IL-1ra polymorphisms and risk of epidural-related maternal fever (EPIFEVER-2): study protocol for a multicentre, observational mechanistic cohort study.

BACKGROUND: Laboratory data suggest that insufficient circulating levels of the anti-inflammatory cytokine interleukin-1 receptor antagonist (IL-1ra) are associated with intrapartum inflammation and epidural-related maternal fever, both of which increase the rate of obstetric interventions and antibiotic use during labour. Genetic polymorphisms strongly influence IL-1ra levels in the general population. We aim to examine the association between IL-1ra polymorphisms and epidural-related maternal fever using Mendelian randomization analysis. METHODS: EPIFEVER-2 is a multicentre UK trial enrolling 637 women receiving epidural analgesia for labour. Blood samples obtained no later than four hours after epidural insertion will provide deoxyribonucleic acid for Taqman single-nucleotide polymorphism genotyping for presence/absence of rs6743376, rs1542176 alleles for IL-1ra, to establish the genetic score. The absence of both alleles is associated with the lowest IL-1ra levels. The primary outcome is pyrexia (>38°C) or intrapartum antibiotic administration. Secondary outcomes include mode of delivery, maternal and neonatal healthcare interventions. RESULTS: The EPIFEVER-2 study was prospectively registered (ISRCTN99641204) following ethical approval. Participant recruitment began in May 2021, with 221 women recruited across three centres as of November 21, 2021. CONCLUSIONS: EPIFEVER-2 will generate the largest prospective dataset detailing the incidence and consequences of epidural-related maternal fever. Using Mendelian randomisation analysis, a causative role for lower IL1-ra levels in determining the risk of epidural-related maternal fever and/or antibiotic administration before delivery will be examined.

Rapid parallel measurements of macroautophagy and mitophagy in mammalian cells using a single fluorescent biosensor.

Mitochondrial dysfunction is implicated in many human diseases and occurs in normal aging. Mitochondrial health is maintained through organelle biogenesis and repair or turnover of existing mitochondria. Mitochondrial turnover is principally mediated by mitophagy, the trafficking of damaged mitochondria to lysosomes via macroautophagy (autophagy). Mitophagy requires autophagy, but is itself a selective process that relies on specific autophagy-targeting mechanisms, and thus can be dissociated from autophagy under certain circumstances. Therefore, it is important to assess autophagy and mitophagy together and separately. We sought to develop a robust, high-throughput, quantitative method for monitoring both processes in parallel. Here we report a flow cytometry-based assay capable of rapid parallel measurements of mitophagy and autophagy in mammalian cells using a single fluorescent protein biosensor. We demonstrate the ability of the assay to quantify Parkin-dependent selective mitophagy in CCCP-treated HeLa cells. In addition, we show the utility of the assay for measuring mitophagy in other cell lines, as well as for Parkin-independent mitophagy stimulated by deferiprone. The assay makes rapid measurements (10,000 cells per 6 seconds) and can be combined with other fluorescent indicators to monitor distinct cell populations, enabling design of high-throughput screening experiments to identify novel regulators of mitophagy in mammalian cells.

Systemic gene transfer of binding immunoglobulin protein (BiP) prevents disease progression in murine collagen-induced arthritis.

Summary Recombinant human binding immunoglobulin protein (BiP) has previously demonstrated anti-inflammatory properties in multiple models of inflammatory arthritis. We investigated whether these immunoregulatory properties could be exploited using gene therapy techniques. A single intraperitoneal injection of lentiviral vector containing the murine BiP (Lenti-mBiP) or green fluorescent protein (Lenti-GFP) transgene was administered in low- or high-dose studies during early arthritis. Disease activity was assessed by visual scoring, histology, serum cytokine and antibody production measured by cell enzyme-linked immunosorbent assay (ELISA) and ELISA, respectively. Lentiviral vector treatment caused significant induction of interferon (IFN)-γ responses regardless of the transgene; however, further specific effects were directly attributable to the BiP transgene. In both studies Lenti-mBiP suppressed clinical arthritis significantly. Histological examination showed that low-dose Lenti-mBiP suppressed inflammatory cell infiltration, cartilage destruction and significantly reduced pathogenic anti-type II collagen (CII) antibodies. Lenti-mBiP treatment caused significant up-regulation of soluble cytotoxic T lymphocyte antigen-4 (sCTLA-4) serum levels and down-regulation of interleukin (IL)-17A production in response to CII cell restimulation. In-vitro studies confirmed that Lenti-mBiP spleen cells could significantly suppress the release of IL-17A from CII primed responder cells following CII restimulation in vitro, and this suppression was associated with increased IL-10 production. Neutralization of CTLA-4 in further co-culture experiments demonstrated inverse regulation of IL-17A production. In conclusion, these data demonstrate proof of principle for the therapeutic potential of systemic lentiviral vector delivery of the BiP transgene leading to immunoregulation of arthritis by induction of soluble CTLA-4 and suppression of IL-17A production.

