Prostaglandin E2 receptor EP1 (PGE2/EP1) deletion promotes glomerular podocyte and endothelial cell injury in hypertensive TTRhRen mice.

Prostaglandin E2 receptor EP1 (PGE2/EP1) promotes diabetic renal injury, and EP1 receptor deletion improves hyperfiltration, albuminuria, and fibrosis. The role of EP1 receptors in hypertensive kidney disease (HKD) remains controversial. We examined the contribution of EP1 receptors to HKD. EP1 null (EP1-/-) mice were bred with hypertensive TTRhRen mice (Htn) to evaluate kidney function and injury at 24 weeks. EP1 deletion had no effect on elevation of systolic blood pressure in Htn mice (HtnEP1-/-) but resulted in pronounced albuminuria and reduced FITC-inulin clearance, compared with Htn or wild-type (WT) mice. Ultrastructural injury to podocytes and glomerular endothelium was prominent in HtnEP1-/- mice; including widened subendothelial space, subendothelial lucent zones and focal lifting of endothelium from basement membrane, with focal subendothelial cell debris. Cortex COX2 mRNA was increased by EP1 deletion. Glomerular EP3 mRNA was reduced by EP1 deletion, and EP4 by Htn and EP1 deletion. In WT mice, PGE2 increased chloride reabsorption via EP1 in isolated perfused thick ascending limb (TAL), but PGE2 or EP1 deletion did not affect vasopressin-mediated chloride reabsorption. In WT and Htn mouse inner medullary collecting duct (IMCD), PGE2 inhibited vasopressin-water transport, but not in EP1-/- or HtnEP1-/- mice. Overall, EP1 mediated TAL and IMCD transport in response to PGE2 is unaltered in Htn, and EP1 is protective in HKD.

Intracellular patterns of sialophorin expression define a new molecular classification of breast cancer and represent new targets for therapy.

BACKGROUND: Sialophorin is a transmembrane sialoglycoprotein. Normally, the molecule is only produced by white blood cells where it regulates functions such as intercellular adhesion, intracellular signalling, apoptosis, migration and proliferation. METHODS: Normal breast tissue and primary breast tumours were analysed by immunohistochemistry for sialophorin expression. The sialophorin-positive breast cancer cell line MCF7 was engineered to stably express either non-targeted or sialophorin-targeted small interfering RNA (siRNA). Assays were then performed in vitro to assess apoptosis, intracellular adhesion, transendothelial migration and cytotoxicity. An orthotopic mouse model assayed ability to produce tumours in vivo. RESULTS: Normal breast epithelial cells exhibit expression of the N-terminal domain of sialophorin in the cytoplasm but not the nucleus. The majority of these normal cells are also negative for expression of the C-terminal domain. In contrast, malignant breast epithelial cells exhibit N-terminal expression both in the cytoplasm and nucleus and the majority express the C-terminus in the nucleus. Using differential patterns of intracellular expression of the N and C termini of sialophorin, we define six subtypes of breast cancer that are independent of histological and receptor status classification. Targeting sialophorin with siRNA resulted in the MCF7 breast cancer cell line exhibiting increased homotypic adhesion, decreased transendothelial migration, increased susceptibility to apoptosis, increased vulnerability to lysis by natural killer cells and decreased ability to produce tumours in mice. CONCLUSION: Our results indicate that intracellular patterns of sialophorin expression define a new molecular classification of breast cancer and that sialophorin represents a novel therapeutic target.

Neurocognitive effects of CNS tumors.

There is ample evidence that many children treated for brain tumors experience long-term neurocognitive deficits. The severity of those deficits is determined by a complex interaction of the child's genetic make-up and age, neuroanatomical damage caused by tumor and surgery, radiotherapy and chemotherapy, the psychosocial environment, and the intensity of targeted rehabilitation. The consequences of neurocognitive deficits are moderated by the number and severity of other deficits, including neurological and endocrine impairments, and this wider context must be considered. The impact of intellectual decline on academic functioning is evident, and underlies, for example, poor reading, writing, and mathematical skills. The effects of early brain damage on development are cumulative as more functions are expected to mature. Many survivors of CNS tumors can be expected to grow into deficits that have far-reaching consequences not only for academic achievement but also for their psychological and social development and their ability to be self-sufficient. Because the problems typically only become apparent over time, surveillance for their detection is an essential prerequisite for early educational and other interventions to support learning and successful transition to independent adult life.

