Paranoid Thoughts in Adolescents with Social Anxiety Disorder.

Recently, social anxiety disorder (SAD) and paranoia have been demonstrated to be closely related. However, data were primarily drawn from adult community samples or patients with schizophrenia. The present study used a cross-sectional design to evaluate a sample of adolescents with SAD (n = 30, mean age 15.3 ± 0.9 years) compared with an age- and sex-matched group of healthy controls (n = 26, mean age 15.9 ± 1.6 years). The SAD group displayed more frequent and intense paranoid thoughts than the control group (t = 4.16, p < 0.001). The level of paranoid thoughts was significantly predicted by the degree of social phobia, even after adjusting for sex and other anxiety disorders, although adjusting for depression slightly reduced the extent and significance of the prediction. A lack of awareness about the association between SAD and paranoia may lead to incorrect diagnoses (e.g. misdiagnosis of psychotic disorders), or it may negatively influence the (psycho)therapeutic process and patient outcomes.

A case of Klinefelter syndrome, mosaicism (46,XY/47,XXY), associated with anorexia nervosa.

We report the case of a 12.4-yr-old boy who presented Klinefelter syndrome (KS) mosaicism (46,XY/47,XXY), associated with mental retardation and anorexia nervosa (AN). KS was undiagnosed before hospitalization in a psychiatric unit. The patient was referred to a child psychiatric unit for restrictive eating. The medical history showed long standing feeding difficulties and failure to thrive. The patient was pre-pubertal and other clinical characteristics were: microcephaly, short stature and dysmorphic traits. Cytogenetic analysis revealed a mosaicism, 46,XY[11] and 47,XXY[19] karyotype. The psychiatric assessment demonstrated the presence of AN and low mood. No specific pathophysiological links between the alterations of KS and the development of AN should be hypothesized on the basis of this case report. In pre-pubertal boys with mental disorders, the possibility of KS should be considered, independently of the presence of eating disorders. Nevertheless, the case shows that KS can be first detected during an assessment for eating disorders. Few cases of the association of KS with AN have been previously reported in literature. This is the first description of KS, mosaicism (46,XY/47,XXY), associated with AN and mental retardation. This case report illustrates the need, for clinicians who work with eating disorders, to investigate the possible association between AN and KS, a rare but intriguing one.

Skeletal myogenic potential of human and mouse neural stem cells.

Distinct cell lineages established early in development are usually maintained throughout adulthood. Thus, adult stem cells have been thought to generate differentiated cells specific to the tissue in which they reside. This view has been challenged; for example, neural stem cells can generate cells that normally originate from a different germ layer. Here we show that acutely isolated and clonally derived neural stem cells from mice and humans could produce skeletal myotubes in vitro and in vivo, the latter following transplantation into adult animals. Myogenic conversion in vitro required direct exposure to myoblasts, and was blocked if neural cells were clustered. Thus, a community effect between neural cells may override such myogenic induction. We conclude that neural stem cells, which generate neurons, glia and blood cells, can also produce skeletal muscle cells, and can undergo various patterns of differentiation depending on exposure to appropriate epigenetic signals in mature tissues.

Inadequate dietary intake but not renal tubular acidosis is associated with bone demineralization in primary biliary cirrhosis.

BACKGROUND: Metabolic bone disease associated with primary biliary cirrhosis (PBC) is inadequately characterized. Renal tubular acidosis (RTA) may lead to bone loss through chronic mobilization of skeletal calcium salts to buffer increased acid load. AIM: To evaluate the prevalence of RTA in PBC and establish the relationships among bone mineral density (BMD), renal function and nutritional status. METHODS: We enrolled 69 female patients with compensated PBC and 35 control patients with chronic hepatitis C. RTA was searched in all patients, and 24-h dietary recalls were collected at enrolment. BMD was measured by dual-energy X-ray absorptiometry at the femur neck, lumbar spine and radius ultradistalis sites. RESULTS: No patients received a diagnosis of RTA. BMD values (Z-scores) showed only little deviation from normal population with no difference between PBC and controls. Osteopoenic PBC patients (T-score < 1) showed significantly lower daily phosphorus intake [median: 672 (288-1374) vs. 921 (253-1923) mg/day; P = 0.037], with a trend towards lower caloric intake than their nonosteopoenic counterparts. CONCLUSIONS: Renal tubular acidosis is uncommon in compensated PBC. Cholestasis is not associated with an increased risk of bone demineralization. Inadequate dietary intake may be a preventable factor contributing to bone loss in PBC.

Adult neurogenesis in serotonin transporter deficient mice.

