocyte flux fraction reaches a maximum of 28% at 40-60 min and returns to 13% at 80-120 min. In P-selectin-deficient mice, rolling is absent initially and reaches 5% at 80-120 min. Rolling flux fraction in L-selectin-deficient mice is similar to wild type initially and declines to 5% at 80-120 min. In both wild-type and L-selectin-deficient mice, initial leukocyte rolling (0-60 min) is completely blocked by the P-selectin monoclonal antibody (mAb) RB40.34, but unaffected by L-selectin mAb MEL-14. Conversely, rolling at later time points (60-120 min) is inhibited by mAb MEL-14 but not by mAb RB40.34. After treatment with tumor necrosis factor (TNF)-alpha for 2 h, approximately 24% of all passing leukocytes roll in cremaster venules of wild-type and P-selectin gene-deficient mice. Rolling in TNF-alpha-treated mice is unaffected by P-selectin mAb or E-selectin mAb 10E9.6. By contrast, rolling in TNF-alpha-treated P-selectin-deficient mice is completely blocked by L-selectin mAb. These data show that P-selectin is important during the initial induction of leukocyte rolling after tissue trauma. At later time points and in TNF-alpha-treated preparations, rolling is largely L-selectin dependent. Under the conditions tested, we are unable to find evidence for involvement of E-selectin in leukocyte rolling in mice.

Only simultaneous blocking of the L- and P-selectin completely inhibits neutrophil migration into mouse peritoneum.

We have examined the inhibitory effect of monoclonal antibodies against mouse P-, E- and L-selectin on the migration of neutrophils into the chemically inflamed peritoneum of the mouse. For this purpose; monoclonal antibodies were raised against mouse P- and E-selectin, which block cell adhesion. We found that blocking of each selectin alone inhibited neutrophil migration to a similar degree ranging from 63% to 72%. Of the three possible combinations of antibodies against two different selectins only the combination of anti-P- and anti-L-selectin antibodies caused an essentially complete blockade of neutrophil emigration. Only the effects of these two antibodies were additive, while the effect of anti-E-selectin antibodies did not add to the effect of antibodies against P- or L-selectin. Thus, although E-selectin is involved in neutrophil migration into the inflamed peritoneum of the mouse, it cannot compensate the block of the other two selectins which seem to play the dominant role in this process.