Critical role of Nox4-based NADPH oxidase in glucose-induced oxidative stress in the kidney: implications in type 2 diabetic nephropathy.

Molecular mechanisms underlying renal complications of diabetes remain unclear. We tested whether renal NADPH oxidase (Nox) 4 contributes to increased reactive oxygen species (ROS) generation and hyperactivation of redox-sensitive signaling pathways in diabetic nephropathy. Diabetic mice (db/db) (20 wk) and cultured mouse proximal tubule (MPT) cells exposed to high glucose (25 mmol/l, D-glucose) were studied. Expression (gene and protein) of Nox4, p22(phox), and p47(phox), but not Nox1 or Nox2, was increased in kidney cortex, but not medulla, from db/db vs. control mice (db/m) (P < 0.05). ROS generation, p38 mitogen-activated protein (MAP) kinase phosphorylation, and content of fibronectin and transforming growth factor (TGF)-ß1/2 were increased in db/db vs. db/m (P < 0.01). High glucose increased expression of Nox4, but not other Noxes vs. normal glucose (P < 0.05). This was associated with increased NADPH oxidase activation and enhanced ROS production. Nox4 downregulation by small-interfering RNA and inhibition of Nox4 activity by GK-136901 (Nox1/4 inhibitor) attenuated d-glucose-induced NADPH oxidase-derived ROS generation. High d-glucose, but not l-glucose, stimulated phosphorylation of p38MAP kinase and increased expression of TGF-ß1/2 and fibronectin, effects that were inhibited by SB-203580 (p38MAP kinase inhibitor). GK-136901 inhibited d-glucose-induced actions. Our data indicate that, in diabetic conditions: 1) renal Nox4 is upregulated in a cortex-specific manner, 2) MPT cells possess functionally active Nox4-based NADPH, 3) Nox4 is a major source of renal ROS, and 4) activation of profibrotic processes is mediated via Nox4-sensitive, p38MAP kinase-dependent pathways. These findings implicate Nox4-based NADPH oxidase in molecular mechanisms underlying fibrosis in type 2 diabetic nephropathy.

Ethical considerations in the collection of genetic data from critically ill patients: what do published studies reveal about potential directions for empirical ethics research?

Critical illness trials involving genetic data collection are increasingly commonplace and pose challenges not encountered in less acute settings, related in part to the precipitous, severe and incapacitating nature of the diseases involved. We performed a systematic literature review to understand the nature of such studies conducted to date, and to consider, from an ethical perspective, potential barriers to future investigations. We identified 79 trials enrolling 24 499 subjects. Median (interquartile range) number of participants per study was 263 (116.75-430.75). Of these individuals, 16 269 (66.4%) were Caucasian, 1327 (5.4%) were African American, 1707 (7.0%) were Asian Pacific Islanders and 139 (0.6%) were Latino. For 5020 participants (20.5%), ethnicity was not reported. Forty-eight studies (60.8%) recruited subjects from single centers and all studies examined a relatively small number of genetic markers. Technological advances have rendered it feasible to conduct clinical studies using high-density genome-wide scanning. It will be necessary for future critical illness trials using these approaches to be of greater scope and complexity than those so far reported. Empirical research into issues related to greater ethnic inclusivity, accuracy of substituted judgment and specimen stewardship may be essential for enabling the conduct of such trials.

Large scale calcium channel gene rearrangements in episodic ataxia and hemiplegic migraine: implications for diagnostic testing.

