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AIM Neonatal haemolysis increases bilirubin production rates, which can then lead to severe hyperbilirubinaemia and bilirubin neurotoxicity. During haemolysis, haem is degraded by haem oxygenase (HO), which can be induced under stress conditions. It is known that neonatal sepsis is a risk factor for haemolysis and severe hyperbilirubinaemia. Here, we evaluated whether an exposure to lipopolysaccharide (LPS) to induce sepsis can upregulate HO-1, further increasing in vivo bilirubin production rates in mouse pups under haem loads. METHODS Three-day-old pups were given LPS (1250 μg/kg) or saline (controls). Liver HO enzyme activity, HO-1 mRNA and HO-1 protein were measured post-LPS exposure. We then assessed the effects of LPS treatment on in vivo bilirubin production rates after haem loading. RESULTS Liver HO activity significantly increased (142%) over controls 24 hours after treatment with LPS (1250 μg/kg) without mortality. Liver HO-1 mRNA was significantly upregulated 2.47-fold at 24 hours post-LPS administration, while liver HO-1 protein increased 1.29-fold 24 hours post-LPS treatment. After haem loading, pups exposed to LPS had significantly higher bilirubin production rates (1.30-fold) compared with age-matched, saline-treated controls. CONCLUSION Low-dose LPS treatment can upregulate liver HO-1 expression and may underlie the severe hyperbilirubinaemia seen in septic infants, particularly when undergoing haemolysis.

Introduction Sickle cell disease in pregnancy carries a high risk of maternal and fetal adverse outcomes. The use of prophylactic transfusions to reduce the risk of sickle complications is controversial. Current UK standards do not recommend the routine use of transfusion for sickle pregnancy. We examined transfusion episodes during sickle pregnancies in a single centre over an 11-year period. Methods We conducted a retrospective observational study of all pregnancies in patients with sickle cell disease who attended the joint obstetric/haematology clinic over an 11-year period. All pregnancies were managed according to a local protocol, which did not recommend routine transfusion. Results A total of 38 pregnancies (HbSS 22, HbSC 13, Hb S/beta thalassaemia 3) were included, with a mean age at booking of 29 years. A total of 61% of pregnancies required on-demand or emergency transfusion during the course of pregnancy or post-partum. Women requiring a transfusion during pregnancy had a higher mean number of hospital admissions in the previous year (1·11 vs 0·15, P = 0·057), a significantly lower mean steady-state haemoglobin (85·0 vs 99·6 g L-1 , P = 0·003) and a significantly lower mean haemoglobin at the pregnancy booking visit. (86·1 vs 99·5 g L-1 , P = 0·02). Conclusion In sickle pregnancies assigned to standard management in a single centre, a high proportion of women required on-demand transfusion. Possible pre-pregnancy factors predictive of a need for transfusion include lower baseline haemoglobin and number of hospital admissions in the previous 12 months.

Aim Relative contributions of increased production [by end-tidal carbon monoxide concentrations (ETCOc)] and decreased elimination of bilirubin to predischarge hour-specific total bilirubin (TB) levels were assessed in healthy late-preterm and term newborns. Secondly, we report predischarge ETCOc ranges to guide clinical management of hyperbilirubinemia. Methods TB and ETCOc (≤3 timepoints) determinations of newborns aged between six hours and 1.5 (p < 0.00003). Conclusion Near-simultaneous ETCOc and TB measurements in infants with TB >75th percentile accurately identify haemolytic hyperbilirubinemia.

Single-cell technologies have immense potential to shed light on molecular and biological processes that drive human diseases. Mass cytometry (or Cytometry by Time Of Flight mass spectrometry, CyTOF) has already been employed in clinical studies to comprehensively survey patients' circulating immune system. As interest in the "bedside" application of mass cytometry is growing, the delineation of relevant methodological issues is called for. This report uses a newly generated dataset to discuss important methodological considerations when mass cytometry is implemented in a clinical study. Specifically, the use of whole blood samples versus peripheral blood mononuclear cells (PBMCs), design of mass-tagged antibody panels, technical and analytical implications of sample barcoding, and application of traditional and unsupervised approaches to analyze high-dimensional mass cytometry datasets are discussed. A mass cytometry assay was implemented in a cross-sectional study of 19 women with a history of term or preterm birth to determine whether immune traits in peripheral blood differentiate the two groups in the absence of pregnancy. Twenty-seven phenotypic and 11 intracellular markers were simultaneously analyzed in whole blood samples stimulated with lipopolysaccharide (LPS at 0, 0.1, 1, 10, and 100 ng mL(-1)) to examine dose-dependent signaling responses within the toll-like receptor 4 (TLR4) pathway. Complementary analyses, grounded in traditional or unsupervised gating strategies of immune cell subsets, indicated that the prpS6 and pMAPKAPK2 responses in classical monocytes are accentuated in women with a history of preterm birth (FDR

Background Spina bifida is the most common form of neural tube defects (NTDs). Etiologies of NTDs are multifactorial, and oxidative stress is believed to play a key role in NTD development. Heme oxygenase (HO), the rate-limiting enzyme in heme degradation, has multiple protective properties including mediating antioxidant processes, making it an ideal candidate for study. The inducible HO isoform (HO-1) has two functional genetic polymorphisms: (GT)n dinucleotide repeats and A(-413)T SNP (rs2071746), both of which can affect its promoter activity. However, no study has investigated a possible association between HO-1 genetic polymorphisms and risk of NTDs. Methods: This case-control study included 152 spina bifida cases (all myelomeningoceles) and 148 nonmalformed controls obtained from the California Birth Defects Monitoring Program reflecting births during 1990 to 1999. Genetic polymorphisms were determined by polymerase chain reaction and amplified fragment length polymorphisms/restriction fragment length polymorphisms using genomic DNA extracted from archived newborn blood spots. Genotype and haplotype frequencies of two HO-1 promoter polymorphisms between cases and controls were compared. Results: For (GT)n dinucleotide repeat lengths and the A(-413)T SNP, no significant differences in allele frequencies or genotypes were found. Linkage disequilibrium was observed between the HO-1 polymorphisms (D′: 0.833); however, haplotype analyses did not show increased risk of spina bifida overall or by race/ethnicity. Conclusion: Although, an association was not found between HO-1 polymorphisms and risk of spina bifida, we speculate that the combined effect of low HO-1 expression and exposures to known environmental oxidative stressors (low folate status or diabetes), may overwhelm antioxidant defenses and increase risk of NTDs and warrants further study. Birth Defects Research (Part A), 2014. © 2014 Wiley Periodicals, Inc.