Serotonin (5-HT) is a regulator of morphogenetic activities during early brain development and neurogenesis, including cell proliferation, migration, differentiation, and synaptogenesis. The 5-HT transporter (5-HTT, SLC6A4) mediates high-affinity reuptake of 5-HT into presynaptic terminals and thereby fine-tunes serotonergic neurotransmission. Inactivation of the 5-HTT gene in mice reduces 5-HT clearance resulting in persistently increased concentrations of synaptic 5-HT. In the present study, we investigated the effects of elevated 5-HT levels on adult neurogenesis in the hippocampus of 5-HTT deficient mice, including stem cell proliferation, survival, and differentiation. Using an in vivo approach, we showed an increase in proliferative capacity of hippocampal adult neural stem cells in aged 5-HTT knockout mice (approximately 14.5 months) compared to wildtype controls. In contrast, in vivo and additional in vitro analyses of younger adult 5-HTT knockout mice (approximately 7 weeks and approximately 3.0 months) did not reveal significant changes in proliferation of neural stem cells or survival of newborn cells. We showed that the cellular fate of newly generated cells in 5-HTT knockout mice is not different with respect to the total number and percentage of neurons or glial cells from wildtype controls. Our findings indicate that elevated synaptic 5-HT concentration throughout early development and later life of 5-HTT deficient mice does not induce adult neurogenesis in adult mice, but that elevated 5-HT levels in aged mice influence stem cell proliferation.

Non-virally mediated gene transfer into human central nervous system precursor cells.

Lipofectamine-based transfection was used as a method of choice to deliver the bacterial beta-galactosidase gene into human central nervous system (CNS) precursor cells. We achieved a transfection efficiency of 7.4%. beta-Galactosidase expressing cells were shown to display both neuronal and glial phenotypes. We also delivered the temperature sensitive allele of SV40 Large-T antigen and obtained a high level of expression of the immortalizing oncoprotein in the cells. Colonies of Large-T antigen immunoreactive cells were indeed visible 10 days after transfection.

Isolation and intracerebral grafting of nontransformed multipotential embryonic human CNS stem cells.

In this work, we show that the embryonic human brain contains multipotent central nervous system (CNS) stem cells, which may provide a continuous, standardized source of human neurons that could virtually eliminate the use of primary human fetal brain tissue for intracerebral transplantation. Multipotential stem cells can be isolated from the developing human CNS in a reproducible fashion and can be exponentially expanded for longer than 2 years. This allows for the establishment of continuous, nontransformed neural cell lines, which can be frozen and banked. By clonal analysis, reverse transcription polymerase chain reaction, and electrophysiological assay, we found that over such long-term culturing these cells retain both multipotentiality and an unchanged capacity for the generation of neuronal cells, and that they can be induced to differentiate into catechlaminergic neurons. Finally, when transplanted into the brain of adult rodents immunosuppressed by cyclosporin A, human CNS stem cells migrate away from the site of injection and differentiate into neurons and astrocytes. No tumor formation was ever observed. Aside from depending on scarce human neural fetal tissue, the use of human embryonic CNS stem cells for clinical neural transplantation should provide a reliable solution to some of the major problems that pertain to this field, and should allow determination of the safety characteristics of the donor cells in terms of tumorigenicity, viability, sterility, and antigenic compatibility far in advance of the scheduled day of surgery.

Long-term survival of cortical neurones from adult guinea-pig maintained in low-density cultures.

In vitro survival of neurones isolated from adult mammalian brain is normally scarce and the postnatal age limit for obtaining viable cultures of cortical, hippocampal and diencephalic neurones is commonly two weeks. Here we describe a novel procedure for the establishment and long-term maintenance of cortical neurones of the adult mammalian brain in low-density cultures. Neurones isolated from the piriform cortex of 30- to 90-day-old guinea-pigs were initially grown in a chemically defined medium enriched with basic fibroblast growth factor (bFGF); later, a small quantity of foetal bovine serum (FBS) was added to facilitate cell differentiation. Under these conditions cells could be maintained in culture for at least 3 weeks, when indirect immunocytochemistry and whole-cell patch-clamp recordings were performed. Cells exhibiting neuronal morphology expressed the neuronal marker microtubule associated protein-2 (MAP2) and generated action potentials. Moreover, about 70% of the MAP2-immunoreactive cells were simultaneously labelled with anti-gamma-aminobutyric acid (GABA) antibody. Cells expressing neuronal antigens were never labelled by antibody raised against the glial marker glial fibrillary acidic protein (GFAP). These results indicate that long-term survival of adult neurones can be achieved under definite culture conditions.

Differential effects of CGRP on adenylyl cyclase in adult and embryonic rat brain.

