Antenatal depression programs cortisol stress reactivity in offspring through increased maternal inflammation and cortisol in pregnancy: The Psychiatry Research and Motherhood - Depression (PRAM-D) Study.

INTRODUCTION: Antenatal depression is associated with a broad range of suboptimal outcomes in offspring, although the underlying mechanisms are not yet understood. Animal studies propose inflammation and glucocorticoids as mediators of the developmental programming effect of prenatal stress on offspring stress responses, but studies in humans are not yet at this stage. Indeed, to date no single study has examined the effects of a rigorously defined, clinically significant Major Depressive Disorder (MDD) in pregnancy on maternal antenatal inflammatory biomarkers and hypothalamic-pituitary (HPA) axis, as well as on offspring HPA axis, behavior and developmental outcomes in the first postnatal year. METHODS: A prospective longitudinal design was used in 106 women (49 cases vs. 57 healthy controls) to study the effect of MDD in pregnancy and associated antenatal biology (inflammatory and cortisol biomarkers), on offspring stress response (cortisol response to immunization, at 8 weeks and 12 months), early neurobehavior (Neonatal Behavioral Assessment Scale, NBAS, at day 6), and cognitive, language and motor development (Bayley Scales of Infant and Toddler Development at 12 months). RESULTS: Compared with healthy controls, women with MDD in pregnancy had raised interleukin (IL) IL-6 (effect size (δ) = 0.53, p = 0.031), IL-10 (δ = 0.53, p = 0.043), tumor necrosis factor alpha (δ = 0.90, p = 0.003) and vascular endothelial growth factor (δ = 0.56, p = 0.008), together with raised diurnal cortisol secretion (δ = 0.89, p = 0.006), raised evening cortisol (δ = 0.64, p = 0.004), and blunted cortisol awakening response (δ = 0.70, p = 0.020), and an 8-day shorter length of gestation (δ = 0.70, p = 0.005). Furthermore, they had neonates with suboptimal neurobehavioral function in four out of five NBAS clusters measured (range of δ = 0.45-1.22 and p = 0.049-<0.001) and increased cortisol response to stress at one year of age (δ = 0.87, p < 0.001). Lastly, maternal inflammatory biomarkers and cortisol levels were correlated with infant stress response, suggesting a mechanistic link. CONCLUSION: This study confirms and extends the notion that depression in pregnancy is associated with altered offspring behavior and biological stress response, and demonstrates that changes in maternal antenatal stress-related biology are associated with these infant outcomes.

New horizons in comprehensive geriatric assessment.

In this article, we discuss the emergence of new models for delivery of comprehensive geriatric assessment (CGA) in the acute hospital setting. CGA is the core technology of Geriatric Medicine and for hospital inpatients it improves key outcomes such as survival, time spent at home and institutionalisation. Traditionally It is delivered by specialised multidisciplinary teams, often in dedicated wards, but in recent years has begun to be taken up and developed quite early in the admission process (at the 'front door'), across traditional ward boundaries and in specialty settings such as surgical and pre-operative care, and oncology. We have scanned recent literature, including observational studies of service evaluations, and service descriptions presented as abstracts of conference presentations to provide an overview of an emerging landscape of innovation and development in CGA services for hospital inpatients.

Cystathionine γ-lyase is expressed in human atherosclerotic plaque microvessels and is involved in micro-angiogenesis.

Atherosclerotic plaques are classically divided into stable and vulnerable plaques. Vulnerable plaques are prone to rupture with a risk for infarction. High intraplaque microvessel density predisposes to plaque vulnerability. Hydrogen sulfide (H2S) is a proangiogenic gasotransmitter which is endogenously produced by cystathionine γ-lyase (CSE), and is believed to have vasculoprotective effects. However, due to its proangiogenic effects, H2S may result in pathological angiogenesis in atherosclerotic plaques, thereby increasing plaque vulnerability. The aim of this study was to determine CSE expression pattern in atherosclerotic plaques, and investigate whether CSE is involved in micro-angiogenesis in vitro. Endarterectomy plaques were studied for CSE expression, and the role of CSE in micro-angiogenesis was studied in vitro. CSE is expressed in plaques with similar levels in both stable and vulnerable plaques. CSE co-localized with von Willebrand Factor-positive microvessel endothelial cells and alpha-smooth-muscle actin-positive SMCs. In vitro, inhibition of CSE in HMEC-1 reduced tube formation, cell viability/proliferation, and migration which was restored after culture in the presence of H2S donor GYY4137. CSE is expressed in intraplaque microvessels, and H2S is a stimulator of micro-angiogenesis in vitro. Due to this pro-angiogenic effect, high levels of CSE in atherosclerotic plaques may be a potential risk for plaque vulnerability.