Objective A growing body of evidence suggests that increased blood–brain barrier (BBB) permeability can contribute to the development of seizures. The protease tissue plasminogen activator (tPA) has been shown to promote BBB permeability and susceptibility to seizures. In this study, we examined the pathway regulated by tPA in seizures. Methods An experimental model of kainate-induced seizures was used in genetically modified mice, including mice deficient in tPA (tPA−/−), its inhibitor neuroserpin (Nsp−/−), or both (Nsp:tPA−/−), and in mice conditionally deficient in the platelet-derived growth factor receptor alpha (PDGFRα). Results Compared to wild-type (WT) mice, Nsp−/− mice have significantly reduced latency to seizure onset and generalization; whereas tPA−/− mice have the opposite phenotype, as do Nsp:tPA−/− mice. Furthermore, interventions that maintain BBB integrity delay seizure propagation, whereas osmotic disruption of the BBB in seizure-resistant tPA−/− mice dramatically reduces the time to seizure onset and accelerates seizure progression. The phenotypic differences in seizure progression between WT, tPA−/−, and Nsp−/− mice are also observed in electroencephalogram recordings in vivo, but absent in ex vivo electrophysiological recordings where regulation of the BBB is no longer necessary to maintain the extracellular environment. Finally, we demonstrate that these effects on seizure progression are mediated through signaling by PDGFRα on perivascular astrocytes. Interpretation Together, these data identify a specific molecular pathway involving tPA-mediated PDGFRα signaling in perivascular astrocytes that regulates seizure progression through control of the BBB. Inhibition of PDGFRα signaling and maintenance of BBB integrity might therefore offer a novel clinical approach for managing seizures.

Background Heme oxygenase (HO) is the rate-limiting enzyme in the heme degradation pathway that produces bilirubin. The promoter region of human heme oxygenase-1 (HMOX1) contains a polymorphic (GT)n repeat that can regulate gene expression. Here, we investigated the association of (GT)n repeat length in the HMOX1 promoter region with neonatal hyperbilirubinemia in a population of Japanese term neonates. Methods Using polymerase chain reaction and fragment analysis, we determined the number of (GT)n repeats in 149 Japanese neonates. To omit the effects of the G71R mutation in uridine diphosphoglucuronosyltransferase on hyperbilirubinemia, we excluded 41 neonates with the G71R mutation. As a result, 25 neonates with hyperbilirubinemia and 83 non-hyperbilirubinemic controls were included in this prospective case–control study. Allele and genotype frequencies of (GT)n repeats in the HMOX1 gene were compared between hyperbilirubinemic and non-hyperbilirubinemic control neonates. Results The prevalence of short alleles (

Summary Objectives First, create a clinical severity score for patients with chronic lung disease of infancy (CLDi) following neonatal intensive care unit (NICU) stay. Second, using California wide population-based data, identify factors associated with clinical severity of CLDi at 4–9 months corrected gestational age (CGA). Study Design Pediatric pulmonologists ranked and weighted eight factors reflecting clinical severity of CLDi. Utilizing these data we scored and assigned these to 4–9 month old CGA moderate/severe bronchopulmonary dysplasia (BPD) infants, born

Background Findings from studies examining risk of preterm birth associated with elevated prepregnancy body mass index (BMI) have been inconsistent. Methods Within a large population-based cohort, we explored associations between prepregnancy BMI and spontaneous preterm birth across a spectrum of BMI, gestational age, and racial/ethnic categories. We analysed data for 989 687 singleton births in California, 2007–09. Preterm birth was grouped as 20–23, 24–27, 28–31, or 32–36 weeks gestation (compared with 37–41 weeks). BMI was categorised as

UNLABELLED Maternal obesity is a major source of preventable perinatal morbidity, but studies of the relationship between obesity and preterm birth have been inconsistent. This review looks at two major studies covering just under 3.5 million births, from California, USA, and Sweden. CONCLUSION Inconsistent findings in previous studies appear to stem from the complex relationship between obesity and preterm birth. Initiatives to decrease maternal obesity represent an important strategy in reducing preterm birth.

Postinjury inflammation has been implicated in secondary degeneration following injury to the spinal cord. The cellular inflammatory response to injury has not been described in the lateral compression injury model, although various types of compression injuries account for ∼20% of human spinal cord injuries (SCI). Here, we used forceps to induce a moderate compression injury to the thoracic spinal cord of female Sprague-Dawley rats. We evaluated innate and adaptive components of the inflammatory response at various times postinjury using immunohistochemical techniques. We show that components of innate immunity (e.g., macrophages and dendritic cells) peak between 1 and 2 weeks postinjury but persist through 42 days postinjury (dpi). CD163 and CD206 expression, associated with an anti-inflammatory, reparative phenotype, was upregulated on activated macrophages in the injury site, as were MHC class II antigens. The expression of MHC class II antigens is necessary for the initiation of adaptive immunity and was accompanied by an influx of T cells. T cells were initially restricted to gray matter at the injury epicenter but were later observed throughout the lesioned parenchyma. In summary, we demonstrate that lateral forceps compression of the spinal cord produces a neuroinflammatory response similar to that described in human spinal cord trauma and in other experimental models of spinal cord trauma, thus is an appropriate model to study secondary neurodegeneration in SCI.

