Regulation of mRNA expression encoding chaperone and co-chaperone proteins of the glucocorticoid receptor in peripheral blood: association with depressive symptoms during pregnancy.

BACKGROUND: Major depressive disorder during pregnancy associates with potentially detrimental consequences for mother and child. The current study examined peripheral blood gene expression as a potential biomarker for prenatal depressive symptoms. METHOD: Maternal RNA from whole blood, plasma and the Beck Depression Inventory were collected longitudinally from preconception through the third trimester of pregnancy in 106 women with a lifetime history of mood or anxiety disorders. The expression of 16 genes in whole blood involved in glucorticoid receptor (GR) signaling was assessed using real-time polymerase chain reaction. In parallel, plasma concentrations of progesterone, estradiol and cortisol were measured. Finally, we assessed ex vivo GR sensitivity in peripheral blood cells from a subset of 29 women. RESULTS: mRNA expression of a number of GR-complex regulating genes was up-regulated over pregnancy. Women with depressive symptoms showed significantly smaller increases in mRNA expression of four of these genes - FKBP5, BAG1, NCOA1 and PPID. Ex vivo stimulation assays showed that GR sensitivity diminished with progression of pregnancy and increasing maternal depressive symptoms. Plasma concentrations of gonadal steroids and cortisol did not differ over pregnancy between women with and without clinically relevant depressive symptoms. CONCLUSIONS: The presence of prenatal depressive symptoms appears to be associated with altered regulation of GR sensitivity. Peripheral expression of GR co-chaperone genes may serve as a biomarker for risk of developing depressive symptoms during pregnancy. The presence of such biomarkers, if confirmed, could be utilized in treatment planning for women with a psychiatric history.

Molecular signatures of peripheral blood mononuclear cells during chronic interferon-α treatment: relationship with depression and fatigue.

BACKGROUND: Interferon-alpha (IFN-α) treatment for infectious disease and cancer causes high rates of depression and fatigue, and has been used to investigate the impact of inflammatory cytokines on brain and behavior. However, little is known about the transcriptional impact of chronic IFN-α on immune cells in vivo and its relationship to IFN-α-induced behavioral changes. METHOD: Genome-wide transcriptional profiling was performed on peripheral blood mononuclear cells (PBMCs) from 21 patients with chronic hepatitis C virus (HCV) either awaiting IFN-α therapy (n=10) or at 12 weeks of IFN-α treatment (n=11). RESULTS: Significance analysis of microarray data identified 252 up-regulated and 116 down-regulated gene transcripts. Of the up-regulated genes, 2'-5'-oligoadenylate synthetase 2 (OAS2), a gene linked to chronic fatigue syndrome (CFS), was the only gene that was differentially expressed in patients with IFN-α-induced depression/fatigue, and correlated with depression and fatigue scores at 12 weeks (r=0.80, p=0.003 and r=0.70, p=0.017 respectively). Promoter-based bioinformatic analyses linked IFN-α-related transcriptional alterations to transcription factors involved in myeloid differentiation, IFN-α signaling, activator protein-1 (AP1) and cAMP responsive element binding protein/activation transcription factor (CREB/ATF) pathways, which were derived primarily from monocytes and plasmacytoid dendritic cells. IFN-α-treated patients with high depression/fatigue scores demonstrated up-regulation of genes bearing promoter motifs for transcription factors involved in myeloid differentiation, IFN-α and AP1 signaling, and reduced prevalence of motifs for CREB/ATF, which has been implicated in major depression. CONCLUSIONS: Depression and fatigue during chronic IFN-α administration were associated with alterations in the expression (OAS2) and transcriptional control (CREB/ATF) of genes linked to behavioral disorders including CFS and major depression, further supporting an immune contribution to these diseases.

Proton MR spectroscopy in a 1T open MR imaging system.

(1)H-MR spectroscopy is an established noninvasive MR imaging technique that can be helpful in the diagnosis of brain lesions and in treatment planning. Claustrophobia and body habitus preclude some patients from routine MR imaging in a closed-bore system. The development of (1)H-MR spectroscopy for use in an open MR imaging system would enable a more complete characterization of brain lesions in these patients.