Oncocytic choroid plexus carcinoma: case report.

Herein, we report an unusual choroid plexus carcinoma with extensive oncocytic transformation. A 13-month-old girl presented with acute lethargy which quickly progressed to coma. A CT scan of the head revealed impending herniation due to hemorrhage within an intracranial tumor. An MRI scan showed a large, partly cystic and highly vascular left lateral ventricular mass. A near total resection was achieved. Microsections revealed a WHO Grade III choroid plexus carcinoma with extensive oncocyti c transformation. A minor portion of the moderately to poorly differentiated tumor exhibited classical microscopic features of choroid plexus carcinoma, including marked nuclear atypia, brisk mitotic activity (78/10 HPF), a high MIB-1 labeling index (44%) and zones of necrosis. In contrast, the large, eosinophilic, cytologically malignant but granular-appearing oncocytes comprising the majority of the lesion showed scant (1/10 HPF) mitotic activity and only a low MIB-1 labeling index (5%). A subsequent recurrence at 1 year consisted entirely of non-oncocytic tumor. Choroid plexus carcinoma with oncocytic transformation has not been previously reported. The remarkable extent of this alteration and its clinical significance remains to be determined.

A nickel-carbon-fibre composite for large adaptive mirrors: fabrication methods and properties.

We present results from our recent research into carbon-fibre composite (CFC) mirror fabrication for optical and infra-red applications. In particular this research is aimed towards the next generation of extremely large telescopes to offer an alternative to thin glass shell adaptive secondary mirrors. We address the issues involved with CFC mirror production, in particular the accuracy of the form replication process, a suitable surface for polishing to optical quality, no fibre print-through, environmental stability (shape change due to thermal and moisture variations), material uniformity and lifetime. We have performed experiments into the effectiveness of cold electroplating thick nickel coatings to totally encapsulate the CFC base substrate; the manufacturing procedure and properties of the Ni-CFC mirror are described here.

Peripheral blood mononuclear cell proliferative responses to soluble and particulate heat shock protein 65 in health and inflammatory bowel disease.

OBJECTIVES: The aims of this study were to determine, in peripheral blood mononuclear cells (PBMC), whether particulate antigen triggers (i) an amplified cell proliferative response compared to soluble antigen and (ii) a dysfunctional response in cells derived from patients with chronic inflammation and specifically in those with inflammatory bowel disease (IBD). SUBJECTS: Healthy volunteers (n = 17), inflammatory controls (n = 8) and patients with IBD (n = 17) were recruited from St Thomas' and Guys' Hospital, London, UK. METHODS: Following optimisation of experimental conditions (0.1-10.0 mug/ml antigen), PBMC were stimulated with (i) 10.0 mug/ml recombinant soluble heat shock protein 65 (hsp 65) and (ii) 1.0 and 10.0 mug/ml hsp 65 conjugated to microparticles (0.5 mum diameter). PBMC proliferative responses were measured by (3)H-Thymidine incorporation at day 5 and results compared between groups using unpaired t-test. RESULTS: Conjugation to microparticles of low dose hsp 65 significantly increased overall proliferative responses by 2-11 fold compared to soluble antigen alone (p < 0.05). However, no specific PBMC proliferative dysregulation was noted in cells from subjects with IBD. CONCLUSIONS: Low dose antigen, in microparticulate form, leads to amplified cell proliferation in primary human cells, as showed previously in cell lines and animal studies. However there is no abnormal proliferative response in cells from subjects with IBD.