The cancer/testis antigen NY-ESO-1 contains an immunodominant HLA-A2-binding peptide (SLLMWITQC), designated S9C, an attractive target for vaccination against several human cancers. As cysteine contains a reactive -SH, the oxidation status of exogenous synthetic peptide is uncertain. We have designed tolerance-breaking DNA fusion vaccines incorporating a domain of tetanus toxin fused to tumor-derived peptide sequences (p.DOM-peptide), placed at the C-terminus for optimal immunogenicity. In a "humanized" HLA-A2 preclinical model, p.DOM-S9C primed S9C-specific CD8+ T cells more effectively than adjuvanted synthetic peptide. A DNA vaccine encoding the full NY-ESO-1 sequence alone induced only weak S9C-specific responses, amplified by addition of DOM sequence. The analog peptide (SLLMWITQL) also primed peptide-specific CD8+ T cells, again increased by DNA delivery. Importantly, T cells induced by S9C-encoding DNA vaccines killed tumor cells expressing endogenous NY-ESO-1. Only a fraction of T cells induced by the S9L-encoding DNA vaccines was able to recognize S9C and kill tumor cells. These data indicate that DNA vaccines mimic posttranslational modifications of -SH-containing peptides expressed by tumor cells. Instability of synthetic peptides and the potential dangers of analog peptides contrast with the ability of DNA vaccines to induce high levels of tumor-lytic peptide-specific CD8+ T cells. These findings encourage clinical exploration of this vaccine strategy to target NY-ESO-1.

UNLABELLED Haem oxygenase-1 (HO-1), the rate-limiting enzyme in haem degradation, plays a role in angiogenesis and vasculogenesis and is highly expressed in the placenta. Deficiencies in HO-1 are associated with several pregnancy disorders, such as recurrent miscarriages and pre-eclampsia. The unique combination of tissue protective, smooth muscle relaxing and angiogenesis regulatory properties makes HO-1 a key player in the maintenance of a healthy pregnancy through a direct effect on placental structural and vascular development, thus affecting foetal development. CONCLUSION Therefore, we conclude that HO-1 plays an important role in placental vasculature development and a deficiency in HO-1 may contribute to pregnancy complications, such as pre-eclampsia, spontaneous abortions and premature births.

For long-term attack on tumor cells in patients with prostate cancer, induction of cytolytic T cells is desirable. Several lineage-specific target proteins are known and algorithms have identified candidate MHC class I-binding peptides, particularly for HLA-A*0201. We have designed tolerance-breaking DNA fusion vaccines incorporating a domain of tetanus toxin fused to candidate tumor-derived peptide sequences. Using three separate peptide sequences from prostate-specific membrane antigen (PSMA) (peptides PSMA(27) , PSMA(663) , and PSMA(711) ), this vaccine design induced high levels of CD8(+) T cells against each peptide in a HLA-A(*) 0201 preclinical model. In contrast, the full-length PSMA sequence containing all three epitopes was poorly immunogenic. Induced T cells were cytotoxic against peptide-loaded tumor cells, but only those against PSMA(27) or PSMA(663) peptides, and not PSMA(711) , were able to kill tumor cells expressing endogenous PSMA. Cytotoxicity was also evident in vivo. The preclinical model provides a powerful tool for generating CD8(+) T cells able to predict whether target cells can process and present peptides, essential for planning peptide vaccine-based clinical trials.

Aim: To compare risk-adjusted outcomes at 18- to 22-month-corrected age for extremely low birth weight (ELBW) infants who never received phototherapy (NoPTx) to those who received any phototherapy (PTx) in the NICHD Neonatal Research Network randomized trial of Aggressive vs. Conservative Phototherapy. Methods: Outcomes at 18 to 22-month-corrected age included death, neurodevelopmental impairment (NDI) and Bayley Scales Mental Developmental Index (MDI). Regression models evaluated the independent association of PTx with adverse outcomes controlling for centre and other potentially confounding variables. Results: Of 1972 infants, 216 were NoPTx and 1756 were PTx. For the entire 501- to 1000-g-BW cohort, PTx was not independently associated with death or NDI (OR 0.85, 95% CI: 0.60–1.20), death or adverse neurodevelopmental endpoints. However, among infants 501–750 g BW, the rate of significant developmental impairment with MDI

Problem Infection during pregnancy can disrupt regulatory/effector immune system balance, resulting in adverse pregnancy and fetal-neonatal outcomes. Heme oxygenase-1 (HO-1) is a major regulatory enzyme in the immune system. We observed maternal immune response dysregulation during late gestational inflammation (LGI), which may be mediated by HO-1. Here, we extend these studies to examine the immune response of offspring. Method of study Pregnant wild-type (Wt) and HO-1 heterozygote (Het) dams were treated with lipopolysaccharide (LPS) or vehicle at E15.5. Pups' splenic immune cells were characterized using flow cytometry. Results CD3+ CD4+ CD25+ (Tregs) and CD3+ CD8+ (Teffs) T cells in Wt and Het were similar in control neonates and increased with age. We showed not only age- but also genotype-specific and long-lasting T-cell dysregulation in pups after maternal LGI. The persistent immune dysregulation, mediated by HO-1 deficiency, was reflected as a decrease in Treg FoxP3 and CD3+ CD8+ T cells, and an increase in CD4+ /CD8+ T-cell and Treg/Teff ratios in Hets compared with Wt juvenile mice after maternal exposure to LGI. Conclusion Maternal exposure to LGI can result in dysregulation of splenic T cells in offspring, especially in those with HO-1 deficiency. We speculate that these immune alterations are the basis of adverse outcomes in neonates from mothers exposed to low-grade (subclinical) infections.

Antigen-specific CD4(+) T cells are central to natural and vaccine-induced immunity. An ongoing antigen-specific T-cell response can, however, influence surrounding T cells with unrelated antigen specificities. We previously observed this bystander effect in healthy human subjects following recall vaccination with tetanus toxoid (TT). Since this interplay could be important for maintenance of memory, we have moved to a mouse model for further analysis. We investigated whether boosting memory CD4(+) T cells against TT in vivo would influence injected CD4(+) TCR transgenic T cells (OT-II) specific for an unrelated OVA peptide. If OT-II cells were pre-activated with OVA peptide in vitro, these cells showed a bystander proliferative response during the ongoing parallel TT-specific response. Bystander proliferation was dependent on boosting of the TT-specific memory response in the recipients, with no effect in naive mice. Bystander stimulation was also proportional to the strength of the TT-specific memory T-cell response. T cells activated in vitro displayed functional receptors for IL-2 and IL-7, suggesting these as potential mediators. This crosstalk between a stimulated CD4(+) memory T-cell response and CD4(+) T cells activated by an unrelated antigen could be important in human subjects continually buffeted by environmental antigens.

Objectives To assess the influence of clinical status on the association between total plasma bilirubin and unbound bilirubin on death or adverse neurodevelopmental outcomes at 18–22 months corrected age in extremely low birth weight infants.