Endogenous glucocorticoids protect against TNF-alpha-induced increases in anxiety-like behavior in virally infected mice.

Endogenous glucocorticoids restrain proinflammatory cytokine responses to immune challenges such as viral infection. In addition, proinflammatory cytokines induce behavioral alterations including changes in locomotor/exploratory activity. Accordingly, we examined proinflammatory cytokines and open-field behavior in virally infected mice rendered glucocorticoid deficient by adrenalectomy (ADX). Mice were infected with murine cytomegalovirus (MCMV), and open-field behavior (36 h post-infection) and plasma concentrations of tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 (42 h post-infection) were assessed. Compared to sham-ADX-MCMV-infected animals, ADX-MCMV-infected mice exhibited significant reductions in total distance moved, number of center entries, and time spent in center. These behavioral alterations were accompanied by significantly higher plasma concentrations of TNF-alpha and IL-6, both of which were correlated with degree of behavioral change. To examine the role of TNF-alpha in these behavioral alterations, open-field behavior was compared in wild-type (WT) and TNF-R1-knockout (KO), ADX-MCMV-infected mice. TNF-R1-KO mice exhibited significantly attenuated decreases in number of rearings, number of center entries and time spent in center, but not distance moved, which correlated with plasma IL-6. Given the potential role of brain cytokines in these findings, mRNA expression of TNF-alpha, IL-1 and IL-6 was assessed in various brain regions. Although MCMV induced increases in proinflammatory cytokine mRNA throughout the brain (especially in ADX animals), no remarkable differences were found between WT and TNF-R1-KO mice. These results demonstrate that endogenous glucocorticoids restrain proinflammatory cytokine responses to viral infection and their impact on locomotor/exploratory activity. Moreover, TNF-alpha appears to mediate cytokine-induced changes in open-field behaviors, especially those believed to reflect anxiety.

Increased total knee arthroplasty ultra-high molecular weight polyethylene wear using a clinically relevant hyaluronic acid simulator lubricant.

In this study, osteoarthritic and periprosthetic synovial fluid samples were rheologically and biochemically compared to develop a hyaluronic acid (HA) supplemented bovine serum (BS) lubricant that mimicked the properties of human joint synovial fluid. The effect of this BS + HA lubricant (50 per cent bovine calf serum + 1.5 g/l HA) on the wear rate of ultra-high molecular weight polyethylene (UHMWPE) during a total knee replacement wear test was then investigated. In conjunction with biochemical similarities, the rheological analysis showed that the BS + HA lubricant viscosity was not statistically different to aspirated total knee arthroplasty (TKA) revision joint fluid viscosity over a range of physiologic shear rates. Gravimetric results at 5 million wear testing cycles showed that the BS + HA lubricant produced an average of 6.88 times more UHMWPE wear than 50 per cent bovine serum lubricant alone. The BS + HA lubricated CoCr femoral component surfaces revealed pitting and surface roughening that was not observed using standard bovine serum only lubricants, but that was similar to the metallic surface corrosion observed on in vivo CoCr femoral component retrievals. These findings support the hypothesis that the addition of HA to simulator lubricant is capable of producing CoCr femoral component surface damage similar to that observed in vivo.

Limited brain diffusion of the glucocorticoid receptor agonist RU28362 following i.c.v. administration: implications for i.c.v. drug delivery and glucocorticoid negative feedback in the hypothalamic-pituitary-adrenal axis.