TGF-ß is an inducer of ZEB1-dependent mesenchymal transdifferentiation in glioblastoma that is associated with tumor invasion.

Different molecular subtypes of glioblastoma (GBM) have been recently identified, of which the mesenchymal subtype is associated with worst prognoses. Here, we report that transforming growth factor-ß (TGF-ß) is able to induce a mesenchymal phenotype in GBM that involves activation of SMAD2 and ZEB1, a known transcriptional inducer of mesenchymal transition in epithelial cancers. TGF-ß exposure of established and newly generated GBM cell lines was associated with morphological changes, enhanced mesenchymal marker expression, migration and invasion in vitro and in an orthotopic mouse model. TGF-ß-induced mesenchymal differentiation and invasive behavior was prevented by chemical inhibition of TGF-ß signaling as well as small interfering RNA (siRNA)-dependent silencing of ZEB1. Furthermore, TGF-ß-responding and -nonresponding GBM neurospheres were identified in vitro. Interestingly, nonresponding cells displayed already high levels of pSMAD2 and ZEB1 that could not be suppressed by inhibition of TGF-ß signaling, suggesting the involvement of yet unknown mechanisms. These different GBM neurospheres formed invasive tumors in mice as well as revealed mesenchymal marker expression in immunohistochemical analyses. Moreover, we also detected distinct zones with overlapping pSMAD2, elevated ZEB1 and mesenchymal marker expression in GBM patient material, suggestive of the induction of local, microenvironment-dependent mesenchymal differentiation. Overall, our findings indicate that GBM cells can acquire mesenchymal features associated with enhanced invasive potential following stimulation by secretory cytokines, such as TGF-ß. This property of GBM contributes to heterogeneity in this tumor type and may blur the boundaries between the proposed transcriptional subtypes. Targeting TGF-ß or downstream targets like ZEB1 might be of potential benefit in reducing the invasive phenotype of GBM in a subpopulation of patients.

Ethnic differences in serum adipokine and C-reactive protein levels: the multiethnic cohort.

BACKGROUND: Ethnic disparities in metabolic disease risk may be the result of differences in circulating adipokines and inflammatory markers related to ethnic variations in obesity and body fat distribution. SUBJECTS/METHODS: In a cross-sectional design, we compared serum levels of leptin, adiponectin, C-reactive protein (CRP), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in control subjects (321 men and 930 women) from two nested case-control studies conducted within the Multiethnic Cohort Study consisting of whites, Japanese Americans (JA), Latinos, African Americans (AA) and Native Hawaiians (NH). General linear models were applied to evaluate ethnic differences in log-transformed serum biomarker levels before and after adjusting for body mass index (BMI) at cohort entry. RESULTS: In comparison to whites, significant ethnic differences were observed for all biomarkers except TNF-α. JA men and women had significantly lower leptin and CRP levels than whites, and JA women also had lower adiponectin levels. Leptin was significantly higher in AA women (P < 0.01), adiponectin was significantly lower in AA men and women (P = 0.02 and P < 0.001), and CRP and IL-6 were significantly higher in AA men and women. Lower adiponectin (P < 0.0001) and CRP (P = 0.03) levels were the only biomarkers in NH women that differed from whites; no statistically significant differences were seen for NH men and for Latino men and women. When adjusted for BMI at cohort entry, the differences between the lowest and the highest values across ethnic groups decreased for all biomarkers except adiponectin in men indicating that ethnic differences were partially due to weight status. CONCLUSIONS: These findings demonstrate the ethnic variations in circulating adipokine and CRP levels before and after adjustment for BMI. Given the limitation of BMI as a general measure of obesity, further investigation with visceral and subcutaneous adiposity measures are warranted to elucidate ethnicity-related differences in adiposity in relation to disparities in obesity-related disease risk.

Gene delivery to the vasculature mediated by low-titre adeno-associated virus serotypes 1 and 5.