Aim: As phototherapy (PT) devices employ a variety of broadband light sources, we developed and tested a standardized bench method for evaluating the efficacy of some devices. Methods: To evaluate efficacy, we quantified the in vitro photodegradation rate (expressed as t1/2) of unconjugated bilirubin in solution at 37°C during exposure to a given light source at its mean delivered irradiance to the 2D body surface area (BSA) of newborn models. Reproducibility (between-day variation) of the method was determined at irradiance levels from 10 to 70 μW/cm2/nm on three different days. Results: Between-day t1/2 measurements had coefficients of variation from 3% to 10%. When t1/2 values were normalized to the exposable 2D horizontal BSA, halogen lamp devices, without and with fiberoptics, were least effective (t1/2= 60–108 min and 100–126 min for preterm and term models, respectively). Fluorescent tube devices had t1/2= 19–78 min and 25–78 min, for preterm and term models, respectively. Light-emitting diode (LED)-based devices yielded the shortest t1/2 values (16–24 min) for preterm and term newborn models. Conclusions: We demonstrated the applicability of the method through the determination of the efficacy of several commercially available PT devices. This standardized method is reproducible and effectively evaluates the relative in vitro efficacy of various devices and may guide further in vitro and in vivo evaluations of devices.

Neonatal microstructural development in the posterior limbs of the internal capsule (PLIC) was assessed using diffusion tensor imaging (DTI) fractional anisotropy (FA) in 24 very-low-birthweight preterm infants at 37 weeks' gestational age and compared with the children's gait and motor deficits at 4 years of age. There were 14 participants with normal neonatal FA values (seven females, seven males; born at 27.6 weeks [SD 2.3] gestational age; birthweight 1027g [SD 229]) and 10 participants with low FA values in the PLIC (four females, six males; born at 28.4 weeks [SD 2.0] gestational age; birthweight 1041g [SD 322]). Seven of the 10 children with low FA and none of the children with normal FA had been diagnosed with CP by the time of gait testing. Among children with low neonatal FA, there was a strong negative correlation between FA of the combined left and right side PLIC and log NI (r=-0.89, p=0.001) and between FA and GMFCS (r=-0.65, p=0.04) at 4 years of age. There was no correlation between FA and gait NI or GMFCS at 4 years of age among children with normal neonatal FA. This preliminary study suggests neonatal DTI may be an important predictor of the severity of future gait and motor deficits.

BACKGROUND Lactate concentrations are increasingly quantified in dogs using point-of-care instruments, but often without canine-specific method evaluation and instrument-specific reference intervals. OBJECTIVES The objectives of this study were to 1) determine the precision of the Accutrend (Roche Diagnostics) for lactate determination in dogs, 2) determine the accuracy of the Accutrend using the Rapidlab 865 (Bayer Diagnostics) as the reference method, and 3) establish and compare reference intervals for lactate concentration in clinically healthy dogs for both instruments. METHODS Precision was evaluated using low and high control materials, and variable (1 drop) and fixed (25 microL) sample volumes. Accuracy was determined by comparing lactate concentrations obtained with the Accutrend with those from the Rapidlab 865 in 273 heparinized canine jugular venous blood samples from 100 clinically healthy dogs and 107 systemically ill dogs (173 samples). Lactate reference intervals were established for both analyzers using data from the 100 clinically healthy dogs. RESULTS The precision of the Accutrend was good (coefficients of variation, < or = 5.3%) for 25-microL samples but not when a drop was used. Lactate concentrations obtained on the Accutrend correlated poorly with those from the Rapidlab 865 (r = 0.864, mean bias = 0.66 mmol/L, 95% confidence interval [CI] = 0.57-0.76 with 95% limits of agreement = -0.87 (lower limit, 95% CI = -1.03 to -0.71) and 2.20 (upper limit, 95% CI = 2.04 to 2.36). The reference interval for canine lactate concentration on the Accutrend was 1.2-3.1 mmol/L compared with 0.46-2.31 mmol/L on the Rapidlab. CONCLUSION Although precision was good with fixed sample volumes, blood lactate concentrations obtained on the Accutrend were significantly different than those on the Rapidlab 865, with systematic and random errors resulting in a positive bias. Further evaluation of the Accutrend is required before its use in dogs can be recommended.

Background: Lactate concentration often is quantified in systemically ill dogs and interpreted based on human data. To our knowledge, there are no published clinical studies evaluating serial lactate concentrations as a prognostic indicator in ill dogs. Objectives: Our objective was to perform a prospective study, using multivariate analysis, to determine whether serial lactate concentrations were associated with outcome in ill dogs requiring intravenous fluids. Methods: Eighty sick dogs had lactate concentrations evaluated, using an analyzer that measures lactate in the plasma fraction of heparinized whole blood, at 0 hours and 6 hours after initiation of treatment. Severity of illness and outcome (survivor, nonsurvivor) were determined by reviewing the patient’s record 2 weeks after admission. Lactate concentrations, age, body weight, gender, and severity of illness were evaluated using multivariate analysis to determine their effects on outcome. Results: Dogs with lactate concentrations greater than the reference interval at 6 hours were 16 times (95% confidence interval 5 2.32‐112.71 times, P , .01) more likely not to survive compared to dogs with lactate concentrations within the reference interval. Lactate concentrations above the reference interval at 0 hours were not significantly related to outcome. However, hyperlactatemia that did not improve by 50% within 6 hours was significantly associated with mortality (P 5 .024). Conclusion: Dogs with a lactate concentration higher than the reference interval at 6 hours were more likely not to survive. These results indicate an association between lactate concentration and outcome and emphasize the importance of serial lactate concentrations in evaluating prognosis. (Vet Clin Pathol. 2007;36:234‐239) � 2007 American Society for Veterinary Clinical Pathology