The experiments described herein present a method for tracking diffusion of the glucocorticoid receptor agonist RU28362 in brain following i.c.v. drug administration. A useful property of glucocorticoid receptor is that it is primarily cytoplasmic when unbound and rapidly translocates to the nucleus when bound by ligand. Thus, removal of endogenous glucocorticoids by adrenalectomy allows us to identify brain regions with activated glucocorticoid receptor after i.c.v. glucocorticoid receptor agonist treatment by examining the presence or absence of nuclear glucocorticoid receptor immunostaining. We have previously demonstrated that an i.p. injection of 150 microg/kg RU28362 1 h prior to restraint stress is sufficient to suppress stress-induced hypothalamic-pituitary-adrenal axis hormone secretion [Ginsberg AB, Campeau S, Day HE, Spencer RL (2003) Acute glucocorticoid pretreatment suppresses stress-induced hypothalamic-pituitary-adrenal axis hormone secretion and expression of corticotropin-releasing hormone hnRNA but does not affect c-fos mRNA or fos protein expression in the paraventricular nucleus of the hypothalamus. J Neuroendocrinol 15:1075-1083]. We report here, however, that in rats i.c.v. treatment with a high-dose of RU28362 (1 microg) 1 h prior to stressor onset does not suppress stress-induced hypothalamic-pituitary-adrenal axis activity. We then performed a series of experiments to examine the possible differences in glucocorticoid receptor activation patterns in brain and pituitary after i.c.v. or i.p. treatment with RU28362. In a dose-response study we found that 1 h after i.c.v. injection of RU28362 (0.001, 0.1 and 1.0 microg) glucocorticoid receptor nuclear immunoreactivity was only evident in brain tissue immediately adjacent to the lateral or third ventricle, including the medial but not more lateral portion of the medial parvocellular paraventricular nucleus of the hypothalamus. In contrast, i.p. injection of RU28362 produced a uniform predominantly nuclear glucocorticoid receptor immunostaining pattern throughout all brain tissue. I.c.v. injection of the endogenous glucocorticoid receptor agonist, corticosterone (1 microg) also had limited diffusion into brain tissue. Time-course studies indicated that there was not a greater extent of nuclear glucocorticoid receptor immunostaining present in brain after shorter (10 or 30 min) or longer (2 or 3 h) intervals of time after i.c.v. RU28362 injection. Importantly, time-course studies found that i.c.v. RU28362 produced significant increases in nuclear glucocorticoid receptor immunostaining in the anterior pituitary that were evident within 10 min after injection and maximal after 1 h. These studies support an extensive literature indicating that drugs have very limited ability to diffuse out of the ventricles into brain tissue after i.c.v. injection, while at the same time reaching peripheral tissue sites. In addition, these studies indicate that significant occupancy of some glucocorticoid receptor within the paraventricular nucleus of the hypothalamus and pituitary is not necessarily sufficient to suppress stress-induced hypothalamic-pituitary-adrenal axis activity.

Habituation to repeated restraint stress is associated with lack of stress-induced c-fos expression in primary sensory processing areas of the rat brain.

Rats repeatedly exposed to restraint show a reduced hypothalamic-pituitary-adrenal axis response upon restraint re-exposure. This hypothalamic-pituitary-adrenal axis response habituation to restraint does not generalize to other novel stressors and is associated with a decrease in stress-induced c-fos expression in a number of stress-reactive brain regions. We examined whether habituation to repeated restraint is also associated with adaptation of immediate early gene expression in brain regions that process and relay primary sensory information. These brain regions may not be expected to show gene expression adaptation to repeated restraint because of their necessary role in experience discrimination. Rats were divided into a repeated restraint group (five 1-hour daily restraint sessions) and an unstressed group (restraint naïve). On the sixth day rats from each group were either killed with no additional stress experience or at 15, 30 or 60 min during restraint. Immediate early gene expression (corticotrophin-releasing hormone heteronuclear RNA, c-fos mRNA, zif268 mRNA) was determined by in situ hybridization. A reduction in stress-induced hypothalamic-pituitary-adrenal axis hormone secretion (plasma corticosterone and adrenocorticotropic hormone) and immediate early gene expression levels in the paraventricular nucleus of the hypothalamus, the lateral septum and the orbital cortex was observed in repeated restraint as compared with restraint naïve animals. This reduction was already evident at 15 min of restraint. Unexpectedly, we also found in repeated restraint rats a reduction in restraint-induced c-fos expression in primary sensory-processing brain areas (primary somatosensory cortex, and ventroposteriomedial and dorsolateral geniculate nuclei of thalamus). The overall levels of hippocampal mineralocorticoid receptor heteronuclear RNA or glucocorticoid receptor mRNA were not decreased by repeated restraint, as may occur in response to severe chronic stress. We propose that repeated restraint leads to a systems-level adaptation whereby re-exposure to restraint elicits a rapid inhibitory modulation of primary sensory processing (i.e. sensory gating), thereby producing a widespread attenuation of the neural response to restraint.