BACKGROUND: Vascular gene therapy requires safe and efficient gene transfer in vivo. Recombinant adeno-associated virus (AAV) is a promising viral vector but its use in the vasculature has produced conflicting results and serotypes other than AAV2 have not been intensively studied. We investigated the efficiency of alternative AAV serotypes for vascular gene delivery in vitro and in vivo. METHODS: Vascular cell lines were transduced in vitro with AAV vectors. Rabbit carotid arteries were transduced with AAV1, 2 and 5 encoding enhanced green fluorescent protein (eGFP) ( approximately 1.4 x 10(9) DNAse-resistant particles (drp)). Gene transfer in vivo was assessed at 14 and 28 days. High-titre doses of AAV2 encoding beta-galactosidase in vivo were also studied. RESULTS: In vitro, transgene expression was not observed in endothelial cells using AAV2 whereas the use of serotypes 1 and 5 resulted in detectable levels of transgene expression. Coronary artery smooth muscle cells (CASMCs) transduced with AAV2 demonstrated higher levels of GFP expression than AAV1 or 5. Transgene expression in vivo was noted using low-titre AAV1 and AAV5 ( approximately 1.4 x 10(9) drp) in the media and adventitia. Only delivery of AAV1eGFP resulted in neointimal formation (3/7 vessels examined), with transgene expression noted in the neointima. Transgene expression with AAV2 was not detected in any layer of the blood vessel wall using low titre ( approximately 10(9) drp). However, high-titre ( approximately 10(11) drp) AAV2 resulted in transduction of cells in the media and adventitia but not the endothelium. CONCLUSIONS: AAV1 and AAV5 have advantages over AAV2 for vascular gene delivery at low titres.

Gene transfer into rat mesenchymal stem cells: a comparative study of viral and nonviral vectors.

Mesenchymal stem cells (MSCs) have been proposed for use in combinatorial gene and cell therapy protocols for the treatment of disease and promotion of repair. The efficacy of such a therapeutic approach depends on determination of which vectors give maximal transgene expression with minimal cell death. The study was carried out on bone-marrow derived rat MSCs, and a range of vectors was tested on the same stem cell preparation. Adenovirus, adeno-associated virus (AAV; serotypes 1, 2, 4, 5, and 6), lentivirus, and nonviral vectors were compared. Lentivirus proved to be most effective with transduction efficiencies of up to 95%, concurrent with low levels of cell toxicity. Adenovirus also proved effective, but a significant increase in cell death was seen with increasing viral titer. Rat MSCs remained refractory to transduction by all AAV serotypes, in contrast to rabbit MSCs tested at the same time. Lipofection of plasmid DNA gave moderate transfection levels but was also accompanied by cell death. Electroporative gene transfer proved ineffective at the parameters tested and resulted in high cell death. High and moderate levels of cell transduction using lentivirus vectors did not affect the ability of the cells to differentiate down the adipogenic pathway.

Unlicensed and off label drug use in acute lymphoblastic leukaemia and other malignancies in children.

BACKGROUND: The need to use unlicensed and off label drugs in children due to the lack of suitable, licensed formulations with appropriate prescribing information creates many problems on an everyday basis for healthcare professionals, carers and children. PATIENTS AND METHODS: This prospective study was designed to examine the incidence and nature of unlicensed and off label prescribing, in paediatric oncology patients with acute lymphoblastic leukaemia and other malignancies. Inpatient and outpatient prescriptions were analysed for a 4-week period. RESULTS: All patients received at least one unlicensed or off label drug. Fifty-five per cent of prescriptions were licensed, 19% were unlicensed and 26% were licensed drugs used in an off label manner. Unlicensed preparations were used in 40% of prescriptions for cytotoxic agents, due to a lack of commercially available formulations suitable for the paediatric patient. These drugs included mercaptopurine and methotrexate which have been used in the treatment of paediatric leukaemia for many years, their efficacy having been demonstrated by on-going Medical Research Council trials. CONCLUSIONS: It is disappointing that drugs, which are the mainstay of therapy for paediatric leukaemia and other malignancies, are unavailable in appropriate licensed formulations to facilitate their administration to children. This needs to be urgently addressed.

Evidence for a sustained increase in clonal beta-cell basal intracellular Ca2+ levels after incubation in the presence of newly diagnosed Type-1 diabetic patient sera. Possible role in serum-induced inhibition of insulin secretion.