The presence of functional receptors for calcitonin gene-related peptide (CGRP) in the brain of adult rats and on nerve cell cultures was investigated. Neuronal and glial cultures were obtained from mesencephalons of embryos at gestational day 16. The response to CGRP was tested by measuring the adenylyl cyclase (AC) activity on isolated membranes. CGRP binding in adult rat brains was ineffective in activating AC, whereas a dose-dependent stimulation of AC activity was induced by the peptide both in neuronal and glial cultures. This effect was more pronounced in the glial cells where high affinity binding sites for CGRP were detected. The presence of functional CGRP receptors in embryonic mesencephalic cells, suggests a role for CGRP in the development of rat mesencephalon.

Basic fibroblast growth factor supports the proliferation of epidermal growth factor-generated neuronal precursor cells of the adult mouse CNS.

Stem cells isolated from the CNS of both embryonic and adult mice undergo extensive proliferation in the presence of epidermal growth factor (EGF). Removal of EGF determines the differentiation of these cells into neurons and glia. We have recently demonstrated that basic fibroblast growth factor (bFGF) regulates the proliferation of EGF-generated progenitors of the embryonic mouse striatum. We report here that bFGF induces proliferation of some EGF-generated precursors of the adult mouse striatum which, in turn, differentiate in vitro into cells possessing neuron-like morphology and neuronal antigenic properties. These results demonstrate that EGF and bFGF can act sequentially to regulate the de novo generation of neurons from the adult mouse CNS in vitro and suggest the existence of a lineage relationship between EGF- and bFGF-responsive progenitor cells of the adult murine brain.

The neural stem cells and their transdifferentiation capacity.

Stem cells play a critical role during embryo and tissue formation throughout development. Thanks to their multipotentiality - i.e., the ability to give rise to different lineages of mature cells - and to their extensive capacity for self-renewal and expansive growth, stem cells can also contribute to the maintenance of tissue integrity in adulthood. Historically, it has been held that fetal and adult (somatic) stem cells are tissue-specific 'entities' whose differentiation potential is limited to the generation of mature cell types of the tissue/organ in which they reside. Yet, recent years have seen the publication of an impressive sequence of reports dealing with what is now emerging as one of the most striking functional attributes of somatic stem cells, that is, their capacity to undergo transdifferentiation. Thanks to this peculiar characteristic adult stem cells display an unexpected ability to give rise to differentiated cells of tissues and organs different from those in which they reside. This commentary briefly illustrates the characteristics of the neural stem cell and its capacity as a neuroectodermal derivative to undergo transdifferentiation, thus giving rise to differentiated cells that normally originate from the mesoderm, like blood or skeletal muscle cells.

Structure of the lymphatic microcirculation in the human urinary bladder with different intraluminal pressure and distension.

The localization, morphology and fine structure of initial lymphatic vessels in the mucosa of the empty and distended urinary bladder were studied. Endoscopic transurethral biopsies of the empty (collapsed) bladder showed under light and electron microscopy numerous intramural lymphatics with a dilated lumen and thin profile. Contacts between endothelial cells were single, overlapping, interlocking, and open while the perivascular connective tissue was filled by fascicles of collagen fibers. In the most superficial layer (subepithelial mucosa), lymphatics were not seen. Biopsies obtained under elevated intraluminal pressure and distension showed on light and electron microscopy lymphatic vessels with small lumens characteristically reduced to irregular slits. Endothelial cell contacts were simple or overlapping; open junctions were rare. The perivascular connective tissue was dense and collagen and elastic fibers often abutted one another. These findings support that with a distended or expanded urinary bladder, the effect of increased intraluminal pressure on the superficial (mucosal) layer radially pulls on the connective tissue that in turn compresses the initial lymphatics thereby restricting lymph transport.

Structure of the initial lymphatics of the human urinary bladder with invasive urothelial tumors.

The ability of urothelial tumors of the urinary bladder to metastasize via the lymphatic circulation and the extent of metastatic involvement of regional lymph nodes is an important parameter in the staging and prognosis of these neoplasms. Accordingly, we examined the site and morphology of initial lymphatic vessels in the mucosa of the human urinary bladder in patients with invasive transitional cell carcinoma. Lymphatics in the papillary tumoral mass was also examined. Endoscopic transurethral biopsies from the urinary bladder of 120 patients with invasive transitional cell papillary carcinoma were utilized for this study. Biopsy from the uninvolved lateral wall of the same patient was utilized as a control. On histopathology of biopsies of neoplastic tissues, initial lymph vessels were seen in the deeper region of the mucosa but not in the subepithelial layer nor in the stroma of the tumoral papillae. The latter were often associated with arteriolar and venular vessels. When edema and inflammation occurred in peritumoral regions, lymphatics showed a dilated lumen, non-indented wall with dissociated perivascular collagen and elastic fibers. Tumoral permeation or embolization of lymphatics was seen in 12% of patients with invasive tumors, and these lymphatic vessels did not display significant morphologic changes. The absence of initial lymphatics in the stroma of tumoral papillae and in infiltrated subepithelial regions of the urinary bladder may explain the absence of lymph node metastasis in early-stage invasive urothelial tumors.