BACKGROUND: Episodic ataxia type 2 (EA2) and familial hemiplegic migraine type 1 (FHM1) are autosomal dominant disorders characterised by paroxysmal ataxia and migraine, respectively. Point mutations in CACNA1A, which encodes the neuronal P/Q-type calcium channel, have been detected in many cases of EA2 and FHM1. The genetic basis of typical cases without CACNA1A point mutations is not fully known. Standard DNA sequencing methods may miss large scale genetic rearrangements such as deletions and duplications. The authors investigated whether large scale genetic rearrangements in CACNA1A can cause EA2 and FHM1. METHODS: The authors used multiplex ligation dependent probe amplification (MLPA) to screen for intragenic CACNA1A rearrangements. RESULTS: The authors identified five previously unreported large scale deletions in CACNA1A in seven families with episodic ataxia and in one case with hemiplegic migraine. One of the deletions (exon 6 of CACNA1A) segregated with episodic ataxia in a four generation family with eight affected individuals previously mapped to 19p13. In addition, the authors identified the first pathogenic duplication in CACNA1A in an index case with isolated episodic diplopia without ataxia and in a first degree relative with episodic ataxia. CONCLUSIONS: Large scale deletions and duplications can cause CACNA1A associated channelopathies. Direct DNA sequencing alone is not sufficient as a diagnostic screening test.

The EP1 subtype of prostaglandin E2 receptor: role in keratinocyte differentiation and expression in non-melanoma skin cancer.

We have previously demonstrated that the EP1 subtype of PGE2 receptor is expressed in the differentiated compartment of normal human epidermis and is coupled to intracellular calcium mobilization. We therefore hypothesized that the EP1 receptor is coupled to keratinocyte differentiation. In in vitro studies, radioligand binding, RT-PCR, immunoblot and receptor agonist-induced second messenger studies demonstrate that the EP1 receptor is up-regulated by high cell density in human keratinocytes and this up-regulation precedes corneocyte formation. Moreover, two different EP1 receptor antagonists, SC51322 and AH6809, both inhibited corneocyte formation. SC51322 also inhibited the induction of differentiation-specific proteins, cytokeratin K10 and epidermal transglutaminase. We next examined the immunolocalization of the EP1 receptor in non-melanoma skin cancer in humans. Well-differentiated SCCs exhibited significantly greater membrane staining, while spindle cell carcinomas and BCCs had significantly decreased membrane staining compared with normal epidermis. This data supports a role for the EP1 receptor in regulating keratinocyte differentiation.

The ecology of parasites of freshwater fishes: the search for patterns.

Developments in the study of the ecology of helminth parasites of freshwater fishes over the last half century are reviewed. Most research has of necessity been field based and has involved the search for patterns in population and community dynamics that are repeatable in space and time. Mathematical models predict that under certain conditions host and parasite populations can attain equilibrial levels through operation of regulatory factors. Such factors have been identified in several host-parasite systems and some parasite populations have been shown to persist over long time-periods. However, there is no convincing evidence that fish parasite populations are stable and regulated since in all cases alternative explanations are equally acceptable and it appears that they are non-equilibrial systems. It has proved particularly difficult to detect replicable patterns in parasite communities. Inter-specific competition, evidenced by functional and numerical responses, has been detected in several communities but its occurrence is erratic and its significance unclear. Some studies have failed to find any nested patterns in parasite community structure and richness, whereas others have identified such patterns although they are seldom constant over space and time. Departures from randomness appear to be the exception and then only temporary. It appears that parasite communities are non-equilibrial, stochastic assemblages rather than structured and organized.

Reading and communication skills after universal newborn screening for permanent childhood hearing impairment.

BACKGROUND: Birth in periods with universal newborn screening (UNS) for permanent childhood hearing impairment (PCHI) and early confirmation of PCHI have been associated with superior subsequent language ability in children with PCHI. However their effects on reading and communication skills have not been addressed in a population-based study. METHODS: In a follow-up study of a large birth cohort in southern England, we measured reading by direct assessment and communication skills by parent report in 120 children with bilateral moderate, severe or profound PCHI aged 5.4-11.7 years, of whom 61 had been born in periods with UNS, and in a comparison group of 63 children with normal hearing. RESULTS: Compared with birth during periods without UNS, birth during periods with UNS was associated with better reading scores (inter-group difference 0.39 SDs, 95% CI 0.02 to 0.76, p = 0.042) and communication skills scores (difference 0.51 SDs, 95% CI 0.06 to 0.95, p = 0.026). Compared with later confirmation, confirmation of PCHI by age 9 months was also associated with better reading (difference 0.51 SDs, 95% CI 0.15 to 0.87, p = 0.006) and communication skills (difference 0.56 SDs, 95% CI 0.12 to 1.00, p = 0.013). In the children with PCHI, reading, communication and language ability were highly correlated (r = 0.62-0.84, p<0.001). CONCLUSION: Birth during periods with UNS and early confirmation of PCHI predict better reading and communication abilities at primary school age. These benefits represent functional gains of sufficient magnitude to be important in children with PCHI.