UNLABELLED: The present multicenter study analysed the relative impact of maternal and infant factors on serum bilirubin levels at 72 +/- 12 h in exclusively breastfed vs formula-fed term infants. End-tidal carbon monoxide levels corrected for ambient air (ETCOc), an index of bilirubin production, were measured in exclusively breastfed (B = 66) or formula-fed (F = 210) term infants at 2-8 h of age. Inclusion criteria included cesarean section to ensure a 3 d hospitalization, birthweight > or = 2,500 g, gestational age >37 wk and absence of any illness. The ETCOc for B infants and F infants did not differ significantly (1.3 +/- 0.7 ppm vs 1.3 +/- 0.8 ppm). The serum bilirubin level at 72 +/- 12 h was significantly higher in B infants than in F infants (8.5 +/- 3.4mg dl(-1) vs 6.7 +/- 3.4mg dl(-1) p < 0.001), as was the percentage weight loss from birthweight. Serum bilirubin levels were significantly higher in infants who were male, who did not have meconium-stained amniotic fluid, and in those whose mothers were insulin-dependent diabetics or hypertensive. There was no difference between groups in the need for phototherapy or exchange transfusion. CONCLUSION: Although higher bilirubin levels were observed in group B at 72 +/- 12 h compared with group F, this finding was not of clinical or therapeutic consequence in this study. The lack of difference in ETCOc between the groups may be a factor of the timing of ETCOc measurement in this study, or may suggest that early increased bilirubin production is not a significant contributor to jaundice observed in exclusively breastfed infants. Key words: bilirubin, breastfeeding, jaundice

UNLABELLED The aim of this study was to evaluate the change in the treatment of neonatal jaundice following introduction of the "American Academy of Pediatrics' Practice Parameter for the management of hyperbilirubinemia in the healthy term newborn". In a historical control observation cohort study, we examined the rate of phototherapy and exchange transfusions among full-term (> or = 37 wk gestation) and near-term (gestational age between 35 and 37 wk and birthweight > 2000 g) infants in two community hospitals. The study included all consecutive infants born during two 15-mo study periods immediately before and after the introduction of the new guidelines. Data were prospectively recorded in a computerized database. The rate of phototherapy significantly decreased in the second study period from 7.9% (514/6499) to 2.9% (251/8650) (p < 0.0001) among full-term infants, and from 20.9% (102/489) to 9.4% (47/502) (p < 0.0001) in near-term infants. The use of exchange transfusion was significantly higher (p < 0.001) in the first compared to the second period: 0.2% (15/6499) vs 0.03% (3/8650). A significant decrease was found when the data from each hospital were analyzed separately. CONCLUSION A significant decrease in the use of phototherapy and exchange transfusion occurred after the publication of the new practice parameters. This trend was observed for both term and preterm newborns, although the new guidelines were not intended for infants born before term.

The objective of this study was to examine the association of carboxyhemoglobin (COHb) and plasma cotinine levels among pregnant women who smoke cigarettes and their fetuses. Fifteen pregnant women who smoked and their fetuses undergoing cordocentesis had blood samples analysed simultaneously for COHb and cotinine. Linear regression was used to test for associations among study variables. Significant maternal-fetal associations were observed both with COHb (r = 0.72, p = 0.003) and with cotinine (r = 0.96, p = 0.003). Maternal cotinine levels were correlated with maternal and fetal COHb levels (r = 0.96, p = 0.003; r = 0.99, p = 0.0003, respectively). Fetal cotinine levels were correlated with COHb in maternal and in fetal blood (r = 0.81, p = 0.0003; r = 0.88, p

We studied how carbon monoxide (CO) is distributed within the human body through quantitation of CO concentrations in postmortem tissue samples from fatalities including possible CO exposure. Stored, frozen tissues were diced, sonicated in water, and 0.01-8.0 mg wet weight (ww) tissues were incubated with sulfosalicylic acid in CO-purged, septum-sealed vials. CO released into the headspace was quantitated by reduction gas chromatography. Mean tissue CO concentrations (pmol/mg ww) from subjects diagnosed to have no known CO exposure (control, N=14), died from fire (N=13), and CO asphyxiation (N=7), respectively, were: adipose (2;13;9), brain (3;13;65), muscle (15;97;297), heart (30;99;371), kidney (22;432;709, lung (54;690;2638), spleen (73;1366;3548), and blood (162;2238;5070). Carboxyhemoglobin concentrations were 1.4%, 25.2%, and 69.1% of total hemoglobin, respectively. We conclude that measurements of CO concentration in a variety of tissues can be used as markers for the degree of exogenous CO exposure and the identification of possible causes of death. Language: en

Haem oxygenases (HO) are involved in the catalytic breakdown of haem to generate carbon monoxide (CO), iron and biliverdin. It is widely accepted that products of haem catabolism are involved in biological signaling in many physiological processes. Conclusions to most studies in this field have gained support from the judicious use of synthetic metalloporphyrins such as chromium mesoporphyrin (CrMP) to selectively inhibit HO. However, metalloporphyrins have also been found to inhibit other haem-dependent enzymes, such as nitric oxide synthase (NOS), cytochromes P-450 (CYPs) and soluble guanylyl cyclase (sGC), induce the expression of HO-1 or exhibit varied toxic effects. To obviate some of these problems, we have been examining non-porphyrin HO inhibitors and the present study describes imidazole–dioxolane compounds with high selectivity for inhibition of HO-1 (rat spleen microsomes) compared to HO-2 (rat brain microsomes) in vitro. (2R,4R)-2-[2-(4-chlorophenyl)ethyl]-2-[(1H-imidazol-1-yl)methyl]-4-methyl-1,3-dioxolane hydrochloride) was identified as the most selective inhibitor with a concentration of 0.6 μM inhibiting HO-1(inducible) by 50% compared with 394 μM for HO-2 (constitutive). These compounds were found to have no effects on the catalytic activities of rat brain NOS and lung sGC, but were potent inhibitors of microsomal CYP2E1 and CYP3A1/3A2 activities. In conclusion, we have identified imidazole–dioxolanes that are able to inhibit microsomal HO in vitro with high selectivity for HO-1 compared to HO-2, and little or no effect on the activities of neuronal NOS and sGC. These molecules could be used to facilitate studies on the elucidation of physiological roles of HO/CO in biological systems. British Journal of Pharmacology (2006) 147, 307–315. doi:10.1038/sj.bjp.0706555

Vaccination against target antigens expressed by cancer cells has now become a realistic goal. DNA vaccines provide a direct link between identification of genetic markers in tumors and vaccine formulation. Simplicity of manufacture facilitates construction of vaccines against disease subsets or even for individual patients. To engage an immune system that exists to fight pathogens, we have developed fusion gene vaccines encoding tumor antigens fused to pathogen-derived sequences. This strategy activates high levels of T-cell help, the key to induction and maintenance of effective immunity. We have dissected the immunogenic tetanus toxin to obtain specific sequences able to activate antibody, CD4+, or CD8+ T cells to attack selected fused tumor antigens. Principles established in preclinical models are now being tested in patients. So far, objective immune responses against idiotypic antigen of neoplastic B cells have been observed in patients with B-cell malignancies and in normal transplant donors. These responses provide a platform for testing physical methods to improve DNA delivery and strategies to boost responses. For cancer, demands are high, because vaccines have to activate powerful immunity against weak antigens, often in a setting of immune damage or tolerance. Vaccination strategies against cancer and against microbes are sharing knowledge and technology for mutual benefit.