Composition and organization of the SCZ: a large germinal layer containing neural stem cells in the adult mammalian brain.

Two known germinal zones continue to generate new neurons and glia in the adult mammalian brain: the subventricular zone (SVZ), lining the lateral walls of the lateral ventricle, and the subgranular zone of the dentate gyrus. Here we describe a region we will refer to as the subcallosal zone (SCZ). The SCZ is a caudal extension of the SVZ that is no longer associated to an open ventricle. It lies between the hippocampus and the corpus callosum. Cells isolated from the SCZ and cultured as neurospheres behave as neural stem cells in vitro. Using electron and light microscopy, we describe the cell types present in this region and how their organization differs from that of the SVZ. Using retroviral labeling and homotypic-homochronic microtransplantation techniques, we show that the majority of cells born in the SCZ migrate into the corpus callosum to become oligodendrocytes in vivo. This study defines the organization and fate of cells born in a large germinal region of the adult forebrain.

Hypercholesterolaemia is not associated with early atherosclerotic lesions in primary biliary cirrhosis.

BACKGROUND: Hypercholesterolaemia often occurs in primary biliary cirrhosis (PBC) as a result of chronic cholestasis, but whether these patients are exposed to greater cardiovascular risk is unknown. AIM: To establish whether hypercholesterolaemia is associated with subclinical atherosclerosis in PBC. PATIENTS: 103 consecutive patients with PBC (37 with total cholesterol > or =6.21 mmol/l) and 37 controls with hypercholesterolaemia, and 141 matched controls with normocholesterolaemia. METHODS: Ultrasound imaging of carotid artery to determine intima-media thickness (IMT) and stenosis. RESULTS: Controls with hypercholesterolaemia had higher IMT and prevalence of carotid stenosis compared with patients with hypercholesterolaemic PBC (mean (SD) 0.850 (0.292) mm v 0.616 (0.137) mm, p(c)<0.001; 43% v 19%, p(c) = 0.129) who, in turn, were similar to the 66 patients with normocholesterolaemic PBC (0.600 (0.136) mm; 5%). Compared with subjects with normocholesterolaemia, controls with hypercholesterolaemia, but not patients with hypercholesterolaemic PBC, had an increased risk of raised IMT (odds ratio (OR) 5.4, 95% confidence interval (CI) 2.5 to 11.9, p<0.001; and 0.7, 0.3 to 2.0, p = 0.543) or carotid stenosis (8.2, 3.4 to 20, p<0.001; and 2.5, 0.9 to 6.9, p = 0.075). In PBC, compared with younger patients without hypertension, the risk of increased IMT was OR (CI) 3.1 (0.6 to 17; p = 0.192) in patients with hypertension or old age, but not hypercholesterolaemia, and 4.6 (0.8 to 27; p = 0.096) in patients who also had hypercholesterolaemia. The corresponding figures for risk of stenosis were 3.6 (0.4 to 36; p = 0.277) and 15.8 (1.8 to 141; p = 0.014). CONCLUSIONS: Hypercholesterolaemia is not consistently associated with subclinical atherosclerosis in PBC, but should be treated if other risk factors for cardiovascular disease are also present. The search for factors that may protect patients with hypercholesterolaemic PBC against atherosclerosis should be encouraged.

The insulin tolerance test and ovine corticotrophin-releasing-hormone test in episodic cluster headache. II: Comparison with low back pain patients.

