Adrenal function under long-term raloxifene administration.

The aim of the present study was to evaluate the effect of long-term (12 months) administration of raloxifene hydrochloride (60 mg/day) on the steroid production of the adrenal cortex and on the hypothalamic-pituitary-adrenal axis in postmenopausal women. We performed a basal evaluation, a corticotropin releasing factor (CRF) (100 microg i.v. bolus) test and a dexamethasone (DXM) (0.25 mg) suppression-adrenocorticotropic hormone (ACTH) (10 microg i.v. bolus) stimulation test in 11 postmenopausal women, before and after 3, 6 and 12 months of raloxifene treatment. Raloxifene administration significantly modified circulating levels of adrenal steroids, decreasing cortisol (-24%), dehydroepiandrosterone (DHEA) (-36%), and its sulfate (DHEAS) (-41%), and androstenedione (-29%), and increasing circulating allopregnanolone (+39%) levels. Progesterone and 17OH-progesterone levels remained unmodified, while estradiol and estrone levels showed a significant decrease (-51% for estradiol and -61% for estrone). We also observed an increase in circulating ACTH (+58%) and beta-endorphin (+120%). No modifications in the hormonal responses to CRF were observed during the treatment. DXM significantly suppressed circulating steroids at any time with a lower suppression of cortisol from the third month and a higher suppression of DHEA at 12 months. ACTH administration was associated with a significantly blunted cortisol response from the sixth month and a significantly increased response of allopregnanolone from the third month. The present data exclude a raloxifene effect on pituitary sensitivity to CRF and demonstrate a reduced adrenal sensitivity to ACTH, sustained by the opposite changes in basal cortisol and Delta5 androgens, which were reduced, and in ACTH and beta-endorphin, which were increased, as well by the reduced response of cortisol to the direct ACTH stimulus. The reduction of circulating cortisol levels and cortisol response to the ACTH challenge suggests that raloxifene protects against the neurotoxic effects of endogenous glucocorticoids. Furthermore, the progressive increase in basal allopregnanolone and its increased response to ACTH indicate that chronic raloxifene administration exerts direct effects on the pattern of adrenal enzymes, leading to specific changes in the circulating levels of this anxiolytic progesterone metabolite. The important reduction in the circulating levels of estradiol and estrone under long-term raloxifene administration may represent a further mechanism by which this molecule may exert a protective effect against breast and endometrial malignancies.     

Pursuit eye movement dysfunction in obsessive-compulsive disorder.

Disturbances in neural circuitry including the basal ganglia and prefrontal cortex have been hypothesized to be a cause of obsessive-compulsive disorder (OCD). Because eye movements are often impaired in neurologic diseases affecting these brain areas, oculomotor functioning was assessed in 17 unmedicated patients with OCD and in 25 normal controls. As compared with control subjects, patients with OCD demonstrated low-gain (slow) pursuit eye movements and an increased frequency of square wave jerk intrusions, but no increase in anticipatory saccades. In addition, several OCD patients showed an unusual pattern of intrusive, brief epochs of high-gain (fast) pursuit lasting on the order of 50 to 130 msec. These epochs of fast pursuit moved the eyes ahead of the target being tracked, and were terminated by corrective reversal saccades. Studies of eye movement abnormalities may provide an informative neurophysiologic approach for studying disturbances in basal ganglia and frontal cortical function that have been observed in functional neuroimaging and neuropsychological studies of OCD.     

Stimulus effects of ibogaine in rats trained with yohimbine, DOM, or LSD.

The stimulus effects of ibogaine were compared with those of yohimbine, an alpha 2-adrenoceptor antagonist, 2,5-dimethoxy-4-methylamphetamine (DOM), a 5-hydroxytryptamine2 (5-HT2) agonist, and lysergic acid diethylamide (LSD), a nonspecific 5-HT agonist. Rats were trained with either yohimbine (6 mg/kg), DOM (0.6 mg/kg), or LSD (0.1 mg/kg) vs. no treatment in a two-lever discrimination task. Tests of generalization were then conducted with ibogaine. In yohimbine-trained animals, 39.7% of responses following ibogaine (15 mg/kg) were on the drug-appropriate lever, but this response level was not significantly different from no treatment-appropriate responding. A response distribution that was significantly different from responding under both drug and no treatment training conditions was observed in DOM-trained rats after administration of 15 mg/kg ibogaine. Pizotyline (BC-105) blocked all DOM-appropriate responding produced by ibogaine. In LSD-trained animals, 20 mg/kg ibogaine mimicked LSD. Pizotyline blocked LSD-appropriate responding produced by ibogaine in five of six animals. The present data suggest the involvement of 5-HT2 receptor activity, and the possibility of a 5-HT1A contribution, in the stimulus properties of ibogaine.     

Expression of c-fos, fos B, and egr-1 in the medial preoptic area and bed nucleus of the stria terminalis during maternal behavior in rats

The spatial and temporal pattern of expression of the protein products of immediate early genes (IEGs) c-fos, fos B, and egr-1 were mapped in medial preoptic area (MPOA) and ventral bed nucleus of stria terminalis (VBST) during maternal behavior in rats. Immunocytochemical analysis indicated significant increases in the number of cells expressing c-Fos after 2 h of pup exposure, while Fos B levels showed a delayed response, reaching maximal levels after 6 h.     