Novel N-glycosylation sites are introduced by somatic mutation into the V genes of the majority of follicular lymphomas. Sites are positively selected and rare in normal memory B cells, indicating a potential role in tumour survival in the germinal centre (GC). The incidence of c. 40% in diffuse large B-cell lymphomas (DLBCL) parallels the known heterogenity of the disease. Immunodeficiency-related non-Hodgkin's lymphomas (NHL) include post-transplant lymphoproliferative disorders (PTLD) and acquired immunodeficiency syndrome-related NHL (AIDS-NHL). Most PTLD derive from B cells that carry mutated VH genes and that have completed the GC reaction. All AIDS-NHL carry mutated VH genes and variable features of GC or post-GC cells. To determine if N-glycosylation is a feature of immunodeficiency-related lymphomas, we analysed the VH genes of 19 PTLD and 36 AIDS-NHL. Novel sites were rare in PTLD (4/19), similar to memory B cells (P = 0·15). AIDS-NHL, including DLBCL and Burkitt's lymphomas (BL), showed heterogeneity with 16 of 36 (44%) having novel sites. The findings indicate no selection of N-glycosylation sites in PTLD, consistent with post-GC features. The variable incidence of N-glycosylation sites in AIDS-NHL mirrors that in DLBCL and sporadic BL of immunocompetent hosts, supporting the known heterogeneity of these disorders, and possibly pointing to distinct routes of tumour development.

Recently, a high incidence of novel N-glycosylation sites introduced by somatic mutation was observed in the immunoglobulin variable region genes of follicular lymphoma. As these are positively selected and are uncommon in normal B cells, they may have a role in tumour growth and behaviour. Sites are not characteristic of chronic lymphocytic leukaemia or myeloma, but are detectable in approximately 50% of diffuse large cell lymphomas. Another feature of the variable region genes of certain lymphomas is ongoing somatic mutation. To determine whether glycosylation is associated with this phenomenon, we analysed variable region gene sequences of Burkitt's lymphoma (BL) and mucosa-associated lymphoid tissue (MALT) lymphoma. Novel sites were common in endemic BL (82%) and in 4/5 patients with Iranian BL. However, sporadic BL had a lower incidence (43%). Patients with MALT lymphoma had a low frequency (9%) of novel sites, comparable to normal B cells. These findings distinguish glycosylation sites from ongoing mutation and may reflect different environmental influences on these tumours.

DNA vaccines induce CTL attack on target tumor epitopes, but tumor elimination in vivo also requires sufficient effector CTL to enter the site, guided by inflammatory chemokines. Many herpesviruses contain genes for chemokine and chemokine receptor-like proteins to protect infected cells from immune attack. To assess if this evasion strategy could protect tumor cells, we used a model where CTL specific for a single epitope were the only effectors. Following DNA vaccination, CTL eliminated tumor cells from a subcutaneous site. However, introducing a viral gene encoding a secreted broad-spectrum chemokine-binding protein (M3) into tumor cells completely blocked CTL attack. Transduced tumor cells also protected neighboring non-transduced tumor. These findings confirm the importance of chemokines for migration of CTL to a non-lymphoid site. They may have relevance for escape of human virus-associated malignancies, and raise the question of whether analogous molecules might contribute to the failure of CTL to eliminate tumors.

DNA vaccines induce immune responses against encoded proteins, and have clear potential for cancer vaccines. For B-cell tumours, idiotypic (Id) immunoglobulin encoded by the variable region genes provides a target antigen. When assembled as single chain Fv (scFv), and fused to an immunoenhancing sequence from tetanus toxin (TT), DNA fusion vaccines induce anti-Id antibodies. In lymphoma models, these antibodies have a critical role in mediating protection. For application to patients with lymphoma, two questions arise: first, whether pre-existing antibody against TT affects induction of anti-scFv antibodies; second, whether individual human scFv fusion sequences are able to fold consistently to generate antibodies able to recognize private conformational Id determinants expressed by tumour cells. Using xenogeneic vaccination with scFv sequences from four patients, we have shown that pre-existing anti-TT immunity slows, but does not prevent, anti-Id antibody responses. To determine folding, we have monitored the ability of nine DNAscFv-FrC patients' vaccines to induce xenogeneic anti-Id antibodies. Antibodies were induced in all cases, and were strikingly specific for each patient's immunoglobulin with little cross-reactivity between patients, even when similar VH or VL genes were involved. Blocking experiments with human serum confirmed reactivity against private determinants in 26-97% of total antibody. Both immunoglobulin G1 (IgG1) and IgG2a subclasses were present at 1.3 : 1-15 : 1 consistent with a T helper 2-dominated response. Xenogeneic vaccination provides a simple route for testing individual patients' DNAscFv-FrC fusion vaccines, and offers a strategy for production of anti-Id antibodies. The findings underpin the approach of DNA idiotypic fusion vaccination for patients with B-cell tumours.

A retrospective study was undertaken on 56 dogs treated for nasal tumours by megavoltage radiotherapy with a hypofractionated schedule consisting of four doses of 9 Gy given at intervals of seven days. The dogs were followed until they died or were euthanased. The clinical signs had improved in 53 of the 56 dogs by the end of the treatment schedule. Mild acute radiation side effects were observed in the majority of the dogs but late radiation side effects were rare. Kaplan-Meier survival analysis revealed a median survival time after the final dose of radiation of 212 days. The one- and two-year survival rates were 45 per cent and 15 per cent. Fifty of the dogs were euthanased because the initial clinical signs recurred.