Human immature oocytes grow during culture for IVM.

BACKGROUND: Oocyte competence for maturation and embryogenesis is associated with diameter in many mammals. We aimed to test whether this relationship exists in humans and to quantify its impact upon in vitro maturation (IVM). METHODS: We used computer-assisted image analysis daily to measure average diameter, zona thickness and other parameters in oocytes. Immature oocytes originated from unstimulated patients with polycystic ovaries, and from stimulated patients undergoing intracytoplasmic sperm injection (ICSI). Some were cultured with meiosis activating sterol (FF-MAS). Matured oocytes were inseminated using ICSI and embryo development was monitored. In vivo matured oocytes were also measured. RESULTS: Immature oocytes were smaller at collection than in vivo matured oocytes. Maturation was related to oocyte diameter and many oocytes grew in culture. FF-MAS stimulated growth in oocytes derived from ICSI patients, but only stimulated growth in PCO derived oocytes if they matured in vitro. Degenerating oocytes showed cytoplasmic shrinkage. Neither zona thickness, perivitelline space, nor the total diameter of the oocyte plus zona were informative regarding maturation capacity. CONCLUSIONS: Immature oocytes grow during maturation culture. FF-MAS promotes oocyte growth in vitro. Oocytes from different sources have different growth profiles in vitro. Measuring oocytes in clinical IVM may provide additional non-invasive information that could potentially avoid the use of growing oocytes.

The pathogenic helminth parasites of eels.

Although 63 and 55 species of helminths have been reported from each species of Atlantic eel and from 29 to 19 for each species of Pacific eel only the monogeneans Pseudodactylogyrus bini and P. anguillae and the nematode Anguillicola crassus, originally specific to species of Pacific eels, can be considered serious pathogens. None of the three are normally pathogenic to their preferred natural eel host species in the wild. Pseudodactylogyrus spp. only cause serious local gill damage when present on a host in large numbers under optimal conditions that facilitate transmission. This is the case in eel aquaculture, where infections can be controlled by drugs. Anguillicola crassus is only pathogenic to Anguilla anguilla and A. rostrata when Atlantic eels are introduced to the far east or when the parasites have been introduced to Europe. Here the parasite life cycle differs in that A. crassus can infect a wide range of intermediate hosts, employ paratenic hosts and survive as larvae for months in the swimbladder wall. This makes it an excellent colonizer. Its major pathogenic effects on eels result from haemorrhaging in, and thickening of, the swimbladder wall. It reduces the oxygen concentration in the swimbladder, reducing its ability to function as a hydrostatic organ, and increases the stress response of eels. In shallow lakes at warm temperatures this can result in mass mortalities. It is also feared that the parasite affects the ability of eels to migrate to the Sargasso Sea and so contributes to the decline in eel populations. Control by drug treatment is possible in culture, but not in the wild.

Risk management in IVF.

Patient safety incidents occur in approximately 10% of hospital admissions in the UK. Although robust data are not available, assisted conception is unlikely to be any less prone to adverse incidents; indeed there have been several high-profile cases which have drawn attention to this problem. Recently established national reporting systems for adverse events are collecting data which will inform trends in patient safety. Because of the nature of the work undertaken in assisted conception, there is the potential to affect not only future generations but also many patients simultaneously because of storage of biological material. It is therefore important to implement strategies to reduce the likelihood of patient safety incidents. Established methodologies exist for the reactive (root cause analysis) and the proactive assessment of risk (failure mode effects analysis). Furthermore, establishing the detail of a process and its context through process mapping is an important prerequisite for understanding its risk. The knowledge gained through these enquiries enables the implementation of an effective risk management programme which this chapter examines in detail.