An investigation of auto-reactivity after head injury.

Murine models of CNS injury show auto-reactive T cell responses directed at myelin antigens, associated with improved neuronal survival and functional recovery. This pilot study shows, for the first time, that similar immune responses against myelin occur in human traumatic brain injury (TBI), with an expansion of lymphocytes recognising myelin basic protein observed in 40% of patients studied. "Reactive" patients did not have greater contusion volume on imaging, but were younger than the "unreactive" subgroup and tended towards a more favorable outcome. These findings are consistent with the concept of "beneficial autoimmunity".

Brain metastases in paediatric solid tumours.

Brain metastases in children with cancer are rare and their incidence is significantly lower (5-10%) than that reported in adults. The development of metastatic brain tumours in children is usually a manifestation of advanced disease and commonly occur after, or at the time of progression at other sites. This review summarises the salient clinical features of the most common paediatric solid tumours that metastasize to the brain including neuroblastoma, musculoskeletal sarcomas, germ cell tumours and melanoma.

Morbidity of second-look surgery in pediatric central nervous system tumors.

The extent of brain tumor resection affects survival. Second-look surgery (resection of residual tumor before radiographic progression) may improve survival by reducing the tumor burden, but the morbidity of the procedure is not known. On chart review of 280 patients with two or more brain tumor operations treated between January 1985 and June 1998, we identified 47 patients with second-look surgery. Lansky and Eastern Cooperative Oncology Group (ECOG) performance scores, as well as perioperative complications were recorded. There were 21 gliomas (6 malignant), 12 medulloblastomas, 3 craniopharyngiomas, 3 ependymomas and 8 miscellaneous tumors. Median time to second surgery was 50 days. Perioperative complications occurred in 45% of patients. There was no significant change in the mean Lansky and ECOG scores 4 and 24 weeks after surgery. Gross total resection (GTR) was achieved in 62% of patients and near total resection (NTR) in 23%, and 15% of patients had subtotal resection. GTR or NTR was achieved in 66% of medulloblastomas and 100% of gliomas. We conclude that second-look surgery by experienced pediatric neurosurgeons has an acceptable morbidity and should be considered in patients with residual tumors.

Dietary methionine is involved in the etiology of neural tube defect-affected pregnancies in humans.

Research has provided evidence of the role of multivitamin supplementation in the prevention of neural tube defects (NTD). Failure of the neural tube to close is one of the most frequent and severe human developmental defects. The etiology of NTD is complex, encompassing genetic, dietary and environmental factors. The purpose of this study was to explore the relationship between maternal dietary intake of methionine and the risk of having a NTD-affected pregnancy. We hypothesized that women with high maternal dietary methionine intake were at a decreased risk for a NTD. Combinations of methionine, folate and vitamin B-12 intakes and NTD risk were also examined. Data from a 5-y, population-based, case-control study of 170 NTD-affected pregnancies and 269 controls were provided by the South Carolina NTD Surveillance, Prevention, and Research Project. There was a 30-55% lower NTD risk among women whose average daily dietary intake of methionine was greater than the lowest quartile of intake (>1580 mg/d). The odds ratios associated with the three quartiles of methionine intake > 1580 mg/d after adjusting for energy, race and body mass index were 0.72 (P < 0.07), 0.68 (P < 0.07) and 0.45 (P < 0.06), respectively. These findings indicate that a reduction in the risk of having a NTD-affected pregnancy is associated with maternal dietary intake of methionine (3 mo pre- to 3 mo postconception). This finding is consistent with the hypothesis that methionine plays a role in the etiology of NTD and suggests the need for further research in the area of maternal diet and pregnancy.

Human aromatase in high yield and purity by perfusion chromatography and its characterization by difference spectroscopy and mass spectrometry.