Interspecies conservation of gene order and intron-exon structure in a genomic locus of high gene density and complexity in Plasmodium.

A 13.6 kb contig of chromosome 5 of Plasmodium berghei, a rodent malaria parasite, has been sequenced and analysed for its coding potential. Assembly and comparison of this genomic locus with the orthologous locus on chromosome 10 of the human malaria Plasmodium falciparum revealed an unexpectedly high level of conservation of the gene organisation and complexity, only partially predicted by current gene-finder algorithms. Adjacent putative genes, transcribed from complementary strands, overlap in their untranslated regions, introns and exons, resulting in a tight clustering of both regulatory and coding sequences, which is unprecedented for genome organisation of PLASMODIUM: In total, six putative genes were identified, three of which are transcribed in gametocytes, the precursor cells of gametes. At least in the case of two multiple exon genes, alternative splicing and alternative transcription initiation sites contribute to a flexible use of the dense information content of this locus. The data of the small sample presented here indicate the value of a comparative approach for Plasmodium to elucidate structure, organisation and gene content of complex genomic loci and emphasise the need to integrate biological data of all Plasmodium species into the P.falciparum genome database and associated projects such as PlasmodB to further improve their annotation.

Acute exposure to a novel stressor further reduces the habituated corticosterone response to restraint in rats.

The present study sought to identify dishabituation of the hypothalamic-pituitary-adrenal(HPA) axis response to different psychological stressors. Young adult male Sprague Dawley rats were exposed to five, 1 h sessions of restraint stress on five consecutive days. On the sixth day, and 2 h before additional exposure to restraint, animals were subjected to 30 min of a small (27cm square), elevated open field stressor (pedestal), which served as the dishabituating stimulus. We predicted HPA axis response dishabituation in chronically restrained rats exposed to the novel pedestal. Rats which underwent five days of restraint stress showed significantly blunted plasma corticosterone levels to restraint (habituation) as compared to restraint-nai've rats. However, rats which underwent five sessions of restraint responded with an enhanced habituation response when confronted with restraint shortly after exposure to the novel pedestal. Instead of HPA axis response dishabituation, we observed enhanced habituation. Subsequent experiments determined that a 1.25 mgkg corticosterone injection could substitute for pedestal exposure to produce enhanced restraint habituation.Combined treatment with both the glucocorticoid receptor antagonist RU40555 (30 mgkg)and the mineralocorticoid receptor antagonist RU283 18 (50 mgkg) blocked the expression of enhanced habituation after pedestal exposure. Thus, the delayed corticosterone negative feedback produced by novel stress led to enhanced expression of corticosterone response habituation.

Selective blockade of the mineralocorticoid receptor impairs hypothalamic-pituitary-adrenal axis expression of habituation.