How old is too old for fertility treatment?

Women are delaying starting their families. High-profile women such as Cherie Blair and Madonna are having successful pregnancies with healthy babies in their forties. Is this sending out messages to women that delaying conception during their twenties and thirties is a good idea? Fertility treatment can overcome certain age-related hurdles but is certainly not the magic wand some expect it to be.

Upregulation of adiponectin receptor 1 and 2 mRNA and protein in adipose tissue and adipocytes in insulin-resistant women with polycystic ovary syndrome.

AIMS/HYPOTHESIS: Polycystic ovary syndrome (PCOS) is a multifaceted metabolic disease linked with insulin resistance (IR) and obesity. Adiponectin, which is lower in IR states, exerts its glucose-lowering and anti-inflammatory effects by activating two receptors, ADIPOR1 and ADIPOR2. There are no data on the relative expression of these receptors in adipose tissue of PCOS women. METHODS: We investigated the expression of adiponectin receptors from corresponding s.c. and omental (o.m.) adipose tissue in women with PCOS compared with matched non-PCOS women. As there is a disturbance in the steroid milieu in PCOS women, we also assessed the effects of testosterone and oestradiol on adiponectin receptors using adipocytes and adipocyte explants. Real-time RT-PCR and western blotting were used to assess the relative adiponectin receptor mRNA expression and protein production, respectively. Biochemical measurements were performed in our hospital's laboratory. RESULTS: We are the first to describe adiponectin receptor expression and production, in corresponding s.c. and o.m. human adipose tissues at the mRNA and protein level. We demonstrate the upregulation of mRNA expression and protein production of adiponectin receptors in women with PCOS, in s.c. and o.m. adipose tissue. Treatment of adipose tissue explants and adipocytes with testosterone and oestradiol induced the expression of adiponectin receptor mRNA and protein. There was a significant positive association between ADIPOR1/R2 expression and homeostasis model assessment, testosterone, oestradiol and triglycerides and a negative relationship with sex hormone-binding globulin. CONCLUSIONS/INTERPRETATION: The precise reason for the upregulation of adiponectin receptors seen in PCOS women, a pro-diabetic state, is unknown, but it appears that sex steroids may play a role in their regulation in adipose tissue.

FSGS-associated alpha-actinin-4 (K256E) impairs cytoskeletal dynamics in podocytes.

Mutations in the ACTN4 gene, encoding the actin crosslinking protein alpha-actinin-4, are associated with a familial form of focal segmental glomerulosclerosis (FSGS). Mice with podocyte-specific expression of K256E alpha-actinin-4 develop foot process effacement and glomerulosclerosis, highlighting the importance of the cytoskeleton in podocyte structure and function. K256E alpha-actinin-4 exhibits increased affinity for F-actin. However, the downstream effects of this aberrant binding on podocyte dynamics remain unclear. Wild-type and K256E alpha-actinin-4 were expressed in cultured podocytes via adenoviral infection to determine the effect of the mutation on alpha-actinin-4 subcellular localization and on cytoskeletal-dependent processes such as adhesion, spreading, migration, and formation of foot process-like peripheral projections. Wild-type alpha-actinin-4 was detected primarily in the Triton-soluble fraction of podocyte lysates and localized to membrane-associated cortical actin and focal adhesions, with some expression along stress fibers. Conversely, K256E alpha-actinin-4 was detected predominantly in the Triton-insoluble fraction, was excluded from cortical actin, and localized almost exclusively along stress fibers. Both wild-type and K256E alpha-actinin-4-expressing podocytes adhered equally to an extracellular matrix (collagen-I). However, podocytes expressing K256E alpha-actinin-4 showed a reduced ability to spread and migrate on collagen-I. Lastly, K256E alpha-actinin-4 expression reduced the mean number of actin-rich peripheral projections. Our data suggest that aberrant sequestering of K256E alpha-actinin-4 impairs podocyte spreading, motility, and reduces the number of peripheral projections. Such intrinsic cytoskeletal derangements may underlie initial podocyte damage and foot process effacement encountered in ACTN4-associated FSGS.