Expression of human cytochrome P450 aromatase (CYP19A1, aromatase) was accomplished at a high level using a baculovirus expression system in an insect cell suspension culture. Using the relatively new chromatographic technique of perfusion chromatography, a very rapid procedure for purification of the protein from solubilized cells was developed. At extraordinary flow rates of between 3 and 9 column volumes per minute, all chromatographic procedures could be performed, including setup, equilibration, and column regeneration steps, in less than 2 h, not including brief dialysis periods. Total yields were 40-52% and resulted in preparations with specific content values of 17.1 nmol aromatase/mg protein. Final purified preparations showed virtually no typical P450 spectra under standard conditions, but displayed full activity with typical enzyme kinetic parameters. These unusual results suggest that standard methods of P450 measurement are inappropriate when applied to aromatase. The findings are fully consistent with those encountered previously for purified preparations from a human placental source and led us to a new aromatase quantification method based on ligand-induced difference spectroscopy. A new HPLC assay is described which rapidly separates heme and apoprotein while measuring total heme content. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry was employed with both glycosylated and deglycosylated forms of the final purified product to confirm its identity as a glycosylated cytochrome P450.

Distinct Albino3-dependent and -independent pathways for thylakoid membrane protein insertion.

The homologous proteins Oxa1, YidC, and Alb3 mediate the insertion of membrane proteins in mitochondria, bacteria, and chloroplast thylakoids, respectively. Depletion of YidC in Escherichia coli affects the integration of every membrane protein studied, and Alb3 has been shown previously to be required for the insertion of a signal recognition particle (SRP)-dependent protein, Lhcb1, in thylakoids. In this study we have analyzed the "global" role of Alb3 in the insertion of thylakoid membrane proteins. We show that insertion of two chlorophyll-binding proteins, Lhcb4.1 and Lhcb5, is almost totally blocked by preincubation of thylakoids with anti-Alb3 antibodies, indicating a requirement for Alb3 in the insertion pathway. Insertion of the related PsbS protein, on the other hand, is unaffected by Alb3 antibodies, and insertion of a group of SRP-independent, signal peptide-bearing proteins, PsbX, PsbW, and PsbY, is likewise completely unaffected. Proteinase K is furthermore able to completely degrade Alb3, but this treatment does not affect the insertion of these proteins. Among the thylakoid proteins studied here, Alb3 requirement correlates strictly with a requirement for stromal factors and nucleoside triphosphates. However, the majority of proteins tested do not require Alb3 or any other known form of translocation apparatus.

Influence of family structure on health among youths with diabetes.

Type 1 diabetes mellitus is a chronic metabolic disorder and is one of the most common chronic diseases in childhood. The study discussed in this article examined the extent to which family structure is significantly associated with health in youths with Type 1 diabetes. A convenience sample of 155 children with diabetes and their mothers completed face-to-face interviews; multiple regression analyses were conducted. Findings demonstrated that family structure remains a significant predictor of youths' health when statistically controlling for race, child's age, family socioeconomic status, and adherence. Social workers in outpatient medical settings are in a unique position to develop family-oriented strategies targeting this neglected area of primary care.

Multiple pathways used for the targeting of thylakoid proteins in chloroplasts.

The assembly of the chloroplast thylakoid membrane requires the import of numerous proteins from the cytosol and their targeting into or across the thylakoid membrane. It is now clear that multiple pathways are involved in the thylakoid-targeting stages, depending on the type of protein substrate. Two very different pathways are used by thylakoid lumen proteins; one is the Sec pathway which has been well-characterised in bacteria, and which involves the threading of the substrate through a narrow channel. In contrast, the more recently characterised twin-arginine translocation (Tat) system is able to translocate fully folded proteins across this membrane. Recent advances on bacterial Tat systems shed further light on the structure and function of this system. Membrane proteins, on the other hand, use two further pathways. One is the signal recognition particle-dependent pathway, involving a complex interplay between many different factors, whereas other proteins insert without the assistance of any known apparatus. This article reviews advances in the study of these pathways and considers the rationale behind the surprising complexity.

Bacillus cereus bacteremia and meningitis in immunocompromised children.