We have previously reported that newly diagnosed Type-1 diabetic patient sera potently suppressed insulin secretion from a clonal rat pancreatic beta-cell line (BRIN BD11) but did not alter cell viability. Here, we report that apoptosis in BRIN BD11 cells incubated in various sera types (fetal calf serum (FCS), normal human serum and Type-1 diabetic patient) was virtually undetectable. Although low levels of necrosis were detected, these were not significantly different between cells incubated in sera from different sources. ATP levels were reduced by approximately 30% while nitrite production increased twofold from BRIN BD11 cells incubated for 24 h in the presence of Type-1 diabetic patient sera compared with normal human sera. Additionally, ATP levels were reduced by approximately 40% and DNA fragmentation increased by more than 20-fold in BRIN BD11 cells incubated in FCS in the presence of a pro-inflammatory cytokine cocktail (interleukin-1beta, tumour necrosis factor-alpha and interferon-gamma), compared with cells incubated in the absence of cytokines. Nitric oxide production from BRIN BD11 cells was markedly increased (up to 10-fold) irrespective of sera type when the cytokine cocktail was included in the incubation medium. Type-1 diabetic patient sera significantly (P<0.001) raised basal levels of intracellular free Ca(2+ )concentration ([Ca(2+)](i)) in BRIN BD11 cells after a 24-h incubation. The alteration in [Ca(2+)](i) concentration was complement dependent, as removal of the early complement components C1q and C3 resulted in a significant reduction (P<0.01) of sera-induced [Ca(2+)](i )changes. We propose that the mechanism of Type-1 diabetic patient sera-induced inhibition of insulin secretion from clonal beta-cells may involve complement-stimulated elevation of [Ca(2+)](i) which attenuates the nutrient-induced insulin secretory process possibly by desensitizing the cell to further changes in Ca(2+).

Ultraviolet radiation induces p16CDKN2A expression in human skin.

The loss of the tumor suppressor gene product p16 in melanoma is well documented, although the normal physiological function of p16 in skin melanocytes is unknown. In this report, we demonstrate that when human skin was irradiated with suberythemal doses of UV radiation, levels of p16 were dramatically increased by 16 h postirradiation, peaking at 24 h, and declining by 72 h. p16 was expressed in the nucleus and cytoplasm of melanocytes and keratinocytes within the epidermis, and the pattern of p16 expression within the epidermis was dependent on the penetrative ability of the different UV wavebands. The existence of a UV-induced response pathway involving up-regulated p16 expression may provide a mechanism linking the loss of p16 and UV exposure with the development of melanoma.

Generation and characterization of human smooth muscle cell lines derived from atherosclerotic plaque.

The study of atherogenesis in humans has been restricted by the limited availability and brief in vitro life span of plaque smooth muscle cells (SMCs). We describe plaque SMC lines with extended life spans generated by the expression of the human papillomavirus (HPV)-16 E6 and E7 genes, which has been shown to extend the life span of normal adult human aortic SMCs. Resulting cell lines (pdSMC1A and 2) demonstrated at least 10-fold increases in life span; pdSMC1A became immortal. The SMC identity of both pdSMC lines was confirmed by SM22 mRNA expression. pdSMC2 were generally diploid but with various structural and numerical alterations; pdSMC1A demonstrated several chromosomal abnormalities, most commonly -Y, +7, -13, anomalies previously reported in both primary pdSMCs and atherosclerotic tissue. Confluent pdSMC2 appeared grossly similar to HPV-16 E6/E7-expressing normal adult aortic SMCs (AASMCs), exhibiting typical SMC morphology/growth patterns; pdSMC1A displayed irregular cell shape/organization with numerous mitotic figures. Dedifferentiation to a synthetic/proliferative phenotype has been hypothesized as a critical step in atherogenesis, because rat neonatal SMCs and adult intimal SMCs exhibit similar gene expression patterns. To confirm that our pdSMC lines likewise express this apparent plaque phenotype, osteopontin, platelet-derived growth factor B, and elastin mRNA levels were determined in pdSMC1A, pdSMC2, and AASMCs. However, no significant increases in osteopontin or platelet-derived growth factor B expression levels were observed in either pdSMC compared with AASMCs. pdSMC2 alone expressed high levels of elastin mRNA. Lower levels of SM22 mRNA in pdSMC1A suggested greater dedifferentiation and/or additional population doublings in pdSMC1A relative to pdSMC2. Both pdSMC lines (particularly 1A) demonstrated high message levels for matrix Gla protein, previously reported to be highly expressed by human neointimal SMCs in vitro. These results describe 2 novel plaque cell lines exhibiting various features of plaque SMC biology; pdSMC2 may represent an earlier plaque SMC phenotype, whereas pdSMC1A may be representative of cells comprising an advanced atherosclerotic lesion.

Elevation of IL-6 in transgenic mice results in increased levels of the 90 kDa heat shock protein (hsp90) and the production of anti-hsp90 antibodies.