Background We describe an in vitro tumour model for targeted radiotherapy and gene therapy that incorporates cell population heterogeneity. Materials and methods Transfectant mosaic spheroids (TMS) and transfected mosaic monolayers (TMM) are composed of two cell populations derived from a single cell line. The cells of one population were transfected with the noradrenaline transporter gene (NAT), allowing active uptake of a radiolabelled targeting agent meta-[131I]iodobenzylguanidine ([131I]MIBG); the other population of cells was derived from the same parent line and transfected with a marker gene – green fluorescent protein (GFP). After treatment with [131I]MIBG, cell kill was determined in TMM by clonogenic assay and in TMS by clonogenic assay and spheroid growth delay. Results We have used the TMS model to assess the ‘radiological bystander effect’ (radiation cross-fire) conferred by the β-emitting radiopharmaceutical [131I] MIBG whose cellular uptake is facilitated by the transfected gene encoding NAT. We show that cell killing by [131I]MIBG in both TMS and TMM cultures increased in direct proportion to the fraction of NAT-transfected cells and that the degree of cell killing against fraction transfected was greater in TMS, suggestive of a greater bystander effect in the three-dimensional culture system. Conclusions TMS provide a useful model for assessment of the effectiveness of targeted radiotherapy in combination with gene therapy when less than 100% of the target cell population is expressing the NAT transgene. Further, this novel model offers the unique opportunity to investigate radiation-induced bystander effects and their contribution to cell cytotoxicity in radiotherapy and other gene therapy applications. Copyright © 2002 John Wiley & Sons, Ltd.

DNA vaccination is a novel approach for inducing immunity against target antigens. It provides a direct link between identification of genes encoding these antigens and incorporation of the gene sequences into a vaccine vehicle. Identification of candidate genes is proceeding very rapidly both for infectious organisms and for cancer cells. One advantage is that DNA appears to activate all pathways of immunity, especially cytotoxic T-cell responses, which have been difficult to induce with protein vaccines. For viruses, including those which have caused problems for blood transfusion, DNA vaccination could be used for prevention. However, for chronic infection, or for cancer, vaccination will be performed in a therapeutic setting. For this situation, it is probable that immune-activating sequences will have to be included in the vaccine. The ease of manipulation of gene sequences, together with the increasing knowledge of the operation of the immune system, means that we now have the tools to take vaccines into the next exciting stage of development.

Background Although [I-131]meta-iodobenzylguanidine (MIBG) is currently one of the best agents available for targeted radiotherapy, its use is confined to a few neural crest derived tumours which accumulate the radiopharmaceutical via the noradrenaline transporter (NAT). To determine whether this drug could be used for the treatment of non-NAT expressing tumours following genetic manipulation, we previously showed that plasmid mediated transfection of NAT into a non-NAT expressing glioblastoma cell line, UVW, endowed the host cells with the capacity to actively accumulate [I-131]MIBG. We now present data defining the conditions required for complete sterilisation of NAT transfected cells cultured as multicellular spheroids and treated with [I-131]MIBG. Methods NAT transfected UVW cells, grown as monolayers and spheroids, were treated with various doses of [I-131]MIBG and assessed for cell kill by clonogenic survival and measurement of spheroid volume over time (growth delay). Spheroids were left intact for different time periods to assess the effect of radiation crossfire on cell death. Results and Conclusions Total clonogen sterilisation was observed when the cells were grown as three-dimensional spheroids and treated with 7 MBq/ml [I-131]MIBG. The, added benefit of radiation crossfire was demonstrated by the improvement in cell kill achieved by prolongation of the maintenance of [I-131]MIBG treated spheroids in their three-dimensional form, before disaggregation and clonogenic assay. When left intact for 48 h after treatment, spheroid cure was achieved by exposure to 6 MBq/ml [I-131]MIBG. These results demonstrate that the efficiency of cell kill by [I-131]MIBG targeted therapy is strongly dependent on beta -particle crossfire irradiation. This gene therapy/targeted radiotherapy strategy has potential for [I-131]MIBG mediated cell kill in tumours other than those derived from the neural crest.

Pneumococcal serotype-specific anti-capsular polysaccharide antibodies protect against invasive pneumococcal disease. Within an individual the diversity of these antibodies is limited. To evaluate the repertoire of antibodies to pneumococcus and determine whether oligoclonality is seen both between serotypes and between individuals, we sampled the B cell repertoire induced by polysaccharide and conjugate vaccine in adult volunteers. Fifteen hybridomas secreting pneumococcus-specific monoclonal antibodies were generated from five volunteers. Ten were isotype switched, six were IgG2 and four were IgA. These included two isotype switch variants of the same clone. V(H)3 and V(kappa)2 were used by 10/15 and 7/13 of the sequenced clones, respectively, with identical genes, V(H)3-48 and V(kappa)2-A17 used by a number of volunteers to a variety of serotypes. VDJ junctional characteristics and complementarity-determining region (CDR) 3 length were variable. High levels of somatic mutation in CDR1 and 2, inconsistent with a primary response, were found in 10/11 of the isotype-switched antibodies, including those induced by plain polysaccharide antigens. These data suggest that wild-type infection or nasopharyngeal carriage of Streptococcus pneumoniae in adults may induce memory and the response to subsequent immunization with plain polysaccharide or conjugate pneumococcal vaccines may have the characteristics of a secondary response.

We investigated 16 patients with elevated serum monoclonal IgG and a leukaemic B-cell lymphocytic disorder different from multiple myeloma. Their clinical history was that of a non-aggressive disease with dominant splenomegaly and long survival. Whereas abnormal blood and bone marrow cells were predominantly small lymphocytes with a few lymphoplasmacytoid cells, histopathological features included a lymphoplasmacytic infiltrate in eight cases. Most frequently, abnormal blood cells displayed a CD19+CD5-CD23+/- immunophenotype different from that of chronic lymphocytic leukaemia, except in two cases with a CD19+CD5+CD23+ phenotype. Interestingly, a coexistent serum monoclonal IgM and/or surface IgMG+ with identical light chain was identified in 10 patients, whereas in the remaining six patients only IgG expression was determined. VH gene analysis was performed in eight patients to investigate the clonal origins of tumour cells. All cases utilized the VH3 family, with evidence of extensive somatic mutations and intraclonal homogeneity in all cases. VH gene analysis indicated a clonal relationship between cells expressing IgM and IgG, with one case being biclonal. Cytogenetic evaluation showed a high incidence of trisomy 12 (60%) and 13q14 deletion (40%). In conclusion, we have described an unusual subset of low-grade lymphoma with high-serum IgG and frequent lymphoplasmacytoid features in which tumour cells derive from post-follicular memory B cells undergoing isotype switching with some cases arrested at both the IgM and IgG stage and others as IgG-positive cells only.