The presenting features of brain tumours: a review of 200 cases.

OBJECTIVE: To determine the presenting features of brain tumours in children. DESIGN: Retrospective case note review. SETTING: Paediatric and neurosurgical services at the Wessex Neurology Centre and Southampton General Hospital, UK. PATIENTS: 200 patients presenting with a CNS tumour between 1988 and 2001. RESULTS: The commonest first presenting symptoms were headache (41%), vomiting (12%), unsteadiness (11%), visual difficulties (10%), educational or behavioural problems (10%), and seizures (9%). The commonest symptoms occurring at any time were headache (56%), vomiting (51%), educational or behavioural problems (44%), unsteadiness (40%), and visual difficulties (38%). Neurological signs were present at diagnosis in 88%: 38% had papilloedema, 49% cranial nerve abnormalities, 48% cerebellar signs, 27% long tract signs, 11% somatosensory abnormalities, and 12% a reduced level of consciousness. The median symptom interval was 2.5 months (range 1 day to 120 months). A short symptom interval was significantly associated with high grade tumours and patient age of 3 years or younger. CONCLUSIONS: The well known predominance of headache in children with CNS tumours is confirmed. Visual, behavioural, and educational symptoms were also prominent. With the exception of seizures, every initial symptom was accompanied by other symptoms or signs by the time of diagnosis. Questions about visual symptoms and educational or behavioural difficulties, as well as the more widely recognised symptoms of raised intracranial pressure and motor dysfunction, are important in the diagnosis of brain tumours, as are vision assessment and the appropriate plotting of growth and head size.

European eels, Anguilla anguilla (L.), infected with Anguillicola crassus exhibit a more pronounced stress response to severe hypoxia than uninfected eels.

The parasite, Anguillicola crassus is a non-native species that infects naive European eels, Anguilla anguilla, and causes pathological damage to the swimbladder, potentially compromising their ability to cope with hypoxic conditions. This study aimed to elucidate whether anguillicolosis exacerbates the stress responses to exposure to hypoxic water, conditions that have been implicated in mass mortalities of wild infected European eels. Blood parameters in infected and uninfected eels were measured during exposure to severe hypoxia over an 8-h period. Infected fish showed significantly higher levels of plasma cortisol compared with uninfected eels after 4 h of hypoxia. Uninfected fish showed an almost twofold increase in plasma glucose after 8-h exposure to hypoxia but infected fish showed no significant change, so that the plasma glucose concentration was significantly higher in uninfected eels than in infected eels. Both groups showed similar elevations in blood haematocrit, suggesting a similar catecholamine response in infected and uninfected eels. The lack of a hyperglycaemic response in infected eels, despite indirect evidence of a catecholamine response to hypoxia, may reflect an increase in glucose turnover. The data suggest that anguillicolosis results in a significantly greater corticosteroid stress response to hypoxia accompanied by a higher metabolic cost.

Dynamics and predicted decline of Anguillicola crassus infection in European eels, Anguilla anguilla, in Neusiedler See, Austria.

The eel population in Neusiedler See has been maintained by regular massive stocking since 1958. After the establishment of the National Park Neusiedler See-Seewinkel in 1993, eel stocking was prohibited and the population, together with the specific parasites of eels, was predicted to decline to extinction within 10 years. This investigation was undertaken to document the decline and extinction of the Anguillicola crassus population in eels. From 1994 to 2001, 720 eels were collected from two sites in the lake. Prevalence and abundance of A. crassus were lower in spring than in summer and autumn and larger eels harboured more parasites than smaller ones. Neither year of study nor sampling site were correlated with parasite infection levels. No significant trend in the population parameters of A. crassus was detected over the 8 years of the survey. This suggested that there had been no significant decline in the eel population. This suggestion was confirmed by investigations of the fishery, which also found evidence of regular illegal stocking. The stability of the A. crassus population over the past decade seems to reflect the lack of change in eel population density. No mass mortalities of eels occurred over the period despite the many similarities between Neusiedler See and Lake Balaton in Hungary. Differences in eel size, eel diet and the lack of large-scale insecticide use are discussed as possible explanations for the absence of eel mass mortalities in Neusiedler See.