Treatment of human peripheral blood lymphocytes (PBL) in vitro with the cytokine interleukin-6 (IL-6) induces increased levels of the 90 kDa heat shock protein (hsp90). Hsp90 levels are also elevated in PBLs of human patients with systemic lupus erythematosus (SLE) and in MRL/lpr mice with autoimmune disease. Although IL-6 is elevated in both these situations it has not been shown that it is involved in stimulating elevation of hsp90 levels in vivo. Here we show directly that the elevation of IL-6 in vivo either in mice transgenic for the IL-6 gene or in knock-out mice lacking a functional gene for the transcription factor C/EBP beta (NF-IL-6) does indeed result in elevated hsp90 levels. This overexpression is associated with the specific production of autoantibodies to hsp90 in these mice which is also observed in SLE patients and MRL/1pr mice. Hence IL-6 is likely to play a critical role in the regulation of hsp90 levels both in autoimmune disease states and potentially in normal cells in vivo. In turn the elevated levels of hsp90 produced in autoimmune diseases are likely to be responsible for the observed production of anti-hsp90 autoantibodies.

Evidence that nerve growth factor plays a role in long-term potentiation in the rat dentate gyrus.

An inbred strain of Wistar rat (GH), which is deficient in nerve growth factor (NGF), was used to assess the possible role of NGF in the generation of long-term potentiation in perforant path-granule cell synapses. The data show that NGF was significantly decreased in the dentate gyrus of GH rats, that this deficit was accompanied by an impairment in long-term potentiation (LTP) and that intraventricular injection of NGF substantially reversed this impairment. Analysis of depolarization-induced glutamate release in synaptosomes prepared from dentate gyrus of control rats revealed that NGF alone was without effect, but in combination with the metabotropic glutamate receptor agonist, aminocyclopentane-1,3-dicarboxylic acid (ACPD), NGF induced a significant increase in release. This effect was occluded by prior induction of LTP, suggesting that the interaction between these agents may be required to enhance transmitter release which accompanies LTP in dentate gyrus. In contrast to the effect of NGF and ACPD on glutamate release in control rats, the combination of these agents had no effect on release in synaptosomes prepared from GH rats, which might be explained by the marked decrease in trk receptors in dentate gyrus of GH rats. It was concluded that the impaired ability of GH rats to sustain LTP is associated with a reduction in NGF concentration, a reduction in stimulated release of NGF and a decrease in trk receptors in dentate gyrus. It is proposed that these data indicate a role for NGF in the generation of long-term potentiation in perforant path-granule cell synapses.

Antibodies to heat-shock protein 27 are associated with improved survival in patients with breast cancer.

The overexpression of the heat-shock proteins hsp90, hsp70 and hsp27 in human mammary carcinomas has previously been shown to correlate with reduced overall survival. Moreover, antibodies to hsp90 were detectable in the serum of a large proportion of breast cancer patients but they were not found in normal controls. High antibody levels also correlated with reduced survival. Here, we show that antibodies to hsp27 were also detectable in the sera from breast cancer patients but not from normal controls, whereas antibodies to hsp70 were detectable in approximately one-third of both groups. The presence of antibodies to hsp27 was correlated with an improved rather than a reduced survival, particularly beyond the first 5 years. Hence, the overexpression of hsps in breast cancer cells does not provoke a generalized immune response to all the hsps. Moreover, the presence of antibodies to different hsps has distinct associations with survival. These effects are discussed in terms of the mechanisms that provoke an immune response to the hsps and the protective/non-protective effects of such a response.

Do heat shock proteins have a role in breast cancer?

It is clear therefore that hsps are overexpressed in patients with malignant tumours compared with healthy controls and this overexpression does show some correlation with disease features. Furthermore, expression of hsps has been reported on the cell surface of tumour cell lines. This could be associated with the immune response which has been reported with hsp90 and which also correlates with some disease features. It now appears that hsps may be involved in the presentation of tumour antigens leading to the possibility of hsps being used as a means of therapy. Hsp65 expression has not been investigated in patients with breast cancer. However, transfection of bacterial hsp65 into a tumour cell line resulted in the hsp65-expressing tumour cells losing their tumorigenicity in mice (Lukacs et al., 1993). Thus, hsps and the immune response to them are of interest as diagnostic and prognostic tools as well as a novel form of immunotherapy.

Characterization of human aortic smooth muscle cells expressing HPV16 E6 and E7 open reading frames.

A comparative study of human papillomavirus type 16 E6E7-transfected and normal human aortic smooth muscle cells by morphological, electron microscopic, immunofluorescent, and biochemical analyses demonstrated that the E6E7-expressing cells retained much of the phenotype of normal aortic smooth muscle cells, including expression of smooth muscle markers and appropriate growth responses to PDGF and heparin. These cells differed from normal vascular smooth muscle cells in that they had slightly altered morphology and a higher growth rate that was not due to an autocrine response to secreted PDGF, and they contained more polyribosomes than normal smooth muscle cells.