Immunoglobulin E (IgE)-dependent mechanisms play a pivotal role in mediating allergic disease. Previously, VH-Cepsilon transcripts from blood or spleen of atopic asthmatics have been analysed for VH gene usage and patterns of somatic mutation. An over-representation of the minor VH5 family has been observed, consistent with a superantigen drive. As local mucosal events in IgE production may be more significant in the disease process, we have analysed VH-Cepsilon transcripts from a bronchial biopsy of a patient with severe asthma. VH5 predominance was confirmed with 10 of 30 unique clones derived from this family. Repeated sequences, some with intraclonal variation, revealed clonal expansion and continuing mutational activity at the site. Unexpectedly, three unmutated VH-Cepsilon sequences were found, indicating that isotype switching to IgE can occur without mutation. Detection of a sister clone with extensive mutations was again consistent with local mutational activity. Evidence for local isotype switching was obtained by identification of clonally related immunoglobulin M (IgM), immunoglobulin G (IgG) and immunoglobulin E (IgE) sequences. However, in contrast to findings in blood, no IgG4 transcripts clonally related to IgE were detected, suggesting that the balance between synthesis of IgG4 and IgE may differ between systemic and local sites. These data confirm a VH5 bias in IgE, and support the concept that IgE-synthesizing B cells arise via local differentiation.

The prevalence of malaria and other infections in tropical Africa provides a setting for the emergence of B-cell tumours distinct from that in Western countries. Attempts to draw comparisons with Western lymphomas have led to difficulties, with so-called African chronic lymphocytic leukaemia (CLL) having a different pattern of incidence from Western CLL. Splenomegaly is common in African CLL, and this has posed diagnostic problems in differentiating the tumour from malaria-associated hyper-reactive malarial splenomegaly (HMS). One feature of the splenomegalic form of African CLL is that the tumour cells often possess short but fine cytoplasmic projections reminiscent of those observed in Western splenic lymphoma with villous lymphocytes (SLVL). Analysis of Ig VH genes both facilitates discrimination between clonal B-cell tumours and HMS, and reveals the differentiation status of the cell of origin. This study indicated that VH genes of nine cases of clonal splenic B-cell tumours with villous lymphocytes from Ghana were relatively unmutated, consistent with an origin from a naive B cell. These features differ from SLVL which arises from a post-follicular antigen-selected B cell. One possibility is that these splenic B-cell tumours derive from a splenic T-independent B cell, with malaria infection as a potential influence.

Cases of Burkitt's lymphoma (BL) from north-western Iran were investigated for the usage and somatic mutational pattern of their immunoglobulin variable region genes. Potentially functional V(H) genes were amplified from 6/12 of the tumour masses and all of these were derived from the V(H)3 family, with 4/6 being derived from the most commonly used V(H)3 family member, V3-23. All of the tumour sequences were mutated from their germline counterparts, to varying degrees, with a mean level of 5.8%, indicating that the cell of origin had encountered the germinal centre. Intraclonal sequence heterogeneity was also evident in 4/6 of the lymphomas, showing that the tumour cells had undergone further somatic mutation following neoplastic transformation. Analysis of the five potentially functional mutated V(H) sequences showed a significant clustering of replacement mutations in the complementarity-determining region 2, consistent with a role for antigen in selection of tumour cell sequences. The pattern of extensive somatic mutation, and intraclonal variation, in these mainly EBV+ve tumours, was similar to that previously reported in V(H) sequences of EBV+ve endemic BL (eBL) and EBV-ve sporadic BL (sBL), with the mean level of somatic mutation lying between those reported for eBL (7.7%) and sBL (4.0%). However, VH gene bias and the distribution of mutations in the Iranian cases showed features which differed from those reported for endemic or sporadic BL.

Isotype switching to IgE contributes to atopic asthma, therefore strategies to divert this process to alternative isotypes could have therapeutic relevance. It is known that patients with allergic disease have serum IgE and IgG4 antibodies with similar specificities, and that cytokines such as IL-4 mediate switching to both of these isotypes. Availability of variable region gene analysis has allowed us to probe isotype variants at the single-cell level. An earlier report described identification in a single atopic patient of short transcripts with a common complementarity-determining region "clonal signature" in combination with C mu, C gamma4 and C epsilon. We have extended this analysis, and have identified V(H)-Cgamma4 transcripts with clear clonal relationship to IgE-derived sequences in blood lymphocytes from three of four patients with atopic asthma. No other IgG subclasses were detected, confirming the link between IgE and IgG4. Full sequences were obtained from each clonally related isotype in all patients, and showed extensive somatic mutation. As previously found for IgE, the IgG4 isotypes had evident intraclonal variation. There were shared mutations between isotypes, but also many differences, indicative of separate cell populations with divergent mutational histories. These findings indicate that, in atopic patients, an individual B cell commonly switches to either IgE or IgG4. Cells producing each isotype then co-exist in the recirculating pool, and the balance between them may influence the disease process.

Conventional brain-imaging modalities may be limited by high cost, difficulty of bedside use, noncontinuous operation, invasiveness or an inability to obtain measurements of tissue function, such as oxygenation during stroke. Our goal was to develop a bedside clinical device able to generate continuous, noninvasive, tomographic images of the brain using low-power nonionizing optical radiation. We modified an existing stage-based time-of-flight optical tomography system to allow imaging of patients under clinical conditions. First, a stationary head-band consisting of thin, flexible optical fibers was constructed. The headband was then calibrated and tested, including an assessment of fiber lengths, the existing system software was modified to collect headband data and to perform simultaneous collection of data and image reconstruction, and the existing hardware was modified to scan optically using this headband. The headband was tested on resin models and allowed for the generation of tomographic images in vitro; the headband was tested on critically ill infants and allowed for optical tomographic images of the neonatal brain to be obtained in vivo.