Two cases of Bacillus cereus meningitis in immunocompromised children at our hospital within a 2-month period prompted us to review B. cereus--related invasive disease. We identified 12 patients with B. cereus isolated in blood cultures from September 1988 through August 2000 at our institution. Three of these patients also had B. cereus isolated from CSF specimens; 1 additional patient had possible CNS involvement (33%, group A), whereas 8 patients had no evidence of CNS involvement (67%, group B). Patients in group A were more likely to have neutropenia at the onset of sepsis and were more likely to have an unfavorable outcome. They were also more likely to have received intrathecal chemotherapy in the week before the onset of their illness. Two patients from group A died. One survived with severe sequelae. The fourth patient had mild sequelae at follow-up. No sequelae or deaths occurred among patients in group B. In patients with unfavorable outcomes, the interval from the time of recognition of illness to irreversible damage or death was short, which demonstrates a need for increased awareness, early diagnosis, and more-effective therapy, particularly that which addresses B. cereus toxins.

Immediate neurocognitive effects of methylphenidate on learning-impaired survivors of childhood cancer.

PURPOSE: To test if methylphenidate (MPH) has an objective beneficial effect on immediate performance on tests of neurocognitive functions among learning-impaired survivors of childhood acute lymphoblastic leukemia (ALL) and malignant brain tumors (BT). PATIENTS AND METHODS: From July 1, 1997 through December 31, 1998, 104 long-term survivors of childhood ALL or a malignant BT completed neurocognitive screening for learning impairments and concurrent problems with sustained attention. Eligibility criteria for the MPH trial included an estimated intelligence quotient greater than 50, academic achievement in the 16(th) percentile or lower for age in reading, math, or spelling, and an ability to sustain attention on a computerized version of the Conners' Continuous Performance Test (CPT) in the 16(th) percentile or lower for age and sex. Of the 104, 32 (BT, n = 25; ALL, n = 7) were eligible on the basis of these a priori criteria for a randomized, double-blinded, placebo-controlled trial of MPH. The patients ingested a placebo (lactose) or MPH (0.6 mg/kg; 20 mg maximum) and repeated selected portions of the screening battery 90 minutes later. RESULTS: Compared to the 17 patients randomized to the placebo group, the 15 patients randomized to the MPH group had a significantly greater improvement on the CPT for sustained attention (errors of omission, P =.015) and overall index (P =.008) but not for errors of commission (indicative of impulsiveness) nor reaction times. A trend for greater improvement in the MPH group on a measure of verbal memory failed to reach statistical significance. No trend was observed for MPH effectiveness in improving learning of a word association task. No significant side effects from MPH were observed. CONCLUSION: MPH resulted in a statistically significant improvement on measures of attention abilities that cannot be explained by placebo or practice effects.

Assessment of a two-generation reproductive and fertility study of mercuric chloride in rats.

Effects of mercuric chloride (MC) on the reproductive performance of two successive generations of rats was evaluated. F(0) rats were exposed to 0.0:0.0 (males:females), 0.50:0.75 (males:females), 1.00:1.50 (males:females) and 1.50:2.50 (males:females) mg/kg/day MC. Selected parental F(1) males and females were exposed to the same doses received by their parents (F(0)). Significant differences resulting from exposure of the F(0) generation to MC were found in implantation efficiency, fertility, live births and day 4 survival indices, litter size, and the body weight of F(1) pups. However, the continued exposure of the F(1) generation to MC did not affect fertility index or litter size, but did significantly affect implantation efficiency, live births and day 4 survival indices. In F(0) males, body weight and weights of the kidneys, testes, epididymides, prostate and seminal vesicles were significantly different, while in F(1) males, body weight, kidney weight, brain weight, liver weight and the weights of the testes, prostate and seminal vesicles were significantly different. In F(0) females, body weight and the weights of the kidneys, brain and liver were significantly different, while in F(1,) females, body weight, as well as the weights of the kidneys, liver, adrenals, uterus and ovaries were significantly different. These data showed that exposure to MC resulted in more adverse reproductive effects in the first generation and that these effects moderated in the second generation.

Comparison of single-mother and two-parent families on metabolic control of children with diabetes.