Attachment and spreading of fibroblasts on an RGD peptide-modified injectable hyaluronan hydrogel

Hyaluronan (HA) hydrogels resist attachment and spreading of fibroblasts and most other mammalian cell types. A thiol-modified HA (3,3'-dithiobis(propanoic dihydrazide) [HA-DTPH]) was modified with peptides containing the Arg-Gly-Asp (RGD) sequence and then crosslinked with polyethylene glycol (PEG) diacrylate (PEGDA) to create a biomaterial that supported cell attachment, spreading, and proliferation. The hydrogels were evaluated in vitro and in vivo in three assay systems. First, the behavior of human and murine fibroblasts on the surface of the hydrogels was evaluated. The concentration and structure of the RGD peptides and the length of the PEG spacer influenced cell attachment and spreading. Second, murine fibroblasts were seeded into HA-DTPH solutions and encapsulated via in situ crosslinking with or without bound RGD peptides. Cells remained viable and proliferated within the hydrogel for 15 days in vitro. Although the RGD peptides significantly enhanced cell proliferation on the hydrogel surface, the cell proliferation inside the hydrogel in vitro was increased only modestly. Third, HA-DTPH/PEGDA/peptide hydrogels were evaluated as injectable tissue engineering materials in vivo. A suspension of murine fibroblasts in HA-DTPH was crosslinked using PEGDA plus PEGDA peptide, and the viscous, gelling mixture was injected subcutaneously into the flanks of nude mice; gels formed in vivo following injection. After 4 weeks, growth of new fibrous tissue had been accelerated by the sense RGD peptides. Thus, attachment, spreading, and proliferation of cells is dramatically enhanced on RGD-modified surfaces but only modestly accelerated in vivo tissue formation.     

Genotype-phenotype relationship in human ATP6i-dependent autosomal recessive osteopetrosis

Autosomal-recessive osteopetrosis is a severe genetic disease caused by osteoclast failure. Approximately 50% of the patients harbor mutations of the ATP6i gene, encoding for the osteoclast-specific a3 subunit of V-ATPase. We found inactivating ATP6i mutations in four patients, and three of these were novel. Patients shared macrocephaly, growth retardation and optic nerve alteration, osteosclerotic and endobone patterns, and high alkaline phosphatase and parathyroid hormone levels. Bone biopsies revealed primary spongiosa lined with active osteoblasts and high numbers of tartrate-resistant acid phosphatase (TRAP)-positive, a3 subunit-negative, morphologically unremarkable osteoclasts, some of which located in shallow Howship lacunae. Scarce hematopoietic cells and abundant fibrous tissue containing TRAP-positive putative osteoclast precursors were noted. In vitro osteoclasts were a3-negative, morphologically normal, with prominent clear zones and actin rings, and TRAP activity more elevated than in control patients. Podosomes, alphaVbeta3 receptor, c-Src, and PYK2 were unremarkable. Consistent with the finding in the bone biopsies, these cells excavated pits faintly stained with toluidine blue, indicating inefficient bone resorption. Bone marrow transplantation was successful in all patients, and posttransplant osteoclasts showed rescue of a3 subunit immunoreactivity.     

Osteocyte dendrogenesis in static and dynamic bone formation: an ultrastructural study

The present ultrastructural investigation into osteocyte dendrogenesis represents a continuation of a previous study (Ferretti et al., Anat. Embryol., 2002; 206:21-29), in which we pointed out that, during intramembranous ossification, the well-known dynamic bone formation (DBF), performed by migrating osteoblast laminae, is preceded by static bone formation (SBF), in which cords of stationary osteoblasts transform into osteocytes in the same site where they differentiated. The research was carried out on the perichondral center of ossification surrounding the mid shaft level of various long bones of chick embryos and newborn rabbits. Transmission electron microscope observations showed that the formation of osteocyte dendrites is quite different in the two types of osteogenesis, mainly depending on whether or not osteoblast movement occurs. In DBF, osteoblasts transform into small ovoidal/ellipsoidal osteocytes and their dendrites form in an asynchronous and asymmetrical manner in concomitance with, and depending on, the advancing mineralizing surface and the receding osteogenic laminae. In SBF, stationary osteoblasts give rise to big globous osteocytes, located inside confluent lacunae, with short and symmetrical dendrites that can radiate simultaneously all around their cell body because they are completely surrounded by unmineralized matrix. Contacts and gap junctions were observed between all osteocytes (both SBF- and DBF-derived) and between osteocytes and osteoblasts. Finally, a continuous osteocyte network extends throughout the bone, regardless of its static or dynamic origin. This network has the characteristic of a functional syncytium, potentially capable of modulating, by wiring transmission, the cells of the osteogenic lineage covering the bone surfaces.     

Model for assessment of proficiency of human immunodeficiency virus type 1 sequencing-based genotypic antiretroviral assays

Use of sequencing-based genotyping as a diagnostic assay for human immunodeficiency virus (HIV) antiretroviral resistance is increasing. Periodic evaluation of the proficiency of laboratories performing this assay should be established. It is important to identify components of the assay that influence the generation of reliable sequencing data and that should and can be monitored. A model was developed to determine what parameters were reasonable and feasible for assessing the performance of genotyping assays. Ten laboratories using the genotyping platform, HIV-1 Genotyping System (HGS) v. 1 and software versions 1.1 or 2.0, participated in two rounds of testing. For each round, each group was sent a panel consisting of three clinical samples to sequence in real time. Six months later, seven laboratories using the TRUGENE HIV-1 Genotyping Kit participated in a separate round, working with both panels at the same time. Analysis of the data showed that one main indicator of genotyping proficiency was achievement of > or =98% sequence homology of a sample tested to a group consensus sequence for that sample. A second was concordant identification of codons at sites identified with resistance mutations in the sample, although scoring of these criteria is still undetermined from this study. These criteria are applicable to all sequence-based genotyping platforms and have been used as a baseline for assessing the performance of genotyping for the determination of antiretroviral resistance in our ongoing proficiency program.