The present study investigated the role of mineralocorticoid receptors (MR) and glucocorticoid receptors (GR) in the expression of habituation of the hypothalamic-pituitary-adrenal (HPA) axis response to stress. Male rats were restrained for 1 h per day for six consecutive days. On day 6, 1 h prior to restraint stress, both restraint-naive and repeatedly restrained rats were injected s.c. with either vehicle (propylene glycol) or one of three corticosteroid receptor antagonist treatments: selective MR antagonist (RU28318 or spironolactone), selective GR antagonist (RU40555), or both MR and GR antagonists combined (RU28318 + RU40555). Blood samples were collected for corticosterone measurement at the beginning of stress, during stress, and 1 h after stress termination. Repeated restraint stress produced significant habituation of corticosterone responses. Acute treatment with the combined MR and GR antagonists prevented the expression of habituation. When tested alone, the MR antagonist also blocked the expression of corticosterone-response habituation, whereas the GR antagonist had no effect. Neither the MR, nor the GR antagonists alone, significantly altered the corticosterone response to restraint in rats exposed to restraint for the first time. The final experiment examined the corticosterone response to a corticotropin releasing hormone (CRH, 3 microg/kg i.p.) challenge. Neither previous exposure to restraint or acute pretreatment with the combined MR and GR antagonists (RU28318 + RU40555) altered the corticosterone response to CRH challenge. This result indicates that the expression of habituation and its blockade by corticosteroid receptor antagonists is not a result of altered pituitary-adrenal response to CRH. Overall, this study suggests that MR plays an important role in constraining the HPA axis response to restraint stress in restraint-habituated rats. The dependence of the HPA axis on MR-mediated corticosteroid negative feedback during acute stress may be an important mechanism that helps maximize the expression of stress habituation and thereby minimize exposure of target tissues to corticosteroids in the context of repeated stress.

Clinical evaluation of a new total hip prosthetic design: 100 consecutive cementless total hip arthroplasties using Sulzermedica's "Natural Hip" with two- to six-year clinical and radiographic follow-up.

One hundred consecutive total hip arthroplasties were performed on 96 patients (age range 30 to 82 years) using the "Natural Hip" design of Sulzermedica (Austin, Texas). Follow up of the clinical and radiographic results with a minimum of 2 years is reported. Twelve patients were excluded for the lack of follow-up data, leaving 88 hips to study. All but three of the stems were used cementless, regardless of patient age or osteoporosis, as long as surgical stability was obtained. No stems were revised for loosening or pain. The only revisions were for infection (2), and minimal bone loss occurred with removal of this proximal ingrowth stem. Thigh pain was present in only one patient at 2 years, and none with longer follow-up. There were no cases of osteolysis, proximal stress shielding, or aseptic radiolucent lines with the cementless stems.

Targeted terminal deletions as a tool for functional genomics studies in Plasmodium.

We describe a transfection system that induces terminal deletions at specific chromosome ends in malaria parasites using a linear construct containing telomeric repeats at one end and plasmodial sequences able to drive homologous recombination at the other. A site-specific deletion was generated at one extremity of chromosome 5 of Plasmodium berghei, which was stably maintained in the parasite population selected after transfection. The telomeric repeat array introduced with the construct reached the average length observed in natural telomeres of Plasmodium, indicating that in vivo telomere addition occurred at the newly formed extremity. The expression of a mutant dhfr/ts gene conferring pyrimethamine resistance, used as a selectable marker, was not affected by the proximity to the telomeric sequences, either in the presence or absence of drug pressure. In addition, no transcriptional silencing was observed on insertion of the mutant dhfr/ts gene either in subtelomeric or internal positions that are transcriptionally silent in blood-stage parasites. This suggests that the activity of its promoter is not affected by the chromatin organization of the chromosomal context.

An experimental examination of learned helplessness in older adolescents and young adults with long-standing asthma.

OBJECTIVE: To examine the effects of experimentally induced learned helplessness in older adolescents and young adults with long-standing asthma. METHODS: Thirty-nine participants (18-24 years of age) with histories of long-standing asthma (AS) and an age-matched healthy cohort (HC) (N = 94) received either contingent or noncontingent feedback on an experimental task. Participants' anagram-solving performance was assessed following the experimental procedure. Participants also completed a measure of depression and pretest-posttest measures of mood, expectancy, and attributions related to experimental task performance. RESULTS: The AS participants demonstrated significantly greater problem-solving deficits following response-noncontingent feedback, compared to the HC group. Further, whereas both AS and HC participants made more internal performance attributions when given response-contingent feedback, only AS participants demonstrated a pattern of increased internal attributions (i.e., self-focus) following response-noncontingent failure. In addition, 21% of AS participants met DSM-IV criteria for major depression, compared to only 5% of the HC group. CONCLUSIONS: Individuals with long-standing asthma may be at increased risk for depression and for learned helplessness deficits, specifically impaired problem solving, in response to environmental noncontingency. Results are discussed in terms of both learned helplessness theory and perseverative self-focus conceptualizations of depression. The implications for both short- and long-term management of pediatric asthma are also discussed.