OBJECTIVE: To understand the impact of family structure on the metabolic control of children with diabetes, we posed two research questions: 1) what are the differences in sociodemographic, family, and community factors between single-mother and two-parent families of diabetic children? and 2) to what extent do these psychosocial factors predict metabolic control among diabetic children from single-mother and two-parent families? RESEARCH DESIGN AND METHODS: This cross-sectional study included 155 diabetic children and their mothers or other female caregivers. The children were recruited if they had been diagnosed with diabetes for at least 1 year, had no other comorbid chronic illnesses, and were younger than 18 years of age. Interviews and self-report questionnaires were used to assess individual, family, and community variables. RESULTS: The findings indicate that diabetic children from single-mother families have poorer metabolic control than do children from two-parent families. Regression models of children's metabolic control from single-mother families indicate that age and missed clinic appointments predicted HbA1c levels; however, among two-parent families, children's ethnicity and adherence to their medication regimen significantly predicted metabolic control. CONCLUSIONS: This study suggests that children from single-mother families are at risk of poorer metabolic control and that these families have more challenges to face when raising a child with a chronic illness. Implications point to a need for developing strategies sensitive to the challenges of single mothers.

Associations between self-reported and objective physical environmental factors and use of a community rail-trail.

BACKGROUND: To effectively promote physical activIty, researchers and policy makers have advocated for greater use of environmental approaches, such as the construction of community paths and trails. However, research on the use of these facilities is limited. METHODS: In this cross-sectional community study, we examined associations between self-reported and objective physical environmental variables and use of the Minuteman Bikeway (Arlington, MA) in a random sample of 413 adults. Sociodemographic and perceived environmental variables were measured with a mail survey during September 1998. Geographic information system (GIS) data were used to geocode survey respondents' homes and create three objective environmental variables: distance to the Bikeway, steep hill barrier, and a busy street barrier. RESULTS: In logistic models, age and female gender showed statistically significant inverse associations with Bikeway use over the previous 4-week period. Increases in self-reported (OR = 0.65) and GIS distance (OR = 0.57) were associated with decreased likelihood of Bikeway use. Absence of self-reported busy street (OR = 2.01) and GIS steep hill barriers (OR = 1.84) were associated with Bikeway use. CONCLUSIONS: Environmental barriers such as travel distance and hilly terrain should be considered when planning community trails. A better understanding of such factors may lead to more effective promotion of trail use.

Purification and characterization of hexahistidine-tagged cyclohexanone monooxygenase expressed in Saccharomyces cerevisiae and Escherichia coli.

Cyclohexanone monooxygenase (CMO) is a soluble flavoenzyme originally isolated from Acinetobacter spp. which carries out Baeyer-Villiger reactions with cyclic ketone substrates. In the present study we cloned the Acinetobacter CMO gene and modified it for facile purification from heterologous expression systems by incorporation of a His(6)-tag at its C-terminus. A single purification step employing metal (Ni(2+))-affinity column chromatography provided essentially homogeneous enzyme in yields of 69-72%. The properties of the purified, recombinant enzymes (rCMO) were compared with that of native CMO (nCMO) isolated from Acinetobacter cultures grown in the presence of cyclohexanone. The specific activities of His(6)-tagged rCMO and nCMO toward their index substrate, cyclohexanone, were similar and ranged from 14 to 20 micromol/min/mg. nCMO and rCMO from the Escherichia coli expression system exhibited molecular masses, determined by electrospray mass spectrometry, of 60,800 and 61,615 Da, respectively, an increase for the recombinant enzyme equivalent to the mass of the His(6)-tag. However, rCMO expressed in Saccharomyces cerevisiae consistently exhibited a mass some 50 Da larger than rCMO expressed in bacteria. Edman degradation confirmed that rCMO purified from the E. coli system and nCMO shared the same N-terminal sequence, whereas no sequence information could be obtained for rCMO expressed in yeast. Therefore, the yeast-expressed enzyme possesses an additional posttranslational modification(s), possibly acylation, at the N-terminus. Expression in E. coli is the preferred system for future site-directed mutagenesis studies and crystallization efforts.