Hypothalamic involvement has been invoked to explain the periodicity of the cluster periods and rhythmicity of the pain attacks in cluster headache. To explore this hypothesis the ovine corticotrophin-releasing headaches sufferers during both cluster period and remission. A group of low back pain patients and healthy subjects comprised the control populations. For the o-CRH test, 7 healthy subjects, 7 low back pain patients, 6 cluster headache patients in remission, and 12 in cluster period were studied. Five healthy subjects, 7 low back pain patients, 6 cluster headache patients in remission, and 9 cluster period were administered the insulin tolerance test. Significantly increased basal cortisol levels were found in cluster headache patients in both illness phases (p < 0.0001), but not in low back pain patients. Significantly reduced cortisol response to the o-CRH test was observed in cluster headache patients in both phases compared to healthy controls (p < 0.02). A blunted ACTH and cortisol response (p < 0.0001 and p < 0.003 respectively) to the insulin tolerance test was present in cluster headache patients in both phases of the illness compared to healthy subjects and low back pain patients. On the contrary, the ACTH surge after insulin induced hypoglycemia was significantly increased in the low back pain patient group (p = 0.02). These results suggest that the altered hypothalamic-pituitary-adrenal axis responsiveness in cluster headache patients is not a consequence of the pain, and point to a central, probably hypothalamic derangement in this pathology.

Effect of hyperthermia on rhodamine 123 cytotoxicity in doxorubicin-sensitive and doxorubicin-resistant human breast carcinoma cell lines in vitro.

Rhodamine 123 (Rh123) cytotoxicity and intracellular accumulation were studied in normothermic and hyperthermic conditions in a human breast carcinoma cell line (MCF7/WT) and its doxorubicin-resistant subline (MCF7/DoxR). MCF7/DoxR cells were resistant to hyperthermia and Rh123. Hyperthermic potentiation of Rh123 cytotoxicity was present in MCF7/WT but not in MCF7/DoxR cells. Results suggest that the effect observed in MCF7/WT cells is related to a heat-induced increased accumulation of Rh123 and not to a heat-induced activation of the drug. A low basal uptake and a fast release of Rh123 could explain the resistance of MCF7/DoxR to the drug. Resistance to Rh123 and lack of a heat-induced increased uptake account for the lack of hyperthermic enhancement of Rh123 cytotoxicity in the resistant cells.

Regulation of neuronal differentiation in human CNS stem cell progeny by leukemia inhibitory factor.

The generation of diverse types of neural cells during development occurs through the progressive restriction of the fate potential of neuroepithelial progenitor cells. This process is controlled by factors intrinsic and extrinsic to the cell. While the effect of extrinsic cues on multipotent stem cells of the murine central nervous system (CNS) is becoming clearer, little is known of neural stem cells of human origin. We sought to establish the roles played by two cytokines, leukemia inhibitory (LIF) and ciliary neurotrophic factor (CNTF), and by nerve growth factor (NGF) and platelet-derived growth factor (PDGF) in regulating neuronal and astroglial differentiation in cultured embryonic diencephalic human stem cells. While NGF did not influence either neuronal or glial formation, PDGF surprisingly decreased the percentage of stem cell-generated neurons, an effect opposite to that observed in murine progenitors. Furthermore, while we confirmed the known ability of LIF and CNTF to support astroglial differentiation, we also observed that, in contrast with their murine counterparts, the fraction of CNS stem cell-generated neurons in human cultures was enhanced twofold in the presence of both cytokines. These findings highlight important differences between humans and rodents in regard to the way epigenetic cues regulate the function of neural stem cells.

Excitable properties in astrocytes derived from human embryonic CNS stem cells.

Although it is widely believed that astrocytes lack excitability in adult tissue, primitive action potential-like responses have been elicited from holding potentials negative to -80 mV, in cultured and injury-induced gliotic rodent astrocytes and in human glia under pathological conditions such as glioblastomas and temporal lobe epilepsy. The present study was designed to investigate the properties of astrocytes (identified by immunoreactivity for glial fibrillary acidic protein) derived from multipotent human embryonic CNS stem cells and cultured for 12-25 days in differentiating conditions. We describe here for the first time that brief (1 ms) current pulses elicit spikes from a resting potential (VREST) of approximately -37 mV and, more interestingly, that spontaneous firing can be occasionally recorded in human astrocytes. A voltage-clamp study revealed that in these cells: (i) the half-inactivation of the tetrodotoxin (TTX)-sensitive Na+ channels is around VREST; (ii) the delayed rectifier K+ current is very small; (iii) the ever-present transient outward A-type K+ channels are paradoxically capable of inhibiting the action potentials elicited from a negative membrane potential (-55 to -60 mV); and (iv) inwardly rectifying currents are not present. The responses predicted from a simulation model are in agreement with the experiments. As suggested by recent studies, the decrease of Na+ channel expression and the changes of the electrophysiological properties during the postnatal maturation of the CNS seem to exclude the possibility that astrocytes may play an excitable role in adult tissue. Our data show that excitability and firing should be considered an intrinsic attribute of human astrocytes during CNS development. This is likely to have physiological importance because the role of astrocytes during development is different from the [K+]o-buffering role played in adult CNS, namely the glutamate release and/or the guiding of migrating neurons.