The putative gene for the first enzyme of glutathione biosynthesis in Plasmodium berghei and Plasmodium falciparum.

The putative gene for gamma-glutamylcysteine synthetase, the rate-limiting enzyme in glutathione biosynthesis, has been characterized both in Plasmodium berghei and Plasmodium falciparum. Protein sequence comparison between these two species reveals large conserved regions sharing more than 80% similarity, separated by less conserved portions. When the comparison is extended to known gamma-glutamylcysteine synthetases from other eukaryotes, a number of high similarity blocks are observed which may help in identifying sequence essential for protein function.

Developmental regulation of a Plasmodium gene involves the generation of stage-specific 5' untranslated sequences.

The B7 gene of Plasmodium berghei, highly conserved within the genus Plasmodium, encodes a nuclear protein most likely involved in chromatin assembly. In this study we describe the transcription pattern of B7 during asexual multiplication and sexual differentiation of the parasites in the blood of the vertebrate host. Two alternative transcripts have been identified: one, 1.4 kb in length is specific for asexual blood stages; the other, 1.8 kb in length is specific for sexually differentiated cells (gametocytes). The processed mRNAs are identical in their coding region and differ only in their 5' untranslated regions (5' UTRs). We show here that the differences in 5' UTRs are the result of two mechanisms: (1) the use of alternative transcription initiation sites mapped at least 1.4 kb apart, which imply the existence of separate, stage-specific promoters; (2) the splicing of a 765 bp gametocyte-specific intron at the 5' UTR of the 1.8 kb transcript.

Synovial tissue examination by frozen section as an indicator of infection in hip and knee arthroplasty in community hospitals.

Twenty-five surgical synovial sections were examined in 18 consecutive patients undergoing revision hip or knee arthroplasty (9 hips and 9 knees). All cases were performed in either of two community hospitals, with frozen-section tissue examined by multiple general pathologists. By protocol, acute inflammation was defined as more than five neutrophils per 60x high-power fields on multiple areas. A positive culture was defined as-organism growth from any surgical specimen. In each case, three surgical cultures and three frozen-section specimens were harvested from the synovium at corresponding periprosthetic surgical sites before antibiotics were administered. The average age of the patients was 68 years (range 40-87 years). There were 11 positive surgical cultures, 9 with positive frozen sections of synovium for acute inflammation (sensitivity, 82%; 95% confidence interval, 78-100%). There were 14 negative cultures; 13 had negative surgical frozen sections (specificity, 93%; 95% confidence interval, 83-100%). The positive predictive value of the test was 82%. There was accurate correlation between frozen section and culture in 22 of 25 cases (88%). In this community hospital setting, frozen section examination of surgical synovial tissue proved to be a reasonably sensitive and specific predictor of deep infection in revision hip and knee arthroplasty.

Illness uncertainty, attributional style, and psychological adjustment in older adolescents and young adults with asthma.

Examined psychological adjustment in a college sample of older adolescents and young adults (n = 49) with histories of childhood asthma. A substantial number of subjects evidenced clinically significant levels of overall distress. In addition, greater perceived asthma uncertainty and increased stable attributions for negative events were significantly associated with poorer psychological adjustment after controlling for demographic and disease variables. Further analyses revealed a moderating influence of uncertainty on attribution-adjustment relationships. These findings provide initial support for a cognitive diathesis-stress view of adjustment in long-standing asthma. Results also support a growing body of evidence suggesting that the focus of efforts to enhance adjustment to asthma need to be expanded beyond childhood and early adolescence.